WO2019048697A1 - Macro- and microporous composite cryogel biomaterial for use in bone regeneration - Google Patents

Abstract

The invention relates to synthetic grafts and their use in bone diseases. In particular, the invention relates to porous composite scaffolds comprising a ceramic phase and a biopolymer phase, which composite scaffold is non- immunogenic, osteoinductive, osteoconductive and osteogenic. The scaffold suitable as carrier for bone active and other bioactive agents for use in treating bone diseases, in particular enhancing bone regeneration. The invention provides a scaffold that can be molded in any desired shape for example to mimics lost bone structures prior to application in the patient.

Description

MACRO- AND MICROPOROUS COMPOSITE CRYOGEL

BIOMATERIAL FOR USE IN BONE REGENERATION

FIELD OF INVENTION

The invention relates to synthetic grafts and their use in treatment of bone defects or trauma, such as bone diseases and bone fractures. In particular, the invention relates to biomateriais for use in bone regeneration comprising a porous composite scaffold and bioactive agents, which scaffold comprises a ceramic phase and a biopolymer phase and is non-immunogenic,

osteoinductive, osteoconductive and osteogenic. The scaffold is suitable as carrier for bioactive agents for use in treating bone defects, in particular enhancing bone regeneration. The invention provides a composite cryogel bone scaffold for incorporation of bioactive agents that can be molded in any desired shape for example to mimics lost bone structures prior to or during application to the patient for bone repair.

BACKGROUND OF THE INVENTION

One of the major challenges in modern orthopedic surgery is to replace and regenerate bone (bone tissue). There is an increasing demand for bone substitutes that can not only augment, but also treat and repair bone defects caused by trauma, bone infections, non-unions and bone tumor resection. In joint prosthetic revision surgery and in fracture mal-union additional measures to regenerate bone and fill empty space and cover cortical defects especially in infected cases is critical for the outcome. The ideal bone graft should be non-immunogenic, osteoinductive, osteoconductive and osteogenic. Autologous bone fulfills these requirements but the availability is limited. Further, surgery is required for its harvesting, which in turn is associated with donor site morbidity and in the worst-case, infection. To overcome these drawbacks synthetic bone grafts are being developed, mainly ceramic, polymeric or composite. Ideally, these materials should not just act as structural support but also be able to carry bone active agents to initiate and enhance tissue regeneration, but only a few such materials are in clinical use today. A possible solution to this problem has been addressed by tissue engineering approaches in bone regeneration, which aim at exploring the triad of biomaterials, cells and growth factors to regenerate large volumes of bone. Biomaterials for bone tissue engineering can be polymeric, composites of polymer with inorganic components like calcium phosphate and calcium sulphate or purely inorganic. Furthermore, they can be classified as macro- porous or micro-porous based on their pore structure.

Solid and injectable bone cements have been used for many years. Calcium sulphate hemihydrate (CSH), known as "Plaster of Paris", calcium sulphate dihydrate (CSD; "Gypsum") and different hardenable calcium phosphate cements (CPC) are known in the art. CSH sets in the presence of water to form a microporous structure in form of interlocked needle shaped calcium sulphate dihydrate (CSD) crystals. The setting reaction also referred to as a hardening reaction can be made before application to the patient (ex vivo), in which case preset CSD is applied to the patient; or after application to the patient (in vivo) in which case CSH is applied to the patient as a paste after mixing with water immediate before use. In the same way, hardenable CPC can be made to set ex vivo or in vivo. CaP exists in many forms. In particular a tricalcium phosphate (a-TCP) is often used as a hardenable component in injectable CPC; and preset CaP, such as β-tricalcium phosphate (β-TCP), hydroxyapatite (HA) and calcium-deficient hydroxyapatite (CDHA), are known to be used as components in injectable CPC compositions or as a solid implantable body for treatment of bone defects. Mixtures of calcium sulphate (CSH/CSD) and CaP are also known in the art as di- or bi-phasic ceramics for use as synthetic bone substitutes. Either of the calcium salts or both can be hardened ex vivo or in vivo. If for example HA is present during the calcium sulphate setting reaction, it will be embedded within the microporous CSD structure. Injectable biphasic ceramics containing HA in combination with CSH are commercial available from Bone Support AB, Sweden (Cerament®) and have been shown to be able to deliver bioactive agents locally, such as antibiotics to a defect site and enhance bone tissue regeneration (WO

2003/053488; WO 2014/128217; WO 2016/150876). CSD resorbs quickly by means of dissolution in body fluids (as early as within 4-12 weeks), enlarging the micropores and thus creating a porous matrix of HA particles. HA is known to be biocompatible and osteoconductive. The carrier eluting drug capacity of Cerament® has been verified in vitro and in vivo in short and long term clinical pharmacokinetic studies of antibiotics. Cryogels are polymeric gels comprising macropores, sometimes also referred to as supermacroporous polymeric gels that have been known for about 40 years and are now gaining more and more attention in technical fields of biotechnology and biomedical engineering (see Kumar et al., Materials Today 2010, Vol. 13, pp. 42-44; Kumar, Ashok. "Supermacroporous Cryogels:

Biomedical and biotechnological applications", CRC Press, 2016).

Supermacroporous implies a pore size of 100 μιη and above. Cryogels doped with hydroxyapatite have been studied as potential alternatives to bone grafts for bone regeneration as described by Hixon et al. Acta Biomaterialia, Vol 62 (2017) p.29-41. Cryogels are formed similarly to hydrogels, however, including a freezing and thawing step. In the cryogelation process, gel precursors, such as natural or synthetic polymers, are crosslinked, either physically or chemically in a solvent and subsequently frozen. The gel-like formation of matrices occurs in the frozen system, typically between -5 and - 20°C, at which temperature the solvent, for example water, crystalizes. The solvent crystals act as porogens during the matrix formation and may remain in the cryogel after thawing or removed for example by use of freeze drying or by drying at temperatures above the freezing point. The final treatment may alter the structure and properties of the cryogel. The use of cryogels as scaffolds for carrying and delivering BMP and Zoledronic acid is disclosed in Hixon et al.

Preparation of cryogels is commonly known in the art and for example disclosed by Murphy et al. Acta Biomaterials, Vol. 10 (2014) p. 2250-2258; Hixon et al. and Kumar (2010; 2016). The content of these disclosures are incorporated by reference.

Bone morphogenic protein-2 (BMP-2) is one of the most potent osteoinductive molecules from the BMP family and can induce bone at both entopic and ectopic locations. The biological half-life of BMPs, especially BMP-2, is very low and this necessitates the development of carrier materials that can enhance the bioactivity of BMP in-vivo and perform site-specific delivery of BMP-2. The only food and drug administration (FDA) approved medical device for the delivery of recombinant human BMP-2 (rhBMP-2) is Infuse™ Bone Graft produced by Medtronic as an absorbable collagen sponge (ACS) containing rhBMP-2. Clinical usage of this medical device for indications like instance lumbar spine fusion has been documented but with rather divided clinical outcomes. Despite these drawbacks, the potency of rhBMP-2 in small animal models of bone formation have kept researchers active in development of efficient carrier systems that can provide a sustained spatiotemporal release of this molecule over to avoid delivery of large doses that are suspected to be responsible for BMP-2 associated side-effects and skepticism towards its usage. New research indicates that rhBMP-2 does not only accelerate bone formation but also induces osteoclast mediated premature bone resorption via the RANKL-RANK signaling, a drawback often not given sufficient attention.

The research has been focusing not only on long-term sustained delivery of rhBMP-2 using biomaterials, but also tackle the BMP-2 induced

osteoclastogenesis by co-delivering 3rd generation bisphosphonate, such as zoledronic acid (ZA) locally (Little et al., J. Bone and Mineral Research 20(11) (2005) 2044-2052; Doi et al., Bone 49(4) (2011) 777-782; Raina et al., Scientific reports 6 (2016); Roelofs et al., Clinical Cancer Research 12(20) (2006) 6222s-6230s). ZA is known to induce osteoclast cell death mediated by the mevalonate pathway and is used for several indications including osteoporosis, osteogenesis imperfecta and several metastatic tumors. By harnessing the cytotoxicity of ZA towards osteoclasts, it is possible to prevent premature bone resorption induced by excessive rhBMP-2 doses. Systemic ZA administration has been the most preferred route in the clinics but there have been reported side effects like osteonecrosis of the jaw, reduced skeletal remodeling, flu like symptoms etc., emphasizing the need of local delivery of ZA.

Raina et a/., Scientific reports, Vol. 6, (2016) pp. 1-13, discloses an injectable biphasic calcium sulphate/hydroxyapatite carrier containing recombinant human Bone Morphogenic Protein-2 (rhBMP-2) or rhBMP-2 and zoledronic acid (ZA). The carrier has mainly two components, one being micro to nanoparticles of HA that is incorporated into an injectable in situ setting of the second component, alfa hemihydrate calcium sulphate, forming solid calcium sulphate dihydrate (CSD). The CSD is water soluble and resorbs in 6-12 weeks during which time added bone active proteins like BMP-2 is slowly eluted and a porous matrix is created for cells to infiltrate and differentiate into osteoblasts. Bisphosphonates incorporated in the ceramic material binds due to high affinity towards calcium ions with HA decreasing its osteoclast mediated resorption. Added Zoledronic acid is eluted up to 20 % during the first weeks and the rest is bound to HA and can stay for many months or even years.

WO 2016/150876 discloses injectable biphasic calcium sulphate/

hydroxyapatite carrier containing bone active protein and an anti-catabolic agent.

WO 03/024316 discloses a bone precursor composition comprising a cement mixture and a pore-forming agent which may be a resorbable biocompatible polymer preferably a polymer absorbable by enzymatic degradation; or calcium sulphate. Contrary to the present invention, the pores are only formed in vivo continuously due to delayed resorption of the pore-forming agent. There are no pores present from the start.

A lot of natural polymer based biomaterials have been pursued in order to develop scaffolds for bone regeneration and gelatin has gained a lot of popularity due to its natural origin, biocompatibility, biodegradability, cell binding motifs and absence of immunogenic reactions. Gelatin is obtained by the partial hydrolysis of collagen. Gelatin is also known to interact with growth factors like BMP-2 by means of electrostatic interactions. Teotia et a/., Appl Mater Interfaces 2016, Vol. 8, pp. 10775-87, discloses injectable bone cement comprising 60% calcium sulphate and 40 %

hydroxyapatite modified with up to 1% gelatin to enhance cellular interaction. A bioactive fraction of native cell derived proteins from Saos-2 cells or Zoledronic Acid (ZA) was incorporated in the gelatin-cement for use in animal experiments. Kim et al., Spine, Vol. 42 (7S), p. S9, discloses an investigation of the safety and efficacy of hyper crosslinked carbohydrate polymers (HCCP) carrying Bone Morphogenic Protein-2 for repair of critical size bone defects.

Kemenge et a/., J Tissue Eng Reg Med 2017, Vol 11, pp 20-33, discloses biodegradable gelatin- and hydroxyapatite-based cryogels to be used as scaffolds in bone tissue regeneration. Composites cryogels with

gelatin : hydroxyapatite ratios of between 90: 10 and 50: 50 were prepared. The cryogels do not contain any additional bioactive agents.

Fassina et a/., Conf Proc IEEE Eng Med Biol Soc 2010, pp. 247-50, discloses the use of bovine gelatin cryogels as a biocompatible and biodegradable scaffold in in vitro differentiation of human bone marrow stroma cells. The scaffold does not comprise any ceramic.

Murphy et al. Acta Biomaterials, Vol. 10 (2014) p. 2250-2258, discloses a spongy collagen-hydroxyapatite scaffold produced by cryogelation and comprising rhBMP-2.

Raina et a/., J Control Release 2016, Vol 10, pp. 265-78, discloses bio- composite materials in a cryogel scaffold comprising silk-fibroin, chitosan, agarose, and hydroxyapatite with and without bioactive glass. The cryogel scaffolds were combined with recombinant human Bone Morphogenic Protein- 2 (rhBMP-2) or rhBMP-2 and Zoledronic Acid (ZA) and tested for in-vitro cell interactions and bone formation in an in-vivo muscle punch model. However, compared to this biomaterial, a marked difference in new bone area formation can be seen for the biomaterial according to the present invention.

