NZ787544A

NZ787544A - Methods and compositions for the treatment of degenerate bone

Info

Publication number: NZ787544A
Application number: NZ787544A
Authority: NZ
Inventors: Colin D White; Edward S Ahn; Chia En Lin
Original assignee: Anika Therapeutics Inc
Filing date: 2017-04-26
Publication date: 2022-04-29

Abstract

The present disclosure relates to methods and compositions for the treatment of degenerate bone in a patient. In some embodiments, the methods and compositions disclosed herein are useful in the treatment, prevention, or in delaying the progression of a bone disease linked to bone degeneration, such as osteoarthritis ("OA"), rheumatoid arthritis, and a vascular necrosis.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF DEGENERATE CROSS REFERENCE TO RELATED APPLICATIONS This application claims the beneﬁt of ty of US. Provisional Application No. ,313, ﬁled April 27, 2016, the contents of which are hereby incorporated by reference.

FIELD OF INVENTION The present disclosure relates to methods and itions for the treatment of degenerate bone in a patient. In some embodiments, the methods and compositions disclosed herein are useful in the treatment, prevention, or in delaying the ssion of a bone disease linked to bone degeneration, such as osteoarthritis ("OA"), rheumatoid arthritis, and lar necrosis.

BACKGROUND Areas of degenerate bone can lead to a host of issues for patients. For example, the onset and progression of symptomatic OA, toid arthritis, and avascular necrosis are thought to be linked to areas of degenerate bone in or adjacent to the affected area. While the etiologies of these diseases are different, each is often associated with signiﬁcant pain and loss of function. Slowing, arresting, and repairing bone degeneration can reduce pain and slow, prevent, or reverse disease progression.

One example of a pathology thought to be linked to degenerate bone is OA.

Osteoarthritis is the most common form of arthritis, affecting the hands, knees, hips, spine, and other joints, and is a leading cause of lost productivity, estimated to affect approximately 27 n Americans. Arthritis Foundation: What is Osteoarthritis, available at http :.//'Www. arthriti s.orgfalz‘outuarthriti s/types/osteoarthriti s/whatui s—osteoarthr‘i tiSphp (last visited April 12, 2017), the contents of which are incorporated herein by reference in their entirety. OA results in damage to cartilage in the joint, pain, ng, and movement ms. As OA progresses, bone in the region begins to degenerate, resulting in bone spurs and further inﬂammation. The etiology of OA is not fully understood, but is thought to include causes such as trauma (e.g., fractures), degeneration, inﬂammation, ischemia, congenital joint abnormalities, metabolic defects, endocrine and neuropathic diseases, and infections.

Patients who initially present with painful bone disease linked to bone ration are usually treated non-surgically. rgical treatments are modestly effective at temporarily relieving pain, but are not risk free. For example, pharmacologic intervention (e.g., non-steroidal nﬂammatory drugs) has been reported to be associated with significant complications, such as gastric ulcers, strokes and heart attacks. Generally speaking, non-surgical interventions are only eff1cacious for alleviating the pain caused by bone disease and do not slow or prevent disease ssion.

When patients fail rgical ent for bone disease, surgical ention, whether invasive or minimally invasive, is often recommended. Current ve surgical approaches aim to alter the biomechanical forces on areas of the affected joint, either by shifting weight from an area of damaged cartilage to an area of healthy cartilage by osteotomy or other means, or by completely replacing the joint and restoring biomechanical function with the use ofjoint replacement hardware. Minimally invasive surgical approaches include the treatment of areas of degenerate bone, such as bone marrow lesions ("BMLs"), whose presence has been associated with the onset and progression of OA. See, e.g., Sharkey, P. F. el al. Am. J. Orthop. (Belle MeadNJ) 2012, 41(9), 413-17, the contents of which are incorporated herein by reference in their entirety. Minimally ve prior art treatments for bone degeneration include the injection of a variety of m ate cements ("CPCs") into the area of degenerate bone, such that the CPCs biomechanically stabilize the joint. See, e.g., Hisatome, T. el al., J. Biomed. Mater. Res. 2002, 59(3), 490-98 (creating subchondral access into the femoral condyle while preserving articular cartilage by using an augmentation material such as a CPC because of its mechanical strength and using the CPC to fill a large defect to prevent collapse and provide the necessary t to preserve the articular cartilage), rjee, D. el al. Clin. Orthop. Relat. Res. 2015, 473(7), 2334-42 (disclosing injection of a CPC with a pore size of 150-500 um into the subchondral bone in order to improve its structural integrity and biomechanical th), the contents of all of the foregoing of which are incorporated herein by reference in their ties. Other prior art CPCs have been used for fracture ﬁxation or to ﬁll bony voids or gaps of the skeletal system (e.g., extremities, craniofacial, spine, and pelvis). See, e. g., Nishizuka, T. el al. PLoS One 2014, 9(8), e104603, the contents of which are incorporated herein by reference in their entirety.

Importantly, these prior art treatments have signiﬁcant drawbacks when used to treat bone diseases such as 0A. For example, invasive surgical approaches carry considerable risk, including infection, deep vein thrombosis, and — in extreme cases — death.

Moreover, total joint replacements are effective for only approximately 20 years. Prior art lly invasive treatments for bone disease have also been shown to be ineffective in patients with more advanced bone degeneration. See, e.g., Chatterj ee, D. el al. Clin. Orthop.

Relat. Res. 2015, 473(7), 2334-42, the contents of which are incorporated herein by reference in their entirety. Finally, use of both invasive and non-invasive prior art ents that provide for biomechanical stabilization of bone result in signiﬁcant pain post-operatively. rmore, prior art treatments that provide for biomechanical stabilization of bone also do not address the causative factors of bone disease characterized by bone degeneration. During the onset and progression of bone disease, bone in the ed area is subject to insult by inﬂammatory and/or non-inﬂammatory mediators. These mediators emanate from the joint space and pass through channels in the subchondral bone plate that link the joint space and the affected area of bone. The inﬂux of these mediators causes degeneration of the bone and ﬂuid accumulation within the weakened trabecular structure, and results in intense pain due to activation of nociceptors in the subchondral bone. The insult to the bone in the affected area is worsened in more advanced cases of bone e because of the destruction of at least a portion of the articular cartilage, which in turn results in an increased ﬂow of mediators from the joint space, through the cortical bone plate, and into the affected area of bone.

CPCs used in the treatment of bone e require l features in order to effectively treat the affected area of bone, including injectability, ﬂowability, settability, cohesion, and adhesion to bone. Unfortunately, tional CPCs typically are lacking with respect to one or more of the d characteristics, which has hindered the development of CPCs capable of being stered to a desired ical location in a minimally invasive manner. CPCs are typically formed by mixing a solid and a liquid to obtain a paste suitable for injection which later sets and cures after administration into the affected area of bone.

Prior art CPCs are designed to have a high compressive strength and elastic modulus so as to provide biomechanical stabilization to the affected area of bone. Such CPCs are generally made with high solid-to-liquid ratios, which results in high compressive strengths and c moduli and generally lower porosity, but these CPCs offer poor injectability due to the required high injection res and poor ﬂowability such that the materials do not adequately ﬁll the space in the affected area. CPCs made with these high solid-to-liquid ratios can also dewater during injection, leaving cement solid in the mentation and preventing the CPC from setting and curing in situ. Attempts to address these issues by preparing CPCs with lower solid-to-liquid ratios have ed in poor cohesiveness and a lack of setting and/or curing post-administration due to the hilic nature of the CPC and its tendency to miX with body ﬂuids. Additionally, even when als made with these lower to-liquid ratios are capable of setting, they do not maintain on or adhesion to bone, such that that they do not remain in the affected area after administration, and instead ﬂow through the porous bone structure.

As a result of these challenges, a very limited number of CPCs are available that have the desired combination of providing biomechanical stability to the affected area while maintaining injectability and ﬂowability to fill the affected area of bone. See, e. g., Subchondroplasty® Procedure Acchill® Bone Substitute al (B SM), available at http:/.I’SL1E9chondroplasty.com/l":ealthcarewpmfessionai s~bsmhtmi (last visited April 18, 2017), see also Tof1ghi, A. ei al. J. Biomimeiics Biomai. Tissue Eng ’g 2009, 2, 39-28, the contents of all of the foregoing of which are incorporated herein by reference in their entireties.

Unfortunately, these CPCs, which are used to treat bone disease, cure to form biomaterials with a high degree of porosity, resulting in signiﬁcant pain post-operatively. See, e. g., Farr, J., Cohen, S. B. Oper. Tech. Sports Med. 2013, 21(2), 138-43, Eliaz, N., Metoki, N.

Materials 2017, 10, 334, the contents of all of the foregoing of which are incorporated herein by reference in their entireties.

While the prior art has provided certain CPCs that include a carbohydrate, these materials have short setting times or are made with high powder-to-liquid ratios and are accordingly insuff1ciently intermixable to provide for facile ation and administration directly from syringes. See, e.g., Pek, Y. S. ei al. Biomai. 2009, 30, , Ahmadzadeh- Asl, S. ei al. Adv. Applied Ceramics 2011, [10(6), 340-45, the contents of all of the foregoing of which are incorporated herein by reference in their entireties.

Accordingly, there is a need in the art for more safe and eff1cacious treatment options that address the underlying causes of bone e associated with degenerate bone with less risk and side s than prior art methods.

WO 89733 2017/029651 SUMMARY OF ION Methods and compositions for the treatment of degenerate bone in a patient in need thereof are disclosed herein.

In one aspect, disclosed herein is an able biomaterial comprising a solid component and a liquid component comprising a carbohydrate, wherein the injectable biomaterial sets and cures to form an apatitic crystal structure after mixing of the solid component and the liquid component.

In r aspect, disclosed herein is a method for making an injectable biomaterial comprising creating the liquid component by providing a liquid on, adjusting the pH of the liquid solution with a pH adjusting agent, and dissolving the carbohydrate in the liquid solution to form a the liquid component, providing the solid component, and mixing the liquid component and the solid component to form the injectable biomaterial.

In some embodiments, the injectable biomaterial sets over a period of time. In some embodiments, the injectable biomaterial cures over a period of time. In some embodiments, the injectable biomaterial sets prior to completely curing.

In some embodiments, the solid component comprises at least one of a metal phosphate and a metal carbonate. In some embodiments, the solid component comprises a ve calcium phosphate. In some embodiments, the solid ent comprises at least one of d-tricalcium phosphate (Ca3(PO4)2), calcium carbonate (CaCO3), and monocalcium phosphate monohydrate PO4)2 H20). In some embodiments, the solid ent comprises 70-90% alpha tricalcium phosphate, 10-20% calcium carbonate, and O.5-2% calcium phosphate monobasic monohydrate mass). In some embodiments, the solid component comprises 80-89% alpha tricalcium phosphate, 11-19% calcium carbonate, and 075-1 .5% calcium ate monobasic monohydrate (mass/mass). In some embodiments, the solid component comprises 82-86% alpha tricalcium phosphate, 13-16% calcium carbonate, and 09-12% calcium phosphate monobasic monohydrate (mass/mass). In some embodiments, the solid component comprises 84.3% alpha tricalcium phosphate, 14.7% calcium carbonate, and 1.02% calcium phosphate monobasic monohydrate (mass/mass). In some embodiments, the solid component further comprises at least one ionic compound of at least one oligoelement occurring naturally in a human body. In some embodiments, the at least one ionic compound comprises a cation selected from the group consisting of Na+, K+, WO 89733 Mg", Ca", Sr", H+, and mixtures thereof. In further embodiments, the at least one ionic compound comprises an anion selected from the group consisting of P043: , H2PO4‘, P2074: C0322 HCO3‘, 8042—, HSO4‘, Cl‘, OH‘, F‘, SiO44_, and mixtures hereof.

In some embodiments, the liquid component further comprises a salt. In some embodiments, the salt is a metal salt. In some embodiments, the salt is selected from a phosphate salt, a silicate salt, a chloride salt, a hydroxide salt, and es thereof. In some embodiments, the salt comprises at least one of sodium ate dibasic, sodium silicate, sodium chloride, and calcium hydroxide.

In some embodiments, the carbohydrate is selected from the group consisting of dextran, alginate, carboxymethylcellulose, and hyaluronic acid. In some embodiments, the carbohydrate is hyaluronic acid, or an ester, ea, acyl isourea, disulf1de, or amide thereof. In some embodiments, the hyaluronic acid is selected from the group consisting of hyaluronan, sodium hyaluronate, potassium hyaluronate, magnesium onate, calcium hyaluronate, ammonium hyaluronate, and combinations thereof. In some embodiments, the hyaluronic acid comprises at least one cross-link. In some embodiments, the hyaluronic acid is derived from bacteria or s. In some embodiments, the hyaluronic acid ses a sulfated hyaluronic acid, or ester, acylurea, acyl isourea, carbomer, disulf1de, or amide thereof. In some embodiments, the onic acid ses an N—sulfated hyaluronic acid, or ester, acylurea, acyl isourea, carbomer, disulf1de, or amide thereof. In some embodiments, the onic acid comprises a hyaluronic ester. In some ments, the hyaluronic ester is a esterif1ed in an amount from about 20 to 100%. In some embodiments, the non-esterif1ed hyaluronic acid is salif1ed with an organic or an inorganic base.

In some embodiments, the carbohydrate is water-soluble. In some embodiments, the liquid component is in the form of a hydrogel.

In some embodiments, the carbohydrate is present in the injectable biomaterial at a concentration of about 0.1 to about 100 mg/mL. In some embodiments, the carbohydrate is present in the injectable biomaterial at a concentration of about 0.1 to about 50 mg/mL. In some embodiments, the carbohydrate is present in the injectable erial at a concentration of about 0.1 to about 10 mg/mL. In some embodiments, the carbohydrate is present in the injectable biomaterial at a concentration of about 1 to about 10 mg/mL. In some embodiments, the carbohydrate is present in the injectable erial at a concentration of about 2 to about 10 mg/mL. In some embodiments, the carbohydrate is present in the injectable biomaterial at a concentration of about 4 to about 8 mg/mL. In some embodiments, the carbohydrate is t in the injectable biomaterial at a concentration of about 5 to about 7 mg/mL.

In some embodiments, the carbohydrate has a molecular weight of from about 0.90 X 106 Da to about 1.0 X 107 Da. In some embodiments, the carbohydrate has a molecular weight of from about 0.90 X 106 Da to about 5.0 X 106 Da. In some embodiments, the ydrate has a molecular weight of from about 0.90 X 106 Da to about 4.0 X 106 Da. In some embodiments, the carbohydrate has a molecular weight of from about 0.90 X 106 Da to about 3.0 X 106 Da. In some embodiments, the carbohydrate has a molecular weight of from about 1.5 X 106 Da to about 3.0 X106 Da. In some ments, the carbohydrate has a molecular weight of from about 1.7 X 106 Da to about 2.5 X 106 Da. In some embodiments, the carbohydrate is onic acid haVing a molecular weight of about 0.90 X 106 Da and is present at a concentration of about 6.0 mg/mL. In some ments, the carbohydrate is hyaluronic acid haVing a molecular weight of about 1.7 X 106 Da and is present at a concentration of about 6.0 mg/mL. In some embodiments, the carbohydrate is hyaluronic acid haVing a molecular weight of about 2.6 X 106 Da and is present at a concentration of about 6.0 mg/mL.

In some embodiments, the molecular weight of the carbohydrate is stable for at least 3 months. In some embodiments, the molecular weight of the carbohydrate is stable for at least 6 months. In some ments, the molecular weight of the carbohydrate is stable for at least 1 year. In some embodiments, the molecular weight of the carbohydrate is stable for at least 2 years. In some embodiments, the molecular weight of the carbohydrate is stable for at least 3 years. In some embodiments, the molecular weight of the carbohydrate is stable for at least 4 years. In some embodiments, the molecular weight of the carbohydrate is stable for at least 5 years.

In some embodiments, the ratio of solid component to liquid component is about 3 to about 1 by mass. In some embodiments, the ratio of solid ent to liquid component is about 2 to about 1 by mass. In some embodiments, the ratio of solid component to liquid component is about 1.5 to about 1 by mass. In some embodiments, the ratio of solid component to liquid component is about 1 to about 1 by mass.

In some embodiments, the able biomaterial is injectable through a needle or cannula prior to initially setting. In some embodiments, the needle or cannula has a size of at least 21 gauge. In some embodiments, the needle or cannula has a size of at least 20 gauge.

In some embodiments, the needle or cannula has a size of at least 18 gauge. In some embodiments, the needle or cannula has a size of at least 16 gauge. In some embodiments, the needle or cannula has a size of at least 15 gauge. In some embodiments, the needle or cannula has a size of at least 14 gauge. In some embodiments, the needle or cannula has a size of at least 12 gauge. In some embodiments, the needle or cannula has a size of at least gauge.

In some embodiments, the injectable biomaterial does not dewater when being dispensed through a needle or a. In some embodiments, the injectable biomaterial does not seize when being sed through a needle or cannula.

In some embodiments, the injectable biomaterial is cohesive. In some embodiments, the injectable biomaterial remains cohesive during its initial setting time.

In some embodiments, the injectable biomaterial adheres to bone. In some embodiments, the injectable biomaterial remains adhesive to the bone during its l setting time.

In some embodiments, the injectable biomaterial is workable for less than about 60 minutes after the mixing of the solid ent and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 50 minutes after the mixing of the solid component and the liquid ent. In some embodiments, the injectable biomaterial is workable for less than about 40 minutes after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 30 minutes after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 20 minutes after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 10 minutes after the mixing of the solid ent and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 5 minutes after the mixing of the solid component and the liquid ent. In some embodiments, the injectable biomaterial is workable for less than about 4 minutes after the mixing of the solid component and the liquid component. In some embodiments, the injectable erial is le for less than about 3 minutes after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 2 s after the mixing of the solid ent and the liquid component. In some embodiments, the injectable biomaterial is workable for less than about 1 minute after the mixing of the solid component and the liquid component.

In some embodiments, the able biomaterial initially sets in less than about 60 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 50 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 40 minutes after mixing the solid ent and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 30 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 20 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 10 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 5 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable erial lly sets in less than in less than about 4 minutes after mixing the solid component and the liquid component. In some embodiments, the injectable biomaterial initially sets in less than in less than about 3 minutes after mixing the solid component and the liquid ent. In some embodiments, the able biomaterial initially sets in less than in less than about 2 minutes after mixing the solid component and the liquid component.

In some embodiments, the injectable biomaterial initially sets in less than in less than about 1 minute after mixing the solid component and the liquid component.

In some embodiments, the injectable biomaterial cures completely in less than about 96 hours after the mixing of the solid component and the liquid component. In some ments, the injectable biomaterial cures completely in less than about 72 hours after the mixing of the solid component and the liquid component. In some embodiments, the able biomaterial cures completely in less than about 48 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 24 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 12 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures tely in less than about 6 hours after the mixing of the solid ent and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 5 hours after the mixing of the solid ent and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 4 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 3 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures completely in less than about 2 hours after the mixing of the solid component and the liquid component. In some embodiments, the injectable biomaterial cures tely in less than about 1 hour after the mixing of the solid component and the liquid component.

In some embodiments, the initial setting and curing of the injectable biomaterial does not result in a gaseous release.

In some ments, the able biomaterial does not cantly alter the pH of the adjacent ﬂuids when disposed in a patient.

