WO2018071319A1 - Compositions and methods for the treatment of tissue defects - Google Patents

Abstract

Provided herein are compositions and methods for the treatment of tissue defects from recruited progenitor cells, such as mesenchymal stem cells.

Description

TITLE OF INVENTION

COMPOSITIONS AND METHODS FOR THE TREATMENT OF

TISSUE DEFECTS CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application Serial No. 62/407,605, filed 13 October 2016, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number AR065023 and grant number DE0231 12 awarded by the National Institutes of Health. The government has certain rights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

Not Applicable.

FIELD OF THE INVENTION

The present teachings relate to methods and compositions for treatment of tissues.

BACKGROUND OF THE INVENTION

Certain bone defects fail to heal, despite clinical use of various bone grafts. Platelet Rich Plasma (PRP) has been used broadly in medicine ranging from pain relief to tissue repair and regeneration, without a clear understanding of its active components or mechanisms of action. A position paper commissioned by the American Academy of Orthopedic Surgeons found little reliable clinical evidence to guide the use of PRP.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision of compositions and methods for the treatment of tissue defects from recruited progenitor cells, such as mesenchymal stem cells.

Other objects and features will be in part apparent and in part pointed out hereinafter. DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a series of illustrations and images depicting platelet-rich plasma (PRP) isolation and activation. Upon IRB approval, whole blood was drawn from 8 healthy human donors (age: 25-38 years old) followed by clinically compatible PRP preparation. PRP was degranulated by 10% CaCI2 and thrombin (1 U/ml_).

FIG. 2A-FIG. 2C are a series of images, graphs, and illustrations depicting proteomics and bioinformatics.

FIG. 2A is an image of immunofluorescent probes for detection of protein- protein interactions, according to an exemplary embodiment.

FIG. 2B is a pie chart of the proteomics and bioinformatics analysis of multiple datasets to elucidate potential functions of individual PRP components in multiple putative functional clusters, according to an exemplary embodiment.

FIG. 2C is an illustration of the proteomics and bioinformatics analysis of multiple datasets to elucidate potential functions of individual PRP components in multiple putative functional clusters, according to an exemplary embodiment.

FIG. 3A is a bar graph depicting bioinformatics analysis to identify individual proteins of rank significance and putative functional clusters, according to an exemplary embodiment.

FIG. 3B is a table depicting bioinformatics analysis to identify individual proteins of rank significance and putative functional clusters, according to an exemplary embodiment.

FIG. 3C is a table depicting ELISA data further confirming enriched molecules concentrations in PRP, compared to donor-matched PPP, according to an exemplary embodiment.

FIG. 3D is an image of a Western blot showing Cxcl16's osteogenic capacity benchmarked to BMP2 (Western blot), according to an exemplary embodiment. FIG. 3E is a chart showing the chemokine signaling pathway, according to an exemplary embodiment.

FIG. 4A is a line and scatter plot depicting Cxcl16 robustly induced MSC migration in a dose-dependent manner and benchmarked to PDGF and TGFpi , according to an exemplary embodiment.

FIG. 4B is a line and scatter plot depicting, surprisingly, Cxcl16 had little effect on MSC proliferation, according to an exemplary embodiment.

FIG. 4C is a bar graph depicting Cxcl16 robustly promoted osteogenic differentiation of MSCs, with augmented OCN, Runx2, BSP, and Col1 a1 at both mRNA and protein levels, according to an exemplary embodiment.

FIG. 4D is an image depicting Cxcl16 acts via ERK/MAPK, rather than

BMP2's Smad1/5/8 pathway, according to an exemplary embodiment.

FIG. 5A-FIG. 5E and FIG. 5A'-FIG. 5E' are images depicting Cxcl16 induced robust mineralization, which was attenuated by an ERK1/2 inhibitor, U0126.

FIG. 5F is a bar graph depicting Cxcl16's osteogenic effect was attenuated by the ERK1/2 inhibitor at both mRNA and protein levels.

FIG. 5G is an image depicting Cxcl16's osteogenic effect was attenuated by the ERK1/2 inhibitor at both mRNA and protein levels. DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery that Cxcl16, a component of platelet-rich plasma (PRP), is a novel chemotactic and osteogenic factor, without previously known association with bone regeneration. Elucidation of the protein components present in PRP can identify the molecules responsible for PRP's therapeutic effects, as well as increase the potential applications of PRP.

As shown herein, this technology utilizes proteomic and bioinformatics analysis to identify all protein components found within PRP (see e.g., Example 3). These findings can be applied to identify specific molecules for the treatment of both orthopaedic and non-orthopaedic disorders. Specifically, this technology has successfully identified Cxcl16, a protein involved in osteogenesis (see e.g., Example 4). Mesenchymal stem cells exposed to Cxcl16 were observed to undergo migration, proliferation, and osteogenic differentiation, suggesting the potential use of Cxcl16 for bone regeneration (see e.g., Examples 2-4). Furthermore, with respect to identification of all protein components in PRP, this technology may be useful in identifying other active proteins for wound healing and regeneration applications.

As shown herein, Cxcl16 promotes osteogenesis through ERK1/2 signaling, rather than BMP2's Smad1/5/8 pathway.

BMP2 was shown to be the 1 15th among all proteins in Platelet-rich Plasma

(PRP) (see e.g., FIG. 3B).

One aspect of the present disclosure provides an original demonstration of proteomics and bioinformatics analysis of detectable protein/peptide in PRP and benchmarking with known proteins. Several instructive findings can provide insight for clinical PRP preparation and use.