Additionally, silk-fibroin possess immunogenic properties which makes the scaffold according to Raina et. al. unsuited for clinical use.

SUMMARY OF THE INVENTION The present invention provides a solution to the above discussed problems by providing a new type of biomaterials comprising a porous biodegradable composite scaffold material comprising at least one ceramic and a crosslinked biopolymer and bone active/bioactive agents. The new composite bone scaffold is in the form of a preset solid, spongy, macro- and microporous material prepared by cryogelation where the composite cryogel biomaterial is especially suited for treatment of large critical defects, anatomical sensitive regions like the face mandible, etc., and also for treatment of the spine (e.g. compression fractures) where a minimal leakage is important. The cryogel composite scaffold comprises in addition to the biopolymer, which in a crosslinked forms a macropores structure, a ceramic comprising calcium phosphate (CaP) and calcium sulphate in the form of calcium sulphate dihydrate (CSD), which provides a microporous structure in addition to the macroporous structure. The presence of the ceramic provides mechanical strength as well as osteoinductive and osteoconductive properties to the scaffold. The present invention also provides an additional improvement of the bone regenerative potential when bone anabolic agents, such as rhBMP-2, are present in the final composite biomaterial. The combined macroporous and microporous cryogel composite biomaterial according to the present invention comprises a crosslinked non-immunogenic biopolymer/ceramic composite cryogel scaffold and at least one bone active agent, such as at least one anabolic agent and preferably additionally at least one anti-catabolic agent, which is highly suitable for use as a bone generating biodegradable bone scaffold. The macroporosity is obtained by preparing the composite biomaterial by cryogelation to prepare a cryogel. The micro- porosity is formed during the cryogelation when CSH in the ceramic powder also comprising CaP, e.g. HA, sets in the presence of water to form CSD in the composite comprising polymer and ceramic as described above. A part of the CaP particles will be embedded in the CSD structure and a part will be embedded in the polymer. Similarly a part of the CSD, with or without embedded CaP particles, will be embedded in the polymer and a part will to be present on the surface of the polymer. The inclusion of the ceramic adds several advantageous properties to the scaffold, such as increasing the mechanical strength, improving the osteoconductivity, the osteoinductivity and the carrier properties for bioactive agents, being relatively fast resorbed in bodily fluids for release of osteoinductive calcium and phosphate ions as well as any added bioactive agents in a delayed manner. The biomaterial in the scaffold according to the present invention provides the best conditions for fulfilling the requirements for osteoinduction, including a macrostructure, a microstructure and an optimal chemical composition. The cryogelated polymer provides the macrostructure and a scaffold for the ceramic. The ceramic provides the microstructure. Together they form the final osteoconductive and osteoinductive scaffold. The macro- and micro-structures contain concavities or pores in which bone formation takes place. Osteoinduction and bone formation takes place on the surface inside the pores where these are present (Habibovic, J. Tissue Eng Reg Med, Vol 1 (2007) p. 25-32) and the rough microstructures inside the micro-pores formed by the interlocked needle shaped CSD crystals) are thought to further enhance osteoinduction and bone formation. Because of the relatively fast resorption of CSD, calcium and phosphate ions are released which further promote osteoinduction and bone formation. The resorption of CSD forming the microstructure over time leads to an enlargement of the micropores whereby ingrowth of bone tissue can accelerate. As CSD is a good carries for many bioactive agent, such as BMP, it also acts as a carrier with both immediate and delayed release of the agent. This is highly advantageous for many agents involved in osteoinduction and bone formation. The CaP component of the ceramic, e.g. crystalline HA particles, which also acts osteoconductive and bioactive, remain to a high degree in the scaffold and later in the newly-formed bone after resorption of the polymer. CaP is also known to bind some biological agents, such as bisphosphonates (inhibitors of osteoclast activity), on its surface. If the binding is strong, such as being the case for the bisphosphonates, the release of activity, e.g. anti-osteoclast activity leading to an inhibition of resorption of newly-formed bone, may be maintained over a long time which strengthens the formation of new bone.

The present invention provides a composite cryogel biomaterial leading to a desired bone regeneration / scaffold degradation ratio where the scaffold may ultimately be completely resorbed in 4-6 months, leaving the patient's own healthy vascularized bone. The present composite cryogel scaffold is an exceptional carrier for many bioactive agents, including bone anabolic agents (e.g. BMP-2, growth factors, etc.) and anti-catabolic agents (e.g.

bisphosphonates), biological drugs and cytostatics, antibiotics, cells, marrow aspirate concentrate, platelet rich plasma (PRP), cell factory derived proteins, and hemostatic agents. Further, the present composite biomaterial can be prepared in many formulations, such as granules, cubes, wedges, sheets, and custom made, for example as exact 3D custom fitted anatomic forms mimicking the bone to be replaced. Because of its solid spongy structure the cryogels may be fitted for a specific use by for example cutting and adapting a standard/uniform cryogel to a desired 3-dimensional form and structure. The composite material may also be sutured and it has some memory as well as tensile strength. Furthermore, the porosity and modulus of elasticity of the composite biomaterial may be differentiated as desired depending on use in a specific anatomic structure by adjusting the different components and their contents as well as the method of preparation.

Compared to the biomaterial of Raina et al. (2016), comprising the same doses of rhBMP-2 and ZA in the same model, a marked difference in the new bone area can be seen for the biomaterial according to the present invention. The new scaffold + rhBMP-2 group shows approximately 17 times more bone area compared to the rhBMP-2 comprising scaffold in the earlier study, while the scaffold + rhBMP-2+ZA group shows nearly 3-times higher bone area compared to the rhBMP-2+ZA comprising scaffold in the earlier study. These differences could be due to a much slower release of rhBMP-2 from the gelatin based composite cryogel scaffold according to the invention compared to the previous study (3.2% vs. 24% after 4-weeks in-vitro). Another important factor could be the difference in the degradation rates of the two scaffolds in- vivo with gelatin-CSD-HA based scaffold degrading faster than a composite scaffold of chitosan-agarose-silk fibroin with inorganic components. Scaffold degradation and bone formation are tightly coupled processes since scaffold degradation creates more room for the new bone tissue to grow. In addition silk-fibroin possesses immunogenic properties which make the scaffold unsuited for clinical use. In a particular embodiment of the present invention, a macro and micro- porous composite cryogel scaffold consisting of gelatin, calcium sulphate (CSD) and hydroxyapatite (HA) is prepared by using the cryogelation technology. By incorporating both the polymeric as well as ceramic

components into the scaffold, a tool for long term co-delivery of bone anabolic agents (e.g. BMP-2, such as rhBMP-2, growth factors, etc.) and anti-catabolic agents (e.g. bisphosphonates, such as ZA) is created, which can act as an off- the-shelf alternative to existing bone grafts. The porous materials mimic the structure of trabecular bone thereby presents a better scaffolding system in terms of osteoconductivity. Addition of CSH, which sets to form CSD in the composite scaffold, is relatively fast resorbed in vivo leading to a fast increase in the porosity of the biomaterial as well as a faster sustained delivery of unbound bone anabolic agents, such as bone active molecules. Gelatin, CSD and HA can all interact with bone active molecules such as rhBMP-2 while HA can also interact with bisphosphonates, such as zoledronic acid (ZA). This will provide local and long-term sustained delivery of bone anabolic and/or anti- catabolic agents, which will synergistically orchestrate the bone forming potential of the bone active molecules (e.g. rhBMP-2) and protection of e.g. rhBMP-2 induced premature bone resorption by the presence of

bisphosphonate, e.g. ZA.

Accordingly, in a first aspect, the present invention concerns a macro- and microporous biodegradable composite cryogel biomaterial comprising at least one ceramic, a crosslinked non-immunogenic biopolymer and at least one bone active agent, such as at least one anabolic agent and/or at least one anti-catabolic agent. The ratio of ceramic to crosslinked biopolymer is between 10 : 1 and 1 : 1 (w/w), preferably between 8 : 1 and 2 : 1, such as between 6: 1 and 2: 1. The ratio may be 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2 : 1 or 1 : 1 (w/w) or about 8 : 1, about 7: 1, about 6 : 1, about 5: 1, about 4: 1, about 3 : 1, about 2 : 1 or about 1 : 1 (w/w). When gelatin is used as polymer, the ratio is preferably between 6: 1 and 2: 1. In a specific embodiment of the present invention gelatin is used as polymer, Cerament^® is used as the ceramic and the ratio is 4: 1 (w/w). The ceramic is preferably mixed with the polymer in the form of a powder, e.g. Cerament^®. Cerament^® comprises 60 wt% CSH and 40 wt% crystalline HA particles. Other ratios of calcium sulphate and CaP can also be used. The calcium sulphate powder can be any mixture of CSH and CSD and the CaP powder can be any hardenable or pre-set CaP or any mixture thereof. In the mixture of polymer, ceramic powder and liquid, the polymer may for example constitute 5% (w/v) and the ceramic between 5% and 50% (w/v), preferably between 10% and 40% (w/v), such as about 15, 20, 25, 30 or 35 % (w/v), the rest being a fluid such as water. If the amount of polymer is more or less than 5%, such as 3, 4, 6, 7, 8, 9 or 10 % (w/v), the content of ceramic can be varied accordingly to prepare a suitable spongy composite product after cryogelation. Different properties of the selected polymer may change the preferred ratio of ceramic to polymer. In selecting the content and ratio of components in the composite mixture, it should be taken into account that a too high ceramic content will retard ice crystal formation and thus the pore formation in the scaffold and a too high ceramic to polymer ratio may result in a too brittle product for molding purposes. A too high crosslinked polymer content will cause a too slow in vivo degradation of the scaffold and a low ceramic to polymer ratio may result in the product being mechanically similar to a pure polymeric gel.

The ceramic in the composite cryogel scaffold is preferably a biphasic ceramic comprising CSD and a CaP, preferably particulate crystalline HA in micro and nano meter size. The ceramic may also be a tri- or more phasic ceramic comprising CSD and two or more CaPs, preferably comprising particulate crystalline HA. Other CaPs may be selected from a-TCP, β-TCP, calcium- deficient hydroxyapatite (CDHA) and the like. In the process of preparing the cryogel, CSD may be the results of a setting process where CSH is mixed with the CaP and water. Other components may be present as part of the ceramic or the scaffold.

The biopolymer may be one or a mixture of more than one non-immunogenic biopolymers, such as for example gelatin, hyaluronic acid, alginate or chitosan, preferably gelatin, and the crosslinked biopolymer is prepared by mixing the biopolymer(s) with a suitable crosslinker, such as glutaraldehyde, EDC, N-hydroxysuccinimide or genipin, preferably glutaraldehyde, in the presence of the ceramic. Some polymer units, such as polyvinyl alcohol (PVA), do not need addition of a crosslinker to produce a crosslinked polymer.

In one embodiment of the composite bone biomaterial according to the present invention comprises at least one bone active agent, preferably at least one bone anabolic agent and/or at least one anti-catabolic agent. The bone anabolic agent(s), such as at least one bone activating agent is selected from the group of bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), transforming growth factor-ps (TGFPs), parathyroid hormone (PTH), sclerostin, cell factory derived bone active proteins and ECM proteins; or strontium. In a preferred embodiment, the bone anabolic agent is BMP-2 or BMP-7, such as human BMP (hBMP-2 or hBMP-7), and is preferable

recombinant human BMP-2 (rhBMP-2 or rhBMP-7). The at least one anti- catabolic agent is preferably in the form of a bone resorption inhibitor, such as an osteoclast activity inhibitor. The osteoclast activity inhibitor may preferably be selected from the group of bisphosphonic acids and their salts. Other biological drugs inhibiting osteoclast activity like RANKL inhibitors or similar biological molecules may also be used. In another embodiment of the present invention, the composite bone biomaterial further comprises at least one additional bioactive agent selected from vitamins, antibiotics, antifungal drugs, bone healing promotors, chemotherapeutics, cytostatics, hormones, bone marrow aspirate, platelet rich plasma, demineralized bone, Mabs, native derived proteins and

exosomes. Bone marrow stem cells, induced pluripotent stem cells (MSCs) and patients own cells cultured to differentiate into bone cells may also be transplanted back into the patient via the scaffold of the present invention. It is an advantage of the present macro- and microporous scaffold that it can support a fast ingrowth of cells due to the presence of large pores right from the time of incorporation of the composite cryogel.