In some embodiments, the l setting curing of the injectable biomaterial does not signiﬁcantly alter the ature of the adjacent ﬂuids when disposed in a patient.

In some embodiments, the curing of the injectable biomaterial yields an apatitic crystal structure substantially consistent with that of hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an apatitic crystal structure that is at least about 90% hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an apatitic crystal structure that is at least about 95% hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an apatitic crystal structure that is at least about 96% hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an apatitic crystal structure that is at least about 97% hydroxyapatite. In some embodiments, the injectable erial yields an apatitic crystal structure that is at least about 98% hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an ic crystal structure that is at least about 99% hydroxyapatite. In some embodiments, the curing of the injectable biomaterial yields an apatitic l structure that is greater than about 99% yapatite.

In some embodiments, the fully set and cured injectable biomaterial has a molar Ca/P ratio of about 1 to about 2. In some embodiments, the fully set and cured injectable biomaterial has a molar Ca/P ratio of about 1.3 to about 1.8. In some embodiments, the fully set and cured injectable biomaterial has a molar Ca/P ratio of about 1.4 to about 1.7. In some embodiments, the fully set and cured injectable biomaterial has a molar Ca/P ratio of about 1.5 to about 1.7. In some embodiments, the fully set and cured able biomaterial has a molar Ca/P ratio of about 1.5 to about 1.667.

In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 20 MPa. In some embodiments, the fully set and cured able biomaterial has a compressive th of less about 15 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 10 MPa. In some embodiments, the fully set and cured injectable biomaterial has a ssive strength of less about 9 MPa. In some embodiments, the fully set and cured able biomaterial has a compressive strength of less about 8 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 7 MPa. In some ments, the fully set and cured injectable biomaterial has a compressive strength of less about 6 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 5 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 4 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 3 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 2 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 1 MPa.

In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 5 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 4 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic s of less than about 3 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 2 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 1 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 0.5 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 0.25 GPa.

In some embodiments, the injectable biomaterial has a viscosity of about 5 Pas and about 30 Pas immediately after mixing the solid component and the liquid component, when measured at room temperature. In some ments, the injectable biomaterial has a viscosity of about 5 Pas and about 20 Pa~s immediately after mixing the solid component and the liquid ent, when ed at room temperature. In some embodiments, the injectable erial has a viscosity of about 5 Pas and about 18 Pa~s immediately after mixing the solid component and the liquid component, when measured at room temperature.

In some ments, the injectable biomaterial does not biomechanically stabilize bone.

In some embodiments, the fully set and cured injectable biomaterial has a true y of about 1 g/cm3 to about 4 g/cm3. In some embodiments, the fully set and cured injectable biomaterial has a true density of about 1.5 g/cm3 to about 3.5 g/cm3. In some embodiments, the fully set and cured injectable biomaterial has a true density of about 1.83 g/cm3 to about 3.14 g/cm3. In some embodiments, the fully set and cured injectable biomaterial has a true density of about 2 g/cm3 to about 3 g/cm3.

In some embodiments, the fully set and cured injectable erial ses a median pore diameter of less than about 1 um. In some embodiments, the fully set and cured injectable biomaterial comprises a median pore diameter of less than about 0.8 um. In some embodiments, the fully set and cured injectable erial comprises a median pore diameter of less than about 0.6 um. In some embodiments, the fully set and cured injectable biomaterial comprises a median pore diameter of less than about 0.5 um. In some embodiments, the fully set and cured injectable biomaterial comprises a median pore diameter of less than about 0.4 um. In some embodiments, the fully set and cured injectable biomaterial comprises a median pore diameter of less than about 0.2 um. In some embodiments, the fully set and cured able biomaterial comprises a median pore diameter ofless than about 0.15 um.

In some embodiments, the fully set and cured injectable biomaterial comprises a total porous area of less than about 4 m2/g. In some embodiments, the fully set and cured injectable biomaterial comprises a total porous area of less than about 3 m2/g. In some embodiments, the fully set and cured injectable biomaterial ses a total porous area of less than about 2 mZ/g.

In some embodiments, the fully set and cured injectable biomaterial comprises a porosity sufﬁcient to prevent diffusional e of at least one of inﬂammatory mediators and non-inﬂammatory mediators.

In some embodiments, the fully set and cured injectable biomaterial is osteoinductive.

In some embodiments, the fully set and cured injectable erial is osteoconductive.

In some embodiments, the fully set and cured injectable biomaterial is resorbable.

In some embodiments, the curing of the injectable erial yields less than about 5% calcium oxide. In some embodiments, the curing of the injectable biomaterial yields less than about 4% m oxide. In some embodiments, the curing of the injectable biomaterial yields less than about 3% calcium oxide. In some ments, the curing of the injectable biomaterial yields less than about 2% calcium oxide. In some ments, the curing of the injectable biomaterial yields less than about 1% calcium oxide.

In some embodiments, the liquid component is sterile. In some embodiments, the solid component is sterile.

In some embodiments, the injectable biomaterial is intermixable.

In some embodiments, the pH adjusting agent is selected from an organic acid and an inorganic acid. In some embodiments, the pH adjusting agent is selected from the group consisting of citric acid, formic acid, acetic acid, and mixtures thereof. In some embodiments, the pH adjusting agent s selected from the group consisting of hydrochloric acid, phosphoric acid, nitric acid, and mixtures thereof.

In some embodiments, providing the solid component further comprises drying the solid component. In some embodiments, the drying comprises exposing the solid component to heat over a period of time. In some embodiments, the heat comprises at least about 165 °C. In some embodiments, the period of time comprises at least about 12 hours.

In another aspect, disclosed herein is a method of ng an affected area of a bone in a patient in need thereof, the method comprising identifying the affected area in the bone of the patient, creating in the bone an incision through a cortical wall of the bone to provide access to a degenerate cancellous space in the affected area of the bone, stering a volume of an injectable biomaterial of any preceding claim through the incision through the cortical wall of the bone and into the degenerate lous space.

In some embodiments, the affected area of bone is adjacent to a joint of the patient in which the patient is experiencing a joint pathology. In some embodiments, the joint pathology is a pathology of the knee, shoulder, wrist, hand, spine, ankle, elbow, or hip. In some embodiments, the joint ogy is selected from the group consisting of pain, osteoarthritis, rheumatoid arthritis, avascular necrosis, and ations thereof. In some ments, the method is for the treatment of osteoarthritis in a joint of the patient. In some embodiments, the osteoarthritis has a Kellgren Lawrence (KL) grade of 1-3. In some embodiments, the joint pathology is not d to joint instability.

WO 89733 In some embodiments, the affected area exhibits at least one of inﬂammatory or degradative changes as a result of at least one of atory mediators and non- inﬂammatory mediators.

In some embodiments, the inﬂammatory or degradative changes are identiﬁed by MRI. In some embodiments, the MRI is a T2 MRI.

In some embodiments, the inﬂammatory or degradative changes are ed in cancellous bone.

In some embodiments, the affected area is disposed between about 0 inches and about 5 inches from the joint of the patient. In some embodiments, the affected area is disposed between about 0 inches and about 4 inches from the joint of the patient. In some embodiments, the affected area is disposed between about 0 inches and about 3 inches from the joint of the patient. In some embodiments, the affected area is disposed between about 0 inches and about 2 inches from the joint of the pat In some embodiments, the affected area is disposed between about 0 inches and about 1 inch from the joint of the patient. In some embodiments, the affected area is disposed between about 0 inches and about 20 mm from the joint of the patient. In some embodiments, the ed area is disposed between about 0 mm and about 10 mm from the joint of the patient. In some embodiments, the affected area is disposed between about 0 mm and about 5 mm from the joint of the patient. In some ments, the ed area is disposed between about 0 mm and about 1 mm from the joint of the patient.

In some embodiments, the incision is percutaneous.

In some embodiments, providing the access to the cancellous space comprises creating a channel in the bone of the patient to couple the incision in the cortical wall of the bone to the cancellous space comprising the ed area. In some embodiments, the channel is perpendicular to the long axis of the bone. In some embodiments, the channel is not dicular to the long axis of the bone. In some embodiments, the l is within about 5 inches from the proximal subchondral plate. In some embodiments, the channel is within about 4 inches from the proximal subchondral plate. In some ments, the channel is within about 3 inches from the proximal subchondral plate. In some embodiments, the channel is within about 2 inches from the proximal subchondral plate. In some embodiments, the channel is within about 1 inches from the proximal ndral plate. In some embodiments, the channel is within about 20 mm of the proximal subchondral plate. In some embodiments, the channel is within about 10 mm of the proximal subchondral plate. In some embodiments, the channel is within about 5 mm of the proximal subchondral plate. In some embodiments, the channel is within about 1 mm of the proximal subchondral plate. In some embodiments, the channel is accessed by a a that is positioned and inserted without the need for onal targeting instrumentation.

In some embodiments, the method further comprises decompressing and aspirating the contents of the affected area prior to administration of the injectable erial to the ed area. In some embodiments, the decompression and aspiration reduces zed inﬂammation in the affected area. In some embodiments, the decompression and aspiration reduces intraosseous pressure in the affected area. In some embodiments, the contents comprise a ﬂuid. In some embodiments, the ﬂuid comprises at least one of inﬂammatory mediators and non-inﬂammatory mediators.

In some embodiments, the at least one inﬂammatory mediator comprises at least one of inin, histamine, prostaglandins, lactic acid, substance P, vasoactive intestinal peptide, calcitonin gene related peptide , and mixtures thereof. In some embodiments, the at least one inﬂammatory mediator comprises an inﬂammatory cytokine.

In some embodiments, the inﬂammatory cytokine is selected from the group consisting of AIMPI (SCYEI), BMPZ, CD40LG (TNFSFS), CSFl (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FASLG (TNFSF6), , IFNAZ, IFNG, IL-1, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, IFN—y, LTA (TNFB), LTB, MIF, NAMPT, OSM, SPPl, TGF-B, TNF, , TNFSFlO (TRAIL), TNFSFll (RANKL), TNFSF13, TNFSF13B, TNFSF4 (OX4OL), VEGFA, and mixtures thereof. In some embodiments, the at least one non-inﬂammatory mediator comprises a proteolytic . In some embodiments, the proteolytic enzyme is selected from the group consisting of matrix metalloproteinases (MMPs), tissue inhibitors of oproteinases (THVIPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS), and mixtures f. In some embodiments, the inﬂammatory or comprises an inﬂammatory chemokine. In some embodiments, the inﬂammatory chemokine is selected from the group consisting of C5, CCLl (1-309), CCLll (eotaxin), CCLl3 (MCP- 4), CCL15 (MIP-ld), CCL16 (HCC-4), CCL17 (TARC), CCL2(MCP-1), CCL20 (MIP-3a), CCL22 (MDC), CCL23 (MPIF-l), CCL24 (MPIF-Z, Eotaxin-2, MPIF-Z, Eotaxin-2), CCL26 (eotaxin-3), CCL3 (MIP-IA), CCL4(MIP-1B), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CX3CL1, CXCLl (GROl, GRO-alpha, , CXCL10(INP10), CXCLll (I- TAC, 113—9), (SDF1), CXCL13, CXCL2 (GROZ, GRO-beta, , CXCL3, CXCLS (ENA-78, LIX), CXCL6 (GCP-2), CXCL9 (MIG), and mixtures thereof. In some embodiments, the inﬂammatory mediator comprises an interleukin. In some embodiments, the interleukin is selected from the group ting of ILl3, IL15, IL16, IL17A, ILl7C, IL17F, IL1A, IL1B, ILlRN, IL21, 1L27, 1L3, IL33, 1L5, IL7, CXCL8, 1L9, and mixtures thereof. In some embodiments, the inﬂammatory mediator comprises an inﬂammatory or ed from the group consisting of bradykinin, calcitonin gene related peptide (CGRP), histamine, lactic acid, nerve growth factor (NGF), prostaglandins, substance P, vasoactive intestinal peptide, and mixtures thereof.

In some embodiments, the injectable biomaterial is administered through a cannula or needle. In some embodiments, the needle or cannula has a size of at least 21 gauge. In some embodiments, the needle or a has a size of at least 20 gauge. In some embodiments, the needle or cannula has a size of at least 18 gauge. In some embodiments, the needle or cannula has a size of at least 16 gauge. In some embodiments, the needle or cannula has a size of at least 15 gauge. In some embodiments, the needle or cannula has a size of at least 14 gauge. In some embodiments, the needle or cannula has a size of at least 12 gauge. In some ments, the needle or cannula has a size of at least 10 gauge.

In some ments, the injectable biomaterial does not r when being dispensed through the needle or cannula.

In some embodiments, the injectable biomaterial does not seize when being dispensed through the needle or cannula.

In some embodiments, the injectable biomaterial is administered h a steerable cannula to minimize surgical damage.

In some embodiments, the injectable biomaterial is injected into the affected area while minimally disrupting the subchondral plate.

In some embodiments, the injectable biomaterial is ed into a layer between about 0 mm and about 20 mm above or below the affected area while lly disrupting the subchondral plate. In some embodiments, the injectable biomaterial is injected into a layer between about 0 mm and about 10 mm above or below the affected area while minimally disrupting the subchondral plate. In some embodiments, the injectable biomaterial is injected into a layer between about 0 mm and about 5 mm above or below the affected area while minimally disrupting the subchondral plate. In some embodiments, the injectable biomaterial is injected into a layer between about 0 mm and about 1 mm above or below the affected area while minimally disrupting the subchondral plate.

In some embodiments, the able biomaterial is administered to an area that is not intrinsic to the ural stability of the bone.

In some embodiments, the method further comprises arthroscopically examining the joint space post-injection to ensure an absence of the injectable biomaterial in the joint.

In some embodiments, the injectable biomaterial ﬂows into the porosity of cancellous bone during stration into the affected area.

In some embodiments, the injectable erial remains cohesive and substantially ﬁlls bone voids during administration into the affected area.

In some embodiments, the injectable biomaterial at least partially coats the interface between the cancellous space and an adjacent joint to provide a protective layer upon g.

In some embodiments, the able biomaterial prevents diffusional passage of at least one of inﬂammatory mediators and non-inﬂammatory mediators from the adjacent joint space into the affected area.

In some embodiments, the protective layer provides a sacrificial layer for osteoclasts to consume during bone remodeling.

In some embodiments, the administration of the injectable biomaterial does not cause stress shielding resulting in the weakening of the ed bone.

In some embodiments, the method does not cause substantial post-operative pain.

In some embodiments, the method ses pain in the joint.

In some embodiments, the method slows the progression of rthritis in the joint.

In some embodiments, the method is for the treatment of rheumatoid arthritis in a joint of the patient. In some embodiments, the method slows the ssion of rheumatoid arthritis in the joint.

In some embodiments, the method slows the progression of avascular necrosis in the joint.

In another aspect, disclosed herein is a kit comprising a solid component and a liquid component for preparing an injectable biomaterial as disclosed herein and instructions for use of the same.

In some embodiments, the instructions are for a method of treating an affected area of a bone in a patient in need thereof.

In some embodiments, the treatment is for pain, osteoarthritis, rheumatoid arthritis, avascular necrosis, or combinations thereof.

In some embodiments, the solid component and the liquid component are disposed in separate sterile ners.

In some embodiments, the packaging conﬁguration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about three months. In some ments, the packaging conﬁguration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about siX . In some embodiments, the packaging conﬁguration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about one year. In some embodiments, the packaging conﬁguration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about two years. In some embodiments, the packaging conﬁguration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about three years. In some embodiments, the packaging conﬁguration allows the solid ent and the liquid component to remain stable at 2 oC — 25 0C for at least about four years. In some ments, the packaging ration allows the solid component and the liquid component to remain stable at 2 oC — 25 0C for at least about ﬁve years.

In some embodiments, the liquid component is disposed in a sterile syringe.

In some embodiments, the solid component is disposed in a syringe sing an integrated mixing deVice for in silu mixing of premeasured portions of the solid component and the liquid component to form the injectable biomaterial. In some embodiments, the syringe is sterile.

In some ments, the kit further comprises a Luer-Lock.

In some ments, the kit further comprises an end cap.

BRIEF DESCRIPTION OF THE DRAWINGS shows a schematic of a human knee comprising an area of degenerate bone.

FIGs. 2A-E show a schematic callout of an area comprising a portion of the degenerate bone of FIGS. 3A-B show an injectable biomaterial ing to the present disclosure as compared to an injectable biomaterial lacking a carbohydrate, each in phosphate buffered saline.

FIGs. 4A-D show injectable biomaterials according to the present disclosure as compared to an injectable biomaterial lacking a carbohydrate, each in phosphate buffered saline.

FIGs. SA-D show an able biomaterial according to the t disclosure as compared to an injectable biomaterial lacking a carbohydrate, after removal of excess phosphate buffered saline.

FIGs. 6A-D show cross-sections of sawbone injected with able erials according to the present disclosure as contrasted with sawbone injected with an injectable biomaterial lacking a carbohydrate.

FIGs. 7A-B show cross-sections of sawbone injected with injectable biomaterials according to the present disclosure as contrasted with sawbone ed with an injectable biomaterial lacking a carbohydrate.

FIGs. 8A-B show the results of a diffusion r experiment comparing control with an able biomaterial lacking a carbohydrate and an injectable biomaterial according to the present sure. shows an X-ray powder diffractogram of an injectable erial, post setting and curing, according to the present disclosure. shows a Fourier Transform Infrared ("FT-IR") spectrograph of an injectable erial according to the present disclosure.

FIGs. llA-C show scanning electron microscopy ("SEM") images of an injectable biomaterial ing to the present disclosure after setting and curing.

FIGs. 12A-B show micro-computed tomography ("micro CT") images from different planes taken 6 weeks after administration of an injectable biomaterial according to the present disclosure into degenerate bone generated in skeletally mature New Zealand White rabbits. shows an image of an injectable biomaterial according to the present disclosure injected into canine femoral e. shows and image of an injectable biomaterial according to the present disclosure ed into human cadaver bone.

DETAILED DESCRIPTION OF THE INVENTION The t disclosure relates to methods and itions for the treatment of degenerate bone in a patient. In some embodiments, the methods and compositions disclosed herein are useful in the treatment, tion, or in delaying the progression of a bone disease linked to bone ration, such as osteoarthritis ("OA"), rheumatoid arthritis, and avascular necrosis.

The inventors have surprisingly discovered that the addition of a carbohydrate to an injectable biomaterial provides for an injectable, intermixable, ﬂowable, settable, curable, cohesive composition that adheres to bone. Further, the injectable erials disclosed herein possess a low porosity and high dimensional stability that is desirable for use in the minimally ve treatment of various bone diseases, a property absent in the biomaterials of the prior art.

Without wishing to be bound by theory, the inventors posit that a low porosity and high dimensional stability injectable biomaterial serves to arrest biochemical ication involving the joint space and the adjacent bone. The inventors posit that this biochemical communication is responsible for the onset of a number of symptoms of bone degeneration, including pain and ﬂuid lation, and that continued bone degeneration can facilitate the progression of bone diseases linked to bone ration, such as OA, rheumatoid arthritis, and avascular is. By way of example, as the cartilage and synovium become d, whether from trauma or e, a milieu of inﬂammatory and/or non-inﬂammatory mediators is released from both tissues. These mediators enter the bone through channels in the cortical bone plane, stimulating pain through nociceptor activation, ﬂuid lation, and degenerative changes in the ed area of bone (such as by the formation of a bone marrow lesion). The inventors hypothesize that, as a result of this biochemical communication between the joint space and the adjacent bone, the homeostasis of the affected area of bone is disturbed, resulting in dysregulation in the synthesis and degradation of bone proteins, and a degenerative positive feedback loop responsible for the progression of pathologies such as 0A is formed. Blocking the effect of the inﬂammatory and/or non- inﬂammatory mediators causing bone degeneration is therefore ial to treating, preventing, and/or delaying the progression of bone disease.

