WO2006057011A2 - Use of chitosan for stimulating bone healing and bone formation - Google Patents

Abstract

A use and a composition based on chitosan are provided for stimulating bone healing and bone formation. Chitosan is demonstrated to be a potent osteoinductor and substantially accelerates new bone formation through the endochondrial ossification pathway, by forming cartilage which is subsequently vascularised and mineralized and transformed into a normal healthy bone tissue.

Description

COMPOSITION AND A METHOD FOR BONE HEALING

Background of the invention

Biomaterials

Biomaterials are generally defined as materials that can safely be implanted into the human body and left there for an extensive period of time without causing an adverse reaction. In clinical practice, biomaterials are generally applied in implantable medical devices comprising combinations of materials and chemicals and compositions used to support or regenerate damaged or injured tissues. The development of biostable biomaterials started in the 1950s and became very active during 1960s and 1970s. This first generation of biomaterials, although having acceptable mechanical strength, yielded only passive contribution to tissue healing. Hence, their function was confined to support tissue replacement. Further, the above materials released degradative debris which, in turn, led to the onset of chronic inflammation, pain, and effusion.

Development of bio-resorbable (degradable) polymeric materials started in the late 1970s and further extended into the 1980s and 1990s. The main purpose of the development of the second generation of biodegradable biomaterials was to eliminate the chronic, long-term problems that resulted from the continuous release of debris particles originating in the first generation materials. Consequently, the new generation of implantable biodegradable materials offered tissue-supporting properties for the desired length of time (months), and thereafter they underwent local degradation by the surrounding tissues and were subsequently excreted outside the body via urine and/or feces. The above biodegradation occurred either in situ and/or in more remote organs such as the liver and kidneys. An example of this technology is biodegradable vascular sutures in use today.

Despite the good biocompatibility properties of the second generation of biomaterials, their contribution to the promotion of tissue healing was limited, due to their passive role in the biologic processes involved in tissue healing and regeneration. The idea of developing a more modern set of a third generation of biomaterials resides in the desire to develop compounds that will actively contribute to the process of tissue healing and regeneration. Such a desired property can be obtained via the linkage of bioactive molecules to biodegradable materials. Typical bioactive additives are compounds such as growth factors, drugs (antibiotics), and bioactive ceramics. As such compounds undergo biodegradation; they release the active additives into their immediate adjacent tissues and thereby facilitate the healing and regeneration. However, even the above compounds possess only a limited capacity to induce tissue regeneration in an injured or damaged site. This fact enhanced the new concept of tissue engineering within the biomaterial sciences. The latter discipline comprises of a new interdisciplinary approach for tissues and organs to overcome deleterious events (mutagenic, traumatic, and elective-iatrogenic) and restore their original architecture and function.

Tissue engineering as related to bones, cartilage, joints and tendons, integrates biodegradable scaffolding and cells; and, therefore, encompasses disciplines such as cell and molecular biology, material sciences and surgery. Hence, current tissue engineering approaches provide new opportunities for surgical treatments of damaged tissues; while eliminating the limitations experienced in the task while using previous technologies. Modem breakthrough biomaterial formulations therefore address the entire aspect of tissue engineering providing structural support, scaffolds for neo-tissue formation and biologically active materials capable of inducing tissue specific progenitor cells to initiate formation of new and functional tissue while avoiding fibrous non functional tissue formation in the damaged side.

Current options in orthopedics Current bone graft devices are supplemental bone materials used to replace existing natural bone that has been damaged by trauma or disease. The available bone graft devices are divided into 2 main categories depending on principal mode of action of the graft composite; osteoconductive and osteoinductive materials.

Osteoconductive (OC) devices provide a scaffold for bone growth and are characterized as materials that fill voids and have no intrinsic properties to cause the body to generate new bone. These materials act as scaffolds that allow host bone to migrate into and anchor in place. There are many well established commercially available OC materials including allografts (donor bone),

Demineralized Bone Matrix (DBM) and synthetic bone substitutes typically composed of calcium phosphate-based materials.

Donor bone, a bone from a cadaver, is referred to as allograft bone and has only the osteoconductive property. It does not contain bone cells or proteins, and has only calcium scaffolding. Similar to the patient's own bone, structural allograft bone comes fully mineralized so the osteoinductive proteins are not exposed and readily active. Bone allografts are sourced from same species donors and typically are processed to remove antigens, diseases and preserved typically by cryogenic process. Allografts typically contain both the inorganic mineral and organic matrix phases intact as harvested bone and are typically considered non viable materials as the osteoblast cells have been removed with the processing and preservation. This is an important aspect as any bone inducing proteins are encapsulated in the composite bone and are not available to participate in new bone formation. Allografts are typically used to fill large bone defects where autograft bone is either not sufficient or not of proper density. Allografts are incorporated into the recipient defect typically by osteoconduction where host bone simply grows into the allograft anchoring it in place.

Demineralized bone matrix is similar to bone allografts as they originate from same species donor in a similar fashion as allografts. However, the DMB materials are usually cleaned with alcohols, ground in a frozen state and treated with harsh chemicals such as hydrochloric acid and to remove the inorganic phase resulting in an organic phase, mostly comprised of collagen. DMB has been readily available for over ten years. This is a manufactured product that includes demineralized pieces of cortical bone to expose the osteoinductive proteins contained in the matrix. These proteins include the family of bone morphogenetic proteins (BMP) known to be able to induce new bone formation de novo. These activated demineralized bone particles are usually added to a substrate or carrier (e.g. glycerol or a polymer). DBM is mostly an osteoconductive product, lacking enough induction to be used on its own in challenging healing environments such as posterolateral spine fusion. It is almost always used as a bone graft extender (not as a substitute) for posterolateral spine fusion surgery and is generally intended to allow the use of less autogenous bone.

Several papers have been presented at the North American Spine Society since 2000 showing that some, but not all, brands of commercially available DBM do enhance bone growth in experimental tests. There is great variability between the efficacy (osteoinductive) of different brands of DBM and few have been properly validated in stringent animal models. Because these materials are tissue rather than devices, clinical trials are not required and there are very limited human data available. One exception to this is Grafton Matrix DBM, made by Osteotech. This is the first commercially available DBM product that has been validated in a non-human primate spine fusion model and the first shown to increase the fusion success rate above that seen with autogenous bone graft.

Banked bone taken from cadavers for demineralization (allogenic bone) must be harvested under rigid standards and conditions to prevent possible immunologic complications or possible transmission of viral or bacterial pathogens. Gamma radiation, one method for sterilization of demineralized bone, may alter the physio-chemical properties critical for bone induction. It is recognized that irradiation of demineralized bone powder before implantation weakens the osteogenic response by 20%.

Osteoinductive (OI) biomaterials or devices promote bone growth by inducing proliferation of progenitor cells capable of developing into cartilage and/or bone tissue and are characterized as materials that not only fill voids but also contain intrinsic properties that cause or induce the body to produce new bone within the material. These materials not only act as a scaffold, but also contain proteins or other substances that induce the formation of new bone. Autografts have been used in orthopedic surgical procedures for many years, and are the most common method of assisting the body's regenerative ability. Only one commercially available product demonstrates true OI properties, the recently introduced device from Medtronic called InFUSE^® incorporating a recombinant version of human bone morphogenetic protein (BMP).

The gold standard for bone grafts, used particularly for lumbar spine fusion, has been autograft bone harvested from the patient's pelvis, which is a surgical procedure performed at the time of the spine fusion surgery. Bone that is harvested from the patient (autologous bone graft, or autograft bone) has two of these properties because it has both the calcium scaffolding (osteoconduction) and it is estimated that some 15% of the bone cells survive the transplantation (osteogenicity). However, the third property osteoinduction may not be sufficiently available in the patient's own bone. Although small amounts of osteoinductive proteins are present in all bone matrix, since autograft is mineralized bone, these osteoinductive proteins are not exposed and may have very limited activity.