Another aspect of the present disclosure identified all protein components of PRP and benchmarked PRP with several known growth factors that the clinical community has suspected to be the primary active ingredients for PRP. Another aspect of the present disclosure provides several ongoing projects to identify novel molecules from the PRP proteomics analysis database for the treatment of multiple orthopedic and non-orthopedic disorders such as osteoarthritic and rheumatoid arthritis, ACL healing, bone fracture healing, and cartilage regeneration.

Another aspect of the present disclosure allows for the present understanding that specific novel molecules in PRP can be identified for the treatment of a given orthopedic (or non-orthopedic) disorder that 1 ) are more efficacious than PRP as a whole and 2) differ from other molecules in PRP.

PRP is a concentrated source of autologous platelets which is more safe and convenient for clinical use. PRP has the advantage of free FDA clearance, also the enriched growth factors and cytokines. All in all PRP is preferable by surgeons to apply in multiple areas, such as nerve injury; tendinitis; osteoarthritis; cardiac muscle injury; bone repair and regeneration; plastic surgery; wound healing; or oral surgery.

As shown herein, screening and analyzing the proteomics and bioinformatics data was used to search for the specific novel molecules in PRP, which applied alone can be more efficacious than whole PRP for the treatment of a given orthopedic (or non-orthopedic) disorder.

SCAFFOLD

Various embodiments described herein employ a scaffold or matrix material (e.g., a substrate). For example, a composition including CTGF or TGF 3 can be included in or on a scaffold. A scaffold or matrix material, as described herein, can be introduced at a defect site in a cartilaginous tissue. A defect site can include an injury, a tear, or deterioration. A defect site can be at least partially located in the inner or avascular region of a cartilaginous tissue.

The scaffold optionally does not comprise a transplanted mammalian cell, i.e. , no cell is applied to the scaffold; any cell present in the scaffold migrated into the scaffold.

A scaffold can be fabricated with any matrix material recognized as useful by the skilled artisan. A matrix material can be a biocompatible material that generally forms a porous, microcellular scaffold, which provides a physical support for cells migrating thereto. Such matrix materials can: allow cell attachment or migration; deliver or retain cells or biochemical factors; enable diffusion of cell nutrients or expressed products; or exert certain mechanical or biological influences to modify the behavior of the cell phase. The matrix material generally forms a porous, microcellular scaffold of a biocompatible material that provides a physical support or an adhesive substrate for recruitment or growth of cells during in vitro or in vivo culturing.

Suitable scaffold or matrix materials are discussed in, for example, Ma and Elisseeff, ed. (2005) Scaffolding In Tissue Engineering, CRC, ISBN 1574445219; Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of

Replacement Organs and Tissues, Oxford ISBN 019514130X. For example, matrix materials can be, at least in part, solid xenogenic (e.g., hydroxyapatite) (Kuboki et al. 1995 Connect Tissue Res 32, 219-226; Murata et al. 1998 Int J Oral Maxillofac Surg 27, 391 -396), solid alloplastic (polyethylene polymers) materials (Saito and Takaoka 2003 Biomaterials 24 2287-93; Isobe et al. 1999 J Oral Maxillofac Surg 57, 695-8), or gels of autogenous (Sweeney et al. 1995 . J Neurosurg 83, 710-715), allogenic (Bax et al. 1999 Calcif Tissue Int 65, 83-89; Viljanen et al. 1997 Int J Oral Maxillofac Surg 26, 389-393), or alloplastic origin (Santos et al. 1998 . J Biomed Mater Res 41 , 87-94), or combinations of the above (Alpaslan et al. 1996 Br J of Oral Maxillofac Surg 34, 414-418).

The matrix comprising the scaffold can have an adequate porosity and an adequate pore size so as to facilitate cell recruitment and diffusion throughout the whole structure of both cells and nutrients. The matrix can be biodegradable providing for absorption of the matrix by the surrounding tissues, which can eliminate the necessity of a surgical removal. The rate at which degradation occurs can coincide as much as possible with the rate of tissue or organ formation. Thus, while cells are fabricating their own natural structure around themselves, the matrix can provide structural integrity and eventually break down, leaving the neotissue, newly formed tissue or organ which can assume the mechanical load. The matrix can be an injectable matrix in some configurations. The matrix can be delivered to a tissue using minimally invasive endoscopic procedures.

The scaffold can comprise a matrix material having different phases of viscosity. For example, a matrix can have a substantially liquid phase or a

substantially gelled phase. The transition between phases can be stimulated by a variety of factors including, but limited to, light, chemical, magnetic, electrical, and mechanical stimulus. For example, the matrix can be a thermosensitive matrix with a substantially liquid phase at about room temperature and a substantially gelled phase at about body temperature. The liquid phase of the matrix can have a lower viscosity that provides for optimal distribution of growth factors or other additives and injectability, while the solid phase of the matrix can have an elevated viscosity that provides for matrix retention at or within the target tissue.

The scaffold can comprise one or more layers, each with the same or different matrix materials. For example, a scaffold can comprises at least two layers, at least three layers, at least four layers, or more. As another example, a scaffold can comprise a first layer comprising a first matrix material and a second layer

comprising a second matrix material. As another example, a scaffold can comprise a first layer comprising a chemotactic factor or profibrogenic factor and a second layer comprising a chondrogenic factor. As another example, a scaffold can comprise a first layer comprising a chemotactic factor or profibrogenic factor and a chondrogenic factor and a second layer comprising a chondrogenic factor. As another example, a scaffold can comprise a first layer comprising a chemotactic factor or profibrogenic factor and a second layer comprising a chondrogenic factor and chemotactic factor or profibrogenic factor. As another example, a scaffold can comprise a first layer comprising CTGF and a second layer comprising TGFP3. As another example, a scaffold can comprise a first layer comprising CTGF and TGFP3 and a second layer comprising TGFP3. As another example, a scaffold can comprise a first layer comprising CTGF and a second layer comprising TGFP3 and CTGF.