Accordingly, the inventors set out to produce an injectable biomaterial with low porosity and high dimensional stability, but which were able to be prepared using lower solid-to-liquid ratios, allowing for their use in the minimally invasive ent of bone disease linked to bone degeneration. During their research, the inventors found that ng techniques used to produce such injectable materials generally resulted in materials demonstrating poor cohesion, adhesion, setting and curing properties, rendering such materials able for use in the minimally invasive treatment of bone disease. Similarly, those prior techniques generally resulted in materials with high porosity that would be poorly suited to arresting biochemical communication between the affected area of bone and the adjacent joint space. See, e. g., Eliaz, N., , N. Materials 2017, 10, 334, at 13, the contents of which are incorporated by reference herein in their entirety. The inventors then surprisingly discovered that the addition of a carbohydrate to an injectable biomaterial allowed for a material that could be prepared with lower solid-to-liquid ratios but was nonetheless able to set, cure, maintain veness and adherency to bone, and that possessed low porosity and high dimensional ity as compared to injectable biomaterials prepared without an added carbohydrate. In other words, the inventors discovered that the addition of a carbohydrate provided for an injectable biomaterial having the critical combination of ability, intermixability, lity, settability and curability, while maintaining the desired ties of cohesivity, adhesivity, low porosity, and high dimensional stability. Accordingly, in one embodiment, the present disclosure provides injectable biomaterials that can be prepared using a lower solid-to-liquid ratio than usly possible while ining the cohesiveness, adherency to bone, low porosity, and high dimensional stability of materials traditionally made with a higher to-liquid ratio.

Accordingly, the disclosed injectable biomaterials are able to stem the ﬂow and prevent ingress of atory and non-inﬂammatory mediators into the affected area of bone from an adjacent joint space while not sacrificing the desirable ability to intermiX, be ed, ﬂow into, remain and set and cure in the area of degenerate bone to be treated without being cleared by the function of normal body ﬂuid exchange. These als provide this surprising and unique combination of properties and, contrary to the common knowledge in the art, which focused on the provision of high ssive strength and c modulus materials, unexpectedly allow for superior treatment of bone disease linked to bone degeneration as compared to prior compositions.

Without wishing to be bound by theory, the inventors posit that the injectable biomaterials disclosed herein serve to treat the underlying causes of bone disease linked to bone degeneration by eliminating and preventing recurrence of biochemical communication WO 89733 between the affected area of bone and the adjacent joint space. The injectable biomaterials disclosed herein provide a barrier between the affected area of bone and the adjacent joint space that prevents the ingress of inﬂammatory and/or non-inﬂammatory mediators from the joint space, arresting degradation mediated by, e.g., proteolytic enzymes and inﬂammatory cytokines, and allowing the bone to recover via normal dynamics (bone tion). In contrast, prior injectable biomaterials addressed the ms, but not the underlying causes of bone disease linked to bone degeneration.

Furthermore, the disclosed injectable biomaterials possess lower compressive strength than the als typically used in the prior art. Without wishing to be bound by theory, the ors posit that, r to the prevailing knowledge in the art, provision of an injectable biomaterial that does not biomechanically stabilize an affected area of bone provides for superior es in the treatment, prevention, and slowing the progression of bone disease linked to bone degeneration. Conventional wisdom in the art indicated that injectable biomaterials (such as CPCs) with high compressive strength were required to properly treat joint pathologies by ing biomechanical ization. ry to this tional wisdom, the inventors have surprisingly discovered that by providing a weaker injectable biomaterial that does not provide biomechanical support to the affected area, the disclosed methods and compositions do not artif1cially alter the hanical forces to which the joint is eXposed, providing a superior methodology to address such pathologies.

Without wishing to be bound by theory, the ors posit that the provision of biomechanical support to an area of degenerate bone can be detrimental by shifting stress and strain to otherwise healthy tissue, potentially resulting in the spread of bone e and/or its symptoms in accordance with Wolff’ s Law. The inventors posit that this is due the impossibility of exact recreation of healthy biomechanics, which necessarily relates on undue strain on other locations. For example, areas of high stress will become thicker and stiffer whereas areas of low stress will resorb according to Wolff’ s Law, which states that bone in a healthy person or animal will adapt to the loads under which it is placed. Based on Wolff’ s Law, if loading on a particular bone increases, the bone will remodel itself over time to become stronger to resist that sort of loading. However, these normal biological responses are not possible in bone disease because the degenerative mical feedback loop present in those conditions adversely affect, and can completely cease, normal bone remodeling processes. Accordingly, by not providing biomechanical support, the disclosed methods and compositions allow for physiological conditions that permit the affected area of bone to recover and rebuild through l bone remodeling, thus slowing and/or reversing the progression of bone disease, and preventing the onset and progression of bone diseases such as symptomatic OA, rheumatoid arthritis, and avascular necrosis, as well as preventing spread of the disease or symptoms to adjacent healthy tissues.

In some embodiments, the strength of the fully set and cured injectable biomaterial is less than that provided by the injectable biomaterials of the prior art. For example, certain prior art injectable erials possess a compressive strength of approximately 50 MPa, which is 4-10 times greater than the average 5-15 MPa compressive strength of healthy cancellous bone. See, e. g., Norian® SRS® Tiabia plateau fractures, Synthes®, available at .1","wwwrch.or‘raufupvloadediiiles/Main/{Iontent/ortho/Norian SR5} '37ibia viateau fractur ssprit (last visited April 24, 2017), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the strength of the fully set and cured able biomaterial is characterized by one or more of compressive strength and elastic modulus. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 20 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 15 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 10 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 9 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 8 MPa. In some embodiments, the fully set and cured injectable biomaterial has a ssive strength of less about 7 MPa. In some ments, the fully set and cured injectable biomaterial has a compressive strength of less about 6 MPa. In some ments, the fully set and cured injectable biomaterial has a ssive strength of less about 5 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 4 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 3 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 2 MPa. In some embodiments, the fully set and cured injectable biomaterial has a compressive strength of less about 1 MPa.

In further ments, the c modulus of the fully set and cured injectable erial is less than that provided by the injectable biomaterials of the prior art. In further embodiments, the c modulus of the fully set and cured injectable biomaterial is close to that of healthy subchondral bone. Without g to be bound by theory, the inventors posit that provision of an injectable biomaterial having an c modulus similar to that of healthy subchondral bone reduces the risk of altering natural biomechanics and resulting in further bone degradation in accordance with Wolff’ s Law (1'.e., stress shielding). See, e. g., Eliaz, N., Metoki, N. als 2017, 10, 334, at 3. For example, the elastic modulus of samples of human subchondral bone has been reported to be about 1.15 GPa. See, e.g., Choi, K. el al. J.

Biomech. 1990, 23(11), 1103-13, see also Brown, T. D., Vrahas, M. S. J. Orthoped. Res. 1984, 2(1), 32-38 (reporting an nt elastic modulus of 1.372 GPa for machined caps of subchondral bone), Mente, P. L., Lewis, J. L. J. Orlhopea’. Res. 1994, 12(5), 637-47 (reporting an elastic modulus calculated from "pure" bovine subchondral bone beams of 2.3 :: 1.5 GPa), the contents of all of the foregoing of which are incorporated herein by reference in their entireties. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 5 GPa. In some embodiments, the fully set and cured injectable biomaterial has an c modulus of less than about 4 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 3 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 2 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic s of less than about 1 GPa. In some ments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 0.5 GPa. In some embodiments, the fully set and cured injectable biomaterial has an elastic modulus of less than about 0.25 GPa. er, the ors singly discovered that the disclosed injectable biomaterials set and cure without signiﬁcant s emission. Without wishing to be bound by theory, the inventors posit that the absence of gaseous release during g and curing post-administration results in an injectable biomaterial does not expand during setting and , thus reducing or ating post-operative pain as compared with prior art als.

Furthermore, the inventors posit that the absence of gaseous release during curing and setting post-administration facilitates the formation of a structure with the desired decreased porosity as compared with prior art materials. The inventors posit that the provision of an injectable biomaterial that does not comprise bicarbonate may serve to prevent gaseous release and thus results in a biomaterial with decreased porosity that does not cause post-operative pain.

WO 89733 tions The term "adherent to bone" as used herein with reference to an injectable biomaterial refers to the materials demonstration of sufﬁcient afﬁnity for bone such that it is not readily cleared away from the site of injection by bodily ﬂuids.

The term "bone disease" as used herein refers to a disease, ion, or pathology in a patient that is linked to bone ration. For example, the bone disease can affect a joint adjacent to a degenerate bone.

The term "cohesive" as used herein with reference to an injectable biomaterial refers to the ability of a material to adhere to itself and be molded such that it non-transiently maintains its shape over a period of time and ﬁlls an affected area of degenerate bone after injection.

The term "cure" as used herein with reference to an injectable biomaterial refers to the process whereby the components of the injectable biomaterial chemically and ally react to form the ﬁnal desired crystal structure. A material that is "cured" no longer oes appreciable changes in compressive strength or porosity. The terms "cure time" and "curing time" as used herein with nce to an able biomaterial refer to the time at which the able biomaterial is fully cured. In some embodiments, the injectable biomaterials disclosed herein cure to form an apatitic crystal structure.

The term "decompression" as used herein refers to a procedure to remove pressure on a structure.

The term "degenerate tissue" as used herein refers to tissue that has undergone a change to a lower or less functionally active form.

The term "degenerate bone" as used herein refers to an area of bone that has undergone a change to a lower or less functionally active form. In some embodiments, degenerate bone exhibits at least one change selected from (1) formation of higher volume fraction trabeculae relative to normal bone, (2) decreased mineral-to-matriX and carbonate-to- matriX values relative to normal bone, (3) increased intraosseous ﬂuid accumulation relative to normal bone, and (4) increased inﬁltration into the marrow space of a ﬁbrous collagen network relative to normal bone.

The term "dewater" as used herein with reference to an injectable biomaterial refers to separation of the solid component and the liquid component.

The term "ﬂowable" as used herein with reference to an injectable biomaterial refers to any generally incompressible material which may be caused to ﬂow under pressure or gravity.

The term "inﬂammatory mediator" as used herein refers to a biological component that induces an inﬂammatory response in an animal or a human. Inﬂammatory mediators include, but are not limited to, chemokines, such as C5, CCLl (1-309), CCL11 (eotaXin), CCL13 (MCP-4), CCL15 (MlP-ld), CCL16 (HCC-4), CCL17 (TARC), CCL2 (MCP-l), CCL20 (MIP-3a), CCL22 (MDC), CCL23 (MPIF-l), CCL24 (MPIF-2, EotaXin-2, , EotaXin-2), CCL26 (eotaXin-3), CCL3 (MlP-IA), CCL4(MIP-1B), CCLS (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CX3CL1, CXCL1 (GROl, GRO-alpha, SCYBl), CXCL10(INP10), CXCL11 (I-TAC, 113—9), CXCL12 (SDF1), CXCL13, CXCL2 (GR02, GRO-beta, SCYB2), CXCL3, CXCLS (ENA-78, LIX), CXCL6 (GCP-2), CXCL9 (MIG), interleukins, such as IL13, IL15, 1Ll6, 1Ll7A, IL17C, 1Ll7F, IL1A, lLlB, ILlRN, IL21, IL27, 1L3, 1L33, 1L5, 1L7, CXCL8, 1L9, cytokines such as A1MP1 (SCYEl), BMP2, CD4OLG (TNFSFS), CSF1 (MCSF), CSF2 F), CSF3 , FASLG (TNFSF6), GM-CSF, IFNA2, IFNG, 1L-1, 1L-6, 1L-8, 1L-15, IL-16, IL-17, IL-18, IFN—y, LTA (TNFB), LTB, MIF, NAMPT, OSM, SPP1, TGF-B, INF, TNF-u, TNFSFIO (TRAIL), 1 (RANKL), TNFSF13, 3B, TNFSF4 (OX4OL), VEGFA, and other atory mediators, such as bradykinin, calcitonin gene related peptide , histamine, lactic acid, nerve growth factor (NGF), prostaglandins, nce P, and vasoactive intestinal peptide, and mixtures thereof. Other inﬂammatory mediators are known to those d in the art.

The term "initial setting time" as used herein with reference to an injectable biomaterial refers to the st amount of time between (1) the miXing of the solid component and the liquid component and (2) the point where a 1 lb. (454 g) Gilmore Needle, having a tip diameter of 1/24 inch (1.06 mm) does not penetrate a sample of the injectable biomaterial of uniform thickness of 3/16 inch (5 mm), the length of the needle tip, in less than 1 minute. See ASTM C414-O3 at 7.2, 8.2 (reapproved 2012), the contents of which are incorporated herein by reference in their entirety. The term "set" or "settable" as used herein with reference to an injectable biomaterial refers to the ability of the injectable material to transition from that state ed at point (1) to point (2) described above.

The term "injectable" as used herein with reference to an able biomaterial refers to a material that is capable of being extruded from a syringe using no more than acceptable hand pressure applied to a r rod. Acceptable hand pressure is known to WO 89733 those of skill in the art. In some ments, the injectable biomaterial must be capable of being ed from a syringe without the use of mechanical advantage, such as a screw- driven syringe. In some embodiments, the syringe is a 3 mL syringe. In some embodiments, the syringe is a 5 mL e. In some embodiments, the syringe is a 10 mL syringe. In some embodiments, the syringe is a 14 mL syringe. In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 15 lb. extrusion force at a rate of 6 ute. In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 10 lb. extrusion force at a rate of 6 ute. In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 7.5 lb. ion force at a rate of 6 mL/minute. In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 5 lb. extrusion force at a rate of 6 mL/minute. In some embodiments, the syringe is coupled to an 11 gauge cannula.

In some embodiments, the syringe is coupled to an 14 gauge cannula. In some embodiments, the syringe is coupled to a 15 gauge cannula. In some embodiments, the syringe is d to an 18 gauge cannula. In some embodiments, the syringe is coupled to a 21 gauge cannula. In some embodiments, the e is coupled to a a and the injectable biomaterial must ﬂow down the length of the cannula in its ty without seizing or dewatering to be considered injectable. In some embodiments, the cannula is about 11 cm long. In some embodiments, the cannula is about 15 cm long.

The term "intermixable" as used herein with reference to an injectable biomaterial refers to the ability to achieve full mixing of the solid component and the liquid component when each is disposed in a 5 mL syringe, wherein the syringes are coupled and the contents extruded between the syringes for 1 minute using hand pressure with no visible seizing or dewatering.

The term "macroporous" as used herein with reference to an injectable erial refers to a material that possess pores which are e macroscopically.

The term "median pore diameter" as used herein refers to the pore diameter corresponding to 50% total intrusion volume from a cumulative intrusion volume vs. diameter plot. See Webb, P. An introduction to the physical characterization ofmaterials by y intrusion porosimetry with emphasis on reduction andpresentation ofexperimental data, Micromeritics Instrument Corp. (2001), the contents of which are incorporated herein by reference in their entirety.

The term "osteoconductive" as used herein with nce to an injectable biomaterial refers to the ability of an osteogenic material to serve as a ate, scaffold or ork supporting new bone growth that is perpetuated by the native bone.

The term "osteogenic" as used herein with reference to an injectable biomaterial refers to the ability of a al to promote the growth of new bone tissue. Exemplary osteogenic materials include, but are not limited to, bone marrow aspirate, bone marrow aspirate concentrate, platelet-rich plasma, platelet-poor plasma, somatic cell autografts, stem cell autografts, stem cell afts, and mixtures thereof. Other exemplary osteogenic materials are known to those skilled in the art.

The term inductive" as used herein with nce to an injectable biomaterial refers to the ability of an osteogenic material to recruit cells from the host that have the potential for forming new bone and repairing bone tissue. Osteoinductive injectable biomaterials according to the present disclosure stimulate osteoprogenitor cells to differentiate into osteoblasts that then begin new bone formation. Exemplary osteoinductive materials include, but are not limited to, BMP2, BMP7, BMP9, PDGF, and P15. See, e.g., Neiva, R. F. er a]. J. Periodontal. 2008, 79(2), 291-99, the contents of which are orated herein by reference in their entirety. Other osteoinductive materials are known to those skilled in the art.

The term "proteolytic enzymes" as used herein refers to enzymes capable of breaking down various proteins. Proteolytic enzymes include, but are not limited to, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases s), a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS), and es f.

Other proteolytic enzymes are known to those skilled in the art.

The term "patient" as used herein refers to humans and non-humans such as primates, pets and farm animals.

The term "resorbable" as used herein with reference to an injectable biomaterial refers to the ability of a material to be broken down and lated back into the body over time.

The term "self-setting" as used herein with reference to an injectable biomaterial refers to the ability of the material to form an apatitic crystal structure as a result of the mixing of the solid component and the liquid component. In contrast, certain prior art materials provide an apatitic crystal structure, e.g., by mixing pre-formed hydroxyapatite with a cement-forming material. Moreover, n such -forming materials (e.g., swellable polymers) lack dimensional stability and are permeable, and are thus inappropriate for preventing ingress of inﬂammatory and/or non-inﬂammatory mediators from an adjacent joint space.

The term "seize" as used herein with reference to an injectable biomaterial refers to the rapid sion of the injectable biomaterial to a material that is uninjectable.

The term "stable" as used herein with reference to an able biomaterial refers to the ability of the able biomaterial, or its precursors, to maintain suff1cient physical and/or chemical properties such that it still functions for its intended purpose in accordance with the present sure after a period of time has elapsed. For example, stability includes, but is not limited to, the ability of the able biomaterial to miX, set, and/or cure.

The terms "subchondral bone plate" and "cortical bone plate" are used herein interchangeably, and refer to the thin cortical lamella lying ately beneath the calciﬁed cartilage.

The term "total porous area" as used herein refers to the total sum of all pore wall area d from the volume of each incremental intrusion step. ng cylindrical pores, the wall area of each step i is At: 4 Vi/Di, where Vi = pore volume and Di = pore diameter. See Webb, P. An introduction to the physical characterization ofmaterials by mercury intrusion porosimetry with emphasis on reduction andpresentation ofexperimental data, Micromeritics Instrument Corp. (2001), the contents of which are orated herein by reference in their entirety.

The terms "treatment," "treating, 77 (L treat, 77 (Ltherapy, 77 (Ltherapeutic," and the like are used herein to refer lly to attempting to obtain a desired pharmacological, biological, and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing or delaying the onset of a condition or symptom thereof and/or may be therapeutic in terms of a partial or te stabilization, amelioration, or remedying of the condition or symptom.

The term "true density" as used herein refers to the mass divided by the solid volume or true skeletal volume. True density is usually determined after the substance has been reduced to a particle size so small that it accommodates no internal voids. See Webb, P.

An uction to the physical characterization ofmaterials by mercury intrusion porosimetry with emphasis on reduction andpresentation ofexperimental data, Micromeritics Instrument Corp. (2001), the ts of which are incorporated herein by WO 89733 2017/029651 reference in their entirety. True density can be measured, e.g., by helium pycnometry (such as by use of an AccuPyc II 1340 pycnometer).