In a further embodiment of the present invention, the composite bone biomaterial further comprises a X-ray contrast agent selected from the group of water soluble non-ionic X-ray contrast agents and biodegradable X-ray contrast agents. In a further aspect of the invention, the composite cryogel bone scaffold is prepared with different gradients of the inorganic component and different modulus of mechanical strength and elasticity.

DRAWINGS

Figure 1. A shows a composite cryogel scaffold according to the present invention in a monolith form. B and C show the spongy nature of the material under compression and after release, respectively. D, E shows a cross- sectional view of the interconnected pores at low and high magnifications, respectively. F,G shows porous structure in a longitudinal manner both at high and low magnifications. Needle like CSD and small apatite particles can be seen in the high magnification images (E and G).

Figure 2 shows a SEM image of a composite cryogel scaffold. Dotted white arrows point at macro pores in the scaffold. Solid white arrows point at micropores (< 10 μιη) in the scaffold.

Figure 3 A shows energy dispersive X-ray spectrum from the surface of a composite cryogel scaffold characterized by peaks for Calcium and Phosphate. B shows the FTIR spectroscopy results indicating crosslinking of gelatin by glutaraldehyde. C shows XRD spectra of the composite cryogel with peaks corresponding to the presence of calcium sulfate dihydrate and

hydroxyapatite. The characteristic x-ray diffraction pattern generated in the XRD analysis provides a unique "fingerprint" of the crystals present in the sample. Figure 4. A shows MC3T3-el cell viability on composite cryogel scaffolds. B and C shows the attachment of cells to the scaffold and scaffold + rhBMP-2, respectively 3 days post cell seeding via scanning electron microscopy. D shows the in-vitro release of rhBMP-2 from the scaffolds. E, F and G show the differentiation of MC3T3-el cells seeded on rhBMP-2 functionalized scaffolds via ALP assay and qPCR analysis of OSX and OCN genes, respectively. Cells differentiated on the scaffold functionalized with rhBMP-2 and not on the control scaffolds without rhBMP-2.

Figure 5 shows a SEM image of a composite cryogel scaffold implanted in the rat muscle pouch for 4-weeks. White solid arrows point to hydroxyapatite particles in the material. Note the absence of needle like calcium sulphate structures.

Figure 6 shows a SEM image of a composite cryogel scaffold implanted in the rat muscle pouch for 4-weeks showing that the macro and micro porous structure of the material is preserved even after in-vivo implantation. White solid arrows point to the micro pores in the material and dotted white arrows point to the macro pores in the material. Figure 7. A, B and C show radiographs of composite scaffold explants harvested 4 weeks later from the abdominal muscle pouch model. A shows Scaffold alone, B shows Scaffold + rhBMP-2 and C shows Scaffold + rhBMP- 2+ZA. D shows quantitative micro-CT results from 3 groups used in the experiment. Data is expressed as highly mineralized volume.

Figure 8. A, D and G show histology of composite scaffold only group from the abdominal muscle pouch model, which did not lead to any bone formation in this ectopic model. (Β,Ε,Η) show the histology of scaffold + rhBMP-2 group at different magnifications and different locations in the scaffold. (C,F,I) shows histology results from the Scaffold + rhBMP-2+ZA group at different locations and magnifications. J shows the difference in the area of bone formation between the scaffold + rhBMP-2 and scaffold + rhBMP-2+ZA group.

Figure 9 shows tartarate resistant acid phosphatase (TRAP) staining of tissue specimens from composite scaffold + rhBMP-2 (A) and Scaffold + rhBMP-2+ZA (B) groups. rhBMP-2 treated samples were rich in TRAP positive area indicating osteoclast mediated premature bone resorption. Very few areas of TRAP positive tissue were seen in the Scaffold + rhBMP-2+ZA group, indicating that the use of ZA induces apoptosis of osteoclasts and thus reduces TRAP staining. Figure 10 shows the release of I-rhBMP-2 from composite scaffold in-vivo over a period of 4 weeks in the abdominal muscle pouch model. A gradual but prolonged release was detected using single photon emission computed tomography (SPECT).

Figure 11 shows the in-vitro (left) and in-vivo (right) (abdominal muscle pouch model) release of ¹⁴C-ZA from composite scaffold. Figure 12 shows an X-ray image of the explants harvested 4-weeks post- surgery from the abdominal muscle pouch. Arrows in top and middle panels on the left side indicate the only areas of mineralization. Note that the areas surrounding the arrows is plain muscle and not bone. Scale bars represent 3 mm.

Figure 13 shows micro-CT quantification of the highly mineralized volume across the 6 groups post harvest at 4 weeks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a macro- and microporous biodegradable composite biomaterial, including a cryogel scaffold comprising at least one ceramic, a crosslinked non-immunogenic biopolymer; and at least one bone active agent, such as at least one anabolic agent and/or at least one anti- catabolic agent, which composite biomaterial is useful in treatment of bone defects and in particular in the treatment of large critical defects, in anatomical sensitive regions like the face mandible, etc., and also in treatment of the spine wherein a minimal leakage is important. The outer surface of the composite biomaterial is defined by the (outer) form of the solid composite macro- and microporous scaffold which form confines the regeneration of new bone material to this form and thus limits undesired bone formation outside the scaffold. In this way, the composite cryogel bone scaffold is excellent as an artificial bone tissue scaffold mimicking and maintaining the 3-dimensional form and structure of the original bone in replacement or reconstruction of damaged, diseased or lost bone tissue. The composite cryogel bone scaffold of the biomaterial according to the present invention is an excellent carrier for a sustained delivery of bioactive agents involved in generating and protecting new bone at a place of a bone defect. The material comprised in and the structure of the composite cryogel bone scaffold allows for a local and long-term sustained delivery of bioactive agents. Because of the solid but workable nature of the composite cryogel bone scaffold, it can be formed in any 3-dimentional form as desired either in a suitable customized mold or by adapting the form as desired after its preparation, for example by manual or computer guided cutting, grinding or sharpening based on a real or artificial imprint of the bone to be replaced.

According to the present invention, the composite bone scaffold is in the form of a cryogel prepared by cryogelation of a mixture of the components. An advantage in using cryogelation for the preparation of the composite bone scaffold is that the method in a simple way provides a natural formation of a mixture of large and smaller pores in the resulting skeleton or scaffold which pores allows a fast ingrowth and formation of new bone cells and bone mass inside the scaffold. The large pores are created by liquid (e.g. water) crystals formed in the freezing step a being present during cross-linking reaction of the polymer to form the skeleton or scaffold. The present ceramic is embedded in the cross-linked skeleton and/or deposited on the surface of the crosslinked polymer and provides microporous structures in the scaffold which becomes enlarged with the fast resorption of the soluble CSD. Biologically active additives are absorbed in or on the ceramic part of the composite cryogel for sustained release upon contact with bodily fluids. Typically the agents are added to the scaffold by soaking the scaffold in a solution comprising the agent(s). If desired, the scaffold can be saturated with the solution. It is a further advantage of the present scaffold that the modulus of mechanical strength and elasticity can be varied as desired by varying the ratio of ceramic to polymer and/or the type of polymer.

The composite cryogel scaffold comprises varying sizes highly of pores ranging from less than 1 micron up to about 200 microns. This range of pores is considered to be suitable for osteonal bone formation as suggested by Itala et al. (A.I. Itala, Journal of Biomedical Materials Research Part A 58(6) (2001) 679-683). The larger pores are beneficial in allowing body fluids with its content of cell growth supporting substances as well as cells and ingrowth of cells to penetrate the scaffold and support a fast in- and regrowth of new bone. The smaller pores are beneficial in allowing body fluids to release bioactive ingredient embedded in the scaffold in a sustained way over time. The smaller pores formed by the ceramic part provide a large increase in surface area inside the pores due to the roughness of the surface as a result of CSD formation which improves the conditions for cell seeding and cell growth. The micro-pores become larger with time due to dissolution of the ceramic thus creating more space over time for the ingrowing and expanding bone cells and at the same time delivers calcium and other ions for bone formation. The multiporous composite cryogel scaffold according to the present invention provides moldable means for a synergistic effect in a fast regeneration of natural bone in bone traumas which is highly appreciated for use at specifically vulnerable areas of the bodily skeleton.

The ceramic comprised in the composite may comprise any suitable CaP, such as a- or β-tricalcium phosphate (TCP), tetracalcium phosphate, calcium- deficient apatite or hydroxyapatite. Particulate crystalline hydroxyapatite is the preferred CaP for use in the present invention. The ceramic also comprises CSD in the composite. The calcium sulphate may be used in the preparation of the composite cryogel in the form of pure CSH or pure CSD in particulate form or as a mixture thereof. If CSH is used as the calcium sulphate powder in the preparation of the cryogel, CSD will be formed during the cryogelation by contact of CSH with water.

The weight/weight (w/w) ratio between calcium sulphate and CaP in the biphasic ceramic is between 5:95 and 95: 5, between 10:90 and 90: 10, between 20 :80 and 80: 20, between 30: 70 and 70 : 30, or between 40: 60 and 60 :40, such as for example 60:40 which is the content in Cerament® powder.

The ceramic is preferably introduced into the composite bone scaffold as a CSH/CaP powder mixed with a solution of the biopolymer during the preparation of the composite cryogel scaffold. Bisphosphonates are known to bind strongly to CaP leading to a slow and long-term local release of these anti-catabolic agents in the patient. A part of the anti-catabolic agents may be carried in the CSD leading to a faster release and an initial boost release of parts of the anti-catabolic agents locally in the patient. Other bioactive agents, such as BMP and antibiotics, are mainly carried in the CSD phase, which allows for a boost release and a relative fast release over the first 10 weeks following dissolution of the CSD phase. Some bioactive agents, such as BMP, have a more or less strong affinity for the CaP phase, i.e. the crystalline CaP, e.g. crystalline hydroxyapatite, which will sustain release of some of these agents loaded in the composite cryogel scaffold.

The biopolymer part of the composite cryogel scaffold is formed by

crosslinking of suitable biopolymers. The term "biopolymer" should be understood to mean one biopolymer or a mixture of different biopolymers. The biopolymer is selected among biopolymers possessing no immunogenic properties. The term "biopolymer" encompasses polymeric biomolecules produced by living organisms and contains monomeric units that are covalently bonded to form larger structures, such as for example polypeptides and polysaccharides. These biopolymers must be biodegradable, meaning that the biopolymer is slowly degraded in situ. Up to about 50% of the scaffold disappears over the first 8-10 weeks, mainly because of resorption of the ceramic, and the rest will disappear over the next 6-12 months. Suitable biopolymers include gelatin, hyaluronic acid, alginate, chitosan and agarose In a preferred embodiment, the biopolymer is gelatin. Alternatively, synthetic polymers prepared from natural monomeric biomolecule units may be used as long as they can be crosslinked and are non-immunogenic.

The biopolymer is crosslinked in the presence of the ceramic, thereby forming a composite biopolymer scaffold wherein the ceramic is incorporated onto and embedded in the porous network and more or less accessible to the body fluid once the biomaterial is inserted into the patient.