The scaffold can comprise a matrix material formed of synthetic polymers. Such synthetic polymers include, but are not limited to, polyurethanes,

polyorthoesters, polyvinyl alcohol, polyamides, polycarbonates, polyvinyl pyrrolidone, marine adhesive proteins, cyanoacrylates, analogs, mixtures, combinations or derivatives of the above. Alternatively, the matrix can be formed of naturally occurring biopolymers. Such naturally occurring biopolymers include, but are not limited to, fibrin, fibrinogen, fibronectin, collagen, or other suitable biopolymers. Also, the matrix can be formed from a mixture of naturally occurring biopolymers or synthetic polymers. Another example of a matrix material is an injectable citrate- based mussel-inspired bioadhesive (iCMBA).

The scaffold can include one or more matrix materials including, but not limited to, a collagen gel, a polyvinyl alcohol sponge, a poly(D,L-lactide-co-glycolide) fiber matrix, a polyglactin fiber, a calcium alginate gel, a polyglycolic acid mesh, polyester (e.g., poly-(L-lactic acid) or a polyanhydride), a polysaccharide (e.g.

alginate), polyphosphazene, polyacrylate, or a polyethylene oxide-polypropylene glycol block copolymer. Matrices can be produced from proteins (e.g., extracellular matrix proteins such as fibrin, collagen, fibronectin), polymers (e.g.,

polyvinylpyrrolidone), or hyaluronic acid. Synthetic polymers can also be used, including bioerodible polymers (e.g., poly(lactide), poly(glycolic acid), poly(lactide-co- glycolide), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates), degradable polyurethanes, non-erodible polymers (e.g., polyacrylates, ethylene-vinyl acetate polymers or other acyl substituted cellulose acetates and derivatives thereof), non- erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,

poly(vinylimidazole), chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon®, or nylon.

The scaffold can further comprise any other bioactive molecule, for example an antibiotic or an additional chemotactic growth factor or another osteogenic, dentinogenic, amelogenic, or cementogenic growth factor. In some embodiments, the scaffold is strengthened, through the addition of, e.g., human serum albumin (HSA), hydroxyethyl starch, dextran, or combinations thereof. Suitable

concentrations of these compounds for use in the compositions of the application are known to those of skill in the art, or can be readily ascertained without undue experimentation.

The concentration of a compound or a composition in the scaffold can vary with the nature of the compound or composition, its physiological role, or desired therapeutic or diagnostic effect. A therapeutically effective amount is generally a sufficient concentration of therapeutic agent to display the desired effect without undue toxicity. For example, the matrix can include a composition comprising CTGF or TGF 3 at any of the above described concentrations. The compound can be incorporated into the scaffold or matrix material by any known method. In some embodiments, the compound can be imbedded in a gel, e.g. , a collagen gel incorporated into the pores of the scaffold or matrix material or applied as a coating over a portion, a substantial portion, substantially all of, or all of the scaffold or matrix material.

Alternatively, chemical modification methods can be used to covalently link a compound or a composition to a matrix material. The surface functional groups of the matrix can be coupled with reactive functional groups of a compound or a

composition to form covalent bonds using coupling agents well known in the art such as aldehyde compounds, carbodiimides, and the like. Additionally, a spacer molecule can be used to gap the surface reactive groups and the reactive groups of the biomolecules to allow more flexibility of such molecules on the surface of the matrix. Other similar methods of attaching biomolecules to the interior or exterior of a matrix will be known to one of skill in the art.

Pores and channels of the scaffold can be engineered to be of various diameters. For example, the pores of the scaffold can have a diameter range from micrometers to millimeters. In some embodiments, the pores of the matrix material include microchannels. Microchannels generally have an average diameter of about 0.1 pm to about 1 ,000 pm, e.g., about 50 pm to about 500 pm (for example about 50, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, or about 550 pm), or any other ranges between about 0.1 pm and about 1 ,000 pm. One skilled in the art will understand that the distribution of microchannel diameters can have any distribution including a normal distribution or a non-normal distribution. In some embodiments, microchannels are a naturally occurring feature of the matrix material(s). In other embodiments, microchannels are engineered to occur in the matrix materials.

Several methods can be used for fabrication of porous scaffolds, including particulate leaching, gas foaming, electrospinning, freeze drying, foaming of ceramic from slurry, and the formation of polymeric sponge. Other methods can be used for fabrication of porous scaffolds include computer aided design (CAD) and

synthesizing the scaffold with a bioplotter (e.g., solid freeform fabrication) (e.g., Bioplotter™, EnvisionTec, Germany).

Biologic drugs that can be added to compositions and methods as described herein can include immunomodulators and other biological response modifiers. A biological response modifier generally encompasses a biomolecule (e.g., peptide, peptide fragment, polysaccharide, lipid, antibody) that is involved in modifying a biological response, such as the immune response or tissue or organ growth and repair, in a manner that enhances a particular desired therapeutic effect, for example, the cytolysis of bacterial cells or the growth of tissue- or organ-specific cells or vascularization. Biologic drugs can also be incorporated directly into the matrix component. Those of skill in the art will know, or can readily ascertain, other substances which can act as suitable non-biologic and biologic drugs.