The term ng time" as used herein with reference to an injectable biomaterial refers to the maximum amount of time between (1) the mixing of the solid component and the liquid component and (2) the point where the injectable biomaterial is no longer workable. The injectable biomaterial is workable if, after the elapsed time after mixing the solid component and the liquid component for one minute and injecting into an area of degenerate bone (or a substitute such as a e model), the injectable biomaterial ﬂows to fill the porosity of the area of administration prior to setting. The injectable biomaterial is no longer workable if, after the d time after mixing the solid component and the liquid component for one minute and injecting into an area of degenerate bone (or a substitute such as a sawbone model, the injectable biomaterial sets prior to ﬁlling the porosity of the area of stration. Tests for working time are known to those skilled in the art.

See, e.g., ASTM C414-03 at 7.2, 8.2 (reapproved 2012), the contents of which are incorporated herein by reference in their entirety.

Bone Structure Bone e often affects the whole of the adjacent joint. shows a schematic of a joint affected by bone disease. Femur 101 comprises cartilage 102 and femoral articular surface 103, while a 106 is shown above the joint space 110. The lower n of shows fibula 109 and tibia 108, the latter of which comprises an area of degenerate bone 105 into which a cannula 104 is inserted. A portion of tibial articular surface 107 is encompassed by callout 111, which is shown in greater detail in the schematics of FIGs. 2A-E, discussed in more detail as follows.

Subchondral bone plays a crucial role in the initiation and progression of bone diseases such as 0A. The term "subchondral bone" as used herein refers to the bony components lying distal to 1ed cartilage. Subchondral bone comprises the subchondral bone plate (aka cortical bone plate) and subchondral trabecular bone (aka cancellous bone).

The ndral bone plate and cancellous bone are not divided by a sharp border, but nonetheless are distinct anatomic entities.

As shown in FIGs. 2A-E, the subchondral bone plate is a thin cortical lamella, lying immediately beneath the calcif1ed cartilage 205. This bone plate is not an impenetrable structure, but rather possesses a marked porosity. It is invaded by channels that provide a direct link between articular calciﬁed age and subchondral trabecular bone. A high number of arterial and venous vessels and/or nerves (collectively 202), penetrate through the channels and send branches into calciﬁed age 205. The distribution and intensity of the channels depend not only on the age of the tissues, but also on the magnitude of the compressive forces transmitting through ed cartilage and subchondral bone within and between joints. These ls are preferentially concentrated in the heavily stressed areas of the joint. Channel shape and diameter also differs with the thickness of the cortical plate.

Channels are narrower and form a tree-like mesh in regions where the plate is thicker, while they tend to be wider and resemble ampullae where the subchondral bone plate is thinner.

Arising from the subchondral bone plate is the supporting trabeculae, which are structures that are part of and support the trabecular bone, and also include deeper bone structure. Subchondral trabecular bone exerts important shock-absorbing and supportive functions in normal , and may also be important for calciﬁed age nutrient supply and metabolism. Relative to the subchondral bone plate 207, ndral trabecular bone (not shown, but disposed distal the joint space from the subchondral bone plate 207) is more porous and lically active, containing blood vessels, sensory nerves, and bone marrow.

Subchondral trabecular bone has an inhomogeneous structure that varies with the distance from the articular surface 203/208. It eXhibits signiﬁcant structural and mechanical anisotropy, that is, the bone trabeculae show preferential spatial orientation and parallelism. ndral bone is a dynamic structure and is uniquely adapted to the mechanical forces imposed across the joint. In addition to bone density patterns and mechanical properties, subchondral bone also dynamically adjusts trabecular orientation and scale parameters in a precise relationship with principal stress. Mechanical stress also modiﬁes the contour and shape of subchondral bone by means of bone ng and remodeling. ndral bone and calciﬁed cartilage are dynamic stress-bearing structures that play complementary roles in load-bearing ofjoints. Subchondral bone supports overlying lar cartilage 210 and butes mechanical loads across joint surfaces with a gradual transition in stress and strain. Stiffened and less pliable subchondral bone tends to transmit increased loads to overlying cartilage, leading to secondary cartilage damage and degeneration. The load transmitted to underlying bone is substantially sed after articular cartilage damage or loss.

Articular cartilage overlies subchondral bone, and provides a vital on of maintaining homeostasis of the joint environment. It encompasses superﬁcial non-calciﬁed cartilage 210 and deeper calciﬁed cartilage 205. Calciﬁed cartilage 205 is permeable to small molecule transport, and plays an important role in the biochemical interaction between non- calciﬁed cartilage 210 and subchondral bone. It is separated from non-calciﬁed cartilage 210 by a boundary called the ark" (204), a dynamic structure that appears as a basophilic line in histological sections. The tidemark 204 represents the mineralization front of calciﬁed age 205, and provides a gradual transition between the two dissimilar cartilage regions.

Continuous collagen ﬁbrils cross the tidemark, indicating the strong link between non- calciﬁed cartilage 210 and calciﬁed cartilage 205. There is also a sharp line between calciﬁed cartilage 205 and subchondral bone, called the "cement line" (206). Unlike the tidemark 204, however, no continuous collagen ﬁbrils cross the cement line.

Given the intimate t between articular cartilage 210 and subchondral bone, they form a closely composited functional unit called the "osteochondral junction" (200).

The osteochondral junction 200 is peculiarly complex, and ts of a layer of non-calciﬁed cartilage 210, the tidemark 204, ed cartilage 205, the cement line 206 and subchondral bone.

In some embodiments, the affected area of bone is disposed about 50 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is ed about 40 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the ed area of bone is disposed about 30 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 20 mm or less from the joint articular surface al to the affected area of bone. In some embodiments, the ed area of bone is disposed about 15 mm or less from the joint articular surface proximal to the ed area of bone. In some ments, the affected area of bone is disposed about 10 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 9 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 8 mm or less from the joint articular e proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 7 mm or less from the joint articular e proximal to the ed area of bone. In some embodiments, the affected area of bone is disposed about 6 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 5 mm or less from the joint articular surface proximal to the ed area of bone. In some embodiments, the affected area of bone is disposed about 4 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed 3 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the affected area of bone is disposed about 2 mm or less from the joint articular surface proximal to the affected area of bone. In some embodiments, the ed area of bone is disposed about 1 mm or less from the joint articular surface proximal to the affected area of bone.

Etiology of Bone Disease Articular cartilage is both aneural and avascular. As such, cartilage is incapable of directly ting pain, stiffness (e.g., either the symptom of pain on moving a joint, the symptom of loss of range of motion, or the physical sign of reduced range of motion), or any of the symptoms that patients with bone disease typically describe. In st, the subchondral bone, periosteum, periarticular ligaments, periarticular muscle spasm, synovium and joint capsule are all richly innervated and can be the source of nociception in bone e. Furthermore, cross-talk , biochemical communication) between subchondral bone, articular cartilage 210, and joint space 211 is l for the initiation and progression of bone disease linked to bone degeneration, in terms of pain, function and pathology.

Alterations of any tissue will modulate the properties and functions of other parts of the osteochondral junction 200. There is intensive stress transfer and biochemical cross-talk across this region which plays a role in maintenance and ration of the joint.

The permeability of calciﬁed cartilage 205 and ndral bone plate 207 allows crossover communication, and provides connecting channels n ndral bone and the joint space 21 1. In vivo studies showed that prostaglandins, leukotrienes and various growth factors released by osteoblasts during subchondral bone remodeling could reach overlying articular cartilage 210. Conversely, inﬂammatory and last stimulation factors released by articular cartilage 210 also leads to subchondral bone deterioration through increased bone remodeling in bone disease.

During bone disease linked to bone degeneration, functional units ofjoints comprising cartilage and ndral bone can undergo uncontrolled lic and anabolic remodeling processes to adapt to local biochemical and biological signals. Changes in cartilage and subchondral bone are not merely secondary manifestations of bone disease but are active components of the disease, contributing to its severity. Increased vascularization and formation of microcracks in joints during bone disease have suggested the facilitation of movement of molecules from the joint space to subchondral bone and vice versa through the synovial and bone marrow ﬂuids. Several biological factors and signaling molecules produced from both tissues may passage from one zone to r, affecting homeostasis of neighboring tissue. Secreted cytokines, growth factors and signaling molecules form cartilage-bone biochemical units play modulatory roles to alter pathophysiology ofjoints during bone disease through pathways such as WNT (wingless type), BMP (bone morphogenic n), TGF-B (transforming growth factor B) and MAPK (mitogen-activated protein ) signaling. The close proximity of cartilage and ndral bone provides an ample unity to induce physical and functional alteration in each other through molecular interaction.

As a result of the mical cross-talk between the joint space 211 and subchondral bone, a degenerative biochemical response is initiated which accelerates as biomechanical changes begin to manifest themselves in patients with bone disease. Both the subchondral cortical plate 207 and cancellous bone show distinct differences in their behavior during ssion of bone disease and hence must be regarded as separate units to understand the joint deformation events. During progression of bone disease, subchondral bone turnover can be 20-fold increase compared to normal bone er. ndral bone in bone disease patients secrete high levels of alkaline phosphatase (ALP), osteocalcin, osteopontin, IL-6, IL-8, and ssive ankylosis protein g (ANKH), urokinase plasminogen tor (uPA), prostaglandin and growth factors, such as IGF-l, IGF-2 and TGF-B and Type I collagen compared to normal subchondral bone. These secreted biochemical factors contribute to bone formation, suggesting an enhanced bone anabolic activity of subchondral bone osteoblasts, exempliﬁed by formation of osteophytes and the sclerosis observed in bone e. However, the bone forming activity of subchondral bone is not necessarily accompanied by equivalent mineralization. Unmineralized immature new bone formation may lead to abundant osteoids in the subchondral bone (both at the level of the cortical plate and at the level of the trabecular bone) resulting in the opposite effect on tissue properties.

Inﬂammatory mediators present in synovial ﬂuid also contribute to lic ties of chondrocytes leading to remodeling of the cartilage extracellular matrix.

Chemokines, cytokines and proteases secreted from chondrocytes and t in the synovial ﬂuid alter biochemical (e.g., catabolic) and functional abilities of cartilage. During bone disease, chondrocytes have been found to secrete TNF-d, IL-l, ILlB converting enzyme (caspase-1) and type 1 IL-1 or. The concentration at which IL-1 is sized by chondrocytes is capable of inducing the activation of proteolytic enzymes, such as matrix metalloproteinases (MMPs), aggrecanases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) and other lic genes in regions of matrix depletion in affected cartilage. Furthermore, under these conditions, chondrocytes are stimulated to express molecules that are associated with ocyte hypertrophy and terminal differentiation, like VEGF, runt-related transcription factor 2 (RUNXZ) and .

Secretion of angiogenic factors such as VEGF increase vascularity within the deep layers of articular cartilage facilitating, together with the presence of microcracks, molecular transport of atory and/or ﬂammatory mediators by diffusion from the joint space and into the articular cartilage and the subchondral bone. Fine, unmyelinated nerves (C-ﬂbers and sympathetic ) accompany also these vessels and te normally aneural tissues, a source of bone disease pain. In addition, IL-6, in combination with other cytokines like ILlB, can switch osteoblasts from a normal phenotype to a tic ype. All these actors potentiate and stimulate the process of bone remodeling, altering the physiology of subchondral bone.

These inﬂammatory and/or non-inﬂammatory mediators also affect nociception in the synovial tissue and the subchondral bone. Nociceptors encompass a broad range of receptors for ligands that change the properties of these neurons, such that they require lower olds to ﬁre action potentials or even ﬂre neously when the receptors are engaged.

These ligands include, but are not limited to, cytokines, chemokines, neuropeptides and prostaglandins, which in some embodiments all form part of the biochemical milieu in the affected joint. As a result of this peripheral sensitization, joint movement within the normal range becomes painful (a phenomenon known as mechanical allodynia).

Furthermore, inﬂammatory mediators such as bradykinin, histamine, prostaglandins, lactic acid, substance P, vasoactive intestinal e, nerve growth factor (NGF), and calcitonin gene related peptide (CGRP) are released into the joint from e.g., synovial ﬁbroblasts and migrate into the subchondral bone and synovium, activating the nociceptors, located in those regions. These mediators reduce the ﬁring threshold of the nociceptors, making them more likely to respond to both non-noxious and noxious painful stimuli. As the disease sses, more and more of these mediators accumulate in the joint migrate into the subchondral space and synovium, y triggering a self-perpetuating cycle of pain generation.

As the damage progresses, degeneration of the subchondral bone s raphically visible. The ty of the joint damage on the radiograph may bear little relation to the severity of the pain experienced. However, utilizing imaging modalities such as magnetic nce imaging ("MRI"), signiﬁcant structural associations such as bone degeneration, sub-articular bone attrition, synovitis and effusion have been related to knee pain.

Without wishing to be bound by theory, the inventors posit that methods and compositions disclosed herein s these issues by breaking the biological communication between the joint space and the affected area of bone by optionally aspirating inﬂammatory and/or non-inﬂammatory mediators from the subchondral bone and subsequently ﬁlling the interconnected pores of the ed area with an injectable erial. shows a schematic of an exemplary embodiment of a diseased joint that can be treated according to the present disclosure. FIGs. 2A-E are a callout of the area of degenerate bone 105 shown in hondral junction 200 comprises an area of degenerate bone 209, surrounded by blood vessels/and or nerves 202. A biological ﬂuid comprising inﬂammatory and/or non- atory mediators 201 have permeated from the joint space 211 past the articular surface 203/208 through articular cartilage 210, tidemark 204, calciﬁed cartilage 205 and have collected in degenerate area of bone 209. In an exemplary ment shown in , cannula 211 is inserted into the area of degenerate bone 209 and the biological ﬂuid 201 is aspirated through the cannula 211 and removed. As shown in , injectable biomaterial 212 prepared according to the present disclosure ﬁlls the area of degenerate bone 209 in the affected area. As shown in , the injectable biomaterial cures to form a low porosity, high dimensional stability material 213, thereby halting biochemical communication to and from the joint space and protecting the affected area and nding cells. Moreover, the injectable biomaterial remains in place for a period of time, preventing the re-inﬁltration of these inﬂammatory and/or non-inﬂammatory mediators. Accordingly, biochemical communication n the affected area of bone and the joint space is arrested by blocking the porosity connecting the affected area of bone and joint space, temporally breaking biochemical communication and allowing the affected area of bone to recover. By shielding the affected area of bone from these actors, tic degeneration of the joint is prevented.

The latter s from the prevention of further biochemical degeneration of the bone and of WO 89733 2017/029651 the adjacent meniscal and cartilage tissues, and alleviation of the corresponding pain in the joint. Furthermore, certain injectable biomaterials according to the present disclosure provide for biomechanical repair of the affected area, such as by the ion of onductive and/or osteoinductive es to encourage natural bone healing processes, shifting the /repair equilibrium toward repair. This exemplary embodiment is shown in , where osteoclasts 214 resorb the cured injectable biomaterial 213 and osteoblasts 215 begin to build new, healthy bone 216. Importantly, the compositions and methods disclosed herein e their intended effects, namely cessation of the underlying causes of bone disease linked to bone degeneration, without further altering the biomechanical stability of the joint being treated or causing signiﬁcant pain post-operatively that can be associated with volume expansion from gas. They therefore offer several advantages over prior art treatments for bone disease.

Identiﬁcation of Bone for Treatment In some embodiments, an affected area for treatment according to the compositions and methods disclosed herein is identiﬁed by the use of MRI. In r embodiments, the MRI is a knee MRI. In further embodiments, the MRI is an ankle MRI. In some embodiments, the MRI is weight-bearing MRI. In some embodiments, the MRI is open MRI. In some embodiments, the MIR is upright open MRI. In further embodiments, the MRI is low ﬁeld strength MRI. In further ments, the MRI is an ultra-high ﬁeld MRI.

In further embodiments, the MRI is extremity MRI. In further embodiments, the MRI is whole body scanner MRI. In some embodiments, the affected area is identiﬁed by hyperintense signals on T2-weighted fat saturated MRI images. In some embodiments, MRI Tlp value, an indicator of early cartilage ation, is elevated in cartilage overlying bone marrow lesions, with the level of cartilage degradation proportional to Tlp signal intensity in a bone marrow lesion. In further embodiments, the affected area is ﬁed using Technetium-99 bone scans. In some embodiments, the affected area is identiﬁed using ﬂuoroscopy.

Without wishing to be bound by theory, the inventors posit that ed areas of bone thought to be associated with tis are ed less than 50 mm, 10 mm or 1 mm from the joint. Accordingly, in some embodiments, the methods and compositions disclosed herein are used to treat affected areas of bone which are disposed between about 0 mm to about 50 mm from the joint. In further embodiments, the affected area of bone is disposed between about 0 mm to about 10 mm from the joint. In still r embodiments, the affected area of bone is disposed between about 0 mm to about 1 mm from the joint. In some embodiments, the affected area of bone is disposed about 40 mm or less from the joint. In some embodiments, the affected area of bone is disposed about 20 mm or less from the joint.

In some embodiments, the affected area of bone is disposed about 15 mm or less from the joint. In some embodiments, the affected area of bone is ed about 10 mm or less from the joint. In some embodiments, the ed area of bone is disposed about 9 mm or less from the joint. In some embodiments, the affected area of bone is disposed about 8 mm or less from the joint. In some embodiments, the affected area of bone is disposed about 7 mm or less from the joint. In some ments, the ed area of bone is disposed about 6 mm or less from the joint. In some embodiments, the affected area of bone is disposed about mm or less from the joint. In some ments, the affected area of bone is disposed about 4 mm or less from the joint. In some embodiments, the affected area of bone is disposed 3 mm or less from the joint. In some embodiments, the affected area of bone is disposed about 2 mm or less from the joint. In some embodiments, the affected area of bone is disposed about 1 mm or less from the joint.

In some embodiments, the affected area of bone comprises a bone marrow lesion.

In further embodiments, the affected area of bone comprises degenerate cancellous bone space.

In some embodiments, the location of administration of injectable biomaterial is determined by ng a previously captured image of the affected area. In further embodiments, the location of administration of injectable erial is determined using additional guidance during surgery. In some embodiments, the additional guidance comprises real-time ﬂuoroscopic imaging. In further embodiments, the additional guidance comprises robotic devices. In further embodiments, the onal ce comprises braces for maintaining the joint in a position consistent with previously captured images of the joint.

In further embodiments, the additional guidance comprises the use of one or more . In some embodiments, the one or more labels comprise radioactive . In some embodiments, the radioactive labels comprise Technetium-99. In some embodiments, the one or more labels comprise radioactive label f1ducial markers.

Injectable Biomaterials The injectable erial disclosed herein is a physiologically compatible material that ﬁlls the porosity in the affected area of bone which is symptomatic of bone disease. Certain ties of the injectable biomaterial, namely that it is injectable, intermixable, ﬂowable, is cohesive, and adherent to bone, allow the biomaterial to ﬁll the porosity that exists in the affected area of bone without being readily cleared away by bodily ﬂuids.