Suitable crosslinkers are known in the art. They can be homobifunctional creating for example amine-to-amine or sulfhydryl-to-sulfhydryl linkages or heterobifunctional creating for example amine-to-sulfhydryl, amine-to- nonselective, carboxyl-to-amine, sulfhydryl-to-carbohydrate or hydroxyl-to- sulfhydryl linkages Examples of crosslinkers include glutaraldehyde, genipin and carbodiimides. Dihydroxythermal crosslinking, which is heat crosslinking under a certain pressure, may also be applied, especially when collagen is crosslinked. Another example is the use of chloride dihydroxide steam. The crosslinker may be added in an amount of between 0.1 and 1% (v/v), such as between 0.2 and 0.85 (v/v). The ratio of ceramic to biopolymer is between 10 : 1 and 1 : 1 (w/w), preferably between 8: 1 and 2 : 1, such as between 6: 1 and 2 : 1 (w/w). The ratio may be about 8: 1, 7 : 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1 or 1 : 1 (w/w). When gelatin is used as polymer, the ratio is preferably between 6 : 1 and 2: 1, such as 4: 1 or about 4: 1

The ceramic preferably constitutes between 50% and 90% (w/w) and the polymeric agent (polymer to be crosslinked) between 10% and 50% (w/w) of the mixture before addition of the liquid prepared for cryogelation to prepare the composite cryogel scaffold. Preferably the mixture is between 60% and 85% (w/w) ceramic and between 15% and 40% (w/w) polymeric agent, such as between 70% and 85% (w/w) ceramic and between 15% and 30% (w/w), for example 80% (w/w) ceramic and 20 polymeric agent (w/w). The mixture is mixed with an aqueous solution, e.g. water, which may constitute up the 80% of the final mixture to be frozen in the cryogelation process. A crosslinker is added at this stage if needed for the crosslinking. The ceramic may for example constitute 20% (w/v), the polymeric agent 5% (w/v) and water 75% of the final mixture for cryogelation. In the mixture of polymer, ceramic powder and liquid, the polymer may for example constitute 5% (w/v) and the ceramic between 5% and 50% (w/w), preferably between 10% and 40% (w/v), such as about 15, 20, 25, 30 or 35 % (w/v), the rest being a fluid such as water. If the amount of polymer is more or less than 5%, such as 3, 4, 6, 7, 8, 9 or 10 % (w/v), the content of ceramic can be varied accordingly to prepare a suitable spongy composite cryogel scaffold product after cryogelation. Different properties of the selected polymer may change the preferred ratio of ceramic to polymer. A too high ceramic content will retard ice crystal formation and thus the pore formation in the scaffold and a too high crosslinked polymer content will cause a too slow in vivo degradation of the scaffold. If the ceramic to polymer is too high the composite cryogel will become too brittle and if the ratio is too low, the mechanical properties will not be sufficiently different to the strength of a cryogel without ceramic.

Bioactive agents to be incorporated in the composite cryogel bone scaffold to prepare the biomaterial according to the present invention for use in regeneration of bone comprise at least one bone active agent, preferably at least one bone anabolic agent and/or at least one anti-catabolic agent.

Additional bioactive agents may also be added to the scaffold. The agent(s) may be incorporated in any suitable way. Typically the scaffold will be soaked in a solution of the agent before use. The absorbency and incorporation capacity of the scaffold can easily be measured as a way of knowing the incorporation capacity and the concentration of the agent in the solution may be adapted for incorporation of a specific amount of the agent(s) in the scaffold as desired. This will be known the skilled artisan. The scaffold doped with a biological agent may be dried and stored before use. The bone anabolic agent is preferably a bone active protein or peptide and may be selected from the group comprising bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), vascular endothelial growth factor (VEGF), transforming growth factor-ps (TGFPs), parathyroid hormone (PTH), sclerostin, cell factory derived bone active proteins and ECM proteins and short peptides of the mentioned growth factors; or may be strontium.

Preferably, the bone active protein or peptide is the bone growth protein BMP- 2 or -7, preferably human BMP (hBMP-2 or 7), such as recombinant human BMP (rhBMP-2 or 7). The bone anabolic agent can be added to the composite scaffold in any of the steps of its preparation as long as it is not inactivated in the

crosslinking/polymerization reaction and/or cryogelation process. Thus, the agent may be mixed with the ceramic powder, the biopolymer (or its solution), or the mixture of ceramic powder and biopolymer before or after the crosslinking or cryogelation step. The agents are preferably incorporated in the composite cryogel scaffold after its preparation in order to avoid any inactivation of the agent. The bone anabolic agent may therefore preferable be added to the composite cryogel scaffold, for example by soaking the dry cryogel scaffold in a solution containing the agent. Further, the agent may be added to and incorporated in the composite cryogel scaffold shortly before. Bone anabolic agent agents with a strong affinity for calcium ions, in particular for hydroxyapatite, may alternatively be soaked into the

hydroxyapatite powder prior to or after mixing with calcium sulphate to prepare the ceramic powder. Proteineous bone active agents should be added after the crosslinking step in order to avoid crosslinking of the proteineous agent and thus any resulting inactivation.

The anti-catabolic agent is preferably a bone resorption inhibitor, and in particular an agent which inhibits osteoclast activity. Suitable anti-catabolic agents may be selected from the group of bisphosphonates (bisphosphonic acids and their salts), a selective estrogen receptor modulator (SERM), denosumab or statins. The group of bisphosphonates includes etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate and zoledronate. Zoledronate (ZA) is the most preferred anti-catabolic agent for use in the composite cryogel bone scaffold of the present invention.

Bisphosphonate molecules can be used to deliver radio labelled drugs for both diagnostic and therapeutic approaches by the present scaffold (Radio- pharmaceutics). By the affinity for radioactive bisphosphonates to bind to apatite, areas with metastasis and high bone turn will receive a higher concentration of tumor eradicating radio emitters. It has previously been shown that the skeleton, especially the spine, is a common site of metastatic disease in about 70-90% of patients with advanced breast and prostatic cancers. In patients with bone metastasis, when 4mg of intravenous

Zoledronate is given, the maximum serum concentration after 15 minutes is O. l-ΙμΜ. The systemic concentration goes down to less than 1% of maximum concentration at 24hours To kill tumour cells in metastatic bone,

bisphosphonates have been also used to deliver β- particle emitting radionuclides (32P, 89Sr, 153Sm). Besides the sensitivity of theranostics, the combination of bisphosphonates with a γ-emitter (Single Photon Emission Computerized Tomography) and/or β-particle emitter and florescent probes in the same molecule gives multimodal imaging approach. The bone anabolic agent and the anti-catabolic agents may be added together in the same step of preparation or separately in the same step or in different steps.

Other bioactive agents which may be included in the composite cryogel biomaterial of the present invention include antibiotics, antifungal drugs, bone healing promotors, chemotherapeutics, cytostatics, vitamins, hormones, bone marrow aspirate, platelet rich plasma and demineralized bone. The

antibiotic(s) may be selected from gentamicin, vancomycin, tobramycin, cefazolin, rifampicin, clindamycin and the antifungal drug(s) selected from the group comprising nystatin, griseofulvin, amphotericin B, ketoconazole and miconazole.

The composite cryogel biomaterial may also comprise a X-ray contrast agent selected from water soluble non-ionic X-ray contrast agents and

biodegradable X-ray contrast agents. Suitable water soluble non-ionic X-ray contrast agents may be selected from iohexol, iodixanol, ioversol, iopamidol, iotrolane, metrizamid, iodecimol, ioglucol, ioglucamide, ioglunide, iogulamide, iomeprol, iopentol, iopromide, iosarcol, iosimide, iotusal, ioxilane, iofrotal, and iodecol. A preferred water soluble non-ionic X-ray contrast agent is iohexol. The X-ray contrast agent is preferably incorporated into the composite by solubilizing it in one of the liquids used during the preparation of the composite scaffold, for example in the liquid, e.g. water, used to solubilize the biopolymer or the crosslinker. The composite cryogel bone scaffold for use in preparing the biomaterial according to the invention is prepared in the form of a cryogel. Cryogelation is known in the art (Hixon et al., Kumar et al. 2010; Raina et. al. 2016).

Preferably the method of preparing a macro- and microporous biodegradable composite cryogel bone scaffold according to the present invention comprises the below steps. Alternative and/or additional steps for the preparation of cryogels will be known to the skilled person and within the present invention. a) solubilizing the biopolymer or biopolymer units or mixture of

biopolymers/units in a suitable solvent, e.g. an aqueous liquid, such as water;

b) providing a ceramic powder comprising calcium sulphate and calcium

phosphate;

c) dispensing the ceramic powder in the biopolymer solution or in an aqueous liquid to be mixed with the biopolymer solution to obtain a dispersion or slurry;

d) optionally adding a crosslinker suitable for crosslinking the biopolymer(s) to the mixture of step c) followed by mixing to obtain a dispersion/slurry having all ingredients evenly distributed;

e) filling the mixture of step d) into a mold and placing it in a freezer

(cryostat) to produce a frozen mixture in the mold;

f) thawing the frozen mixture of step e) to obtain a cryogel; and

g) optionally freezing the cryogel obtained in step f) followed by drying, such as freeze-drying, to obtain a dry cryogel (macro-and micro-porous biodegradable composite cryogel bone scaffold) comprising a microporous ceramic and macroporous crosslinked non-immunogenic biopolymer(s).

The biomaterial according to the present invention is prepared by adding at least one bone active agent, such as at least one anabolic agent and/or at least one anti-catabolic agent to the solution or mixture in one of the steps a), b), c), d) e) or f), provided that the agent is not inactivated by one of these steps); or to the scaffold obtained in step f) or g).

As an alternative to step g, the cryogel obtained in step f) may be air-dried with and without heat. The suitability of this alternative depends on the behavior of the cryogel upon drying and/or rehydration of the dried cryogel for incorporation of additives and/or in use which may sometimes lead to deformation, shrinking and sometimes collapse of the cryogel. Freeze-drying of the cryogen of step f) secures retention of the form and structure of the cryogel. Temperatures, time and/or relative volumes applicable to the different steps in the cryogelation method, including the crosslinking reaction, may differ within practical limits known to the skilled person and/or as disclosed in the prior art disclosed above. If the bone active agent is a protein or protein derivative, e.g. a peptide, it needs to be added after the crosslinking in order to avoid the active agent being crosslinked and thus losing its activity. Alternatively, the bone active agent may be protected against crosslinking. The cryogel bone scaffold or biomaterial of the present invention could be in the form of microspheres that may be dispersed in a carrier such as for example dextran or hyaluronic for local injection to give a sustained release of the microspheres at a local site preferably but not exclusive into bone for a subsequently sustained release of any bone bioactive agents. In such an application, it would still contain one ceramic and at least one bone active agent but it would be possible to have a long term sustained release of for example ZA and in addition to for instance MABs, etc. for for example systemic bone tumors such as myeloma. The composite cryogel biomaterial of the present invention could also be applied subcutaneously for treating of a bone disease.

In a specific embodiment, the composite cryogel biomaterial could have a gradient with TGF-beta in one end and BMP-2 in the other end and a gradient for apatite and dihydrate in for instance deep cartilage defects/fractures and also shaped accordingly using a contralateral joint for digital copied molding.

Macroporous architecture of the cryogel scaffold and physico-chemical characterization The composite cryogel scaffold for use in preparing the composite cryogel biomaterial according to the present invention has a macroporous and a microporous architecture meaning that the pore size ranges between 1 and several hundred micrometers. This is true in both longitudinal and cross sectional directions, which can enable better cell infiltration as verified by scanning electron microscopy. Fourier transform infrared spectroscopy and X- ray diffraction show the chemical cross linking of the polymer gelatin with glutaraldehyde as well as the presence of calcium sulfate dihydrate and crystalline hydroxyapatite. These techniques are widely used in the physico- chemical characterization of biomaterials. It has also been shown that porous scaffolds with a pore size range of even less than 100 micron can lead to osteonal bone formation.

In one embodiment of the present invention, the aim is to produce a trabecular bone like pre-set biomaterial that can perform spatio temporal delivery of bone active molecules. These molecules have been shown to be very effective in bone regeneration in several different animal models.

In another embodiment of the present invention the long term aim of the present invention is to provide an off-the-shelf biomaterial containing at least one bone active factor to stimulate bone repair and also replace the conventional methods of bone grafting. While different methods like cryogelation, thermally induced phase separation (TIPS), freeze drying, electrospinning etc. have been used to produce porous scaffolds previously, only cryogelation in the presence of a ceramic provides an open and interconnected pore structure with excellent osteoconductive and

osteoinductive properties together with high mechanical strength.