Compositions described herein can also be modified to incorporate a diagnostic agent, such as a radiopaque agent. The presence of such agents can allow the physician to monitor the progression of wound healing occurring internally. Such compounds include barium sulfate as well as various organic compounds containing iodine. Examples of these latter compounds include iocetamic acid, iodipamide, iodoxamate meglumine, iopanoic acid, as well as diatrizoate derivatives, such as diatrizoate sodium. Other contrast agents that can be utilized in the compositions can be readily ascertained by those of skill in the art and can include, for example, the use of radiolabeled fatty acids or analogs thereof.

The concentration of an agent in the composition can vary with the nature of the compound, its physiological role, or desired therapeutic or diagnostic effect. A therapeutically effective amount is generally a sufficient concentration of therapeutic agent to display the desired effect without undue toxicity. A diagnostically effective amount is generally a concentration of diagnostic agent which is effective in allowing the monitoring of the integration of the tissue graft, while minimizing potential toxicity. In any event, the desired concentration in a particular instance for a particular compound is readily ascertainable by one of skill in the art.

EXPLANT MODEL

An explant model, as that term is used herein, can be a well-established model for tissue damage or repair. An explant model can be a human explant model or non-human explant model, similar to a human meniscus. For example, an explant model can be mammal, reptile, or avian, more preferably, human, equine, bovine, rabbit, porcine, canine, cat, sheep, chicken, or goat. The non-human explant model can exhibit cross-reaction of human markers. qRT-PCR can be performed to design PCR primers and test for any species where the explant model has no cross reactivity with human markers.

TISSUE REGENERATION

Compositions and methods as described herein can have specific and robust efficacy treatment of tissues, including tissues with defects, disorders, diseases, or conditions. For example, compositions and methods as described herein can treat tissue by tissue regeneration. As an example, tissue regeneration can be

regeneration of tissue or regeneration of missing tissue, tissues with defects, disorders, diseases, or conditions.

Novel PRP molecules, such as Cxcl16, can have specific and robust efficacy for orthopedic or non-orthopedic, musculoskeletal, or non-musculoskeletal defects, disorders, diseases, or conditions.

Compositions and methods as described herein can regenerate a variety of tissues or tissues with defects.

Compositions and methods as described herein can be used in a variety of tissue engineering applications associated with various defects, diseases disorders or conditions.

Defects, diseases, disorders, or conditions can be associated with orthopedic or non-orthopedic, musculoskeletal, or non-musculoskeletal defects, disorders, diseases, or conditions.

Compositions and methods as described herein can be used to treat a tissue defect. For example a tissue defect can be orthopedic or musculoskeletal. As another example, the tissue defect can be a fracture, tear, injury, osteoarthritis, or degeneration. As another example, the tissue defect can be a tear or fracture, a longitudinal or vertical tear or fracture, a radial tear or fracture, a horizontal tear or fracture, a bucket handle tear or fracture, a parrot beak tear, or a flap tear.

The tissue (in which the defect is present) that can be treated with identified components of PRP, such as Cxcl16, can be orthopedic or non-orthopedic, musculoskeletal, non-musculoskeletal, bone tissue, bone, cartilage, tendon, intravertebral discs (IVDs), hair follicles, cardiac infarcts, cartilaginous tissue, cartilage, a meniscus, a knee meniscus, a ligament, a ligament enthesis, a tendon, a tendon enthesis, an intervertebral disc, a temporomandibular joint (TMJ), a TMJ ligament, or a triangular fibrocartilage. Features related to tissue and tissue defect can be combined with other features discussed above and below.

Defects, disorders, diseases, or conditions that can be treated with identified components of PRP, such as Cxcl16, can be nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair or regeneration, plastic surgery, wound healing, or oral surgery. Features related to tissue and tissue defect can be combined with other features discussed above and below.

The clinical or therapeutic applications of the compositions and methods as described herein (e.g., use of Cxcl16) can encompass any bone defects resulting from trauma, congenital anomalies, infections or tumor resection, such as (1 ) oral facial bone defects (e.g., Cxcl16 may be used in conduction with fillers, i.e.

periodontal defects and before implant placement); (2) long-bone defects including bone fractures, with or without irradiation, diabetes, etc.; (3) other types of bone defects or diseases, osteogenesis imperfecta, osteoporosis, hormone related bone loss; or (4) augmentative applications. Features related to tissue and tissue defect can be combined with other features discussed above and below.

FORMULATION

The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

THERAPEUTIC METHODS

Also provided is a process of treating a tissue defect in a subject in need administration of a therapeutically effective amount of a platelet-rich plasma (PRP) component so as to regenerate a tissue. For example, the platelet-rich plasma (PRP) component can be a therapeutic protein.

Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a tissue defect such as an orthopedic or non-orthopedic, musculoskeletal, or non- musculoskeletal defect, disorder, disease, or condition; a fracture, tear, injury, osteoarthritis, or degeneration; a tear or fracture, a longitudinal or vertical tear or fracture, a radial tear or fracture, a horizontal tear or fracture, a bucket handle tear or fracture, a parrot beak tear, or a flap tear; associated with a defect, disease, disorder, or condition associated with bone tissue, bone, cartilage, tendon, intravertebral discs (IVDs), hair follicles, cardiac infarcts, cartilaginous tissue, cartilage, meniscus, knee meniscus, ligament, ligament enthesis, tendon, tendon enthesis, intervertebral disc, temporomandibular joint (TMJ), TMJ ligament, or triangular fibrocartilage; an injury associated with nerve injury, tendinitis,

osteoarthritis, cardiac muscle injury, bone repair or regeneration, plastic surgery, wound healing, or oral surgery; or any bone defects resulting from trauma, congenital anomalies, infections or tumor resection, such as (1 ) oral facial bone defects (e.g., Cxcl16 may be used in conduction with fillers, i.e. periodontal defects and before implant placement), (2) long-bone defects including bone fractures, with or without irradiation, diabetes, etc., (3) other types of bone defects or diseases, osteogenesis imperfecta, osteoporosis, hormone related bone loss, or (4)

augmentative applications.