The injectable biomaterials disclosed herein comprise a solid component and a liquid ent that comprises a carbohydrate. The injectable erials disclosed herein can be prepared by mixing of the solid component and the liquid component. The injectable biomaterials sed herein set and cure to form an apatitic crystal structure after mixing of the solid component and the liquid component. The solid component provides a solid al, or mix of materials, that reacts when combined with the liquid component to form the apatitic crystal structure. The liquid component provides a medium for the ents of the solid component to mix and react to form the apatitic crystal structure. In some embodiments, the solid component comprises a calcium ate. In some embodiments, the liquid component comprises water. In some embodiments, the solid component and/or the liquid component include additional components.

The liquid component of the injectable biomaterials disclosed herein includes a ydrate. While the carbohydrate itself may not be a liquid, when provided as a constituent of the liquid component, it is dissolved or suspended therein. In some embodiments, the liquid component is in the form of a gel or a el.

In some embodiments, the solid component and the liquid component are mixed at a particular ratio to e the desired injectable erial. In some embodiments, the ratio of solid component to liquid component is about 3 to about 1 by mass. In some embodiments, the ratio of solid component to liquid component is about 2 to about 1 by mass.

In some embodiments, the ratio of solid component to liquid component is about 1.5 to about 1 by mass. In some embodiments, the ratio of solid component to liquid component is about 1 to about 1 by mass. In some embodiments, these ratios of solid component to liquid component provide an injectable biomaterial that is injectable. While prior art materials comprising a solid component and a liquid component comprising a carbohydrate are disclosed, these materials e a higher powder-to-liquid ratio than is achievable according to the present sure. See, e.g., Ahmadzadeh-Asl, S. ei a1. Adv. Applied Ceramics 2011, 110(6), 340-45, the contents of which are incorporated herein by reference in their entirety.

Accordingly, these materials are not intermixable and require high extrusion forces to dispense the entirety of the al, resulting in compositions that cannot be readily administered in a lly invasive n with the ease of the presently disclosed injectable biomaterials. In contrast, the able biomaterials of the t sure are readily interrnixable, such that the solid component and liquid component can be provided in separate syringes, which are coupled to one another, the contents intermixed and then directly administered to an area of degenerate bone. This property not only es for an injectable biomaterial with additional convenience, ease of mixing and use, but also reduced chance of contamination. By contrast, prior art materials made using higher powder-to-liquid ratios lack intermixability such that they must be manually mixed in a container and subsequently transferred to a syringe, such as by a a, increasing the changes for contamination and compromising of sterility. See, e.g., Ahmadzadeh-Asl, S. ei a1. Adv. Applied Ceramics 2011, 110(6), 340-45, at 341, the contents of which are incorporated herein by reference in their entirety. The inventors have surprisingly discovered that, contrary to conventional wisdom, the d powder-to-liquid ratios able with the injectable biomaterials according to the present disclosure are still able to set in a commercially feasible time, remain cohesive, adhesive to bone, and provide the reduced strength desired by the inventors to prevent biomechanical stabilization. Moreover, prior art materials that ed carbohydrates began with materials capable of setting, and added a carbohydrate to improve ﬂowability and injectability. In st, the inventors surprisingly discovered that the addition of a carbohydrate to injectable biomaterials that were not capable of setting or ing cohesive surprisingly were able to set and remain cohesive by virtue of the carbohydrate addition.

Exemplary injectable biomaterials according to the present disclosure can comprise calcium phosphate cements, settable polymers such as lysine diisocyanates, proteins, such as collagen, gelatin and their derivatives, and carbohydrates, such as hyaluronic acid, tes, chitosan, cellulose, dextran and their derivatives. In r embodiments, the injectable biomaterial comprises bone, such as autografts, allografts, and artiﬁcial or synthetic bone substitutes. In certain ments, the injectable biomaterial comprises one or more of platelet-rich plasma ("PRP"), platelet-poor plasma ("PPP"), bone marrow aspirate ("BMA"), bone marrow aspirate concentrate ("BMAC"), or cell lysates. In r embodiments, the injectable biomaterial comprises at least one polymeric material.

In some embodiments, the injectable biomaterials disclosed herein are self-setting.

In some embodiments, the etting injectable biomaterials disclosed herein set and cure to form an fully set and cured injectable biomaterial that has a major phase of hydroxyapatite.

In further embodiments, the major phase is at least 95% hydroxyapatite. See ASTM F1185- 03 (reapproved 2014), at 42, ISO 13175-3 (2012), at 4.2.2, the contents of all of the foregoing of which are incorporated herein by reference in their entireties.

In some embodiments, the injectable erials disclosed herein are ﬂowable.

In contrast, prior art materials lack sufﬂcient lity to be able to be injected into an area of degenerate bone such that they ﬂow into and substantially ﬁll the area. While some prior art als were capable of being injected, they cease ﬂowing when backpressure ceases.

Accordingly, these materials tend to stay resident at the immediate location of administration, rather than ﬂowing to ﬁll in the increased porosity present in the area of rate bone.

Additionally, these materials tend to set prior to administration in full, and/or seize in the instrumentation. Accordingly, these materials were incapable of ing the requisite barrier to prevent inﬂux of atory and/or ﬂammatory mediators from the adjacent joint space.

In some embodiments, the solid component can comprise le constituents.

In some embodiments, the constituents react to form an apatitic crystal structure. In some embodiments, the constituents are provided as solid powders. In some embodiments, the le sizes of the powders of the constituents can be adjusted to effect the desired setting and curing times. For example, the particle size of constituents can be reduced to provide for faster reaction with other constituents, consequently shortening the initial setting time. sely, the particle size of constituents can be increased to provide for slower reaction with other constituents, consequently lengthening the initial setting time. See, e.g., Bohner, M. er a]. J. Mater. Chem. 2008, 18, 5669-75, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the solid component is substantially free of bicarbonate.

In accordance with certain aspects of the present invention, the injectable material can be injected into bone to ﬁll the ed area. In some embodiments, the injection volume of biomaterial is from about 1 to about 6 mL. In some ments, the injection volume of biomaterial is about 6 mL. In some embodiments, the injection volume of biomaterial is about 5 mL. In some embodiments, the injection volume of biomaterial is about 4 mL. In some embodiments, the injection volume of biomaterial is about 3 mL. In some embodiments, the injection volume of biomaterial is about 2 mL. In some embodiments, the injection volume of biomaterial is about 1 mL. Without wishing to be bound by theory, the inventors posit that the injectable biomaterial disclosed herein shields the ed area of bone from inﬂammatory and/or non-inﬂammatory mediators, thereby arresting, ting and/or ing degeneration of the joint. These effects result from the prevention of further biochemical degeneration of the bone and of the adjacent meniscal and cartilage tissues, and alleviation of the corresponding pain in the joint. Furthermore, the injectable biomaterials disclosed herein provide for biomechanical repair of the affected area, such as by the provision of osteoconductive and/or osteoinductive surfaces to age natural bone healing processes, shifting the damage/repair equilibrium toward repair. Importantly, the injectable biomaterials sed herein achieve their attended effects, namely cessation of the underlying causes of bone disease, without further altering the biomechanical stability of the joint being treated or causing signiﬁcant pain post-operatively that can be associated with volume expansion from gas.

In some embodiments, the injectable biomaterial has suitable viscosity such that it can be injected into the affected area from a syringe h an 10-21 gauge a. In some embodiments, the injectable biomaterial can be injected using pressure applied by no more than e hand and finger strength. Typical ranges of average hand and ﬁnger strength are known in the art, and are disclosed, for example, in DiDomenico, A, Nussbaum, M. A. Ergonomics 2003, 46(15), 1531-1548, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, an injectable biomaterial is able to be ed from a syringe using no more than 15 lb. extrusion force at a rate of 6 mL/minute.

In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 10 lb. ion force at a rate of 6 mL/minute. In some embodiments, an injectable biomaterial is able to be extruded from a syringe using no more than 5 lb. extrusion force at a rate of 6 mL/minute.

In some ments the ﬂowability comprises suff1cient ﬂowability such that the injectable biomaterial ﬂows into ty in the bone in the affected area prior to initially setting and/or . In some embodiments, the injectable biomaterial ﬂows into ty in the bone as a result of hand pressure applied to the e from which it is expelled.

In some embodiments, the injectable biomaterial disclosed herein is cohesive.

The cohesiveness of the injectable biomaterial manifests in the y of the material to resist phase separation (e.g., dewatering) over the time required to prepare and inject the material into the affected area, while simultaneously maintaining sufﬁcient ﬂowability such that the al can still be injected and caused to ﬂow through the porosity of the degenerate bone.

In some embodiments, the liquid component has a pH that can be modified to achieve the desired working, l setting, and curing times. See, e. g., Bohner, M. er a]. J.

Mater. Chem. 2008, 18, 5669-75, the contents of which are incorporated herein by reference in their entirety. For example, if lower g, initial setting, and curing times are desired, the pH of the liquid ent may be lowered. In some embodiments, the pH of the liquid component is between about 3 and about 8. In some embodiments, the pH of the liquid component is between about 3 and about 7. In some embodiments, the pH of the liquid component is between about 3 and about 6. In some embodiments, the pH of the liquid component is between about 4 and about 6. In some embodiments, the pH of the liquid component is between about 5 and about 6. In some embodiments, the pH of the liquid components is about 6. In some embodiments, the pH of the liquid component is adjusted using a pH adjusting agent. In some embodiments, the pH adjusting agent is selected from an organic acid and an inorganic acid. In some embodiments, the pH adjusting agent is selected from the group consisting of citric acid, formic acid, acetic acid, and mixtures thereof. In some embodiments, the pH adjusting agent s selected from the group ting of hydrochloric acid, phosphoric acid, nitric acid, and mixtures thereof. In some embodiments, the pH adjusting agent is citric acid.

In some embodiments, the liquid component comprises a salt whose concentration may also be modified to modify the desired working, initial setting, and curing times. For example, if lower working, l setting, and curing times are desired, the salt concentration of the setting on may be raised. In some embodiments, the liquid solution comprises a salt t at a concentration of about 0.01 to about 10 M. In further ments, the concentration of the salt is from about 0.1 to about 1 M. In further embodiments, the concentration of the salt is from about 0.2 to about 0.4 M. In further embodiments, the concentration of the salt is from about 0.3 M. In some embodiments, the salt is sodium ate dibasic, sodium silicate, sodium chloride, calcium hydroxide, or mixtures thereof.

In some embodiments, the salt is sodium phosphate dibasic.

In some ments, the liquid component comprises water as the solvent.

In some embodiments, the injectable biomaterial cures to form a al haVing a molar calcium to phosphorus ("Ca ") ratio of about 1 to about 2. In further embodiments, the material has a molar Ca/P ratio of about 1.3 to about 1.8. In further embodiments, the material has a molar Ca/P ratio of about 1.4 to about 1.7. In further embodiments, the material has a molar Ca/P ratio of about 1.5 to about 1.7. In further embodiments, the material has a molar Ca/P ratio of about 1.5 to about 1.667. Ca/P ratios can be determined according to methods known in the art. In some embodiments, the Ca/P ratio is ated theoretically. In some ments, the Ca/P ratio is ated using inductively-coupled plasma mass spectroscopy ("ICP-MS"). In some embodiments, the Ca/P ratio is calculated using ion chromatography.

In some embodiments, the injectable biomaterial disclosed herein is adherent to bone. In some embodiments, injectable biomaterial demonstrates suff1cient adherence to bone such that it remains resident at the location of stration for sufﬁcient time to t the re-inﬁltration of inﬂammatory and/or non-inﬂammatory mediators and to allow the d bone to heal. In some embodiments, the injectable biomaterial s substantially resident at the location of administration for up to about 30 days. In some embodiments, the injectable biomaterial remains substantially resident at the location of administration for up to about 2 months. In some ments, the able biomaterial remains substantially resident at the location of administration for up to about 3 months. In some embodiments, the injectable biomaterial remains substantially resident at the location of administration for up to about 6 months. In some ments, the injectable biomaterial remains substantially resident at the location of administration for up to about one year. In some embodiments, the injectable biomaterial remains substantially resident at the location of administration for up to about 18 months. In some embodiments, the injectable biomaterial remains substantially resident at the location of administration for up to about 2 years. In some embodiments, the injectable biomaterial remains substantially resident at the location of administration for up to about 30 . In some embodiments, the able biomaterial s substantially resident at the location of administration for up to about 3 years. In some embodiments, the injectable biomaterial is resident at the location of administration until the injectable biomaterial is completely resorbed. Resorption is the process by which osteoclasts break down the injectable biomaterial and replace it with healthy bone. See, e. g., Sheikh, Z. er a]. Materials, 2015, 8, 7913-25. In some embodiments, the adherence of the injectable biomaterial to bone ts the permeation of inﬂammatory and/or non- inﬂammatory mediators into the affected area, allowing the degenerate bone to heal and/or . In some embodiments, degenerate bone healing and/or repair prevents further cartilage damage.

In some embodiments, the able biomaterial is resorbable over time. In some embodiments, the injectable biomaterial is completely ed in about 30 days. In some embodiments, the injectable biomaterial is completely resorbed in about 2 months. In some embodiments, the injectable biomaterial is completely ed in about 3 . In some embodiments, the injectable erial is completely resorbed in about 6 months. In some ments, the injectable biomaterial is completely resorbed in about one year. In some embodiments, the injectable biomaterial is tely resorbed in about 18 months. In some embodiments, the injectable biomaterial is completely resorbed in about 2 years. In some embodiments, the injectable biomaterial is completely ed in about 30 . In some embodiments, the injectable biomaterial is completely resorbed in about 3 years.

In some embodiments, the injectable biomaterial disclosed herein is macroporous when set or cured. Macroporosity allows for the inﬁltration of endogenous cells from the host. Without wishing to be bound by theory, the inventors posit that macroporosity allows nous cells to stimulate bone remodeling at the affected area.

In some embodiments, the injectable biomaterial possesses suff1cient cohesion prior to setting and curing such that it remains in the location of administration, but lacks the compressive strength post-curing that would be required to substantially alter or t the existing biomechanics of the joint or biomechanically stabilize the ed area.

Consequently, an injection that changes the stress distribution within a bone will change the structure of the bone as well. Moreover, the provision of biomechanical support to an area of degenerate bone alone fails to address the underlying causes of bone disease and/or its symptoms and can lead to increases in biomechanical instability in other areas of the joint according to Wolff’ s law. Accordingly, the methods and compositions of the present disclosure do not directly prevent further degeneration of the bone by providing biomechanical support, but instead provide a mical environment to the affected area of bone which shields the bone from the effect of inﬂammatory and/or non-inﬂammatory actors and thus allows the bone to naturally heal and restore its original condition without Wolff’ s Law intervention.

In some embodiments, the injectable biomaterial disclosed herein includes an osteoinductive component. Osteoinductive injectable erials according to the present disclosure have the y to cause precursor cells (such as osteoprogenitors or mesenchymal stem cells) to differentiate into osteoblasts that then begin new bone formation in the affected area, thus facilitating rebuilding of healthy bone. Exemplary osteoinductive components suitable for use in the injectable erials disclosed herein include, but are not limited to, bone morphogenetic proteins ("BMP," e.g., thMP-Z), transforming growth factors (e.g., transforming growth factor beta or "TGF-beta"), osteoblast cells, polymers such as hyaluronic acid, poly-hydroxyethylmethacrylate ("Poly-HEMA"), metals such as titanium, various forms of calcium phosphates including hydroxyapatite, tricalcium phosphate, natural ceramics such as hydroxyapatite and yapatite/calcium carbonate, including those derived from coral exoskeleton, synthetic non-sintered calcium ate cs such as tricalcium phosphate, ium phosphate dihydrate, dicalcium phosphate anhydrous, hydroxyapatite, biphasic calcium phosphate, and octacalcium phosphate, synthetic sintered calcium phosphates such as pyrophosphate, hydroxyapatite, biphasic calcium phosphate, tricalcium phosphate, and carbonated apatite, other ceramics such as aluminum oxide, Bioglass®, and Pyrex®, composites such as hydroxyapatite/poly(D,L-lactide), and various other organic species known to induce bone formation by those of skill in the art. In some ments, the injectable biomaterial is prepared using a dilute suspension of type I collagen. In some embodiments, the osteoinductive component is BMP. In some embodiments, the osteoinductive component is TGF-beta. In some embodiments, the osteoinductive component is selected from PRP, PPP, BMA conditional media, BMAC, BMA lysate, cell lysates, and mixtures thereof.

In some ments, the injectable biomaterials disclosed herein including an osteoconductive component. Osteoconductive injectable biomaterials according to the present disclosure provide a ld or framework for new bone growth that is perpetuated by the native bone, thus facilitating rebuilding of healthy bone. Osteoblasts from native bone are supported by osteoconductive injectable biomaterials as they form new bone. Exemplary osteoconductive components le for use in the able biomaterials disclosed herein include, but are not limited to, ralized bone matrix ("DBM"), collagen, autograft, aft, tic scaffolds, and es thereof.

In some embodiments, osteoconductive and/or osteoinductive properties are provided by bone marrow, blood plasma, morselized bone of the patient, or commercially available materials. In some embodiments, osteoconductive and/or osteoinductive properties are provided by hydroxyapatite, cium phosphate, CaSO4, and/or other materials known to those of skill in the art.

In some embodiments, the injectable biomaterials disclosed herein have a working time ent to allow a person skilled in the art to administer the injectable biomaterial to an affected area in a patient after mixing of the solid component and the liquid component prior to the transition of the injectable biomaterial to a al that it is no longer injectable.

In some embodiments, the properties of the injectable erial disclosed herein are obtained after injection. For example, in some embodiments the injectable biomaterial is less adherent to bone prior to ion, but becomes more adherent to bone after injection into the affected area. In some embodiments, the able biomaterial becomes more adherent to bone after lly setting. In some embodiments, the injectable biomaterial s more adherent to bone after curing.

In some embodiments, the setting or curing of the injectable biomaterial is not signiﬁcantly exothermic. In some embodiments, the setting or curing of the injectable biomaterial is rrnic. Release of heat during the setting and/or curing of the injectable biomaterial in silu can result in damage to the surrounding tissues, and thus an injectable biomaterial that sets and/or cures rmically can prevent damage to these tissues.

In some embodiments, the carbohydrate is selected from the group consisting of dextran, alginate, carboxymethylcellulose, and hyaluronic acid. In some embodiments, the carbohydrate is hyaluronic acid. In some embodiments, inclusion of hyaluronic acid in the injectable biomaterial improves the intermixability, lity and cohesion of the injectable biomaterial, while providing a material that sets and cures to form an apatitic crystal structure. In some embodiments, the inclusion of onic acid in the injectable biomaterial weakens the injectable biomaterial to prevent biomechanical stabilization. In some embodiments, the able biomaterial including hyaluronic acid disclosed herein exhibit anti-inﬂammatory properties, which function to dampen the inﬂammatory milieu causing destruction of the subchondral bone.

In some embodiments, the injectable erial bonds to the bone. In further embodiments, the injectable biomaterial attaches to the bone. In further ments, the injectable biomaterial adheres to the bone. In some embodiments, the bonds are formed by biological processes in situ.