There are two main potential problems with harvesting bone from the patient's pelvis:

• Graft site morbidity

Taking the bone graft from the patient's pelvis is a surgical procedure. With proper surgical techniques, bone graft site morbidity can be decreased (see surgical techniques under bone grafts). There is, however, always the potential for a complication. Some of these potential complications include bleeding, infection, and chronic pain at the donor site.

• Failure to fuse (pseudoarthrosis or nonunion)

Even if the spine fusion operation is performed correctly, not every patient will obtain a solid fusion. Spinal instrumentation has to some extent reduced the risk of not getting a solid fusion, but there are some patients who are still at high risk for a pseudoarthrosis (e.g. patients who have had multiple spine surgeries, who are obese, who smoke, or are having a multilevel spine fusion).

The above two issues, graft site morbidity and failure to fuse, are the two primary reasons there has been a great deal of interest in creating a bone graft substitute with improved osteoinductive properties. This is particularly so for use in a spine fusion procedures.

Much interest and research has occurred once the identification of Bone Morphogenetic Protein (BMP) was made by Urist over 3 decades ago. BMP is a class of proteins naturally occurring in bone tissue, where they are actively involved in bone formation. Even outside the bone tissue environment, such as when implanted subcutaneously, the BMPs are capable inducing bone formation, hence this property has been used as an indication for BMP activity. When incorporated into medical devices these proteins are integrated into a synthetic matrix such as collagen sponge, providing a platform for bone regeneration. BMPs (e.g. BMP-2 or BMP-7) have been shown to be excellent at growing bone (osteoinduction) and there are several implant device product being tested. Extensive animal testing has already been undertaken, and human trials are finished or in process for these products. Recent product introduction incorporating BMP is from Medtronic Sofamor Danek. BMP-2 delivered on an absorbable collagen sponge (InFUSE^®) has been used inside titanium fusion cages. In July 2002, following extensive clinical evaluation, the InFUSE brand of BMP received FDA approval for use in certain types of spine fusion. A pilot study with BMP-2 delivered on a ceramic carrier was recently published and reported a 100% successful posterolateral fusion rate. Another BMP, BMP-7 (OP-I) has reported 50-70% successful posterolateral lumbar fusion results in human studies to date. Studies with these and other BMPs are underway.

Extensive testing has been done in Europe on a product (Healos) that is a matrix made up of collagen with hydroxyapitite spun onto it. Microscopically it closely resembles bone and it works by absorbing harvested bone marrow before insertion. Therefore, with marrow it has both osteoconductive and osteogenic properties, yet it would eliminate the need for an open incision (to retrieve bone from the patient's hip) as the patient's bone marrow can be harvested with a needle. It also may be less expensive than BMP, although it may not be as effective. Animal studies have yielded conflicting results with respect to its success in posterolateral spine fusions.

There is clearly a need for a new class of bone graft devices for use in osseous repair including trauma, osteosarcoma, spinal fusion and a myriad of age dependent diseases such as osteoporosis. These new devices must include requisite mechanisms that comprise the following:

• Osteoconduction— this refers to the scaffolding that is needed for new bone to grow on;

• Osteogenicity— this refers to the transmittal of live bone cells or osteoblasts; • Osteoinduction— this is the process whereby proteins and growth factors induce the bone to grow, and;

• Early mechanical weight bearing properties— this is the ability for the accelerated formation of repair that allows for earlier weight bearing. Evidences for a role of carbohydrates and their binding proteins and receptors in bone tissue remodeling and regeneration

Glucosamine (GN or GIcN) is a modified glucose with NH₂ replacing the OH group on the carbon two in the sugar molecule. In animal cells, glucosamine is only found in two forms; as glucosamine-6-phosphate (GN-6-P) and N-acetyl glucosamine (NAG or GIcNAc). The amino sugar GN-6-P is synthesized from glutamine and fructose-6-phosphate (F-6-P). This reaction is catalyzed by glucosamine synthase as the rate limiting step in amino sugar biosynthesis. GN- 6-P is the precursor to all hexosamines and hexosamine derivatives. In the next step, GN-6-P is acetylated by acetyl coenzyme A to N-acetyl glucosamine (NAG or GIcNAc). NAG can subsequently be converted into N-acetyl galactosamine or N- acetyl mannosamine. These three amino sugars are important in glycosylation of proteins as well as building blocks for glycolipids, glycosaminoglycans (GAG), hyaluronan and proteoglycans. Hyaluronan (HA), the backbone of many proteoglycans, is a polysaccharide

(comprising up to 25,000 sugar units) composed of repeating disaccharide units of NAG and glucuronic acid (GIcA) [I]. HA is thought to be the earliest evolutionary form of GAG. HA is not only an important polysaccharide in cartilage, synovial fluid, vitreous humor of the eye and in the skin of vertebrates, but may also play an important role in tissue organization, morphogenesis, cancer metastasis, wound healing and inflammation [I]. HA is synthesized at the inner face of the plasma membrane by HA synthases (HAS), and is directly extruded to the extracellular space. However, HA can also re-enter the cell, and can even translocate to the nucleus, [1, 2] for review. HA is produced in large quantities during wound repair, and is an essential constituent of joint fluid, where it serves as a lubricant [3], NAG stimulates the synthesis of hyaluronan by mesothelial cells and fibroblasts in a dose-dependent manner [4].

HA is secreted from cells by an enzyme complex, named HA synthases (HAS) which are embedded in the plasma membrane [I]. These enzymes are thought to have evolved from chitin synthases [I]. In vertebrates, three HA synthases (HASl, HAS2 and HAS3) encoded by three distinct genes have been identified by complementing HA-deficient cell lines [5-9]. HASl, HAS2 and HAS3 have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis [9].

A mouse HA synthase (HASl) is capable to synthesizing HA in vitro, when it is supplied with UDP-GIcA and UDP-NAG [10], but when the enzyme is incubated with UDP-NAG alone, it synthesizes chitin-oligosaccharides (CHOS) [10]. A demonstration of similar activity of eukaryotic HA synthases in vivo, would suggest novel functions for chitin-oligosaccharides in the vertebrates [I]. Natural chitin oligosaccharides are produced in vivo during the development of vertebrates [Xenopus, zebrafish and mouse), where the chitin synthase-like DG42/HAS subfamily synthesizes both chitin-oligosaccharides and HA during cell differentiation. These natural chitin-oligosaccharides have been shown to be vital for a normal anterior/posterior axis formation in the late gastrula, prior to neurolation [1, 10-15] reviewed in [16]. Chitinase like proteins or the CLPs are proteins that have evolved from the

Chitinase Family 18 (a single chitinase, expressed in all animals from bacteria to mammals). These proteins have conserved their catalytic domain (their ability to bind chitin) but many have lost their activity (the ability to cut chitin) by one or more amino acid substitution. This domain is herein referred to as the chitin binding domain,. In some of these proteins the binding to a chitin structure has been shown to cause conformal changes of the protein molecule. Another important definition of the nature of the CLPs is that they are cell signaling proteins (growth promoters or cytokines) possessing powerful cell and tissue signaling and growth modulating properties. In humans, six CLPs have been described. These are YKL-40 (HC gp-39), YKL-39, ECF-L (YmI), Chitotriosidase, Acidic Mammalian Chitinase (AMCase; two subforms TSA1902-L and -S) and Oviductin. All except AMCase (TSA1902-L) and Chitotriosidase are inactive (silent) as chitinases.

The crystal structure of human and goat YKL-40 (HC gp-39) has been worked out by Houston, [17] and Mohanty, , [18]. The structure of the human YKL-40 along with CHOS (A9) aligning its binding domain and these authors state that unlike the chitinases, binding of the oligosaccharide ligand to the YKL-40 protein induces a large conformational change in the protein structure [17]. The structure of ECF- L (YmI) has been work out by Tasai, [19]. The crystal structure of chitotriosidase has been described by Fusetti [20] but the chitinolytic activity is preserved in this protein, inhibited by allosamidin, a classical inhibitor of the family 18 chitinases.