Methods and compositions as described herein can result in a reduction of pain or promote tissue regeneration in the subject.

A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue.

Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans. For example, the subject can be a human subject.

Generally, a safe and effective amount of PRP component (e.g., Cxcl16) is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of PRP component (e.g., Cxcl16) described herein can substantially regenerate tissue, inhibit degeneration or further injury, slow the progress of degeneration or further injury, or limit the development of diseases disorders or conditions associated with a tissue defect.

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeutically effective amount of PRP component (e.g., Cxcl16) can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to substantially regenerate tissue, inhibit degeneration or further injury, slow the progress of degeneration or further injury, or limit the development of diseases disorders or conditions associated with a tissue defect.

The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be

understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes

preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g. , arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

Administration of PRP component (e.g., Cxcl16) can occur as a single event or over a time course of treatment. For example, PRP component (e.g., Cxcl16) can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a tissue defect such as an orthopedic or non-orthopedic, musculoskeletal, or non-musculoskeletal defect, disorder, disease, or condition; a fracture, tear, injury, osteoarthritis, or degeneration; a tear or fracture, a longitudinal or vertical tear or fracture, a radial tear or fracture, a horizontal tear or fracture, a bucket handle tear or fracture, a parrot beak tear, or a flap tear; associated with a defect, disease, disorder, or condition associated with bone tissue, bone, cartilage, tendon, intravertebral discs (IVDs), hair follicles, cardiac infarcts, cartilaginous tissue, cartilage, meniscus, knee meniscus, ligament, ligament enthesis, tendon, tendon enthesis, intervertebral disc, temporomandibular joint (TMJ), TMJ ligament, or triangular fibrocartilage; an injury associated with nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair or regeneration, plastic surgery, wound healing, or oral surgery; or any bone defects resulting from trauma, congenital anomalies, infections or tumor resection, such as (1 ) oral facial bone defects (e.g., Cxcl16 may be used in conduction with fillers, i.e. periodontal defects and before implant placement), (2) long-bone defects including bone fractures, with or without irradiation, diabetes, etc., (3) other types of bone defects or diseases, osteogenesis imperfecta, osteoporosis, hormone related bone loss, or (4) augmentative applications.

A PRP component (e.g., Cxcl16) can be administered simultaneously or sequentially with another agent, such as an antibiotic, an antiinflammatory, or another agent. For example, a PRP component (e.g., Cxcl16) can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate

compositions, each containing one or more of a PRP component (e.g., Cxcl16), an antibiotic, an antiinflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a PRP component (e.g., Cxcl16), an antibiotic, an antiinflammatory, or another agent. A PRP component (e.g., Cxcl16) can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, a PRP component (e.g., Cxcl16) can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent.

ADMINISTRATION

Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μηι), nanospheres (e.g., less than 1 μιτη), microspheres (e.g., 1 -100 μιτη), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally,

Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

KITS

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to PRP components (e.g., Cxcl16). Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized

component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen.

Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials.

Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current

Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10:

0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises,"

"comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims.

Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. EXAMPLE 1: PRP ISOLATION AND ACTIVATION

The following example shows the platelet-rich plasma (PRP) isolation and Activation.

PRP isolation and activation was performed by drawing whole blood from 8 healthy human donors (age: 25-38 years old) followed by clinically compatible PRP preparation. PRP was degranulated by 10% CaC and thrombin (1 U/ml_) (see e.g., FIG. 1 ).

EXAMPLE 2: PROTEOMICS AND BIOINFORMATICS

The following example shows the proteomic and bioinformatic analysis.

Proteomics Analysis of PRP sample revealed that the most abundant proteins in PRP included those for growth factor binding, cytokine activity, protease binding, and growth factor activity. Subsequent Bioinformatics Analysis unraveled multiple datasets of individual proteins of rank significance and biological affiliation of cellular functions.

The direct protein-protein interaction network in all the components of PRP was constructed. Furthermore, all the proteins were ranked according to their concentrations and then sorted out the extracellular proteins to run the screening for migration related ones. Finally 30 potential candidates were narrowed down for the recruitment of stem cells in multiple tissues regeneration.

An image of the immunofluorescent probes for detection of protein-protein interactions can be seen in FIG. 2A. The proteomics and bioinformatics analysis of multiple datasets to elucidate potential functions of individual PRP components in multiple putative functional clusters can be seen in FIG. 2B and FIG. 2C.

EXAMPLE 3: ENRICHMENT ANALYSIS

The following example shows enrichment analysis of chemotaxis-related proteins by AmiGO and protein screening.

Proteomics and enrichment analysis clustered all plasma-derived proteins and peptides into functional groups such as growth factor binding, cytokine functions, protease binding and growth-factor activity (FIG. 3A).

AmiGO analysis unraveled individual PRP proteins specific for MSC migration, with their concentration ranking listed (FIG. 3B).

Because non-union bone fractures do not heal, here it is suggested that local osteogenic factors including BMP2 are perhaps insufficient. BMP2 is ranked at the 1 15th place (at 1 .52 ng/mL plasma concentration) among most common proteins in plasma per our proteomics data below (see e.g., FIG. 3B). Contrastingly, Cxcl16 is ranked at the 59th place with a plasma concentration of 8.12 ng/mL (FIG. 3B).