In certain embodiments, the injectable biomaterial is in the form of a ﬂuid. In further embodiments, the injectable biomaterial is in the form of a viscous liquid having a ity between about 5 Pas and about 30 Pas at room temperature. In some embodiments, the viscosity is between about 5 Pas to about 25 Pas. In some embodiments, the viscosity is between about 5 Pas to about 24 Pas. In some embodiments, the viscosity is between about 5 Pas to about 23 Pas. In some embodiments, the viscosity is between about Pas to about 22 Pa~s. In some embodiments, the viscosity is n about 5 Pa s to about 21 Pas. In some embodiments, the viscosity is between about 5 Pas to about 20 Pas. In some embodiments, the viscosity is between about 5 Pa s to about 19 Pa s. In some embodiments, the viscosity is between about 5 Pa s to about 18 Pa s. In some embodiments, the viscosity is n about 5 Pa s to about 17 Pa s. In some embodiments, the viscosity is between about 5 Pa s to about 16 Pa~s. In some embodiments, the viscosity is between about Pas to about 15 Pa~s. In some embodiments, the viscosity is between about 5 Pas to about 14 Pa~s. In some embodiments, the viscosity is between about 5 Pas to about 13 Pa~s. In some embodiments, the viscosity is between about 5 Pa s to about 12 Pa s. In some embodiments, the viscosity is n about 5 Pa s to about 11 Pa s. In some embodiments, the viscosity is between about 5 Pas to about 10 Pa~s. In some ments, viscosity is measured immediately after the mixing of the solid component and the liquid component. In r embodiments, the injectable biomaterial is in the form of a semi-solid. In further embodiments, the injectable biomaterial is in the form of a gel. In further ments, the injectable biomaterial is in the form of a hydrogel. In further embodiments, the injectable biomaterial is in the form of a dispersion. In further embodiments, the injectable biomaterial is in the form of a slurry.

In some embodiments, the injectable biomaterial remains in its originally-injected state after preparation and/or injection. In further embodiments, the injectable biomaterial lly sets to a less ﬂuid state after preparation and/or injection.

In some embodiments, the injectable biomaterial converts from a liquid to form a olid after preparation and/or injection. In some ments, the injectable erial converts from a liquid to form a semi-solid after preparation and/or injection over a working time. In some embodiments, the injectable biomaterial ts from a liquid to form a semi-solid after preparation and/or injection over an initial setting time. In some embodiments, the injectable biomaterial converts from a liquid to form a semi-solid after preparation and/or injection over a curing time. In some embodiments, the injectable biomaterial converts from a liquid to form a gel after preparation and/or injection. In some embodiments, the injectable biomaterial ts from a liquid to form a gel after preparation and/or injection over a working time. In some ments, the injectable biomaterial converts from a liquid to form a gel after preparation and/or injection over an initial setting time. In some embodiments, the injectable biomaterial converts from a liquid to form a gel after preparation and/or injection over a curing time. In some embodiments, the injectable WO 89733 2017/029651 biomaterial converts from a liquid to form a solid after preparation and/or injection. In some embodiments, the injectable biomaterial converts from a liquid to form a solid after preparation and/or injection over a working time. In some embodiments, the injectable biomaterial converts from a liquid to form a solid after preparation and/or injection over an initial setting time. In some embodiments, the injectable biomaterial converts from a liquid to form a solid after preparation and/or injection over a curing time.

In some embodiments, the injectable biomaterial is provided in a syringe. In some embodiments, the injectable biomaterial is provided in a syringe that is coupled to a a.

In some embodiments, the injectable biomaterial is injected into the bone so as to form an injectable biomaterial in situ. In some embodiments, an opening is created in the bone prior to injection of the injectable biomaterial.

In some ments, the injectable biomaterial is formed in a syringe. In some embodiments, the solid component is disposed in a ﬁrst syringe. In some embodiments, the liquid ent is disposed in a second e. In some ments, at least one of the ﬁrst and the second syringes comprises an integrated mixing system. In some embodiments, the injectable biomaterial is provided by injecting the contents of the second syringe into the ﬁrst syringe, thereby combining the solid component and the liquid ent. In some embodiments, the solid component and the liquid ent are mixed by repeated extrusion between the ﬁrst and second syringes. In some embodiments, the solid component and the liquid component are mixed by use of the integrating mixing system. Integrated mixing s are known in the art, such as the Medmix® P-System and em. See, e. g., Bone- Cemem Delivery System (P-SysleM), available at hiring? f3¥.3§f;.§il§£iﬂll;?i;.§l?xi’£gﬁi§liﬂ: item/boneucementudeliverywsvgten‘:"pusystem/ (last visited April 20, 2017). In some embodiments, the ﬁrst e is coupled to the second syringe. In some embodiments, the coupling is by Luer lock. In some embodiments, the Luer-Lock is then disconnected and the ﬁrst syringe is capped with an end cap. In some embodiments, the mixture in the ﬁrst syringe is then mixed using the integrated mixing system to form the injectable biomaterial.

In some embodiments, the injectable biomaterial and/or the ners in which it or its precursors are stored are sterile. In some embodiments, the sterility comprises a condition in which an object has a sterility assurance level (SAL) of 10—3 or less. In further embodiments, the sterility comprises a condition in which an object has a SAL of 10—6 or less.

In some embodiments, the SAL is determined in accordance with t FDA guidelines for WO 89733 medical devices. In some ments, the injectable material is sterile for up to up to 5 years.

In some embodiments, the injectable erial has a shelf life of at least about 3 months. In some embodiments, the injectable biomaterial has a shelf life of at least about 6 . In some embodiments, the injectable biomaterial has a shelf life of at least about 1 year. In some embodiments, the injectable biomaterial has a shelf life of at least about 18 months. In some embodiments, the injectable erial has a shelf life of at least about 2 years. In some embodiments, the injectable biomaterial has a shelf life of at least about 3 years. In some embodiments, the injectable erial has a shelf life of at least about 4 years. In some embodiments, the injectable biomaterial has a shelf life of at least about 5 years. s of Treatment The compositions and methods disclosed herein are useful for the treatment of degenerate bone in a patient. In some embodiments, the degenerate bone is disposed in an affected area of bone. In some embodiments, the affected area or bone is a region of bone that eXhibits inﬂammatory and/or degradative changes as a result of inﬂammatory and/or non-inﬂammatory mediators. In some embodiments, the methods and compositions disclosed herein are useful for the treatment of bone disease in a patient.

In some embodiments, the methods and compositions disclosed herein are useful for the treatment ofjoint pain in an affected area. In some embodiments, the methods and compositions disclosed herein are useful for the treatment of bone pain in an affected area. In some ments, the s and compositions disclosed herein are useful for the treatment of arthritic pain in an affected area. In some embodiments, the ed area is a knee. In further embodiments, the affected area is a hip. In further embodiments, the affected area is a shoulder. In further embodiments, the affected area is an ankle. In further embodiments, the affected area is a wrist. In further embodiments, the affected area is an elbow. In further embodiments, the affected area is a vertebrae. In further embodiments, the affected area is a hand.

In some embodiments, the methods and compositions disclosed herein are useful for the treatment of arthritis in an affected joint. In some embodiments, the arthritis is 0A.

In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the ed joint is a knee. In further embodiments, the ed joint is a hip. In further embodiments, the affected joint is a shoulder. In r embodiments, the affected joint is an ankle. In further embodiments, the affected joint is a wrist. In further embodiments, the ed joint is an elbow. In further embodiments, the affected joint is a vertebrae. In further embodiments, the affected joint is a join proximal to a hand.

In some embodiments, the methods and compositions disclosed herein are useful for the treatment of avascular necrosis. In some ments, the affected joint is a knee. In further embodiments, the affected joint is a hip. In further embodiments, the affected joint is a shoulder. In further embodiments, the affected joint is an ankle. In further embodiments, the affected joint is a wrist. In r embodiments, the affected joint is an elbow. In further embodiments, the affected joint is a vertebrae. In further embodiments, the affected joint is a joint proximal to a hand.

In some embodiments, the s and compositions disclosed herein are useful for the treatment of focal osteochondral defects in an affected bone. In some embodiments, the affected bone is a femoral condyle. In some ments, the affected bone is a humeral head. In some embodiments, the affected bone is a talus. In some embodiments, the affected bone is a llum of the humerus. In some ments, the affected bone is an elbow.

In some embodiments, the affected bone is a wrist. In some embodiments, the affected bone is a hand bone. In some embodiments, the affected bone is a toe. In some embodiments, the s and compositions disclosed herein are useful for the treatment of a femoral head. In some embodiments, the methods and compositions disclosed herein are useful for the ent of an acetabulum. In some embodiments, the methods and compositions disclosed herein are useful for the treatment of a tibial u.

In some embodiments, the affected area is a tight, pressure-fllled environment.

Accordingly, in some embodiments, the methods disclosed herein comprise the step of decompressing or ting the affected area. In some embodiments, the step of obtaining access to the affected area provides for ression of the affected area.

In some embodiments, the compositions and methods disclosed herein are used to fill bony voids or gaps that are not intrinsic to the stability of the bony structure. Typically, these bony voids or gaps are not regions with poor biomechanical integrity.

In some ments, the methods disclosed herein break the biochemical communication between the joint space and the affected area of bone. In some embodiments, the methods disclosed herein provide a protective coating around the affected area of bone against inﬂammatory and/or non-inﬂammatory mediators. In some embodiments, the methods disclosed herein decrease pain associated with arthritis. In some embodiments, the methods disclosed herein slow the progression of arthritis. In some ments, the methods disclosed herein stop the progression of arthritis.

In some embodiments, the injectable biomaterial is administered to the affected area via a cannula. In some embodiments, the cannula is between 10 and 21 gauge. In some embodiments, the cannula has an integrated trocar to allow for penetration of the al bone and the ion of a ﬂuid-tight seal in the ed area. In some embodiments, the cannula is fenestrated to allow for directional injection of the injectable biomaterial. In some embodiments, the cannula is non-fenestrated. In some cases, the cannula is designed to facilitate either directional ion of the biomaterial through a fenestrations located in the wall of the cannula or through an opening at the distal end of the cannula. In accordance with this type of system, an outer cannula and two inner cannulas are provided. The outer cannula is provided with fenestrations in the wall of the cannula and an open distal tip. The ﬁrst inner cannula is provided with fenestrations in the wall of the a and a closed distal tip. The second inner cannula includes an g in the distal tip and no fenestrations in the side wall of the a. The user can then select the first inner cannula for insertion into the outer cannula, whereby when the ﬁrst inner cannula is coupled to a syringe and the contents extruded, directional injection is achieved. atively, the user can select the second inner cannula for ion into the outer cannula, whereby when the second inner cannula is coupled to a syringe and the contents extruded, injection through the distal tip is achieved.

Fenestrated and non-fenestrated cannulas, such as the Ranfac Bone Marrow Aspiration and Access s are known in the art. In some ments, the cannula is steerable to minimize surgical damage due to the need to create more intrusive access into affected areas that are in diff1cult-to-access places. Steerable cannulas, such as the Osseon® Osseoﬂex® SN, are known in the art. In some embodiments, after injection the syringe is removed from the cannula and a rod is used to push the remaining injectable biomaterial disposed in the cannula into the affected area.

In some embodiments, the injectable biomaterial is injected without increasing post-operative intra-osseous pressure, resulting in no or l post-operative pain that can be ated with volume ion from gas.

In accordance with certain aspects, the procedure produces short term pain reduction in S 7 days and continue to reduce pain and prevent total joint replacement for Z 2 years, as measured by visual analog scored (VAS) or any other clinical accepted measure for pain and/or function.

In some embodiments, patients are required to maintain partial weight bearing and use ambulatory aids post-operatively. In some embodiments, full weight bearing is permitted post-operatively. In some embodiments, post-intervention physical therapy is required. In some embodiments, patients require routine post intervention care, observation and follow- In some embodiments, the methods disclosed herein further comprise the application of electrical ation to the bone to e bone healing.

In another aspect, the present disclosure provides a kit comprising an injectable biomaterial and instructions for use of the same.

In some embodiments, the kit comprises two syringes. In some embodiments, a solid component is disposed in a ﬁrst syringe and a liquid component is disposed in a second syringe. When mixed together, the solid component and liquid component form the injectable biomaterial. In some embodiments, at least one of the syringes in the kit comprises an integrated miXing device for in silu mixing of premeasured portions of ingredients from each of the ﬁrst and second syringes, wherein the ingredients form the injectable biomaterial upon ation. In some embodiments, the kit comprises a Luer lock. In some embodiments, the kit comprises an end cap. In some embodiments the kit comprises a cannula. In some embodiment, the cannula comprises and inner cannula and an outer cannula. In some ments, at least one of the syringes is disposed in a sealed pouch to protect the contents from moisture. In some embodiments, the pouch is ucted of foil reinforced with nylon.

In some embodiments, the kit ses bone tools. In some embodiments, the bone tools are adapted to provide a channel in the bone into which the injectable biomaterial is injected. In some embodiments, the kit ses a bone ﬁller to seal the open end of the l in the bone in which the injectable biomaterial is injected. In some embodiments, the kit comprising the bone tools is ct from the kit comprising the solid ent and the liquid ent.

In some embodiments, at least a n of the kit and its contents are sterile. In some embodiments, the sterility comprises a condition in which an object has a sterility assurance level (SAL) of 10—3 or less. In further embodiments, the sterility comprises a condition in which an object has a SAL of 10—6 or less. In some embodiments, the SAL is determined in accordance with current FDA guidelines for medical devices. In some embodiments, the syringes are sterile.

EXAMPLES The following es further describe and demonstrate embodiments within the scope of the present disclosure. The examples are given solely for the e of illustration and are not to be construed as tions of the present disclosure, as many variations thereof are possible without departing from the spirit and scope of the disclosure.

Example 1: Diagnosis of a Patient in Need of Treatment Described herein is an exemplary diagnosis of a patient in need of treatment for bone disease according to the present disclosure.

A patient presents with pain in a joint, for example a knee joint. Pain and activity are evaluated using a clinical score such as KOOS, IKDC, and/or Tegner Lysholm Activity Scale, which reveals increased pain and sed function relative to an unaffected joint.

See, e.g., Collins, N. J. el al. Arthritis Care Res. (Hoboken) 2011, 63(011), 28, the contents of which are incorporated herein by reference in their entirety. tional radiography does not reveal an obvious cause thereof. Accordingly, the patient undergoes T2 MRI to fy an area of bone degeneration, visible as an intense white area in the MRI output.

Example 2: Method of Treating a Patient Described herein is an exemplary method of treatment for bone disease in a patient in need thereof according to the present disclosure.

The surgical area is draped and d using standard surgical protocols. The leg of the t is abducted and a mini-ﬂuoroscopy unit is placed so that appropriate anteroposterior and lateral views of the knee can be obtained. The appropriate starting site is identified based on the location of the affected area of bone. Cannula tory is determined and an on is made in the skin of the patient at a location that allows for access to the affected area of bone.

A trocar of a bone marrow aspiration needle is used to access an area adjacent to the affected area and the tip of the bone marrow aspiration needle is inserted and d through the remaining cortex and into or adjacent the affected area of bone. Optionally, ﬂuoroscopy is present in the ing room to allow for veriﬁcation of instrument location.

Optionally, a K-wire is used to drill through the cortex to the site of injection and a cannula is placed over the K-wire. The trocar is removed from the cannula and the contents of the affected area of bone are optionally aspirated, such as by suction. ally, an outer cannula and two inner cannulas are provided. The outer cannula is provided with fenestrations in the wall of the cannula and an open distal tip. The ﬁrst inner cannula is provided with rations in the wall of the cannula and a closed distal tip. The second inner cannula includes an opening in the distal tip and no rations in the side wall of the cannula. The user can then select the ﬁrst inner cannula for insertion into the outer cannula, whereby when the ﬁrst inner cannula is coupled to a e and the contents extruded, directional ion is achieved. Alternatively, the user can select the second inner cannula for insertion into the outer a, whereby when the second inner cannula is coupled to a syringe and the contents extruded, injection h the distal tip is ed.

Next, a e comprising an injectable biomaterial according to the present disclosure is prepared and coupled to the cannula. Pressure is applied to the e causing the injectable biomaterial to be injected through the cannula in an amount sufﬁcient to ﬁll the degenerate area of bone in the affected area. Administration can be perpendicular relative to the long axis of the bone or at an angle relative to the long axis of the bone. The injectable biomaterial is optionally allowed to sit in the affected area for 5-30 minutes before removal of the cannula. During this time, the working time and/or initial setting time of the injectable biomaterial can expire. Initial g of the injectable biomaterial can reduce clearance of the injectable biomaterial from the affected area by ﬂuids, such as bodily ﬂuids or ﬂuids from surgical irrigation. During removal, additional injectable biomaterial can optionally be extruded into the space to ﬁll the space in which the cannula was resident.

Final ﬂuoroscopic images are obtained to conﬁrm the appropriate location of the injected biomaterial and an arthroscope is inserted into the knee to verify that no injectable biomaterial has extravasated into the capsule (e.g., that no extrusion of the injectable biomaterial occurs into the joint space post-implantation, such as via the microcracks).

Arthroscopy is optionally used to address other correctable issues (e.g., meniscal tears, osteophytes, etc.) during the procedure. The cannula is removed from the patient and the incision is closed, such as by simple s.

After clinical evaluation after a period of one month, the patient shows an improvement in clinical scores, such as KOO S, IKDC, and/or Tegner Lysholm Activity Scale.

Example 3: Exemplary Solid Components Described herein are exemplary solid components according to the present disclosure.

Solid Component 1 A 98.5 g batch of solid component was made as s. Separate s of 83.0 g of alpha tricalcium phosphate ("d-TCP," Ca3(PO4)2), 14.5 g calcium ate ), and 1.00 g calcium phosphate monobasic monohydrate ("monocalcium phosphate monohydrate," Ca(H2PO4)2 H20) were weighed out as powders and separately dried at a temperature of at least 165 oC overnight, for at least 12 hours. The dried s were then combined in a jar and mixed by hand shaking for 10 minutes to produce a 98.5 g batch of Solid Component 1 containing 84.3% alpha cium phosphate, 14.7% m carbonate, and 1.02% calcium phosphate monobasic monohydrate (mass/mass).

Aliquots of the resulting solid component were then dispensed into sterile syringes comprising integrated mixing devices (Medmix Systems AG, Rotkreuz, Switzerland). Into 3 mL sterile syringes were dispensed 1.50 g of Solid Component 1. Into 14 mL sterile syringes were dispensed 4.00 g of Solid Component 1.

Particle size analysis was conducted on the component powders using a Malvern MasterSizer 2000 to ensure compliance with a set of exemplary particle size speciﬁcations as detailed in Table 1 below.

Table 1: Exem la ements of Particle Size of Solid Com onent Constituents CaCO3 -5.73 Ca(H2PO4)2 13.17 -75.88 Particle size of alpha-TCP and calcium carbonate were measured Via laser diffraction of a water dispersion. Particle size of calcium phosphate monobasic monohydrate were ed by laser diffraction of an isopropanol dispersion. Particle size measurements were conducted in accordance with USP <429>, the ts of which are incorporated herein by nce in the entirety. Particle size analysis was performed using MasterSizer 2000 software. The particle sizes measured were found to be in the acceptable range for each component.

Solid Component 2 A 100. g batch of solid component was made as follows. Separate amounts of 83.0 g of alpha tricalcium phosphate ("d-TCP," Ca3(PO4)2), 16.0 g m carbonate (CaCO3), and 1.00 g calcium phosphate monobasic monohydrate ("monocalcium phosphate monohydrate," Ca(H2PO4)2 H20) were weighed out as powders and separately dried at a temperature of at least 165 oC overnight, for at least 12 hours. The dried powders were then combined in a jar and mixed by hand shaking for 10 minutes to e a 100. g batch of Solid Component 2 containing 83.0% alpha tricalcium phosphate, 16.0% calcium carbonate, and 1.00% m ate monobasic drate (mass/mass).