The crystal structure of YKL-39, AMCase and Oviductin has not yet been published. Healing of damaged tissue occurs through two main pathways. One is tissue repair, involving inflammation, subsequent proliferation, mainly involving fibroblast proliferation and fibrous tissue formation, and finally maturation involving remodeling of the repair tissue to form a scar, producing architectural inconsistencies in the functional tissue. The other pathway is tissue regeneration, whereby healing occurs through regeneration of the original tissue. Specific tissues do not possess the capacity to regenerate; these include among others both cartilage and bone. It is evident that for both healing processes, extracellular proteins and carbohydrates such as collagens, hyaluronan play a crucial role and recent scientific evidence suggests an equally important role of chitinous oligosaccharides and their binding proteins or receptors. The chitinase like protein YKL-40 plays a role in tissue remodeling, [17, 21-24] especially in connective tissue remodeling and possibly degradation of extracellular matrix [17, 25]. YKL-40 is a growth factor for connective tissue cells increasing the growth of fibroblast cell lines in a dose-dependent way [26, 27]. In this manner the protein is possibly involved in tissue healing through scar tissue formation. It is up regulated in cirrhotic liver diseases such as hepatitis C virus (HCV) [28]; is suspected to trigger fibrosis and is known as a fibrosis serum marker [24, 29-33]. It has proved to be a potent migration factor for endothelial cells [34] and vascular smooth muscle cells [35].

YKL-40 is undetectable in the chondrocytes of normal articular cartilage [36]. YKL-40 in guinea pig chondrocytes (GPC), rabbit chondrocytes (RC), and rabbit synoviocytes (RS) was higher in dividing cells than in confluent cells, suggesting a participation of YKL-40 in cell cycle events [27]. Chondrocyte culture experiments have shown that YKL-40 production increases to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures in response to tissue injury [37]. Chondrocytes differentiate from mesenchymal cells during embryonic development [38], and the phenotype of the differentiated chondrocyte is characterized by the synthesis, deposition, and maintenance of cartilage-specific extracellular matrix (ECM) molecules, including type II collagen and aggrecan [39-41]. The phenotype of differentiated chondrocytes is unstable in culture and is rapidly lost in serial monolayer culture [42, 43]. This process is referred to as "dedifferentiation" and is a major impediment using mass cell populations for cell therapy or tissue engineering of damaged cartilage. When isolated chondrocytes are cultured in a monolayer at low density, the typical globular chondrocytes will transform their morphology into flattened fibroblast- like cells, with profound changes in biochemical and genetic characteristics, including reduced synthesis of type II collagen and other cartilage proteins [44]. When these chondrocytes are transferred and cultured three-dimensionally in a scaffold, such as agarose, collagen, or alginate, they re-differentiated and re- express the chondrocytic differentiation phenotype genes [45, 46]. Among the gene products highly expressed at this stage were leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and YKL-40 (cartilage glycoprotein-39) [46].

The master chondrogenic transcriptor factor Sox9 is expressed in all prechondrogenic and chondrocytic cells during embryonic development. Many lines of evidence have shown that Sox proteins are necessary for chondrogenesis. Sox9, as well as L-Sox5, and Sox6, are members of the Sox family of transcription factors that are characterized by high-mobility-group (HMG)-box DNA-binding domain [47] and [48]. Sox9 is essential for converting cells of the mesenchymal cell condensations into chondrocytes and acts further at every stage of chondrocyte differentiation. Sox9 is expressed in cells of mesenchymal condensations and in proliferating chondrocytes, but not in hypertrophic chondrocytes. In cultured cells, Sox9 stimulates transcription of a number of cartilage matrix genes, including Col2al, Collla2 and aggrecan , for a review see [49] and [50]. Inflammatory agents or cytokines such as lnterleucin-1 (Il-l) and tumor necrosis factor-α (TNF-α) strongly inhibit Sox9 [51], hence cartilage regeneration and endochondral ossification is halted during inflammation caused by infection, injury and in autoimmune diseases such as osteo- and rheumatoid arthritis [51-53]. The down-regulation of Sox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint. diseases [51].

The bone tissue is a highly mineralized and dynamic tissue, comprising type II collagen, crystallized calcium phosphate (hydroxy apatite) and several cell types taking part in turnover of the tissue matrix. Osteoclasts are specialized to resorb the bone matrix but osteoblasts are specialized in reconstructing new bone tissue matrix. In human osteophytic tissue the chitinase-like protein YKL-40 is expressed intensively in end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation [36]. Proliferating osteoblasts express low to moderate YKL-40 levels and mature osteocytes are negative [36]. The authors suggest not only cartilage degeneration but increased osteogenesis in osteoarthritis [36]. In osteoblast cell lines, supplemented with 10% fetal bovine serum (FBS) and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly increased compared to the control culture group, indicating increased osteoblast activity and bone formation [54].

Conclusion

It is concluded herein that the field of applying biologically active biomaterials in bone healing inducing osteoinductive properties of the osseous tissue is a rapidly growing and dynamic field within the orthopedics with the current focus set on bone morphogenic proteins and suitable formulations for their controlled release in situ.

It is also clear that through recently accumulating evidence, carbohydrates and their binding proteins and receptors are now emerging as important players in tissue repair, chondrogenesis, and bone remodeling. These evidences have inspired the inventors to investigate the role of chitinous carbohydrate structures in bone regeneration with the goal of developing new biomaterials possessing osteoconductive as well as osteoinductive properties in a fully biocompatible, clinically and industrially well manageable formulation providing mechanical support to bone defects. References

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Summary of the invention

Surprisingly, the present inventors have realized that chitosan itself acts as an osteoinductive agent in addition to an angiogenic agent and can be used not only as a scaffold or support material for other agents, but as an active agent promoting and enhancing bone formation. Based thereon, the inventors have developed methods and compositions that can be successfully used for tissue repair or tissue regeneration after any kind of trauma or during surgical operations of the connective tissue.

It is an object of the present invention to provide a composition, use and a method for promoting tissue repair or tissue regeneration in large injuries in long bones, vertebrae, and the sternum of a mammal such as in particular in humans.

In a first aspect the invention provides the use of chitosan for the manufacture of a medicament for inducing or enhancing endochondral ossification at a tissue site in a mammal. Chitosan provides tissue regeneration in bone tissue through the endochondrial ossification pathway, which is how bone is originally embryonically formed during the development of bone structures in a mammalian body.

In another aspect of the invention a method is provided for inducing or enhancing endochondrial ossification at a tissue site in a mammal. The method includes supporting the tissue site with a scaffolding mechanism in order to direct the tissue repair or tissue regeneration. A therapeutically effective amount of a composition comprising chitosan is applied to the tissue site and the tissue site is then secured in a fixed position. The chitosan is enzymatically cleaved into chitooligomers, by endogenous chitanses (such as??) which are responsible for the inducing or enhancing endochondral ossification effect of the compound. By placing a chitosan formulation at a tissue site in a mammal, such as in a bone fracture, a continuous and slow release of the pharmaceutically effective therapeutic compound is provided. As will be further demonstrated in the examples, the chitosan is responsible for proliferation and differentiation of bone progenitor cells, vascularisation via angiogenesis, and mineralization of induced hypertrophic cartilage and the formation of endochondral bone, mimicking the essential processes described as endochondral ossification.

In a further aspect of the invention a kit is provided for inducing or enhancing endochondrial ossification at a tissue site in a mammal. The kit is provided sterile and comprises at least chitosan and optionally a support matrix material and a scaffold for supporting osseous tissue formation, regeneration and repair.

Prior art methods fail to show and induce vascularisation of cartilage tissue, thus enhancing mineralization and subsequent ossification of the induced cartilage tissue into bone.

Description of the invention

In an embodiment of the present invention a use of chitosan for the manufacture of a medicament for inducing or enhancing endochondrial ossification at a tissue site, where the medicament is in a form selected from the group consisting of a paste, a cream, a solution, a film, a coating, a gel or solid bars or plates, or a combination thereof.