In another aspect for the present invention, the composite cryogel biomaterial is formulated as a kits-of- parts, comprising the composite cryogel scaffold as a product in one compartment and one or more bioactive (bone active) agents in one or more separate compartments and instructions of how to add the agent(s) to the composite cryogel scaffold before use. The kits-of-parts may also comprise one or more additional bioactive agents and/or a X-ray contrast agent.

Application in treatment (Results/effects)

There are several indications for a preset shaped carrier that regenerates bone and/or exerts antitumor capacity. In the spine metallic cages, rods or plates are often used for creating fusion or stability. Presently used high dose BMP containing collagen is far from optimal and slow resorbing material with a sustained release will be a significant improvement. The material can be pre- shaped to fit the cage or placed as stripes along the dorsal part of the vertebral bodies. In reconstruction of complex anatomy a pre shaped carrier based on digital information has a number of indications in the skeleton especially in maxillofacial, neuro and plastic surgery. The material could be drilled to allow a rod to be passed through in an intra medullary location. For a critical bone defect a longer resorption time may be an advantage. The mechanically stable sponge-like composite cryogel scaffold can be shaped prior to or during surgery to fit the defect, which is in contrast to treatment with an hardenable injectable bone substitute composition. The application of the composite cryogel biomaterial according to the present invention is beneficial in places where long term structural integrity of the construct is necessary for instance in spinal fusion. It is important that the scaffold material holds its shape and does not flow, which may caused bone formation at undesired locations. Known synthetic bone substitutes, such as Cerament remodels faster in-vivo (most likely because CSD resorbs quickly) due to which bone can grow even towards the middle of the scaffold. This does not happen with the composite cryogel scaffold of the present invention despite having micropores, because bone formation and scaffold degradation are couple and it takes the cryogel longer to degrade than e.g. Cerament.

However, in terms of bone formation around the scaffold, the composite cryogel biomaterial performs excellent (results not shown here). In bone the specific possibilities to add tumor specific drugs or radio emitters to the material in the soluble phase or to the bisphosphonates that binds to apatite is a major step in both metastatic disease as well as in for instance myeloma.

Compared to the cryogel biomaterial earlier described in Raina et al. (2016), which is based on a mixture of different polymer systems but mainly chitosan, agarose and silk-fibroin on the organic part and hydroxyapatite and bioactive glass in the ceramic part, a marked difference in the rhBMP-2 release was observed when compared to the present invention. The composite cryogel biomaterial described in the present invention provides much more sustained release of bone active factors. Because of this, the new scaffold+rhBMP-2 group shows approximately 17 times more bone area compared to the rhBMP- 2 treated scaffold in the earlier study, while the scaffold + rhBMP-2+ZA group shows nearly 3-times higher bone area compared to the rhBMP-2+ZA treated scaffold in the earlier study. This is an important finding clearly showing that by having the right combination of a polymer and ceramic and their respective ratios, the release kinetics can be modulated for a better delivery. This can have clinical implications especially since optimal BMP release requires a slow burst pattern in the beginning but a much more sustained release over time. By using a sustained delivery composite cryogel biomaterial according to the present invention the current doses of BMPs delivered in the clinics to overcome the dosage related side effects can be reduce.

EXAMPLES EXAMPLE 1

Preparation of crvoqel scaffold

500 mg gelatin (Gelatin from cold-water fish; Sigma Aldrich (U.S. A)) was dissolved in 9 ml_ deionized water (5 % (w/v) gelatin in the final mixture for preparing the cryogel) overnight at room temperature to prepare a pale but clear solution. The dissolution rate may be increased by increasing the temperature of the water to for example 40 °C. 2 g Ceramic powder (Cerament™ V (60% CSH and 40% HA) kindly provided by Bone Support AB (Sweden)) was mixed into the gelatin solution to prepare a gelatin/powder mixture or slurry with uniformly dispersed solid material (20% w/v in the final mixture for preparing the cryogel). Some of the solid material tends to sink in the water due to its weight and the mixture/slurry should therefore be stirred again prior to use if not used immediately after mixing. The mixture/slurry is precooled to about 4 °C.

A crosslinker solution (0.2% (v/v)) was prepared by mixing glutaraldehyde (Sigma Aldrich (U.S. A)) in 1 ml_ of deionized water. The cold gelatin/powder mixture/slurry was remixed if needed to re-suspend any settled particles, where after the crosslinker solution was added and mixed rigorously with a spatula to avoid bubble formation and to distribute the crosslinker evenly in the mixture. This step is critical and needs to be done quickly to avoid outside crosslinking in the mixture.

The resulting mixture was poured into 2.5 ml_ syringes with closed bottom that act as molds and immediately transferred into a circulating liquid cryostat (Thermo Scientific, U.S. A), and maintained at -20 °C for a period of 12 h. The mold can be of any desired shape. After completion of the cryostat incubation, the syringes were transferred to a beaker containing deionized water at room temperature which will allow the ice in the frozen polymer mixture to thaw. The process was continued until the resulting cryogel monoliths were at room temperature. This may take up to 2 h, but the thawing may be speeded up for example by constantly replacing the water in the beaker with fresh water at room temperature. The cryogel monoliths were frozen again at -80 °C for about 2 h and then transferred the frozen scaffold to a freeze drier

(lyophilizer; Southern Scientific, India). The material is taken out of the freeze drier after 24-36 hrs. and the final dry cryogel monoliths stored in a moisture free sterile environment for later use. The composite cryogel rods produced in the syringes may be cut into discs for further use (Figure 1A). The fabricated monoliths appeared to be spongy when soaked in PBS (Figure 1 A-C) and regained their original shape when the pressure was released. The SEM image in Figure 2 shows that the cryogel scaffold possesses both macro- and micro- porosity.

EXAMPLE 2

Varying the ceramic component in the gelatin-Cerament cryogel.

The preparation according to Example 1 was selected after an initial experiment testing different ceramic to biopolymer ratios. The amount of gelatin and the cross linker (glutaraldehyde) in the reactions were kept constant at 5% (w/v) and 0.2% (v/v) respectively, while the ceramic amount (Cerament^® powder) was varied to investigate what gives the optimal consistency of the final composite cryogel material for this particular choice of ceramic, polymer and crosslinker used to exemplify the present invention. The gross morphology and handleability of the gels were tested. Cerament concentrations of 5%, 10%, 20% and 40% (w/v) together with 5% (w/v) gelatin and 0.2% (v/v) glutaraldehyde were tested and the following observations were made:

5% Cerament (ratio 1 : 1) : -These gels were very similar (mechanically) to plain gelatin only cryogels. Addition of ceramic components did not seem to make a big difference.

10% Cerament (ratio 2 : 1) : - These gels were better than 5% Cerament in terms of its ability to maintain its structure and they had a better

compressibility.

20% Cerament (ratio 4: 1) : - these gels showed a spongy property to the material and the structure of the gels were maintained. Of all the

combinations tested, this particular ratio was chosen for further production and analysis.

40% Cerament (ratio 8: 1) : - The gels were getting to a point where they became brittle.

EXAMPLE 3

For clinical use, sterile composite cryogelated material prepared according to Example 1 may be placed in 2-floor packing. The bottom floor will contain the pre-set material of desired shape in a tight fitting with surrounding packaging material. The top floor will consist of desired amount of rhBMP-2 and ZA in powder form. 30 min- 1 h (no-upper time limitation) prior to surgery, saline solution can be added to the top floor and the contents gently mixed by hand until no powder can be seen. At this time, the central borders between the top and the bottom floor can be opened using a sliding piece of packaging material. This will allow for the growth factor or anti-catabolic drugs to drip on to the scaffold. After sufficient time for interactions (minimum 30 min), the scaffold would be functionalized with necessary molecules and ready to use. Alternatively, ZA can be functionalized to HA particles before the starting of cryogelation process. This can be done either by soaking hydroxyapatite particles with or without calcium sulphate in ZA and then mixing the functionalized apatite particles with or without calcium sulphate into the polymer solution. Alternatively, HA particles can be functionalized with ZA by spraying ZA onto the HA particles before, during or after sintering. Then a mixture of CSH and ZA functionalized HA will be added to the polymer solution and steps for cryogelation will remain the same. This will provide a pre-functionalized scaffold with ZA.

Bioactive agent in form of proteins (e.g. rhBMP-2) or derivatives thereof cannot be added before crosslinking unless suitably protected; because such proteins/peptides will take active part in the crosslinking step, crosslinking all present proteins/peptides thereby rendering the protein/peptide unsuitable for osteoinduction.

EXAMPLE 4

Macroporous architecture of the crvoqel scaffold 4.1 Microarchitecture analysis using scanning electron microscopy (SEM)

Discs of the fabricated cryo-gel material from Example 1 measuring 8 mm in diameter and 2 mm in height consisting of calcium sulfate, HA and a biopolymer (~80% ceramic and ~20% polymer) were cut in a cross-sectional manner as well as longitudinal manner using sterile blades. The scaffolds were dried using increasing ethanol gradient starting with 70% up to 99.5%.

Thereafter, the scaffolds were allowed to dry in a vacuum desiccator overnight and sputter coated with gold (with 40 mA current for 120 s) (Cressington, Watford, U.K). Scanning electron micrographs were taken on a Carl Zeiss EV018 electron microscope (Carl Zeiss GmbH, Germany) at an operating voltage of 5-10 kV.

SEM revealed interconnected pore architecture in the biomaterial both in cross sectional as well as longitudinal images (Figure 1D-G and Figure 2). Particles of CSD and HA could be seen embedded within the polymeric network as well as on the surface of the polymer. The presence of needle like structures of CSD could clearly be seen on the biomaterial surface. The overall surface of the scaffolds appeared to have open pores and the pore size seen in both the cross sectional and the longitudinal images ranged approximately between 30 μιη to 110 μιη with a heterogeneous pore distribution. Smaller pores down to 1 μιη and even smaller within the ceramic covered polymeric walls were also noticeable.

4.2 Physico-chemical characterization 4.2.1 Energy dispersive x-ray (EDX) analysis

The elemental analysis on the surface of the cryogel biomaterial from Example 1 showed the presence of elements corresponding to both organic and inorganic components. Prominent peaks for Carbon, Oxygen, Calcium and Phosphorous could be seen. A total of 21.8±3.5 % Ca was found to be present on the surface of the biomaterial (Figure 3A).

Sample preparation was performed as described in section 4.1. The electron microscope was connected to an elemental analyzer (Oxford instruments, Oxfordshire, UK). Surface calcium distribution was quantified by operating the machine at a constant operating voltage and scanning area. The average weight percentage calcium distribution was calculated using four different specimens and each specimen was scanned at four random locations. The data was then averaged to determine the total calcium distribution on the surface of the scaffolds.

4.2.2 Fourier transform infrared spectroscopy (FTIP

Biomaterial cryogel scaffolds were crushed in a mortar pestle to achieve a fine powder in a clean and sterile environment. In order to confirm the

crosslinking of gelatin with glutaraldehyde, the crushed sample was then loaded on an FTIR spectroscope (Perkin-Elmer 1000 paragon spectroscope, U.S. A). The % transmittance values were plotted against wavelength to identify vibrations from different bonds (Figure 3B).

Amide I (C=0) stretch in the gelatin was observed between 1620-1630 cm-1 wavelength. Amide II peak from the native gelatin could be observed between 1530-1540 cm-1, which refers to the N-H bend and the stretching obtained from the C-H bond. The crosslinking of gelatin with glutaraldehyde (aldehyde and the amino group crosslinking) could be confirmed by formation of the aldimine bond and a strong peak at 1450 cm-1 confirms that.