Cxcl16 at 10- and 50-ng/mL stimulated MSCs to produce common osteogenesis- related factors including Runx2, BSP, OCN and upregulated p-Smad1/5/8 by

Western blot (FIG. 3D). Cxcl16 signaling pathways were explored by systems biology and discovered that it regulates MSC migration and differentiation through MAPK/ERK (FIG. 3E).

BMP2's efficacy in promoting osteogenesis in experimental studies and in human patients suggests that other blood-derived proteins may also have efficacy for bone growth. Proteomics of platelet-rich plasma (PRP) was performed to map out all plasma-derived proteins and peptides. Here, a protein that is novel for

osteogenesis, Cxcl16, was identified and benchmarked Cxcl16 with BMP2 for osteogenesis, and several strong chemotactic inducers for cell migration.

Multiple human PRP samples (N=7) were subjected to proteomics and Gene Ontology/Am iGO analysis. Bioinformatics analysis was performed to identify individual proteins of rank significance and putative functional clusters (see e.g., FIG. 3A and FIG. 3B). ELISA further confirmed enriched molecules concentrations in PRP, compared to donor-matched PPP (see e.g., FIG. 3C). Cxcl16's osteogenic capacity benchmarked to BMP2 (Western blot) (see e.g., FIG. 3D).

Bone-marrow mesenchymal stem/progenitor cells (MSCs) were assayed via high-throughput screening for migration, differentiation, and proliferation in response to bioinformatically screened individual PRP proteins.

EXAMPLE 4: CXCL16 PROMOTES OSTEOGENESIS

The following example shows the discovery of novel proteins in PRP that promote osteogenesis, shows the novel factor, Cxcl16's roles in chemotaxis and osteogenesis, and that Cxcl16 is a novel chemotactic and osteogenic factor, without previously known association with bone regeneration. The following example also shows the elucidation of Cxcl16's signaling pathways and mechanisms of action.

This example shows Cxcl16 is chemotactic and promotes osteogenesis and further shows the promotion of osteogenesis is via ERK/MAPK pathway.

Cxcl16 was benchmarked with PRP, PDGF and TGFpi in transwells for cell migration.

Cxcl16 induced robust MSC migration that was benchmarked to both PRP and PDGF at escalating concentrations and in a dose-dependent manner (see e.g., FIG. 4A), and was superior to another potent chemotactic factor, TGFpi (see e.g., FIG. 4A). Remarkably, Cxcl16 induced chemotaxis with little effect on cell

proliferation (see e.g., FIG. 4B).

Cxcl16 robustly promoted osteogenic differentiation of MSCs, with augmented OCN, Runx2, BSP and Col1 a1 at both mRNA and protein levels (see e.g., FIG. 4C). Cxcl16 further induced dose-response increases of common osteogenesis factors including Runx2, OCN, BSP at both mRNA and protein levels (see e.g., FIG. 4C), along with activation of ERK1/2 signaling, rather than BMP2-associated Smad1/5/8 signaling (see e.g., FIG. 4D). Cxcl16 acts via ERK/MAPK, rather than BMP2's Smad1/5/8 pathway (see e.g., FIG. 4D).

The promotion of osteogenesis via ERK/MAPK was shown in FIG. 5A-E'. It was shown that Cxcl16 induced robust mineralization, which was attenuated by an ERK1/2 inhibitor, U0126 (see e.g., FIG. 5A-FIG. 5E and FIG. 5A'-FIG. 5E'). It was shown that Cxcl16's osteogenic effect was attenuated by the ERK1/2 inhibitor at both mRNA and protein levels (see e.g., FIG. 5F-FIG. 5G).

The above example shows that Cxcl16 promotes osteogenesis through ERK1/2 signaling, rather than BMP2's Smad1/5/8 pathway. pSMAD is the canonical BMP pathway and pERK1/2 is the non-canonical BMP pathway. BMP2 goes through canonical BMP pathway and Cxcl16 goes through non-canonical BMP pathway.

The above example also shows that Cxcl16 provides for osteogenic effects as robust as BMP2, cell migration effect, and showed no effect on proliferation.

MSCs proliferation and differentiation in response to Cxcl16 were evaluated by CCK8, real-time PCR and western blot for in vitro osteogenesis with/without ERK1/2 inhibitor (U0126). All quantitative results were treated by ANOVA and Bonferroni analysis, upon verification of normal data distribution with a value at 0.05.

EXAMPLE 5: BENCHMARKING BETWEEN PRP AND PDGF-BB

The below example shows benchmarking between PRP and PDGF-BB by molecular assays with human mesenchymal stem cells (hMSCs).

Using the transwell migration system, it was found that when PRP at 1 %-90% was compared to PDGF-BB, up to 25% concentration was readily benchmarked with PDGF concentrations from 20 to 500 ng/mL. Surprisingly, hMSCs migration arrested by 50% PRP. It was initially suspected that CaC that was conventionally used to activate PRP was cytotoxic due to excessive calcium, but found that the same MSC migration arrest with 50% PRP by thrombin activation. We then depleted PDGFs (PDGF-AA, PDGF-BB, PDGF-AB) with PDGF antibodies (NAb), and found that hMSCs migration was attenuated by about 1/3 in comparison with 5% PRP, suggesting that PDGF-BB alone accounts for ~1/3 of migratory capacity of PRP. Cell viability assay revealed substantial cell death in response to 50% PRP, whereas 10% and 25% PRPs promoted cell proliferation. Given that angiogenesis arguably is the common initiator for tissue healing, we benchmarked PRPs to PDGF in angiogenesis tubular formation assays and found similar efficacy of PDGF-BB with 5% and 20% PRPs in promoting tubular formation, which exceeded the conventional VEGF-supplemented HUVEC culture medium. Upon exposure of MSCs to PRP and chemically defined medium for osteogenesis, PRPs showed an approximate dose- dependent response in Alizarin Red staining with quantitative PCR data of up- regulation of Runx2, ALP, and collagen 1 a expression.