The preferred method or use of administration of the compositions or medicament of the present invention is by a surgical procedure. The medicament or compositions can also be administered via a medical implant product, defined as a Class I, II or III regulated medical device by the LJS Food and Drug Administration, where the medical implant typically is installed during a surgical procedure either for repair from trauma, disease, resection or revision. Said medical devices may be permanently placed or for temporary use. Further said medical implants may comprise technology that decompose over a desired time frame. The medicament or compositions may be used in conjunction with other medical implants either as a coating or as a component. Further, said medical devices are represented by metallic or plastic joint implants including finger, knee, TMJ, stabilizing plates or other articulating joints. Furthermore, the medicament or compositions may be applied as a coating to similar medical devices. Said example would be as a surface coating of an implantable medical device such as an intramedullary rod or other stabilizing devices such as reconstruction plates or screws. Further application may be administered via catheter for placement into bone structures including vertebral bodies such as for use in vertibralplasty or kyphoplasty.

In an embodiment of the present invention the medicament or compositions comprise a support matrix material selected from the group consisting of, but not limited to calcium phosphates, including hydroxyapatite, tetracalcium phosphate, calcium sulphate and sodium tri-polyphosphate.

In an embodiment of the present invention the medicament or compositions are intended for patients suffering from a disease selected from the group of rheumatoid arthritis, osteoarthritis, osteoporosis, cartilage defect, bone fracture resulting from trauma or disease or damage and dental implants. In another embodiment of the present invention a method for inducing or enhancing endochondrial ossification at a tissue site is provided, including

- supporting the tissue site with a scaffolding mechanism; - placing an therapeutically effective amount of a composition comprising chitosan at the tissue site; and

- securing the tissue site in a fixed position where the medicament is in a form selected from the group consisting of a paste, a cream, a solution, a film, a coating, a gel or solid bars or plates, or a combination thereof.

In another embodiment of the present invention a method for controlling bone formation and/or functional tissue regeneration through the endochondral ossification pathway is provided. The method comprises the following steps:

- placing an therapeutically effective amount of a composition comprising chitosan, a support matrix and optionally chitooligomers, between parts of a sectioned bone or a bone fracture;

- arranging and securing parts of a sectioned bone or a bone fracture composition in a desired position by metal fixtures;

- allowing bone healing through endochondrial ossification; and

- optionally removing the metal fixtures.

The bone healing and/or bone formation involves development of cartilage with well developed vascularisation and subsequent formation of new bone tissue without scar formation.

In another embodiment of the present invention a kit is provided for inducing or enhancing endochondrial ossification at a tissue site. The kit provided sterile and comprises at least:

- chitosan and optionally support matrix material and/or chitooligomers,

- scaffold for supporting tissue formation or regeneration, and

- optionally a support matrix material.

In the present context the term "at a tissue site" refers to in a tissue of a living mammal, such as a human. The site is selected from, but not limited to traumatized tissue such as injured tissue, broken bone or cartilage, skin or any other tissue of the connective tissue system. The medicament or compositions are applied directly on the severed surfaces of bone to be re-joined in cases where bone has been broken or taken apart during a surgical procedure. The medicaments or compositions of the present invention are well suited for tissue repair and tissue regeneration as shown in the examples below. In the present context the term "therapeutically effective amount" refers to the total amount of the active component of the composition that is sufficient to induce a significant benefit, i.e., healing of bone and/or bone formation of new bone or enhance the rate of healing.

The term "endochondral ossification" refers to a bone forming process, whereby cartilage develops first yielding the future format of the final bone, such as vertebrae, long bones, sternum, etc. The cartilaginous tissue needs less local oxygen tension for its development and maintenance than bone tissue and therefore, wherever the blood supply system has not attained its final stage of development, cartilage will supersede bone. Cartilage will only be replaced by new bone after vascularization has reached its more advanced stage, guaranteeing the essential supply of oxygen to the developing tissues.

As described in detail in the accompanying Examples, the present inventors have demonstrated that chitosan based compositions can be successfully used for healing a severed sternum and tibia in mammalian animals. Bone regeneration and reunification of the two halves of the severed sternum is substantially enhanced and healing is induced.

The chitosan composition provides additional advantages:

- Chitosan acts as a haemostatic agent and thereby reduces the risk of post¬ operative bleeding from the severed blood capillaries of the open bone wound.

- Chitosan is a potential antibacterial agent and thus reduces the risk of infection of the surgical wound

Consequently, the invention provides, in a related aspect, the use of chitosan for the manufacture of a medicament for enhancing bone formation and haemostasis in the healing of a fractured or severed bone.

Typically, the medicament is administered directly to the fractioned bone surfaces which are to be joined.

The medicament is in one embodiment in a liquid or semi-liquid form such as not limited to, a salve, a cream, a solution, a syrup, a paste, a gel or a cream. Such formulations may be conveniently applied directly to the bone fracture surface. The medicament is preferably provided in a formulation such as the above which makes use of the tissue-adhesive properties of chitosan, thus adheres well to the wet bone surface, i.e. it should preferably be suitably "sticky" such that it can simply be spread on the surface of the bone wound. Alternatively, the medicament can be injected into the bone wound without rupturing the soft tissues surrounding the bone. However, in alternative embodiments, the medicament is formulated in a soft film or "tape" with plastic characteristics that can be directly laid or rolled on the bone surface. Such film is preferably slightly elastic and sticky such that it can be stretched onto and adhered to the bone surface. Alternatively the chitosan material can be applied directly into the wound as freeze-dried foam.

In another embodiment the medicament is formulated as solid units, e.g. bricks, plates, pellets or bars or similar rigid or semirigid constructs that are fixed to the fractured bone surface, e.g. by surgical wire or thread. Such solid members are used in Example 1 where 1.6x1.6 cm square plates of 2 mm width are used. A plurality of plates are used and placed in a row but with sizable gaps in between (one to a few cm). Such solid form medicament contains in some embodiments a support matrix material such as hydroxy apatite or other calcium phosphate material, calcium sulphate, sodium tripolyphosphate and the like - substantially inert inorganic materials that are biocompatible and osteoconductive and preferably degrade slowly and become integrated within the newly formed bone matrix.

The medicament can in certain embodiments consist of substantially only chitosan. Alternatively the medicament further contains additional components, e.g. water, one or more organic acids which are suitable for protonizing the chitosan amine groups to adjust the solubility and solution characteristics of the chitosan. Such organic acid is suitably selected from pharmaceutically acceptable acids such as acetic acid, citric acid, lactic acid, propionic acid, hydroxyacetic acid, hydroxybenzoic acid etc.

The medicament may additionally contain a diluent, excipient or carrier material. Diluents and excipients can be selected from cellulose derivatives, e.g. methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, polymethacrylates, polyvinylpyrrolidone, pregelatinized starch, alginate, collagen, alginic acids and salts thereof such as sodium alginate, polyethylene glycol and the like.

In certain preferred embodiments, the medicament is formulated to provide sustained extended slow release of chitosan at the bone healing location. Solid state formulation of the medicament, such as the hydroxyapatite plates used in the Examples are of this type, resulting in extended release of chitosan composite is slowly decomposed.

The method provided by the invention for inducing bone healing and bone formation of a severed sternum is in essence based on the above described chitosan composition. The method comprises - placing an therapeutically effective amount of a composition comprising chitosan which is suitably formulated in any of the above described forms, between the fractioned halves of the sternum, bringing together the two halves with said composition in between the bone halves,

- securing the bone halves in fixed position,

- closing the tissues covering the affected bone, wherein the bone healing and/or bone formation involves development of cartilage with well developed vascularisation and subsequent formation of new bone tissue without scar formation emphasizing the endochondrial bone formation.

Preferably, the composition is applied directly on the severed surfaces of the two sternum halves to be re-joined.