4.2.3 X-ray diffraction (XRD) analysis

The presence of ceramic components in the form of CSD and HA in the cryogel from Example 1 was verified using XRD (Figure 3C). Composite cryogel cylinders prepared according to Example 1 were cut by use of a razor blade in two pieces. The inner surface of one part was as best as possible adjusted against a knife edge. The quantification of the crystalline amount was done using the Rietveld method (X-Ray Diffraction, DIN EN 13925-1, 2, 3; DIN EN 1330-11; Mode: Bragg-Brentano; Equipment: STOE Θ/Θ- Diffractometer, Reflection mode; Radiation : Cu- Κα, λ = 1.5418 A;

Parameter: U = 40kV, I = 30mA; Monochromator: Reflected beam (Graphite, plan (002)); Detector: Scintillation counter; Sample holder: Reflection type, rotating; Slits : 0,75mm, 0,35mm, vertical 2 x 6mm (small piece); Angle region : 2Θ = 3 - 70°; Step size: Δ2Θ = 0.04°; Counting time / step: 6s). The results of the qualitative and quantitative phase analysis (in %[m/m]) are shown in the table below. Neither Ca- Sulfate (Anhydrite) nor any

hemihydrates (for example Bassanite) were detected, which means that all CSH had converted to CSD (Gypsum).

EXAMPLE 5

Cell culture experiments 5.1 Effect of In-vitro rhBMP-2 functionalization 5.1.1 Cell viability analysis via MTT assay

Pre-osteoblast MC3T3-el cells from mouse bone were cultured in aMEM with 1 mM sodium pyruvate, 10% FBS and 1% antibiotics (pencillin-streptocillin) with normoxia conditions until sufficient cell number was reached. All the experiments involving cells were performed in sterile conditions at 37 °C and cells were never grown over 80-90% confluence. Moreover, cells were maintained at low passage numbers, typically between 4 and 8 for the in-vitro experimentation. The cryogel scaffolds from Example 1 (8 mm x 2 mm) were sterilized using increasing ethanol gradient (Under sterile conditions the cut scaffolds were immersed in 70% ethanol for at least 20 min with 2 changes (40 min in total). Then in 99.5% ethanol for 20 min x 2 times. The ethanol was evaporated before further processing) and allowed to dry in 6-well plates. To avoid contamination with alcohol, the scaffolds were washed with PBS for 5 min. An aspiration pipette was used to drain the excess PBS from the scaffolds. On the day of the experiment, cells were trypsinized, counted for viability and concentrated to achieve the desired cell number. rhBMP-2 functionalization was performed by solubilizing 4 μg rhBMP-2 in PBS for each scaffold in separate wells of a 24-well, non-tissue culture treated plates and care was taken to ensure that the protein solution does not leak from the scaffold. The scaffolds were then incubated for an hour in a humidified incubator before cell seeding. In case of the scaffold only group, the scaffolds were incubated with same volume of PBS. A total of 1.7 x 105 cells were seeded on each scaffold contained in 30 μΙ_ of culture medium and the culture plates were transferred to a humidified incubator for 15-30 min to allow cell settlement before adding more medium. This step was performed to avoid cell leakage from the scaffolds. Afterwards, 1 ml of osteogenic culture media was added to all wells. The osteogenic media consisted of aMEM with 1 mM sodium pyruvate, 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, 10% FBS and 1% antibiotic solution. Media was replaced every 48 h and fresh osteogenic media was added. Day 3 and 6 post cell seeding, media was aspirated from the wells of each experimental group and the scaffolds were washed with PBS IX for 2-3 min followed by the addition of 500 μΙ_ MTT reagent (0.5 mg/mL in basal medium). The scaffolds were then incubated at 37 °C in a humidified incubator for 3 h to allow the formation of formazan crystals. The purple crystals were then solubilized using 450 μΙ_

dimethylsulfoxide (DMSO) for 10 min and the absorbance were recorded on a spectrophotometer at 595 nm. DMSO was used as a blank background control. The MTT experiments were performed twice with at least three replicates in each treatment.

MC3T3-el cells seeded on both scaffold only and scaffold functionalized with rhBMP-2 remained viable both at day 3 and day 6 with no statistically significant differences between the two groups. It is also noticeable that the viability of cells seeded on the scaffolds shows an increasing trend of viability from day 3 to day 6 indicating that the scaffolds do not have any adverse effects on the cells (Figure 4A). 5.1.2 In-vitro cell attachment via SEM

SEM was performed on the cell seeded cryogel from Example 1 scaffolds to look at the early cell attachment. Day 3 scaffolds from both groups i.e.

scaffold only and scaffold + rhBMP-2 groups were washed with PBS IX followed by cell fixation using 4% formaldehyde in PBS for 1 h at room temperature. Post fixation, the scaffolds were dehydrated and dried as described in

Example 4.1. The samples were then sputter coated using chromium and analyzed on a SEM instrument (JEOL JSM-7800F, U.S. A).

Pre-osteoblast cells MC3T3-el attached to the scaffold surface as early as day 3, which can be seen in Figure 4B, C. Flattened cell morphology as well as extending filopodial structures were observed. The scaffold surface was also covered with extra cellular matrix (ECM) in some places. No big differences in the cell adherence were noted between the two groups using SEM. 5.2. In-vitro rhBMP-2 release

In-vitro detection of rhBMP-2 release was performed using a well-established method via the ELISA technique (n = 3/time point). Scaffolds were sterilized as described in 5.1. land then incubated with 1 μg rhBMP-2 solubilized in PBS for 1 h at 37 °C in a humidified incubator. Care was taken to avoid protein solution from flowing out of the scaffold before and during incubation. After incubation, the scaffolds were moved to lo-bind eppendorf tubes and filled with 1 mL PBS. At each time point (Day 1, 3, 7, 14& 28) the PBS was replenished and stored at -20 °C until the day of the assay. Fresh PBS (1 mL) was added to the scaffolds at each time point. On the day of the ELISA assay, all samples were thawed and diluted 1 : 10 in the sample diluent buffer and the assay was performed as per the manufacturers guidelines. Released protein concentrations were detected using the protein standard curve and the cumulative release was calculated thereafter. The scaffold exhibited a very gradual release of rhBMP-2 over a period of 4- weeks with a small initial burst on the first two time points of day 1 and day 3 followed by a nearly constant release profile thereafter (Figure 4D). In-vitro, the scaffold released 1.7±0.3, 2.7±0.3, 2.8±0.1, 3±0.1, 3.1±0.1, 3.3±0.1% rhBMP-2 on day 1, 3, 7, 14, 21 and 28, respectively.

5.3. In-vitro rhBMP-2 bioactivitv analysis

In order to ensure that the bound rhBMP-2 within the scaffold is bioactive and functional, osteogenic differentiation of the MC3T3-el preosteoblast cells seeded on the rhBMP-2 functionalized scaffolds was studied and compared with seeding on non-functionalized scaffold. Cell seeding on sterile scaffolds was performed as described earlier in Example 5.1.1. Differentiation of the preosteoblasts was studied via ALP assay as well as qPCR analysis of OSX and OCN genes. 5.3.1 ALP activity of MC3T3-el cells seeded on rhBMP-2 functionalized scaffolds rhBMP-2 (4 μg/scaffold) was added as described in Example 5.1.1 and two groups were used during the experiment; scaffold only as a control and scaffold functionalized with rhBMP-2 as the experimental group. The same cell number (1.7 x 105/scaffold) as used for the MTT was used during the ALP assay. Post cell seeding on the scaffolds, the cells were cultured in similar osteogenic medium (αΜΕΜ + lmM sodium pyruvate+ 10 mM β- glycerophosphate+50 μg/mL ascorbic acid+ 10% FBS and 1% antibiotic solution), however, in order to minimize the interference with the color development and the final absorbance analysis during the assay, phenol red free medium was used. At each time-point of the assay (day 3 and 6), the media was aspirated out of the experimental wells and the scaffolds were washed with PBS 2X followed by the addition of 500 μΙ_ pNPP solution on the scaffolds. Incubation of the scaffolds with the substrate solution was performed at 37 °C for 30 min. Following this, the supernatant containing the reaction products were moved to wells of a transparent 96-well plate and the absorbance was read at 405 nm within a short time span (< 5 min). The pNPP solution was used as a blank control. ALP assay was performed twice with at least three replicates in each treatment.

The ALP activity of MC3T3-el cells seeded on rhBMP-2 functionalized scaffold almost doubled 3 days post seeding when compared to the cells seeded on the scaffold only (p<0.001). With the passage of time, the rhBMP-2 functionalized scaffolds had an even stronger effect on the production of ALP at day 6 (Figure 4E). Cells seeded on scaffold+rhBMP-2 expressed five times higher ALP when compared to the scaffold alone (p<0.001).

5.3.2 qPCR analysis of MC3T3-el cells seeded on rhBMP-2 functionalized scaffolds

To perform the qPCR analysis, MC3T3-el cells were seeded on scaffolds with two different treatments; scaffold+cells+osteogenic medium and scaffold functionalized with rhBMP-2+cells+osteogenic medium. Each treatment consisted of 6 scaffolds/treatment. Cell seeding protocol and cell numbers were kept the same as in Example 5.3.1. Media was replenished every 3 days and the qPCR analysis was performed 11 days post cell seeding. On the assay time point, scaffolds were washed 2X in PBS, 2 scaffolds from each treatment were pooled together to enhance the RNA extraction thereby allowing us to have a sample size of 3/treatment. The scaffolds were transferred to homogenization tubes containing ceramic beads and filled with 600 μί of RNA extraction buffer (RLT buffer, Qiagen)+ 6 μί β-mercaptoethanol and allowed to stand for 1 min before homogenization. Mechanical homogenization was performed using TissueRuptor (Qiagen) for 2X 5 min at maximum speed at 4 °C. Thereafter, manufacturer guidelines were followed to extract the RNA and the quality and quantity of the RNA was verified on a nano-drop (Thermo Scientific, U.S. A). RNA integrity was checked using the agarose gel electrophoresis and both 18S and 28S ribosomal RNA bands were confirmed before further processing. cDNA synthesis was performed by using the same amount of RNA for each sample using the Maxima first strand cDNA synthesis kit (Thermo Scientific, U.S. A) by following the guidelines in the kit. Finally to perform the qPCR, the cDNA was diluted 1 : 50 in nuclease free water and the contents of the Maxima SYBR Green/rox qPCR (Thermo Scientific, U.S. A) were mixed in the following order; SYBR Green Master Mix (2x) (10 μΙ_), forward and reverse primer (lOx) (2 μΙ_) for GAPDH, OSX and OCN separately, template cDNA (1 μΙ_) and nuclease free water (7 μΙ_) for a total reaction volume of 20 μΙ_/ gene. GAPDH was used as an endogenous housekeeping gene. The experiment was performed on StepOnePlus (Applied Biosystems, U.S. A) thermal cycler and annealing temperatures were chosen based on the primer supplier's guidelines. Eventually the 2^Λ-ΔΔΟί method was used to quantify the fold-change expression of the target genes between different treatments.

Day 11 post cell seeding, MC3T3-el cells seeded on rhBMP-2 functionalized scaffolds exhibited an increase in the expression of both osteoblast differentiation gene OSX as well as an increase in the expression of osteoblastic maturation marker OCN (Figure 4F, G). The cells seeded on scaffold+rhBMP-2 loaded scaffolds expressed 3.7-fold increase in the expression of OSX (p<0.01) and a 12.9-fold increase in the expression of OCN (p<0.001) when compared to MC3T3-el cells seeded on the control scaffolds.

EXAMPLE 6

6.1 In-vivo bone formation via the abdominal muscle pouch model

A previously published abdominal muscle pouch model was used to evaluate the osteoconductivity and osteoinductivity of the composite cryogel biomaterial and to check whether it is feasible to deliver bone active molecules (rhBMP-2 and ZA) using the scaffold as a carrier [Raina et. al. (2016)] . Scaffolds prepared as described in Example 1 were sterilized using increasing ethanol gradient (70% to 99.5%) and a scaffold size of 5 mm x 2 mm was used. In this first animal trial, the following three groups were used : 1. Scaffold alone, 2. Scaffold + rhBMP-2 (10 μς), 3. Scaffold + rhBMP-2 (10 μg)+ZA (10 μg). Both bioactive molecules were physically absorbed on the scaffolds for 1 h before implantation. Animals were anaesthetized by using a combination of sodium pentobarbital (15 mg/mL) and diazepam (2.5 mg/mL). Male Sprague-Dawley rats (average weight 303 g) (n = 12) were used and a bilateral muscle pouch was created on both sides of the abdominal midline and maintained a minimum distance of 1.5 cm between the two pouches. 6 animals received scaffold alone on the left side of the abdominal midline and scaffold + rhBMP-2 on the right side of the midline. Another 6 animals received scaffold only controls on the left side and 6 samples of scaffold + rhBMP-2+ZA on the right side making a total of 12 samples for the scaffold only group and 6 samples for both scaffold + rhBMP-2 and scaffold + rhBMP-2+ZA groups.