Cxcl16 induced osteogenesis per Alizarin red staining (FIG. 5A-FIG. 5E and FIG. 5A'-FIG. 5E'), more pronounced than conventional osteogenesis induction medium (OIM), and Cxcl16's osteogenesis effect was abolished by ERK1/2 inhibitor (U0126) at both mRNA and protein levels (FIG. 5F, 5G), further confirming that Cxcl16 functions via ERK1/2 pathway.

Cxcl16 previously has not been associated with bone growth. Shown here is an original demonstration that Cxcl16 not only is a potent osteogenesis inducer, but also a capable chemotactic cue. For tissue regeneration by cell homing, chemotaxis, and tissue-specific differentiation are two pivotal cellular processes. Cxcl16 appears to enable both chemotaxis and osteogenic differentiation of MSCs, and therefore a novel factor for osteogenesis. Our finding of Cxcl16's function via ERK1/2 signaling, rather than BMP2's Smad1/5/8 pathway, suggests that Cxcl16 may not be

susceptible to some of the adverse effects of BMP2 in clinical use.

EXAMPLE 6: TISSUE SPECIFIC REGENERATION

The above example demonstrated that the novel factors in plasma can promote tissue-specific regeneration in comparison to PRP as a whole. As such, the above methods can be used a model system for interrogating novel plasma proteins for regeneration of other tissues.

Cxcl16 infused samples for bone regeneration in vivo are to be harvested. In vivo experiments include TCP scaffold + Cxcl16-treated MSC cells in dorsum of mice promoting tissue-specific regeneration.

Further experiments also include testing the novel factors in bone fracture animal model promoting tissue-specific regeneration.

The novel factors regenerate tissue using the novel factors tested in an ectopic bone regeneration in vivo model (implanted into dorsum of a mouse) and a bone fracture model.

REFERENCES

References:

1 . van Baardewijk, L.J. et al. Circulating bone morphogenetic protein levels and delayed fracture healing. Int Orthop 37, 523-527 (2013).

2. Lee, C.H. et al. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet 376, 440-448

(2010).

3. Lee, C.H. et al. Protein-releasing polymeric scaffolds induce

fibrochondrocytic differentiation of endogenous cells for knee meniscus

regeneration in sheep. Sci Transl Med 6, 266ra171 (2014).

4. Lee, C.H. et al. Harnessing endogenous stem/progenitor cells for tendon regeneration. J Clin Invest 125, 2690-2701 (2015).

Claims

Claim 1 . A method for treating a subject having a tissue defect, comprising:

(a) providing a platelet-rich plasma (PRP) component;

(b) delivering the platelet-rich plasma (PRP) component to a tissue defect;

(c) forming tissue at the site of the tissue defect; and

(d) recruiting progenitor cells at the tissue defect site.

Claim 2. The method of claim 1 , wherein providing the PRP component comprises contacting the tissue defect with the PRP component.

Claim 3. The method of any one of claims 1 -2, further comprising providing a scaffold, wherein providing the scaffold comprises contacting the scaffold with the PRP component before delivering the scaffold to the tissue defect.

Claim 4. The method of any one of claims 1 -3, further comprising providing a scaffold, wherein providing the scaffold comprises contacting the scaffold with the PRP component after delivering the scaffold to the tissue defect.

Claim 5. The method of any one of claims 1 -4, wherein the PRP component improves both chemotaxis of MSCs and osteogenic differentiation of MSCs when compared to whole PRP.

Claim 6. The method of any one of claims 1 -5, wherein the PRP component stimulates osteogenesis.

Claim 7. The method of any one of claims 1 -6, wherein delivering the PRP component comprises placing the PRP component in fluid communication with a progenitor cell at the tissue defect site.

Claim 8. The method of any one of claims 1 -7, wherein

the platelet-rich plasma (PRP) component comprises one or more of Cxcl16, GDF2, or HGF; the PRP component is a chemotactic and an osteogenic factor; the progenitor cells comprise mesenchymal stem cells (MSCs); or

the PRP component stimulates production of OCN, Runx2, BSP, or Col1 a1 .

Claim 9. The method of any one of claims 1 -8, wherein the PRP component further comprises one or more selected from the group consisting of albumin, PDGF, PDGF-AA, PDGF-BB, PDGF-AB, IGF, FGF2, MCP1 , SDF-1 , FGF-2, bFGF, CTGF, GDF-5, TGF-βΙ , TGF-P3, IGF-1 , BMP-2, or BMP-7.

Claim 10. The method of any one of claims 1-9, wherein the release duration of the PRP component is for an amount of time sufficient to regenerate tissue.

Claim 1 1. The method of any one of claims 1-10, wherein the tissue defect site or scaffold:

(i) comprises an endogenous progenitor cell;

(ii) comprises an exogenous progenitor cell;

(iii) does not comprise an exogenous progenitor cell;

(iv) comprises a progenitor cell prior to scaffold delivery to the tissue defect site;

(v) does not comprise a progenitor cell until after scaffold delivery to the tissue defect site;

(vi) comprises an endogenous progenitor cell introduced to the scaffold in vivo or ex vivo; or

(vii) comprises an exogenous progenitor cell introduced to the scaffold in vivo or ex vivo.