In another embodiment the invention provides a method for controlling bone formation and/or functional tissue regeneration in a mammal through the endochondral ossification pathway, the method comprising:

- placing an therapeutically effective amount of a composition comprising chitosan and optionally a support matrix between parts of a sectioned bone or a bone fracture,

- arranging and securing the bone parts to be joined in a desired position, e.g use of metal fixtures, surgical thread or a combination thereof,

- allowing bone formation and healing through endochondral ossification, - optionally removing metal fixtures if they have been used for the securing of the bone, wherein the bone healing and/or bone formation involves development of cartilage with well developed vascularisation and subsequent formation of new bone tissue without scar formation.

As can be inferred from above, the compositions and methods of the present invention may be used in various clinical conditions including, but not limited to repair of bone defects and deficiencies, such as closed, open and non-union fractures; inducing bone healing in plastic surgery; attachment of prosthetic joints, limbs and dental implants; elevation of peak bone mass in pre-menopausal women, increase in bone formation during distraction osteogenesis; and treatment of skeletal disorders, such as post-menopausal osteoporosis, senile osteoporosis, glucocorticoid-induced osteoporosis or disease osteoporosis and arthritis. The compositions of the present invention can also be useful in repair of oncological surgical procedures and in cosmetic surgery. The compositions of the present invention can also be used in the treatment of cartilage defects.

The clinical conditions to be treated by the methodologies of the invention are based on treating vertebrate subjects by local administration of the compositions of the invention. The compositions of the invention are designed to be administered locally. When the composition is provided locally, the bioactive substance and optional support matrix provide a supporting environment for the growing cartilage and or bone, where the matrix will be replaced by new bone.

The composition of the present invention may be used to induce growth or differentiation of bone-forming cells or bone-forming cell precursors. The composition of the present invention may further generate an environment, which attracts bone-forming cells to home to such a site and generate new bone tissue at a desired site.

Human diseases are complex and typically are manifested by several factors. Bone diseases such as osteosarcoma not only have malignant tissues, but also require resection of the cancerous area and regeneration of healthy bone. Anti- cancer drugs are prescribed to combat these types of diseases and often have significant side effects not only locally, but also systemically. Adult mature bone is a tissue that demonstrates typically lower turnover than other tissues in the body. This creates a challenge for the caregiver to be able to deliver a therapeutic level of treatment to the affected site, especially if the treatment is delivered systemically. Many bone cements such as polymethylmethacrylate can be formulated with anti-cancer agents to aid in more local delivery of these agents provide more effective therapy. It would be advantageous in addition to the osteoinductive stimulus of the inventive constructs described within to combine these with other agents that can augment the anti-cancer drugs prescribed for the treatment of the cancer.

Endochondral bone formation requires substantial angiogenesis to occur to modify the oxygen tension and is one of the distinguishing factors in this type of new bone formation. There is an advantage to combine additional agents that promote new vascular formation such as vascular endothelial growth factor (VEGF).

Additionally in certain cases other growth factors may be beneficial to consider combining with the inventive constructs described within, one or more additional growth factors such as bone morphogenetic protein, human growth factor, and other growth factors to provide for targeted local delivery presenting the opportunity for a synergistic approach to managing human diseases.

Traumatic bone loose occurs spontaneously due to accidents, violence, war, and other unexpected trauma. The landscape for these traumas is often associated with unsanitary conditions resulting in the potential for infection and disease transmission. Additionally bone and blood loss often occur from these traumas. It would be advantageous to combine additional treatments with the inventive formulations described within. For example, it may be advantageous to combine agents such as a hemostatic agent; an anti-infective agent; an anticancer agent; a protein; bone cement; or an anti-inflammatory agent to obtain improved repair of the affected area.

In a similar fashion, bone loss can be quite extensive and require not only metallic, plastic and ceramic devices for repair, but may also require or benefit from the use of existing other bone devices in the health care provider's armamentarium including autograft bone, allograft bone, demineralized bone, bone marrow, bone cement, bone protein and envision the use of stem cells to augment the use of the inventive constructs described within.

The composition of the present invention may optionally be used with other medicaments selected from the group consisting of: haemostatic agents, an anti- infective agents, anti-inflammatory agents, anti-cancer agents, a protein or bone cement. In the present context, such a protein can be selected from a group of growth factors such as, but not limited to vascular endothelial growth factor

(VEGF), fibroblast growth factor (FGF) and bone morphogenic proteins (BMP's).

Furthermore, the composition of the present invention may optionally be used with other bone graft materials selected from the the group consisting of: an autograft, an allograft, a demineralized bone, a bone marrow, a bone cement, a bone protein, a stem cell.

The term "precursor cell" refers to a cell that is not pre-committed to a certain differentiation pathway, but expresses few or no markers or function as a mature, fully differentiated cell. The term "osteogenic cells" comprises osteoblasts and osteoblast precursor cells.

Surprisingly, the present inventors have realized that chitosan itself acts as an osteoinductive agent and can be used not only as a scaffold or release matrix for other agents, but as an active agent promoting and enhancing bone formation. In addition, the present work shows that chitosan stimulates vascularization of cartilage tissue, thus enhancing mineralization and subsequent ossification of the induced cartilage tissue. Based thereon, the inventors have developed methods and compositions that can be successfully used for enhancing bone healing after bone fractures and bone sectioning during surgical operations.

As demonstrated herein, the invention can be successfully applied for healing chest bone incisions after chest operations such as sternotomy when the chest bone (sternum) has been longitudinally severed in half.

It is an object of the present invention to provide a composition and a method for promoting bone repair in large injuries in long bones and the sternum of a mammal, as well as all bone repairs of bones, which are embryonically developed through endochond al ossification in particular in humans. In a first aspect the invention provides the use of chitosan for the manufacture of a medicament for enhancing bone formation, in particular for healing of a severed sternum after surgical intervention.

In another aspect of the invention a method is provided for inducing bone healing and bone formation of a bisected sternum in a mammal comprising:

- placing an therapeutically effective amount of a composition comprising chitosan between the fractioned halves of the sternum, - bringing together the two halves with said composition in between the halves,

- securing the bone halves in fixed position,

- closing the tissues covering the affected bone,

wherein the bone healing and/or bone formation involves development of cartilage with well developed vascularisation and subsequent formation of new bone tissue without scar formation.

Detailed description of the present invention.

The invention will now be further disclosed in the following drawings with reference to the examples below.

Figure 1; Sterile plates of co- precipitated hydroxyapatite and chitosan in a 50/50 ratio, ready to be implanted into a bone defect. Dimensions in cm: 1.6 x 1,6 x 0.2

Figure 2: Sterile cylinder of co-precipitated hydroxyapatite and chitosan in a 50/50 ratio, ready to be implanted into a bone defect.

Figure 3: X-rays diffraction pattern of the chitosan/hydroxyapatite composite.

Figure 4: EDS spectrum for Chitosan/hydroxyapatite composite

Figure 5: Powder of calcium phosphate in a mixing bowl for blending of the powder with chitosan in a sterile acidic water solution. The mixture has a predetermined setting and hardening time depending on the properties and ration of the individual ingredients.

Figure 6: Sterilized pellets subcutaneously and aseptically inserted into one of the 40 Young Female Sprague Dawley Rats.

Figure 7: A view of the pellet and the adjacent tissues at sacrifice. Figure 8: At two weeks post-implantation the de novo granulation tissue encapsulating the Chitosan implant is usually rich in new blood vessels

Figure 9: A close image of the tissue surrounding the implant at 13 weeks post- implantation, remnants of the implant are indicated by the arrows. Note collagenous tissue (C) attached to the pellet. Farther away the tissue becomes looser (L), and devoid of inflammation.

Figure 10: At 13 weeks post-implantation the surrounding tissues regained their typical morphology. A discrete capsule surrounded the pellets is noted.

Figure 11: A solitary 2 mm unicortical drill hole in the midshaft of a rat femur.

Figure 12: A cross section of the injured bone in a control animal from Group A at week 4. The image shows the fragile bridging of the hole and a lack of trabecular tissue to fill the injury.

Figure 13: A cross section of the injured bone in an animal from Group B at week 3. The image shows the fragile bridging of the hole and a lack of trabecular tissue to fill the injury.