Aliquots of the resulting solid component were then dispensed into sterile syringes comprising integrated miXing deVices (MedmiX Systems AG, Rotkreuz, Switzerland). Into 3 mL sterile syringes were dispensed 1.50 g of Solid ent 2. Into 14 mL sterile syringes were dispensed 4.00 g of Solid ent 2.

Example 4: Exemplary Liquid Components bed herein are exemplary liquid components according to the present disclosure.

Control Liquid Comp_onent A control liquid component lacking a carbohydrate was prepared by dissolVing sodium phosphate dibasic in sterile water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the solution was adjusted to about pH 6 using citric acid.

Aliquots of the resulting liquid ent were then dispensed into sterile syringes n Dickinson, Franklin Lakes, NJ). Into 3 mL sterile syringes were dispensed 1.50 mL of the Control Liquid Component. Into 5 mL sterile syringes were dispensed 4.00 mL of the Control Liquid Component.

Liquid Component 1 A liquid component comprising hyaluronic acid was prepared by dissolving sodium ate c in sterile water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the solution was adjusted to about pH 6 using citric acid. Sodium hyaluronate having an average lar weight of 0.90 X 106 was added to a ﬁnal concentration of 6.0 mg/mL.

Aliquots of the resulting liquid component were then dispensed into sterile syringes (Becton Dickinson, Franklin Lakes, NJ). Into 3 mL sterile syringes was dispensed 1.50 mL of the liquid component. Into 5 mL sterile syringes was dispensed 4.00 mL of the liquid component.

Liquid Component 2 A liquid component comprising hyaluronic acid was prepared by dissolving sodium phosphate dibasic in sterile water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the on was adjusted to about pH 6 using citric acid. Sodium hyaluronate having an average molecular weight of 1.7 X 106 was added to a ﬁnal concentration of 6.0 mg/mL.

Aliquots of the resulting liquid component were then dispensed into sterile syringes (Becton Dickinson, Franklin Lakes, NJ). Into 3 mL sterile es was dispensed 1.50 mL of the liquid ent. Into 5 mL sterile syringes was dispensed 4.00 mL of the liquid component.

Liquid Component 3 A liquid component sing hyaluronic acid was prepared by dissolving sodium phosphate dibasic in sterile water for injection to a tration of 0.30 M. Once fully dissolved, the pH of the solution was adjusted to about pH 6 using citric acid. Sodium onate having an average lar weight of 2.6 X 106 was added to a ﬁnal concentration of 6.0 mg/mL.

Aliquots of the resulting liquid component were then dispensed into sterile syringes n Dickinson, Franklin Lakes, NJ). Into 3 mL sterile syringes was dispensed 1.50 mL of the liquid component. Into 5 mL sterile syringes was dispensed 4.00 mL of the liquid component.

Liquid Component 4 A liquid component comprising alginic acid is prepared by dissolving sodium phosphate dibasic in sterile water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the solution is adjusted to about pH 6 using citric acid. Sodium te -Aldrich, St. Louis, MO) is added to a ﬁnal concentration of 6.0 mg/mL.

Aliquots of the resulting liquid component are then dispensed into sterile syringes (Becton Dickinson, Franklin Lakes, NJ). Into 3 mL sterile es is dispensed 1.50 mL of the liquid component. Into 5 mL sterile syringes is dispensed 4.00 mL of the liquid component.

Liquid Component 5 A liquid component comprising chitosan is prepared by dissolving sodium phosphate dibasic in sterile water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the solution is adjusted to about pH 6 using citric acid. Medium molecular weight chitosan (Sigma-Aldrich, St. Louis, MO) is added to a ﬁnal concentration of 6.0 mg/mL.

Aliquots of the resulting liquid component are then dispensed into sterile syringes (Becton Dickinson, Franklin Lakes, NJ). Into 3 mL sterile syringes is dispensed 1.50 mL of the liquid component. Into 5 mL sterile syringes is dispensed 4.00 mL of the liquid component.

Liquid Component 6 A liquid component comprising cellulose is prepared by dissolving sodium phosphate dibasic in sterile water for injection to a tration of 0.30 M. Once fully dissolved, the pH of the solution is adjusted to about pH 6 using citric acid. rystalline cellulose (Sigma-Aldrich, St. Louis, MO) is added to a ﬁnal tration of 6.0 mg/mL.

Aliquots of the resulting liquid component are then dispensed into sterile syringes n son, Franklin Lakes, NJ). Into 3 mL e syringes is dispensed 1.50 mL of the liquid component. Into 5 mL sterile syringes is dispensed 4.00 mL of the liquid component.

Liquid Component 7 A liquid component comprising n is prepared by dissolving sodium phosphate dibasic in e water for injection to a concentration of 0.30 M. Once fully dissolved, the pH of the on is adjusted to about pH 6 using citric acid. Dextran from Leuconostoc spp. with a relative molecular weight of 450,000-650,000 (Sigma-Aldrich, St.

Louis, MO) is added to a ﬁnal concentration of 6.0 mg/mL.

Example 5: Preparation of Injectable Biomaterials Described herein is the preparation of exemplary injectable biomaterials according to the present disclosure.

The injectable biomaterials indicated in Table 2 below were prepared according to the following procedure. A ﬁrst syringe containing the indicated solid ent described in Example 3 and comprising an integrated mixing deVice was coupled Via Luer lock to a second syringe containing the indicated liquid component bed in Example 4. The contents of the ﬁrst syringe were expelled into the second syringe. The Luer lock and the second syringe were removed and an end cap was coupled to the ﬁrst syringe. The integrated mixing deVice was actuated and the ts of the ﬁrst syringe were mixed for one .

The ant injectable biomaterial can then be dispensed by removal of the end cap and extrusion of the contents, either directly or Via a cannula or syringe coupled to the ﬁrst syringe.

Table 2: Exemplaﬂ Injectable Biomaterials Solid Component Liquid ent (amount) (amount) Control Inj ectable l (1.50 mL) 1(1.50 g) Biomaterial Injectable Biomaterial l l (1.50 g) l (1.50 mL) Injectable erial 2 l (1.50 g) 2 (1.50 mL) Injectable Biomaterial 3 l (1.50 g) 3 (1.50 mL) Injectable Biomaterial 4 l (4.00 g) l (4.00 mL) Injectable Biomaterial 5 l (4.00 g) 2 (4.00 mL) Injectable Biomaterial 6 l (4.00 g) 3 (4.00 mL) Example 6: Comparison of able Biomaterials Including and Lacking a Carbohydrate Described herein is a comparison of exemplary injectable biomaterials according to the present disclosure with materials lacking a ydrate.

Samples of the Control Injectable Biomaterial and Injectable Biomaterial 3 were prepared as indicated in e 5. Immediately after preparation, the syringes containing each ition were coupled to 18 gauge needles. The contents of the tive syringes were each expelled into separate vials, each containing 10.0 mL phosphate buffered saline ("PBS") at 37 oC. The vials were placed on a shaker plate at 125 rpm for 15 minutes. After removal from the shaker plate, the vials were immediately photographed as shown in FIGs. 3A-B. As shown in , the Control Injectable erial (1'.e., lacking a carbohydrate) produced an un-set powder mixture that was partly suspended in on and evenly settled at the bottom of the vial. The m settling of the material indicates a lack of setting and cohesiveness, and indicates this material is unsuitable for treatment of rate bone as disclosed herein. In contrast, demonstrates that an able biomaterial according to the present disclosure (1'.e., Injectable Biomaterial 3) is cohesive and sets to form a material suitable for the ent of degenerate bone as disclosed herein.

Example 7: Second Comparison of Injectable erials Including and Lacking a Carbohydrate Described herein is a comparison of exemplary injectable biomaterials according to the present disclosure with materials lacking a carbohydrate.

In another example comparing the properties of injectable erials according to the present disclosure made using carbohydrates of varying molecular weights with materials lacking a carbohydrate, compositions of the Control Injectable Biomaterial, Injectable Biomaterial 1, Injectable Biomaterial 2, and Injectable Biomaterial 3 were separately prepared as indicated in Example 5. Immediately after preparation, the syringes contain each composition were coupled to 18 gauge needles. The contents of the respective syringes were each expelled into separate vials, each containing 10.0 mL PBS at 37 oC. The vials were placed on a shaker plate at 125 rpm for 15 minutes. After removal from the shaker plate, the vials were immediately photographed as shown in FIGs. 4A-D. As shown in , the Control Injectable Biomaterial (1'.e., lacking a carbohydrate) produces an un-set powder mixture partly suspended in solution and evenly settled at the bottom of the vial. The uniform ng of the material tes a lack of setting and cohesiveness, and tes this material is unsuitable for treatment of degenerate bone as disclosed herein. In contrast, (Injectable Biomaterial 1), (Injectable Biomaterial 2), and (Injectable Biomaterial 3) demonstrate that injectable biomaterials according to the t disclosure made utilizing carbohydrates having a variety of molecular weights are cohesive and set to form materials suitable for the treatment of degenerate bone as disclosed herein.

FIGS. 5A-D show the same materials as FIGS. 4A-D after l of excess PBS by pipette, washing of the material with additional PBS (3 x 10 mL) and drying (100 oC, 3 hours). As shown in , the Control Inj ectable erial (i.e., lacking a carbohydrate) produces an un-set loose powder mixture evenly distributed over the bottom of the vial. The uniform settling of the material indicates a lack of setting and cohesiveness, and indicates this material is unsuitable for treatment of degenerate bone as disclosed herein. In contrast, FIGs. 5B-D demonstrate that injectable erials according to the present sure made utilizing carbohydrates having a range of molecular weights are cohesive and set to form materials suitable for the treatment of degenerate bone as disclosed herein.

Mass of the injectable biomaterials shown in FIGs. 4A-D and FIGs. 5A-D were measured prior to and after removal of excess liquid. The Control Injectable Biomaterial (i.e., g a carbohydrate) shown in and retained only 54% of its mass, representing a considerable loss in the amount of material formed. In contrast, the injectable biomaterials according to the present disclosure shown in FIGs. 4B-D and FIGs. 5B-D retained 92%, 95% and 88% of their masses, respectively, demonstrating a much higher yield of the desired material.

Example 8: Evaluation of Injectable Biomaterials in Sawbone bed herein is the evaluation of exemplary injectable biomaterials ing to the present disclosure when injected into sawbone as compared with materials lacking a carbohydrate. Sawbone provides a useful model for the evaluation of the performance of injectable biomaterials in patient bone as it mimics the porosity of cancellous bone. See, e. g., Patel, P. S. D. el al. BMC Musculoskelelal Disorders 2008, 9, 137, the contents of which are incorporated herein in their entirety.

Sawbones Open Cell Block 15 PCF ( 1522-524, Paciﬁc Research Laboratories, Vashon Island, WA) was ted into several sample blocks using a hacksaw. On a level, ﬂat face of each sample block was d a hole approximately 6 mm in diameter and 12 mm deep to simulate a degenerate area of bone. Each sample block was submerged into PBS at 37 0C. 18 gauge needles were coupled to syringes ning compositions of the Control Injectable Biomaterial, Injectable erial 1, Injectable Biomaterial 2, and Injectable Biomaterial 3, separately prepared as indicated in Example 5. Each needle was inserted into the side of a sample block and into the simulated rate area of bone, and the compositions were injected. After g for 15 minutes in the heated PBS solution, the sample blocks were removed from the medium and washed with deionized water to remove cement debris, in part to mimic normal function of bodily ﬂuids. The blocks were shaken to remove excess water, dried using compressed air and photographed as shown in FIGs. 6A-D.

As shown in , no perceptible amount of the l Injectable Biomaterial (1'. e., lacking a ydrate) remained and set in the defect, leaving the defect substantially unaffected. In st, as shown in FIGs. 6B-D, Injectable Biomaterial l, Injectable Biomaterial 2, and Injectable Biomaterial 3 having various molecular weights demonstrated cohesion and set to substantially ﬁll the defects in the sample blocks of sawbone. These results demonstrate the utility of the injectable biomaterials according to the t sure in retaining cohesiveness and adhering to a bone-like substance to effectively ﬁll and protect a material that mimics an area of degenerate bone in a patient.

Example 9: Second Evaluation of Injectable Biomaterials in Sawbone Described herein is the evaluation of exemplary able biomaterials according to the present disclosure when injected into sawbone as compared with materials lacking a carbohydrate.

In another example comparing the properties of injectable biomaterials according to the present disclosure with materials g a ydrate, compositions of the Control Injectable Biomaterial and Injectable Biomaterial 3 were separately prepared as indicated in Example 5. Two roughly cylindrical samples of es Open Cell Block 15 PCF ( 1522- 524, Pacific Research Laboratories, Vashon Island, WA) were prepared using a hacksaw. On a level, ﬂat face of each sample block was drilled a hole approximately 6 mm in diameter and 12 mm deep to simulate a degenerate area of bone. Each sample block was submerged into PBS at 37 0C. 18 gauge s were coupled to syringes containing compositions of the Control Injectable Biomaterial and Injectable Biomaterial 3. Each needle was inserted into the side of a sample block and into the simulated rate area of bone, and the compositions were injected. After sitting for 15 minutes in the heated PBS solution, the sample blocks were removed from the medium and washed with deionized water to remove cement debris, in part to mimic normal function of bodily ﬂuids. The blocks were shaken to remove excess water, dried using compressed air, cut cross-sectionally through the simulated defect using a hacksaw, photographed as shown in FIGs. 7A-B. As shown in , little of the l Injectable Biomaterial (i.e., lacking a carbohydrate) remained and set in the defect, leaving the defect substantially unaffected. In contrast, as shown in , Injectable Biomaterial 3, prepared according to the present disclosure, was cohesive and set to substantially ﬁll the defect in e. These results demonstrate the utility of the injectable biomaterials according to the present disclosure in retaining cohesiveness and ng to a bone-like substance to effectively ﬁll and protect a material that mimics an area of degenerate bone in a patient.

Example 10: Evaluation of Diffusional Permeability of Injectable Biomaterials Described herein is the evaluation of the diffusional permeability properties of exemplary injectable biomaterials according to the present sure as compared with, inter alia, a control composition lacking a carbohydrate.

In an in vitro experiment, the diffusional permeability of injectable biomaterials according to the present disclosure was compared to injectable biomaterials lacking a carbohydrate. This experiment utilized Transwells® (Corning Inc., Corning, NY), two-part trays assembly that comprise a lower compartment with multiple receiving wells that couple with an upper compartment that includes corresponding wells that engage with the receiving wells but include a membrane at their base. See Transwell® Permeable ts Selection and Use Guide, ble at ht: 3://csmedia;.coming.com/LifeSciences/h/ledia/ dﬁ’transwell Guide. 3th" (last visited April 24, 2017). ells® are useful to measure the permeability test materials deposited on the membranes by ﬁlling the receiving wells with a control liquid, afﬁxing the upper tray to the lower tray, and g a liquid containing a solute (e.g., a color indicator) on top of the test materials. The amount of solute that penetrates the test materials and nes to diffuse into the l liquid in the lower wells can then be assessed, qualitatively or quantitatively (such as by absorption spectroscopy).

In this ment, three permeability tests were conducted: (1) a control membrane including no able biomaterial (shown in column (i) of FIGs. 8A-B), (2) a membrane treated with Control Injectable Biomaterial , lacking a carbohydrate) (shown in column (ii) of FIGs. 8A-B), and (3) a membrane treated with Injectable erial 3 (shown in column (iii) of FIGs. 8A-B). The lower well was ﬁlled with PBS and the upper tray afﬁxed to the lower tray. The Control Injectable Biomaterial and Injectable erial 3 were prepared as disclosed in Example 5. These materials were extruded from the syringe onto each membrane as applicable, and 1 mL of a 0.026 M (0.25 g/4O mL) solution of alizarin red was then added on top of the material in each of the upper tray wells. The tray assembly was then incubated overnight at 37 oC and photographed as shown in . The upper tray was then removed and the results photographed in . As shown in in FIGs. 8A-B, the dyed liquid was able to penetrate the membrane in column (i) as well as the ne d with Control Injectable Biomaterial in column (ii), but was ntially blocked from penetrating the membrane treated with Injectable Biomaterial 3 according to the present disclosure shown in column (iii). These results demonstrate the effectiveness of the diffusional barrier provided by the injectable biomaterials according to the present sure as compared with control compositions lacking a carbohydrate.

Quantification of this experiment is conducted by preparing stock solutions of alizarin red and ng a calibration curve by measuring the absorption of various dilutions at a wavelength of 450 nm. Final solutions passed through Transwells® are then measured at 450 nm and correlated to a speciﬁc absorbance based on extrapolation. Results expressed as tage permeated are expected to results demonstrate the effectiveness of the ional barrier provided by the injectable biomaterials according to the t disclosure as compared with control compositions lacking a carbohydrate.

Example 11: Methods for Testing Working Time and Injectability Described herein are tests for working time and injectability of exemplary able erials according to the present disclosure.

Working time and injectability are ed using procedures detailed in ASTM C4l4-O3 at 7.2, 8.2 (reapproved 2012), the contents of which are incorporated herein by reference in their ty as disclosed above. Brieﬂy, approximately 0.5 oz (15 g) ns of the injectable biomaterial are extruded at desired intervals and troweled onto smooth, clean and dry horizontal surfaces. The injectable erial is considered workable if it stays in the applied position without following the trowel, or without curling behind the trowel while spreading. The injectable biomaterial is no longer workable when it fails to stay in the applied position while spreading.

Working time and injectability were also tested using a 14 mL syringe of Injectable Biomaterial 6, prepared as disclosed in Example 5. After preparation, the syringe was allowed to sit for 5 minutes. The syringe was coupled to a 15 gauge cannula and the contents were able to be expelled using normal hand re. Required pressure for injectability is also measured using an Instron 3342. g time was also tested on a sample of Injectable Biomaterial 3, prepared as indicated in Example 5. The entire volume of the injectable biomaterial was extruded on a clean surface and formed into a ball. The material was considered workable when the surface was tacky to the touch of a dry gloved hand, as indicated by a visible e t on the ﬁnger of the glove. The material was no longer considered workable when the surface was no longer tacky to the touch of a dry gloved hand, as indicated by no visible residue present on the ﬁnger of the glove. g for tackiness was repeated every 15 seconds. The working time was determined to be approximately 6 minutes, 15 seconds.

Example 12: Methods for Testing Viscosity Described herein are tests for viscosity of exemplary injectable biomaterials according to the present disclosure.

A sample of Injectable Biomaterial 1 was prepared as indicated in Example 5.

The resulting material was extruded directly onto a Model AR 1000 ter at 25 0C. A 60 mm 1-degree stainless steel plate was used with a truncation gap of 28 pm at a sheer rate of 1 s'l. Once stabilized after 10 seconds, the viscosity was recorded. The viscosity is ed as 18.69 Pas. e 13: s for g Cohesion Described herein is a test for cohesion of exemplary injectable biomaterials according to the present disclosure.

A sample of Injectable Biomaterial 3 was prepared as indicated in Example 5.

Immediately after preparation, a syringe containing the composition was coupled to an 18 gauge needle. The contents of the syringe was expelled into a vial containing 10.0 mL PBS at 37 oC. The material was visually cohesive and did not break apart in solution.