Claim 12. The method of any one of claims 1-1 1 , wherein the tissue defect comprises

an orthopedic, non-orthopedic, musculoskeletal, or non-musculoskeletal defect, disorder, disease, or condition;

a fracture, tear, injury, osteoarthritis, or degeneration;

a longitudinal or vertical tear or fracture, a radial tear or fracture, a horizontal tear or fracture, a bucket handle tear or fracture, a parrot beak tear, or a flap tear; associated with a defect, disease, disorder, or condition associated with bone tissue, bone, cartilage, tendon, intravertebral discs (IVDs), hair follicles, cardiac infarcts, cartilaginous tissue, cartilage, meniscus, knee meniscus, ligament, ligament enthesis, tendon, tendon enthesis, intervertebral disc, temporomandibular joint (TMJ), TMJ ligament, or triangular fibrocartilage;

an injury or defect associated with nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair or regeneration, plastic surgery, wound healing, or oral surgery; or

any bone defect resulting from trauma, congenital anomalies, infections, tumor resection, oral facial bone defects, periodontal defects, long-bone defects including bone fractures, with or without irradiation, diabetes, other types of bone defects or diseases, osteogenesis imperfecta, osteoporosis, hormone related bone loss, or augmentative applications.

Claim 13. The method of any one of claims 1-12, wherein the treatment results in a reduction of pain or promotes tissue regeneration in the subject.

Claim 14. A composition comprising PRP enriched with an isolated PRP component, optionally Cxcl16.

Claim 15. The composition of claim 14, wherein the PRP component improves both chemotaxis of MSCs and osteogenic differentiation of MSCs when compared to whole PRP.

Claim 16. The composition of any one of claims 14-15, wherein the PRP component stimulates osteogenesis.

Claim 17. The composition of any one of claims 14-16, wherein

the platelet-rich plasma (PRP) component comprises one or more of Cxcl16, GDF2, or HGF;

the PRP component is a chemotactic and an osteogenic factor;

the progenitor cells comprise mesenchymal stem cells (MSCs); or

the PRP component stimulates production of OCN, Runx2, BSP, or Col1 a1 .

Claim 18. The composition of any one of claims 14-17, wherein the PRP component further comprises one or more selected from the group consisting of albumin, PDGF, PDGF-AA, PDGF-BB, PDGF-AB, IGF, FGF2, MCP1 , SDF-1 , FGF- 2, bFGF, CTGF, GDF-5, TGF-βΙ , TGF-P3, IGF-1 , BMP-2, or BMP-7.

Claim 19. The composition of any one of claims 14-18, wherein the release duration of the PRP component is for an amount of time sufficient to regenerate tissue.

Claim 20. The composition of any one of claims 14-19, for application to a tissue defect site or scaffold, wherein the tissue defect site or scaffold:

(i) comprises an endogenous progenitor cell;

(ii) comprises an exogenous progenitor cell;

(iii) does not comprise an exogenous progenitor cell;

(iv) comprises a progenitor cell prior to scaffold delivery to the tissue defect site;

(v) does not comprise a progenitor cell until after scaffold delivery to the tissue defect site;

(vi) comprises an endogenous progenitor cell introduced to the scaffold in vivo or ex vivo; or

(vii) comprises an exogenous progenitor cell introduced to the scaffold in vivo or ex vivo.

Claim 21. The composition of any one of claims 14-20, for use with a tissue defect, wherein the tissue defect comprises

an orthopedic, non-orthopedic, musculoskeletal, or non-musculoskeletal defect, disorder, disease, or condition;

a fracture, tear, injury, osteoarthritis, or degeneration;

a longitudinal or vertical tear or fracture, a radial tear or fracture, a horizontal tear or fracture, a bucket handle tear or fracture, a parrot beak tear, or a flap tear; associated with a defect, disease, disorder, or condition associated with bone tissue, bone, cartilage, tendon, intravertebral discs (IVDs), hair follicles, cardiac infarcts, cartilaginous tissue, cartilage, meniscus, knee meniscus, ligament, ligament enthesis, tendon, tendon enthesis, intervertebral disc, temporomandibular joint (TMJ), TMJ ligament, or triangular fibrocartilage;

an injury or defect associated with nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair or regeneration, plastic surgery, wound healing, or oral surgery; or

any bone defect resulting from trauma, congenital anomalies, infections, tumor resection, oral facial bone defects, periodontal defects, long-bone defects including bone fractures, with or without irradiation, diabetes, other types of bone defects or diseases, osteogenesis imperfecta, osteoporosis, hormone related bone loss, or augmentative applications.

Claim 22. The composition of any one of claims 14-21 , wherein the treatment results in a reduction of pain or promotes tissue regeneration in the subject.

Claim 23. A tissue construct comprising:

(i) a progenitor cell;

(ii) an effective amount of a PRP component having a release duration; and

(iii) optionally, a scaffold comprising a matrix material,

wherein,

the effective amount of the PRP component induces migration of a progenitor cell into or onto the scaffold when the scaffold is in fluid

communication with the progenitor cell; and

the effective amount of the PNP component stimulates osteogenesis.

Claim 24. A method of forming the fibrocartilage tissue construct of claim 23, comprising:

(i) providing a scaffold comprising a matrix material;

(ii) contacting the scaffold with a PRP component;

(iii) placing the scaffold in fluid communication with a progenitor cell; and

(iv) delivering the scaffold to a tissue defect site; wherein the PRP component induces chemotaxis of MSCs and osteogenic differentiation of MSCs at the tissue defect site.