Figure 14: The image shows a complete sealing of the hole and a rich trabecular tissue filling the injury.

Figure 15: A macroscopical image of the extensively injured femur two weeks post op showing a rapid healing of the wound with a strong overreaction to the chitosan

Figure 16: A composite histological cross section of the entire bone at the locus of extensive injury at two weeks, group C. Notice the extensive regeneration of new bone tissue within the injury as well as surrounding the entire bone, forming a new bone girdle and a new periosteum.

Figure 17: A composite histological cross section of the entire bone at the locus of injury at two weeks, group C, extensive injury. Notice the extensive regeneration of new bone tissue.

Figure 18: Endochondral bone formation outside the original cortex, the image reveals a section of a vital new cartilage tissue and new trabecular bone tissue next to the cortex.

Figure 19: The image reveals new bone tissue on both sides of the original cortex. Figure 20: A ring-like piece (6-7 mm in diameter) was extirpated from the tibia, using an electrical saw.

Figure 21: Fixation of the two fragments of the bone with a stainless steel plate, the chitosan cylinder can bee seen inside the marrow space bridging the ostectomized portion of the bone.

Figure 22: Post-operative x-rays of the operated leg were taken at 4 occasions throughout the experimental period : one hour after operation and at 4 weeks, 5.5 months and 10 months post operatively.

Figure 23: At sacrifice 10 months post operatively the tibia was completely healed, showing solid bony union.

Figure 24: At sacrifice 10 months post operatively the bone was completely healed but still remnants of the chitosan-HA composite were left inside the marrow space.

Figure 25. Microscopic image showing polynuclear macrophage resorbing the chitosan-HA composite.

Figure 26: The image shows how chitosan-HA plates are positioned with intervals along the sternum osteotomy, leaving gaps between the individual plates.

Figure 27: An overall overview of the operative field prior to its final closure. The gap in the sternotomy created by the plates is around 2-3 mm.

Figure 28: CT image at day 10 showing that the chitosan-HA plates were stable in place and maintained their intended position.

Figure 29: CT image at day showing bone resorption next to the chitosan-HA plates.

Figure 30: The appearance of the intact sternum following sacrifice, 13 weeks post op. The picture shows exostosis exceeding the dotted line indicating the initial dimensions of the sternum.

Figure 31: Part of the intact sternum showing the chitosan-HA plates imbedded into intact new bone tissue bridging the gap of the sternotomy. Figure 32: A cross section of the sternum at Body 7 showing a complete healing of the sternotomy. No scar formations or abnormalities were observed in the new bone tissue.

Figure 33s A cross section of the sternum at Body 9, showing the remnants of the chitosan-HA plates imbedded in the new bone mass.

Figure 34: A giant polynuclear macrophage resorbing the chitosan-HA composite

Figure 35: The chitosan-HA composite becomes increasingly porous as the material is resorbed, new tissue and blood vessels invade the porous material.

Figure 36: Formation of new and vital cartilage tissue has been induced by the chitosan, now occupying large spaces where the composite was before. Light remnants of the composite can be seen in the centre of the image.

Figure 37: The new cartilage tissue is invaded by new blood vessels, showing the initial signs of cartilage being mineralized and developed into bone tissue.

Figure 38: Hypertrophic cartilage develops into bone tissue. Image shows three cell types; chondrocytes, osteoblasts and osteocytes

Figure 39: Collagen fibres visualized by polarized light microscopy. The fibres have a lamellar orientation with regular structures as would be expected in a bone tissue regenerated through the endochondral pathway.

Figure 40: Collagen fibres within a new regenerated trabecular bone tissue visualized in polarized light. The fibres shown all signs of the regularity expected in a bone tissue generated through the endocondral pathway.

Example 1. Osteoinductive biomaterial compositions.

A) Coprecipitated hydroxyapatite-chitosan composition in the shapes of pellets, plates (Code: G030812 and cylinders (code: G030208)

Materials and methods

Chitosan - The chitosan (Primex; lot number: TM1238), was heterogenously deacetylated to 87% degree of deacetylation. Its apprent viscocity of 1% solution in 1% acedic acid at 25⁰C was 144 cps or equivalent to a molecular weight of 800 KDa, and an ash content of less than 0.5%. The particle size was less than 100 mesh.

Purification Of Chitosan - The solution of chitosan (0.5% chitosan in 1% aqueous acetic acid, pH 3.3 ± 0.1) was stirred for 12 - 24 h, then was filtered through sintered glass funnel, No.l. Aqueous 0.5% sodium hydroxide was added to the clear solution until the pH was 8 and above. After two wash with with distilled water, it was freeze-dried.

Preparation of chitosan solutions - To prepare the chitosan solution, 900 ml of 1% aqueous acetic acid solution was added to 2 g of purified chitosan and stirred for 24 h. The volume was adjusted to give a final concentration of 0.2 % chitosan. Preparation of chitosan/ 'hydroxyapatite(HAp) composite materials by co- precipitation - The ratio of Ca/P in HAp (Caio(P0₄)₆(OH)₂) was 1.67. Stock solutions of CaCI₂.6H₂O (11.1 g; 0.506 M) and NaH₂PO₄-H₂O (8.28 g; 0.600 M) were prepared in 100 mL of bidistilled H₂O. 10 ml of each stock solution was added to 50 ml of 0.2% solution of chitosan in 1% aqueous acetic acid, and mixed thoroughly. Co-precipitation of chitosan and calcium phosphates takes place during the slow, dropwise addition of a 5 % NaOH solution to the chitosan / calcium phosphate mixture. After stabilizing the solution, i.e. at pH 11, the solution was stirred overnight. Subsequently the solution was filtered to obtain the composite in form of a hydrogel, and washed with water until neutral pH.

Drying process - Drying of the composite was conducted in a mold, either square or cylindrical shape, in a ventilated 37⁰C oven (Figure 1 and 2).

The X-rays diffraction profile and EDS spectrum of the composite are shown in Figure 3 and Figure 4. Solid implant of 50/50 at dry had an ultimate compressive strength of more than 35 MPa, but only 2.5 MPa at wet state.

B) Self hardening putty like paste composed of calcium phosphates and chitosan (Code G040121)

Materials and methods

Preparation of chitosan - The chitosan (Primex, lot TM1534) used in this example was heterogenously deacetylated to 70%DD and was used without further purification. Its apparent viscosity ( 1% TM1534 in 1% acetic acid ) and ash content were 1350 cps and <0.5%, respectively. The particle size of this chitosan was less than 100 mesh.

Preparation of putty like composite - The self setting putty-like composite, containing 5% chitosan (TM 1534), was prepared by mixing of a solid phase and a liquid phase, as shown in Table 1. Table 1. Composition of the self-setting composite

The paste was prepared at the operation side just before applying to the bone defect. To prepare the self-setting composite, the liquid phase was added to the solid phase, in a 200 ml bowl (Figure 5). After thorough mixing, the paste was stirred and mixed for additional 1 minute to ensure a proper mixing of all ingredients. Then the paste was applied to the targeted site. The setting time of this paste was 5 min 44 s ± 14 s.

The strain and compression strength after 7 days in 37⁰C water were 20.5 ± 3.5% and 6.5 + 1.0 MPa, respectively. Through X-rays diffraction study, all calcium phosphates transformed into precipitated hydroxyapatite in 7 days.

Example 2« Biocompatibility of 50/50 HA-chitosan composite and absence of osteoinduction in subcutaneous implantation

Materials and methods

Pellets of coprecipitated HA-chitosan composite (G030812) were sterilized in 20% alcohol and implanted subcutaneously in 40 Young Female Sprague Dawley Rats (Figure 6). The animals were followed for 4 months and sacrificed at intervals throughout the 4 month period. Blood samples were drawn for hematological and chemistry analysis. The implanted pellet was harvested and analyzed macroscopically and the adjacent tissues were analyzed macroscopically and histologically. All major internal organs (liver, lungs, heart, spleen, kidneys, adrenal glands, lymph nodes and intestines) were also analyzed for any pathological changes.