Animals were sacrificed 4-weeks post implantation, following which bone formation in the explants was evaluated by SEM, X-ray radiography, micro- computed tomography (micro-CT), histology and histomorphometry.

Upon harvesting the samples 4-weeks post-surgery, all samples appeared to be integrated well with the underlying tissue. The control samples (scaffold only group) were very soft to feel. Scaffold + rhBMP-2 and scaffold + rhBMP- 2+ZA samples appeared to be bigger in dimensions but the scaffold+rhBMP- 2+ZA were harder when palpated. Explants had no well-defined shapes and their geometry was quite heterogeneous.

SEM images are shown in Figure 5 and Figure 6. Figure 5 shows a SEM image of the composite cryogel biomaterial implanted in the rat muscle pouch for 4- weeks. Abundant HA particles were observed in the throughout scaffold.

Needle like calcium sulphate structures/particles were absent. Figure 6 shows that the macro and micro porous structure of the material is preserved even after in-vivo implantation. This shows that even after in-vivo implantation, the micro and macro porous structure of the scaffold is preserved. A lot of connective tissue appears to be growing into the pores of the material too.

6.2. X-ray radiography The explants from each group were placed together in a long plastic tube and were wrapped in cotton gauze and moistened with saline to minimize tissue damage. The radiographs were taken using the scout view of a micro-CT machine (nanoScan, Mediso Medical Imaging Systems, Budapest, Hungary) at an operating voltage of 65 kV and an exposure rate of 1300 ms.

From the radiographs shown in Figure 7A-C, it is clear that the samples from the scaffold only group appear to be the smallest in dimensions with some radiolucency in different parts, which might be due to the presence of CSD/HA in the scaffolds. The samples in the scaffold+rhBMP-2 group appeared to be larger in size than the scaffold only group but no apparent differences in the radiolucency could be seen between the two groups. The samples in the scaffold + rhBMP-2+ZA group seem to be largest and the densest of all the three groups. More homogenous radiolucency was seen in this group.

6.3 Ex-vivo micro-CT at 4-weeks

After the X-rays, samples were individually placed in 5 ml_ eppendorf tubes and wrapped with saline soaked gauze. Samples were then scanned on the micro-CT scanner (nanoScan, Mediso Medical Imaging Systems, Budapest, Hungary) with circular scanning at a voltage of 65 kV, exposure time of 1300 ms and 720 projections. Image reconstruction was performed using a RAMLAK filter with 100 % cutoff and a voxel size of 10 μιη was achieved at the end. The micro-CT image stacks were imported as DICOM sequence on the image analysis software Seg3D (v 2.2.1, NIH, NCRR, Science Computing and Imaging Institute (SCI), University of Utah, U.S. A) and were thresholded at the same Hounsfield value for each sample to compute the highly mineralized volume across each group by quantifying all the pixels above a certain preset threshold. The whole sample was considered as the region of interest (ROI). The micro-CT result of explants shows no significant differences in the highly mineralized volume (HMV) between the scaffold only group compared to the scaffold + rhBMP-2 group (p=0.3) (Figure 7D). The scaffold + rhBMP-2+ZA group had the highest amount of HMV when compared to scaffold only group (p<0.001) and the scaffold + rhBMP-2 group (p<0.001). 6.4 Ex-vivo histology and histomorphometrv at 4-weeks

All samples were fixed in neutral buffered 4% formaldehyde solution for 24 h prior to histology. Specimens were decalcified using 10% (w/v)

ethylenediaminetetracetate (pH 7.3-7.4) in ultrapure water at room temperature. EDTA solution was replenished every 3 days by fresh EDTA solution until sufficient decalcification was achieved. Routine dehydration and embedding procedure was used for embedding of the specimens in paraffin. Embedded tissue was then sectioned to a thickness of 5 μιη using a semiautomatic microtome (HM355S, Thermo Scientific, U.S. A) and sections were allowed to attach to coated glass slides at 37 °C for 48 h before staining. De- paraffinization and hydration was performed using standard procedures and the slides were stained with hematoxylin and eosin by following standard protocol. Stained sections were scanned using an automatic slide scanner (Hamamatsu, Japan) and imported into the histomorphometry software (HALO™, Leica Biosystems, v 1.92) for calculation of the area of new bone. Area of bone formation across all groups was done manually to increase the accuracy of measurement and the observer was blinded during the analysis.

Histologically, the scaffold only group shows no signs of bone formation (Figure 8A, D, G). The scaffold is infiltrated by fibrous tissue both on the periphery as well as towards the middle. In the group where the scaffold is treated with rhBMP-2, a cortical shell is seen on the outer margins of the scaffold (Figure 8B). Much of the scaffold and the new bone is resorbed in the middle and is replaced by marrow like tissue. Some remnants of bone can also be seen towards the middle of the scaffold (Figure 8E,H). In the scaffold + rhBMP-2+ZA group (Figure 8C, F, I), a thicker cortical shell can be seen on the outer margins of the implant. When moving inwards, some areas are infiltrated with new bone while some of the areas had portions of remaining scaffold infiltrated with fibrous tissue. Bone formation could also be seen within the original pores of the scaffold, clearly demarcated by the polymeric borders filled with bone tissue (Figure 8C,F,I). Histomorphometric analysis corroborates the results from micro-CT analysis. Significantly higher area of bone formation is seen in the scaffold + rhBMP-2+ZA group when compared to the scaffold + rhBMP-2 group (p<0.01) (Figure 8J). 6.5 Tartrate resistant acid phosphatase (TRAP) staining

The areas of TRAP positive regions on the new-formed bone tissue were analyzed by staining for TRAP using a commercially available staining kit and by following the manufacturers guidelines. Post-staining, the sections were thoroughly rinsed in de-ionized water for 5-10 min followed by dehydration, clearing and mounting.

Representative TRAP staining images show that the scaffold + rhBMP-2 has abundant regions of TRAP positive activity. All 6 specimens show varying degree of TRAP activity. In case of scaffold + rhBMP-2+ZA group only 2 specimens out of the 6 specimen stained TRAP positive (Figure 9). Moreover, the positive specimens exhibit activity only on a very small region on the periphery.

EXAMPLE 7

In vitro and in vivo release of bioactive agents from the scaffold

7.1 In-vivo 1251 labeled rhBMP-2 release kinetics

The abdominal muscle pouch model (described in Example 6) was used to analyze the release of rhBMP-2 from the composite cryogel scaffolds prepared according to Example 1. 1251 isotope was used to label rhBMP-2 (same batch of rhBMP-2 as used for in-vivo muscle pouch model). The selection of the treatment group was based on the results from first abdominal muscle pouch study. Sterile scaffolds (5 mm x 2 mm) were loaded with 10 μg 125I-rhBMP-2 (specific radioactivity= 2.5 MBq/scaffold) and 10 μg ZA and were allowed to bind to the scaffolds for 1 h before implantation. A total of 6 sprague-dawley male rats (average weight 342 g) were used for the experiment. Animals were allowed to recover from surgical stress overnight following which, single photon emission computed tomography (SPECT) imaging modality was used to follow the same set of animals at different time points (days 1, 3, 7, 14, 21 and 28) over a period of 4-weeks to plot the release kinetics curve. During each imaging session, animals were anaesthetized using a mixture of Isoflurane (4%) mixed with oxygen and nitrous oxide in a sleep induction chamber (flow rate 0.4 L/min). The animals were then moved to the SPECT machine (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary) under controlled anesthesia (during imaging anesthesia : 2% Isoflurane, 02 and N20, flow 0.4 L/min) and were constantly monitored for controlled breathing rate (40-60 breaths/min) using a respirator module (SA Instruments, Stony Brook, NY). This step was performed to avoid imaging artifacts during CT scans. Scaffold identification in the abdominal muscle pouch was performed using micro-CT with 360 projections, 65 kV voltage and 1500 ms exposure time (Voxel size= 73 μιη). After completion of the micro- CT, the SPECT ROI was chosen based on the micro-CT scan and default detector setting for 1251 was chosen. SPECT image reconstruction was performed using HiSPECT: Fast reconstruction module (VivoQuant v2.5p3, inviCRO, U.S. A) on the image processing software. Each scan lasted approximately 10 min and the scan time was constant for all animals and at each time point. Moreover, in order to compensate for the decay in the radioactivity of the tracer, a control tube containing 20 μΙ_ (0.98 MBq) of the 125I-rhBMP-2 was placed on the side of the animal but within the scan range of the device. In order to perform image analysis, firstly, the micro-CT and the SPECT images were superimposed to confirm the anatomical location of the SPECT signal (VivoQuant v2.5p3, inviCRO, U.S. A). A constant circular ROI1 was drawn on all samples and at all time points by using the

quantification module of the imaging software. Same sized ROI2 was also used to quantify the radioactivity signal (counts) from the control tube kept on the side of each animal. Eventually a ratio of counts in ROI1/ROI2 was calculated for each time point to compensate for the decay in radioactivity and a release kinetics curve for rhBMP-2 from the scaffolds was plotted.

Besides image analysis, the scaffolds from the sacrificed animals at 4-weeks were harvested and subjected to γ-counting on a Fidelis radionuclide calibrator to calculate the absolute radioactivity left in the samples post 4- weeks. By comparing the initial radioactivity/scaffold with the remaining radioactivity at 4-weeks, the % rhBMP-2 release during the 4-week period was calculated.

A different trend of rhBMP-2 release was seen in the in-vitro and in-vivo experiments, with more rhBMP-2 being released in-vivo. Using the SPECT-CT imaging modality, a nearly linear but sustained and long term release of rhBMP-2 from the scaffold in-vivo was found (Figure 10). The scaffold released 11.2±5.1, 23.2±7.7, 42± 16.5, 54.5± 16.1 and 65.3± 15.2 % of 125I-rhBMP-2 on day 3, 7, 14, 21 and 28, respectively. Based on the absolute radioactivity left in the scaffolds as measured using γ-counting, 25.2± 12.2% 125I-rhBMP-2 was found remaining (or 74.7± 12.2% released) in the scaffold after a period of 4-weeks.

7.2 In-vitro and In-vivo 14C labeled ZA release kinetics 7.2.1 In-vitro ZA release kinetics

To perform the in-vitro release kinetics of ZA 14C-ZA was used. Discs of sterile scaffolds (n = 6/timepoint) were loaded with 2 μg 14C-ZA and incubated at 37 °C for 1 h following which they were transferred into eppendorf tubes and filled with 1 ml_ PBS. At each time point (days 1, 3, 7, 14 and 28), the PBS was collected and fresh PBS (1 ml_) was added back to all the tubes. The collected PBS was then transferred to a scintillation vial and mixed with 3 ml_ of scintillation cocktail (Optiphase HiSafe 2, Perkin Elmer, U.S. A) and read on a scintillation counter (Perkin Elmer, Wallac 1414 liquid scintillation counter, U.S. A) for a counting period of 120 s. A standard curve was created by adding known amounts of 14C-ZA to a mix of PBS+ scintillation cocktail (1 : 3) and the amount of ZA released at each time point was calculated from the standard curve. Thereafter, a cumulative release kinetics curve of ZA release from the scaffolds was plotted. In-vitro the scaffold released low but sustained amount of 14C-ZA over a period of 4-weeks. There was an early burst release of ZA on day 1 following, which nearly constant release was observed (Figure 11 (left)). The scaffold released 8.8±0.7, 11±0.5, 11.9±0.1, 12.5±0.3, 13±0.1 % of 14C-ZA on days 1, 3, 7, 14 and 28, respectively.