Example 14: Methods for Testing Initial Setting Time Described herein is a test for initial setting time of exemplary able biomaterials according to the present disclosure.

Injectable Biomaterial 3 was made according to Example 5. A 14 gauge cannula was coupled to the syringe and the contents ed onto an aluminum dish. The surface was struck off evenly using a straight-edge spatula. The remainder of the material was spread out in the mixing pan to a uniform thickness of 3/16 inch (5 mm). The pan was submerged in PBS at 37 0C for 15 s and then d. After removal, a 1 lb. (454 g) Gilmore Needle, having a tip diameter of 1/24 inch (1.06 mm) penetrated the sample the length of the needle tip, in greater than 1 minute, establishing an initial setting time of no more than 15 minutes. See ASTM C414-03 at 7.2, 8.2 (reapproved 2012), the contents of which are orated herein by reference in their entirety.

Additional testing at 15 second intervals provides a more reﬁned assessment of initial setting time.

Example 15: Methods for Testing Extrusion Force Described herein is a test extrusion force for exemplary injectable erials according to the present disclosure.

An injectable biomaterial according to the present sure is prepared as disclosed herein. The al is loaded into a 10 mL syringe with an al tip diameter of 800 mm, and the syringe is loaded into a computer-controlled extrusion force testing machine, such as a Zwick/Roell-HCr 25/400, set at a crosshead speed of 5 mm min-1. The injectable biomaterial is extruded by a compressive load vertically mounted on top of the plunger. The able biomaterials according to the present disclosure are expected to be completely extruded using a peak force less than about 150 N.

Example 16: X-Ray Diffraction Testing of Phase Composition Described herein are methods for testing the phase of exemplary injectable biomaterials according to the present disclosure. ASTM and ISO requirements mandate a minimum hydroxyapatite content of 95% of the crystalline phases and a m mass fraction of calcium oxide of 1% of the crystalline phases. See ASTM F1185-03 (reapproved 2014), at 42, ISO 13175-3 (2012), at 4.2.2, the contents of all of the foregoing of which are orated herein by reference in their ties.

Injectable Biomaterial 3 was made according to Example 5. The sample was injected into PBS at 37 oC and allowed to sit for approximately 16 hours. The resulting solid was collected, ground in a zirconia mortar and pestle, and sintered (1 hour, 1,100 0C) before being allowed to cool to room temperature and being ted to analysis by X-ray diffraction ("XRD") at 1.2°/min with a scan range of °20 using a Rigaku Mini ﬂex II desktop X-ray diffraction machine model 2005H302. As shown in only crystalline phases were detected, and an e of amorphous material. onally, characteristic peaks for hydroxyapatite were identified and the percentage hydroxyapatite was ined to be greater than 99%. See International Center for Diffraction Data ("ICDD") 9-342. The amount of calcium oxide was found to be less than 1%. See ICDD 4-777, the contents of which are orated herein by nce in their entirety.

Example 17: FT-IR Testing of Phase Composition Described herein are methods for testing the phase of exemplary injectable biomaterials according to the present disclosure.

Injectable Biomaterial 3 was made according to Example 5. The sample was injected into PBS at 37 oC and allowed to sit for approximately 16 hours. The resulting solid was ted, ground in a ia mortar and pestle, and ed (1 hour, 1,100 0C) before being allowed to cool to room temperature and being and subjected to FTIR. As shown in , characteristic bands were observed for hydroxyapatite as follows: PO43' absorption bands at 563, 598, 961, 1019, and 1086 cm'l, OH' bands are present at 629 and 3570 cm'l.

No other bands were observed. This spectrum indicated that hydroxyapatite was formed with no other mineral phases present.

Example 18: Scanning Electron Microscopy of Injectable Biomaterial Described herein are scanning electron micrographs of injectable biomaterials according to the present disclosure.

A sample of Injectable Biomaterial 3 was prepared as indicated in Example 5.

The resulting material was injected into PBS at 37 °C and allowed to sit for approximately 16 hours. After drying (100 °C for 3 hours), the samples were fractured and imaged by scanning electron microscopy ("SEM"). As shown in FIGs. 11A-C, representative SEM images show ized, interlocking, interconnected lline structures typical to that of a hydroxyapatite crystal structure.

Example 19: Methods for Elemental is Described herein are s for testing the tal composition of exemplary injectable biomaterials according to the present disclosure. ASTM and ISO requirements e maximum content of certain elements for compositions used for bone cement. See ASTM F1185-03 (reapproved 2014), at 4.3, ISO 13175-3 (2012), at 4.1, the contents of all of the foregoing of which are incorporated herein by reference in their entireties.

Injectable Biomaterial 3 was made according to e 5. The sample was injected into PBS at 37 oC and d to sit for approximately 16 hours. The resulting solid was collected and ground in a zirconia mortar and pestle. The material was then diVided, with Sample 1 being sintered (1 hour, 1,100 0C) before being allowed to cool to room temperature, and Sample 2 not being ed. Both samples were then subjected to analysis by ICP-MS. Triplicate tests were run for each sample to conﬁrm the results. Results were substantially the same, and in compliance with speciﬁcation, both with and without ing.

Table 3 es the results of these experiments.

Table 3: Results of ICP-MS Elemental Anal sis of able Biomaterial 3 With and Without Sintering Specification Sample 1 (ppm) Sample 2 (ppm) Element (upper limits ppm) Runl Run3 Runl Run3 Pb 30 0.1 0.1 0.2 0.1 Hg 5 0.03 0.05 0.4 0.4 As 3 0.04 0.04 0.1 0.1 Cd 5 0.02 0.03 <03 <03 Example 20: Methods for Testing Setting Reaction Temperature Described herein are ary test methods for measuring the setting reaction ature of injectable biomaterials according to the t disclosure.

The tic characteristics of the setting reaction are tested on an injectable biomaterial prepared in accordance with Example 5. The resulting material is extruded into an exothermic heat mold made of polytetraﬁuoroethylene (PTFE), poly(ethyleneterephthalate), polyoxymethylene, high density polyethylene, or ultra-high molecular weight polyethylene (UHlVIWPE) and equipped with a No. 24 gage wire thermocouple, or similar , positioned with its junction in the center of the mold at a height of 3.0 mm in the internal cavity. The plunger is immediately seated with a C-clamp or suitable press to produce the 6.0 mm specimen height. Upon producing plunger seating, excess material and the C-clamp or press are removed for the remainder of the procedure.

The ature is uously recorded with respect to time from the onset of mixing the liquid component and the solid component until cooling is observed. The maximum temperature recorded is reported to the nearest 1°C.

At least three independent samples are tested. See ASTM 451-16, at 7.6, the contents of which are incorporated herein by reference in their entirety. The results are expected to te that the setting reaction is substantially isothermic and does not signiﬁcantly change the temperature in the immediate Vicinity of setting material.

Example 21: Methods for Testing Compressive Strength Described herein are exemplary methods for testing the compressive strength of able erials according to the present disclosure.

The compressive strength of Injectable Biomaterial 3 prepared in ance with e 5 was tested. A 14 gauge cannula was d to the syringe and the mixture was expelled into a mold submerged in 37 oC PBS using hand pressure to produce test specimens in the shape of cylinders approximately 12 mm high and 6 mm in diameter. After 24 hours, the samples were removed and ﬁled so that the ends of each sample were plane parallel. The samples were then subjected to compressive strength testing using an Omega Force Gauge Model DFG35-100 used to read the force of failure at 12.7 mm/minute, which provided the compressive strength. See ASTM F45 1-16, at 7.9, the contents of which are incorporated herein by reference in their entirety. A total of three compressive strength gs were recorded for each sample. The results indicated a compressive strength of 5.7 :: 1 MPa.

Example 22: Methods for Testing Dimensional Stability bed herein are ary methods for testing the dimensional stability of injectable biomaterials according to the present disclosure.

An injectable biomaterial is produced according to the Examples disclosed herein.

After ﬁve minutes from the tion of mixing have d, an 14 gauge cannula is coupled to the syringe and the mixture is expelled into a mold approximately 12 mm high and 6 mm in diameter submerged in 37 oC PBS using hand pressure. After 15 minutes, the samples are ejected from the mold and their height and diameter measured using digital calipers. The samples are then submerged in 37 oC PBS for a further 24 hours after which their height and diameter is re-measured. The sample is then dried in an oven (60 °C, 24 hours). A total of three readings are ed for each sample during the ﬁrst, second, and third measurements.

The results are expected to indicate no signiﬁcant change in height or er n the ﬁrst, second, and third readings, indicating high dimensional stability. The maximum change in any dimension over the three tests is expected to be less than 10%, less than 7.5%, less than 5%, or less than 3%.

Example 23: Methods of Testing Bone Formation In Vivo Described herein are in vivo tests of bone formation in rabbits using exemplary injectable biomaterials according to the present disclosure.

The skin of ally mature New Zealand White rabbits was opened and the periosteum reﬂected using a periosteal elevator in the medial aspect of the distal femur. Bi- l critical size defects (6 mm in diameter and 10 mm deep) were created using a burr with a 6 mm ﬁat drill and controlled with a depth indicator. The medial epicondyle was used as an ical landmark. The defects were prepared under saline irrigation to minimize thermal . The defects were ﬂushed with sterile saline during preparation and at completion to remove residual bone. The defects were ﬁlled with approximately 0.3 mL Injectable Biomaterial 6 (prepared according to Example 5) using a spatula to the height of the original cortex. The skin was closed using 3-0 Dexon. s were given post- operative analgesia and returned to their holding cages. The animals were free to mobilize and weight-bear post-operatively as tolerated. Animals were red daily, to include changes in skin, fur, eyes, and mucous membranes, as well as behavior pattern and central s system and somatomotor ty.

At 6-12 weeks post-implantation the animals were euthanized and the right and left femora harvested and photographed with a digital . The general integrity of the skin incision was examined along with the macroscopic and underlining subcutaneous tissues. Internal organs (e.g., heart, liver, lungs, and spleen) were d, photographed, and preserved in 10% neutral buffered formalin (NBF) until further processing. After ﬁxation, the internal organs were embedded, sectioned and stained with hematoxylin and eosin (H&E). The harvested femora were radiographed in the anteroposterior and lateral planes using an HP Faxitron and high resolution mammography ﬁlm at 24 kV for 45 seconds.

Micro-computed aphy ("micro CT") slices were also taken for representative animals using an Inveon in vivo micro-computer tomography scanner in order to obtain high resolution images of bone ion and test article resorption. The distal femurs were scanned and the raw images reconstructed to DICOM data using s software. Images were examined in the axial, sagittal, and coronal planes to assess the overall quality of the healing sites and any local reactions. As shown in FIGs. 12A-B, the injectable biomaterial (shown in solid white) remains resident at the site of administration 6 weeks post- implantation.

Additional analysis is conducted at 6, 12, 18 and 26 weeks post-implantation to determine if new bone formation is detected by micro-computed tomography.

WO 89733 Example 24: Administration of Injectable Biomaterials to Canines Described herein are in vivo tests of exemplary injectable biomaterials ing to the t disclosure in canines.

Animals were anesthetized by intramuscular administration of Acepromazine Maleate Injection as a pre-medication at a dose of 0.5 mg/lb. Subsequently, animals were administered Telazol® (Tiletamine HCl and Zolazepam HCl, Zoetis) intramuscularly as an anesthetic at a dose of 4.5 mg/lb. to allow endotracheal tube insertion prior to isoﬂurane anesthesia at a rate of 1.5-2%. Finally, animals were administered buprenorphine ion uscularly at a dose of 1-3 ug/lb. to ensure pain free surgery. All hair around the stiﬂe joint was shaved, and extra hair was removed from the surgical site using alcohol soaked gauze. Final surgical site cleaning was achieved using a 2% chlorhexidine/70% isopropyl preparation stick with tint to ensure coverage, starting in the middle of the surgical site and applied clockwise ever ing circles until entire site was clean.

Two medial or lateral incisions were made to expose the fascia over the distal femur and proximal tibia. h these incisions, and one at a time, a 15 gauge, four inch cannula was driven into the bone of either the distal femur or proximal tibia by hand pressure.

Live ﬂuoroscopic imaging conﬁrmed appropriate placement. The trocar was removed from the cannula and a syringe containing Injectable Biomaterial 3 prepared as described in Example 5 was d to the cannula, followed by extrusion of the material from the syringe, through the cannula, and into the site of administration. Injection was monitored by copy sful injection was achieved.

The injectable biomaterial was allowed to set for 15 minutes post-initiation of mixing. The a was left in place. Animals were euthanized using intravenous Euthasol® at a dose of 0.1 mL/lb. The fascia were then dissected to expose the bone. Bone was be removed from the animal by sawing above and below the involved joint. The resultant specimens were set in epoxy and ned along the sagittal plane. As shown in , the cured injectable material 1302 intruded into the existing porosity of the cancellous bone in the femoral condyle. The cannula 1301 is also shown still resident at the site of administration.

Example 25: Administration of able Biomaterials to Human Cadaver Bones Described herein are in vivo tests of exemplary injectable biomaterials according to the present sure in human cadaver bones.

Surgeries were performed on the knee joint of human cadavers. Specimens were positioned appropriately and an incision was made on the medial and lateral sides to allow insertion of a cannula into either the distal femur or tibial plateau. An 11 gauge outer cannula was driven into the cancellous bone, the trocar removed, and an inner 15 gauge cannula was inserted into the outer cannula to allow for either injection through an open distal tip or a l fenestration. An arthroscope was positioned in the knee to monitor for extravasation of the cement into the joint space and a ﬂuoroscope was positioned to allow visualization of the positioning of the surgical instrumentation and injectable biomaterial.

A syringe containing Injectable Biomaterial 3 prepared as described in Example 5 was coupled to the inner cannula, ed by extrusion of the material from the syringe, through the cannula, and into the site of administration using normal hand strength, with no back pressure hindering injection. No leakage of the injectable material into the joint space or from the surgical instrumentation was noted. Dissection into the cancellous space post- injection was performed. As shown in , the cured injectable biomaterial 1402 ed into the existing porosity of the cancellous bone of human distal femur cadaver bone. Void 1401 shows the location in which the cannula was placed.

INCORPORATION BY REFERENCE The entire sure of each of the patent documents, including certificates of correction, patent application documents, ific articles, governmental reports, websites, and other references referred to herein is orated by reference in its entirety for all purposes.

ATIONS It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of te embodiments, may also be ed in combination in a single ment.

Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided tely or in any suitable sub-combination. All combinations of the embodiments are speciﬁcally embraced by the present disclosure and are disclosed herein just as if each and every ation was dually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also speciﬁcally embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination of factors was individually and explicitly disclosed .

E UIVALENTS As will be apparent to one of ordinary skill in the art from a reading of this disclosure, the present disclosure can be embodied in forms other than those speciﬁcally disclosed above without departing from the spirit or essential characteristics thereof. The particular embodiments described above are, therefore, to be considered as illustrative and not restrictive or limiting of the t disclosure. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the ic embodiments described herein. The scope of the disclosure is as set forth in the appended claims and equivalents thereof. All changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein, rather than being limited to the es contained in the ing description.

Claims

What is claimed is:

1. A method for making an injectable biomaterial, the method comprising: (a) creating a liquid component comprising a carbohydrate by: (i) providing a liquid solution; (ii) adjusting the pH of the liquid solution with a pH adjusting agent; and (iii) dissolving the ydrate in the liquid solution to form the liquid component; (b) ing a solid component; and (c) mixing the liquid component and the solid component to form the injectable biomaterial; wherein the injectable biomaterial sets and cures to form an apatitic crystal structure after mixing of the solid component and the liquid component; n the ratio of solid component to liquid component is between about 1.5 to about 1 by mass and about 1 to about 1 by mass; and wherein the carbohydrate is sodium hyaluronate.

2. The method of claim 1, wherein the solid component comprises at least one of a metal phosphate and a metal carbonate.

3. The method of any preceding claim, n the solid component comprises at least one of α-tricalcium phosphate (Ca3(PO4)2), calcium carbonate (CaCO3), and monocalcium ate monohydrate (Ca(H2PO4)2 H2O).

4. The method of any preceding claim, wherein the solid component comprises α- tricalcium phosphate and calcium ate.

5. The method of any preceding claim, wherein the solid component comprises α- cium phosphate, m carbonate, and monocalcium phosphate monohydrate.

6. The method of any preceding claim, wherein the solid component comprises 70-90% alpha tricalcium phosphate, 10-20% calcium carbonate, and 0.5-2% calcium phosphate monobasic monohydrate (mass/mass).

7. The method of any preceding claim, wherein the solid component comprises 80-89% alpha tricalcium phosphate, 11-19% calcium ate, and 0.75-1.5% calcium phosphate monobasic monohydrate (mass/mass).

8. The method of any preceding claim, wherein the solid ent comprises 82-86% alpha tricalcium ate, 13-16% calcium ate, and 2% calcium phosphate monobasic monohydrate (mass/mass).

9. The method of any preceding claim, wherein the solid component comprises 84.3% alpha tricalcium phosphate, 14.7% calcium carbonate, and 1.02% calcium phosphate monobasic monohydrate (mass/mass).

10. The method of any preceding claim, wherein the carbohydrate is present in the injectable biomaterial at a concentration of about 1 to about 10 mg/mL.

11. The method of any preceding claim, wherein the carbohydrate has a molecular weight of from about 0.90 x 106 Da to about 1.0 x 107 Da.

12. The method of any preceding claim, wherein the ratio of solid ent to liquid component is about 1.5 to about 1 by mass.

13. The method of any preceding claim, wherein the ratio of solid component to liquid component is about 1 to about 1 by mass.

14. The method of any preceding claim, wherein the curing of the injectable biomaterial yields an apatitic crystal structure that is at least about 90% hydroxyapatite.

15. The method of any preceding claim, wherein the curing of the injectable biomaterial yields an apatitic crystal ure that is at least about 95% hydroxyapatite.

16. The method of any preceding claim, wherein the curing of the injectable biomaterial yields an apatitic l structure that is greater than about 99% hydroxyapatite.

17. The method of any preceding claim, wherein the fully set and cured injectable biomaterial has a molar Ca/P ratio of about 1 to about 2.

18. The method of any ing claim, wherein the fully set and cured injectable biomaterial has a compressive strength of less about 10 MPa.

19. The method of any preceding claim, wherein the fully set and cured injectable erial has a compressive strength of less about 9 MPa.

20. The method of any preceding claim, wherein the injectable biomaterial has a ity of about 5 Pa·s and about 20 Pa·s immediately after mixing the solid component and the liquid component, when measured at room ature.

21. The method of any preceding claim, wherein the fully set and cured injectable erial comprises a median pore diameter of less than about 1 µm.

22. The method of any ing claim, wherein the pH adjusting agent is selected from an organic acid and an inorganic acid.

23. The method of any preceding claim, wherein the pH adjusting agent is selected from the group consisting of citric acid, formic acid, acetic acid, and mixtures thereof.

24. The method of any one of claims 1-22, wherein the pH adjusting agent is selected from the group consisting of hydrochloric acid, phosphoric acid, nitric acid, and mixtures thereof.

25. The method of any preceding claim, n providing the solid component further comprises drying the solid component.

26. The method of claim 25, wherein the drying comprises exposing the solid component to heat over a period of time.

27. The method of claim 26, wherein the heat comprises at least about 165 °C.

28. The method of claim 26, wherein the period of time comprises at least about 12 hours.

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