Results

During the 4 moth experimental period there were no signs of either mortality or morbidity among the treated animals. Throughout the 4 month in vivo duration the composite pellet appeared intact and no pathologic phenomena were noted under the skin and/or along the skeletal muscles underlying the pellets (Figure 7). Blood sample analysis and investigation of the internal organs revealed no pathological changes after the 4 month experimental period. At two weeks, histological samples revealed initial inflammatory response to the implant (Figure 8), and this tissue was apparently very rich in new blood vessels (Figure 9). Granulation tissue was encapsulating the implanted object at week 13 (Figure 10) Throughout the 4 month period all organ tissue samples appeared normal and healthy and no showed no indication of pathology or rejection of the composite nor signs of osteoblast activity.

Conclusions Transient local inflammatory reaction followed the implantation of HA-Chitosan composite pellets. Following 3 months in vivo the local inflammatory response receded and the tissues around the pellets regained their normal morphology. Blood tests at that time also appeared normal. A contributory factor to the above response could be the fact that the pellets were soaked with alcohol prior to their implantation, suggesting an alternative method of sterilization.

No pathologies were noted throughout the animal (macro and micro) following 4 months of implantation and no signs of osteoinduction were noticed in the histological samples indicating that the formulation and its chitosan ingredient does not actively promote bone formation subcutaneously, which is different from the activity of the bone morphogenetic proteins (BMPs), which are known to induce bone growth subcutaneously.

Example 3: Effect of a composite self hardening paste, made of calcium phosphate and chitosan, on localized injury in the rat femur

A bone injury was created in 80 rats through a 2 mm unicortical drill hole at the midshaft of the femur (Figure 11). The animals were divided into 3 treatment groups; Group A - control group, comprising 13 animals receiving no treatment, Group B - control group comprising 26 animals receiving treatment with calcium phosphate and Group C - experimental group comprising 41 animals receiving treatment with the composite (G040121). In Group A, the hole was left untreated and the wound closed by suture. In Group B, the hole was filled with a paste made of inorganic calcium phosphate and water, the surrounding tissues cleaned of any excess paste and the wound closed by suture. In Group C, the hole was filled with the composite G040121, the surrounding tissues cleaned of any excess paste and the wound closed by suture.

In few of the animals in Group B and Group C, an excessive injury was created through widening the diameter of the hole to 5 mm and in some cases, also by expanding the injury by penetrating through both cortices.

Animals were sacrificed after 2, 3, 4, and 5 weeks and the injured bone tissue examined in histological sections. Results

In Group A, a fragile and thin bridge of bone tissue covered the wound after 4 weeks (Figure 12) without any new bone tissue filling the hole even at 5 weeks, indicating non union.

In Group B, a more apparent bridging of the bone gap was evident at week 3 compared to Group A (Figure 13), although the new tissue was still fragile and even at week 5, filling of the hole was still incomplete, indicating non union.

In Group C, a rapid bridging of the wound was apparent after 2 weeks (Figure 14) with a complete sealing and filling of the hole with new cortical and trabecular bone tissue.

Healing of the extended injury, Group B: Following 2 weeks post operative many new trabeculae filled the large hole. The new bone was of a woven bone character and irregular.

Following 5 weeks post operative, the tunnel communicating the two penetration holes was filled with fibrous tissue. Although some cartilage was noted along the tunnel, the overall result indicated toward a fibrous nonunion.

Healing of the extended injury. Group C:

Following 2 weeks post operative, a marked overreaction was noticed to the chitosan treatment (Figure 15). The bone marrow space was filled with new trabecular bone which was continuous with a large mass of new bone outside the original cortex (Figures 16 and 17)

The implanted calcium phosphate-chitosan composite induced a rapid healing, and the histological analysis of the new tissue revealed a pronounced regeneration of new bone tissue. Generation of new cartilage and bone tissue was not only limited to the drill hole itself, but surrounded the bone to create a new girdle and a new periosteum enclosing the new tissue. Large de novo masses of cartilage developed in direct association with the periosteum of the original cortex as well as with that of the detached pieces of bone (Figures 18 and 19).

Conclusion

The new cartilage tissue revealed all stages of differentiation; from cartilage progenitor cells, through young chondroblasts, through mature chondrocytes, through hypochondriac chondrocytes, to matrix mineralization and new bone formation. Hence, our findings strongly indicate towards an immense potential of Chitosan (5%) to induce in vivo chondrogenesis from progenitor cells in the periosteum _f endosteum and marrow tissue. Chitosan possesses a strong osteoinductive potential, the calcium phosphate matrix serves as a good carrier for the active chitosan ingredient and together they yield a manageable product that can be used clinically for bone repair. The formulation provides osteoconductivity and osteoinductivity, essential properties for bone healing through regeneration of lamellar bone tissue in stead of repair, involving fibrous tissue formation by fibroblasts and subsequent ossification producing woven bone architecture.

Example 4: Application of chitosan to a large bone gap in sheep tibia

An adult female sheep was used for this experiment. The right leg tibia was exposed and an ostectomy created by removing a 5-6mm piece of the bone using an electrical saw (Figure 20). A cylinder of chitosan-hydroxyapatite composite

(G030812) (Figure 2) was inserted into the marrow space bridging the ostectomy and the two bone fractions were fixed together with a stainless steel plate using 6 screws (Figure 21). After the soft tissues were sutured, the leg was put into a plaster cast for three weeks.

X-ray images of the tibia were taken at 1 hour after the operation, and at 4 weeks, 5.5 and 10 months postoperatively (Figure 22). Radiographic signs of healing were shown as early as 4 weeks postoperatively. The animal was sacrificed at 10 months post operatively and both macroscopic and microscopic examinations showed a full regeneration of the missing bone. New trabecular bone occupied the gap (Figures 23 and 24). Although microscopic evidence showed resorption of the chitosan-hydroxyapatite composite (Figure 25), remnants of the composite were still left in the marrow space at 10 months post op. No signs of inflammation were seen throughout the experiment.

The experiment revealed that the chitosan-HA composite was effective at regenerating new bone in an ostectomy, creating a complete bone union bridging the bone gap. This indicates an immense osteoinductive potential of the chitosan composite. The absence of any signs of inflammatory reactions, both macroscopic and microscopic indicates anti-inflammatory activity of chitosan.

Example 5: Chitosan as an enhancer of bone tissue formation in sheep sternum after bone surgery

Chitosan was mixed with hydroxyapatite at a 50/50 ratio and was set to form plates (1.6 x 1.6 x 0.2 cm) during hardening (G030812). After sterilization (25 kGy), the plates were ready for implantation (Figure 1).

Sheep were subjected to a sternotomy involving longitudinal dissection of the sternum resulting in opening of the thoracic cavity. The chitosan HA plates were fixed to the parted bone surfaces at intervals of 2-3 cm, leaving gaps between the individual plates (Figure 26). Special attention was given to maintain the plates in their assigned position while the parted sternum was carefully approximated with surgical wires. After the thoracic cavity was closed by tightening the wires, the parted sternum was fixed together into a non-union position where the plates created a distance of 0.2-0.5 cm between the two sections of the dissected bone. This left a bone gap between the plates (Figure 27). After surgery was completed, the animals recovered rapidly and showed no signs of complications.

A non-invasive follow-up examination was carried out through Computed Tomography (CT images) on day 10 and day 33 and the animal was sacrificed in week 13 post operative. At day 10 it was noted that the plates maintained their intended positions (Fig. 28) producing bone gaps between the plates. At day 33, a marked initial bone resorption was noticed around the plates (Fig. 29).

After sacrificing the animal, the sternum was analyzed both macroscopically and microscopically. Macroscopic observations showed that the bone gaps were fully closed with new bone tissue and on top of that a extensive exostosis was observed in junction with the plates (Figures 30-33).