7.2.2 In-vivo ZA release kinetics

In-vivo release kinetics of 14C-ZA was performed in the abdominal muscle pouch model as described above. Sterile scaffolds (5 mm x 2 mm) containing 10 μg 14C-ZA (specific radioactivity= 0.07 MBq/scaffold) and 10 μg rhBMP-2 (incubation time 1 h) were implanted into the abdominal muscle pouch of 24 Sprague Dawley male rats (average weight 332 g). At each time point (days 1, 7, 14 and 28), 6 animals were sacrificed and the scaffold samples containing 14C-ZA were harvested and cleaned from surrounding soft tissue with extreme care to avoid damage to the implanted sample. All samples were transferred to scintillation vials and mixed with 2 mL of 5M hydrochloric acid for 48 h to aid in rapid decalcification of the specimens. At this stage, all samples were mechanically homogenized at 13,500 rpm for 1 min. The homogenate was then mixed with the scintillation cocktail at a dilution of 1 : 10. Same steps were followed at each time point. A standard curve containing known amounts of 14C-ZA in 5M HCI and scintillation cocktail (1 : 10) was used to calculate the percentage of 14C-ZA released from the scaffolds in-vivo during each time point. The data for each time point was averaged to plot the release kinetics curve. The scaffolds released slightly more 14C-ZA after 4-weeks of implantation in the abdominal muscle pouch when compared to the in-vitro release kinetics. However, a similar but larger initial burst release of ZA was seen day 1 post implantation. Thereafter, the scaffold exhibited a nearly constant release all through the experiment up until 4-weeks (Figure 11 (right)). In-vivo, the scaffold released 43.2±7.6, 35±6.2, 39.2± 17.2 and 40±3 % on days 1, 7, 14 and 28, respectively.

EXAMPLE 8

Comparing the bone forming potential of the qelatin-Cerament crvoqel biomaterial with the Medtronic Absorbable Collagen Sponge (ACS).

This Example is an extract from Raina et al., Journal of Controlled Release, Vol. 272 (2018) p.83-96, "Gelatin-hydroxyapatite-calcium sulphate based biomaterial for long term sustained delivery of bone morphogenic protein-2 and zoledronic acid for increased bone formation : In-vitro and in-vivo carrier properties", confirming the potential of the composite cryogel biomaterial according to the present invention.

Method : 5 mm (diameter) x 2mm (height) pieces of sterile cryogel (composite scaffold from Example 1) and the ACS material were implanted in the abdominal muscle pouch model (Example 6). The following six treatment groups were used :

1. ACS + rhBMP-2 (10 μς),

2. Scaffold + rhBMP-2 (10 μς),

3. ACS + rhBMP-2 (10 μς) + ZA (10 μς),

4. Scaffold + rhBMP-2 (10 μς) + ZA (10 μς),

5. Scaffold + rhBMP-2 (5 μς) + ZA (10 μς) and

6. Scaffold + rhBMP-2 (2.5 μς) + ZA (10 μς).

Both materials were functionalized with rhBMP-2 and ZA by pouring appropriate volumes of the solution containing bioactive molecules on top of the materials using a pipette with the doses as mentioned above. Groups 1 and 2 were implanted in the same animal with the ACS group 1 implanted on the left and the Scaffold group implanted on the right. The same procedure was followed for G3 and G4 as well as for G5 and G6. A total of 8

animals/group were used. (For details of the method, see Raina et al.

(2018)). The results are shown in Figure 12 and Figure 13.

The highly mineralized volume was significantly higher in the scaffold + rhBMP- 2 (10μg) group when compared with the ACS + rhBMP-2 (10 μg) group (p < 0.01). Furthermore, addition of ZA to the scaffold in the scaffold+ rhBMP-2 (10 μg) + ZA (10 μg) group led to a significant increase in the highly mineralized volume when compared to ACS + rhBMP-2 (10 μg) (p < 0.001), scaffold + rhBMP-2 (10 μς) (p < 0.01) and ACS + rhBMP-2 (10 μς) + ZA (10 μg) group (p < 0.01). Addition of ZA could also aid in reducing the total rhBMP-2 doses. The highly mineralized volume in the scaffold + rhBMP-2 (5 μς) + ZA (10 μς) and scaffold + rhBMP-2 (2.5 μς) + ZA (10 μς) groups was significantly higher than a full dose rhBMP- 2 delivered via the ACS material (ACS + rhBMP-2 (10 μς) group) (p < 0.001 and p < 0.05, respectively) and no significant differences were found either when a full dose of rhBMP-2 was delivered via the scaffold carrier (scaffold + rhBMP-2 (10 μg) group). The highly mineralized volume was significantly higher in the scaffold +rhBMP-2 (5 μς) + ZA (10 μς) group when compared with the highly mineralized volume in the scaffold+ rhBMP-2 (2.5 μς) + ZA (10 μς) group (p < 0.01).

Statistical analysis

All data is represented as mean±SD. Data from animal experiments was tested for normality using the Shapiro-Wilk test and one-way ANNOVA with Tukey Post-hoc test to compare 3 groups. Comparison of two groups was performed using Welch's t-test. Level of statistical significance was set at 0.05. In-vitro data from multiple time points was tested using the multiple t- test while single time point data was tested using the Welch's t-test. Statistics was performed on Prism 7 for Mac OS X (V7.0a, 2016) (GraphPad Software Inc., CA, USA)

Claims

1. A macro- and microporous biodegradable composite cryogel biomaterial for use in regenerating bone in a patient, comprising

a. a composite cryogel scaffold comprising a ceramic and a crosslinked non-immunogenic biopolymer, wherein the ceramic comprises calcium sulphate dihydrate (CSD); and calcium phosphate (CaP), such as a- or β-tricalcium phosphate (α-TCP/p-TCP), tetracalcium phosphate, calcium-deficient hydroxyapatite (CDHA) or hydroxyapatite (HA), preferably particulate crystalline hydroxyapatite; and

b) at least one bone active agent.

2. The composite cryogel biomaterial according to claim 1, wherein the at least one bone active agent is at least one bone anabolic agent and/or at least one anti-catabolic agent.

3. The composite cryogel biomaterial according to claims 1 or claim 2, wherein the crosslinked biopolymer is crosslinked gelatin, hyaluronic acid, alginate, or chitosan or a combination thereof.

4. The composite cryogel biomaterial according to any one of claims 1 to 3, wherein the crosslinked biopolymer is prepared by mixing the biopolymer or biopolymers with a suitable crosslinker in the presence of the ceramic.

5. The composite cryogel biomaterial according to claim 4, wherein the crosslinker is selected from glutaraldehyde, genipin and carbodiimides.

6. The composite cryogel biomaterial according to any one of claims 1 to 5, wherein the ratio of ceramic to crosslinked biopolymer is between 10: 1 and 1 : 1 (w/w), preferably between 8 : 1 and 2: 1 (w/w), such as between 6 : 1 and 2: 1 (w/w); for example 8 : 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2 : 1 or 1 : 1 (w/w).

7. The composite cryogel biomaterial according to any one of claims 1 to 6, wherein the bone anabolic agent is at least one bone activating agent selected from the group of bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), transforming growth factor-ps (TGFPs), parathyroid hormone (PTH), sclerostine, cell factory derived bone active proteins and ECM proteins; or strontium, preferably BMP-2 or BMP-7, more preferably human BMP (hBMP-2 or hBMP-7), such as recombinant human BMP (rhBMP-2 or rhBMP- 7), VEGF, IGF, PTH, Teripartide, TNF-a and TGF-b.

8. The composite cryogel biomaterial according to any one of claims 1 to 7, wherein the anti-catabolic agent is at least one bone resorption inhibitor, such as an osteoclast activity inhibitor or an RANKL inhibitor.

9. The composite cryogel biomaterial according to claim 8, wherein the anti- catabolic agent is selected from the group of bisphosphonates (bisphosphonic acids and their salts), a selective estrogen receptor modulator (SERM), denosumab or statins

10. The composite cryogel biomaterial according to claim 9, wherein the group of bisphosphonates includes etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate and zoledronate.

11. The composite cryogel biomaterial according to any one of claims 1 to 10, further comprising at least one additional bioactive agent selected from antibiotics, antifungal drugs, bone healing promotors, chemotherapeutics, cytostatics, vitamins, hormones, bone marrow aspirate, platelet rich plasma, stem cells, induced pluripotent cells, patients own bone cells and

demineralized bone.

12. The composite cryogel biomaterial according to claim 11, wherein the antibiotic is selected from gentamicin, vancomycin, tobramycin, cefazolin, rifampicin, clindamycin and the antifungal drug is selected from the group comprising nystatin, griseofulvin, amphotericin B, ketoconazole and miconazole.

13. The composite cryogel biomaterial according to any one of claims 1 to 12 further comprising a X-ray contrast agent selected from water soluble non- ionic X-ray contrast agents and biodegradable X-ray contrast agents, wherein the water soluble non-ionic X-ray contrast agent is selected from iohexol, iodixanol, ioversol, iopamidol, iotrolane, metrizamid, iodecimol, ioglucol, ioglucamide, ioglunide, iogulamide, iomeprol, iopentol, iopromide, iosarcol, iosimide, iotusal, ioxilane, iofrotal, and iodecol.

14. The composite cryogel biomaterial according to any one of claims 1 to 13 for use in the treatment of a bone defect.

15. The composite cryogel biomaterial according to any one of claims 1 to 14 for use as an artificial bone tissue scaffold mimicking the structure of trabecular bone and as a carrier of bioactive agents involved in generating new bone at a place of a bone defect.

16. The composite cryogel biomaterial according to any one of claims 1 to 15, wherein the 3-dimensional form and structure of the scaffold mimics the original 3-D bone form and structure for replacement or reconstruction of damaged, diseased or lost bone tissue.

17. The composite cryogel biomaterial according to any one of claims 1 to 16 for use in local and long-term sustained delivery of bioactive agents.

18. A macro- and microporous biodegradable composite cryogel scaffold comprising a ceramic and a crosslinked non-immunogenic biopolymer, wherein the ceramic comprises calcium dihydrate (CSD); and calcium phosphate (CaP), such as a- or β-tricalcium phosphate (a-TCP/p-TCP), tetracalcium phosphate, calcium-deficient hydroxyapatite (CDHA) or hydroxyapatite (HA), preferably particulate crystalline hydroxyapatite.

19. A method for preparing a macro- and microporous biodegradable composite cryogel bone scaffold according to claim 18 comprising a ceramic and a crosslinked non-immunogenic biopolymer, comprising : a) solubilizing the biopolymer or mixture of biopolymers in a suitable solvent, e.g. an aqueous liquid, such as water; b) providing a ceramic powder;

c) dispensing a ceramic powder in the biopolymer solution or in an

aqueous liquid to be mixed with the biopolymer solution to obtain a dispersion or slurry;

d) optionally adding a crosslinker suitable for crosslinking the

biopolymer(s) to the mixture of step b) followed by mixing to obtain a dispersion/slurry having all ingredients evenly distributed;

e) filling the mixture of step c) into a mold and placing it in a cryostat to produce a frozen mixture in the mold;

f) thawing the frozen mixture of step d) to obtain a cryogel; and g) optionally freezing the cryogel followed by drying, such as freeze- drying, to obtain a dry cryogel (macro-porous and micro-porous biodegradable composite bone scaffold) comprising a ceramic and crosslinked non-immunogenic biopolymer(s).

20. A method for preparing a biodegradable composite cryogel biomaterial for use in regenerating bone, comprising adding at least one bone active agent to the solution or mixture in one of the steps a), b), c) or e) in the method according to claim 19, provided that the agent is not deactivated in any of the steps a)-f); or to the scaffold obtained in step f) or g) in the method according to claim 19.

21. A kits-of- parts, comprising

a. a macro- and microporous biodegradable composite cryogel scaffold according to claim 18 or as prepared according to claim 19, b. one or more bone active agents;

c. optionally one or more additional bioactive agent;

d. optionally a X-ray contrast agent; and

e. instructions of how to add said one or more bone active agents and optionally additional bioactive agents and/or a X-ray contrast agent to said composite cryogel scaffold.