Histological examination of the new bone tissue in various parts of the sternum revealed bone formation through endochondral ossification involving high level of vascularization. Initially chitosan was resorbed by macrophages (Figures 34 and 35) with subsequent formation of new cartilage tissue, both adjacent and within the chitosan (Figure 36). Apparent vascularization of the new chondral tissue (Figure 37) lead to mineralized cartilage which was then followed by ossification (Figure 38). Through microscopic examination of the new bone tissue using polarized light, it appears that the collagen fibrils are oriented like in a normal and healthy bone tissue as would be expected in a bone tissue generated through the endochondral ossification process (Figure 39 and 40). Conclusion The new bone tissue generated during the bone healing was of a regular lamellar structure and was shown to be developed through the endochondral bone formation pathway.

Conclusive remarks

The inventors have conducted animal studies showing that a novel composition comprising controlled amount of biologically and therapeutically active partially deacetylated chitin derivatives contains all three of the requisite mechanisms of the new class of biomaterials for bone grafts. The novel composition fills bone voids providing an osteoconductive scaffold with optimal mechanical properties as well as granting osteoinductive stimulus for generation of new bone growth into the scaffold. This new bone growth takes place through an endogenous process referred to as "endochondral ossification"; similar process as induced by BMP. This bone formation produces normal lamellar bone architecture instead of fibrous and woven bone tissue, produced by fibroblasts in untreated bone injuries. Moreover, the novel composition is able to efficiently and rapidly close and heal bone defects, eliminating the risk of non union repair seen when using many OC devices.

The composition is also applicable for use with the newly adopted balloon vertibralplasty and Kyphoplasty, which both utilize a cement-like material that is injected directly into a fractured osteoporotic vertebral bone. The composition is applicable to replace the currently available cement-like material, by not only offering mechanical properties but also the ability to induce regeneration of new bone to support the osteoporotic vertebrae.

The biological mechanisms behind the apparent osteoinductive activity of the chitinous polysaccharide is suggested to occur through modulation of the activity and expression levels of tissue specific chitinase like proteins as disclosed in a recent patent application filed by the inventors. The chitin derivatives are suggested to exert their activity through continuous release of oligomeric chitinous compounds capable of modulating local inflammation and thereby suppressing the down regulation of the Sox9 transcription factor, a vital component for chondrogenesis, the initial step in endochondral ossification. Through modulation of the activity of YKL-40 expressed by diversifying chondrogenic progenitor cells, we suggest that the in situ released chitinous oligosaccharides generated through hydrolysis by Chitotriosidase and Acidic Mammalian Chitinase expressed locally by phagocytes, play a role as chitinous ligand binding to the YKL-40 protein. After the neonatal stage or infancy, there is no evidence to indicate that endogenous chitin oligosaccharide production occurs that is comparable to what has been shown in early embryonic development as shown by Lee, Semino and Yoshida, Rosa and Bakkers (1, 10-14) and reviewed by van der Hoist [16]. We suggest that this absence of the endogenous chitooligomers might be one of the reasons why the ability of forming new bone through endochondral ossification is lost during the development of the individual from early embryonic stage through infancy. The chitinous ligands provided by the current invention will interact with the YKL-40 protein and thereby induce chondrogenesis through chondrogenic progenitor cells in the bone marrow, the periosteum or in the endosteum.

The present invention presents a state of the art non-protein third generation of fully biocompatible and biodegradable biomaterial providing possibilities for mechanical support to the injured bone tissue releasing osteoinductive molecules through endogenous hydrolysis into the injured site. All ingredients of the current invention are natural to the metabolic pool of the organism, calcium phosphates and sulphates being, resorbed and remodeled by the bone tissue and the chitinous monomers, glucosamine and N-acetyl-glucosamine, being a major constituents of extracellular matrixes and constituents in protein glycosylation throughout the entire body. The major concern in the formulation of the invented compositions is therefore not toxicity of the break-down products but the correct dosage of the osteoinductive chitinous ingredient.

In contrast to BMP or any other osteoinductive protein, our osteoinductive carbohydrate formulations will not induce bone formation outside the bone environment and are less sensitive than the proteins and will tolerate a wide range of pH, temperature and radiation exposure, offering more options in formulation and sterilization compared to proteins.

Claims

1. Use of chitosan for the manufacture of a medicament for inducing or enhancing endochondral ossification at a tissue site.

2. The use according to claim 1, wherein the medicament is in a form selected from the group consisting of a paste, a cream, a solution, a film, a coating, a gel or solid bars or plates, or a combination thereof.

3. The use according to claims 1 or 2, wherein the medicament is formulated in a soft film or tape with plastic characteristics that can be directly laid or rolled on the bone surface

4. The use according to claims 1-3, wherein the medicament is administered by injection, surgical procedure, oral administration.

5. The use according to any claims 1-4, wherein said tissue site is bone or cartilage.

6. The use according to any claims 1-5, wherein said tissue site is traumatized.

7. The use according to claim 6 wherein the medicament is applied directly on the severed surfaces of bone to be re-joined.

8. The use according to any claims 1-7, wherein the medicament is for tissue repair or tissue regeneration.

9. The use according to any claims 1-8 wherein the medicament comprises a support matrix material.

10. The use according to any claims 1-9 wherein the medicament comprises chitooligomers for an immediate initiation of the endochondrial ossification.

11. The use according to claim 9 wherein the support matrix material is selected from the group consisting of calcium phosphates, including hydroxyapatite, tetracalcium phosphate, calcium sulphate and sodium tri-polyphosphate.

12. The use according to any of the preceding claims, wherein the medicament is intended for patients suffering from a disease selected from the group of rheumatoid arthritis, osteoarthritis, osteoporosis.

13. The use according to any of the preceding claims, wherein the medicament is intended for patients suffering from a cartilage defect or damage.

14. A method for inducing or enhancing endochondral ossification at a tissue site:

- supporting the tissue site with a scaffolding mechanism, - placing an therapeutically effective amount of a composition comprising chitosan at the tissue site,

- securing the tissue site in a fixed position.

15. The method of claim 14, wherein the composition comprising chitosan is in a form selected from the group consisting of a paste, a cream, a solution, a film, a gel or solid bars or plates, or a combination thereof.

16. The method according to claims 14-15, wherein said tissue site is bone or cartilage.

17. The method according to claims 14-16, wherein said tissue site is traumatized.

18. The method according to claims 14-17, wherein the composition is applied directly to the severed surfaces of bone to be re-joined.

19. The method according to claims 14-18, wherein the composition comprises a support matrix material selected from the group consisting of calcium phosphates, including hydroxyapatite, calcium sulphate and sodium tri- polyphosphate.

20. A method for controlling bone formation and/or functional tissue regeneration through the endochondrial ossification pathway, the method comprising:

placing an therapeutically effective amount of a composition comprising chitosan and a support matrix between parts of a sectioned bone or a bone fracture, arranging and securing parts of a sectioned bone or a bone fracture composition in a desired position by metal fixtures, allowing bone healing through endochondrial ossification, optionally removing the metal fixtures,

wherein the bone healing and/or bone formation involves development of cartilage with well developed vascularisation and subsequent formation of new bone tissue without scar formation.

21. A kit for inducing or enhancing endochondrial ossification at a tissue site, the kit provided sterile and at least comprising: chitosan scaffold for supporting tissue formation or regeneration, and optionally a support matrix material.

22. Pharmaceutical composition comprising chitosan for inducing or enhancing endochondrial ossification at a tissue site.

23. A pharmaceutical composition according to claim 22, wherein the composition further comprises chitooligomers.

24. A pharmaceutical composition according to claims 22-23, wherein the composition may optionally be used with other medicaments selected from the following classes: a. a hemostatic agent; b. an anti-infective agent; c. an anti-inflammatory agent; d. an anti-cancer agent; e. a protein; f. a bone cement.

25. A pharmaceutical composition according to claims 22-23, wherein the composition may optionally be used with other bone graft materials selected from the following: a. an autograft; b. an allograft; c. a demineralized bone; d. a bone marrow; e. a bone cement; f. a bone protein; g. a stem cell.