3D printed bioactive scaffolds

Abstract

An implantable tissue scaffold is provided, comprising a mixture of a biocompatible organic polymer and chitin embedded within the biocompatible organic polymer. Also provided is a composition for 3D printing, comprising at least one biocompatible organic polymer and chitin, which can be partially deacetylated, wherein the chitin is embedded within the biocompatible organic polymer. Further provided is a method for promoting tissue formation, comprising implanting a tissue scaffold comprising a mixture of a biocompatible organic polymer and chitin at a site requiring regenerative bone tissue formation.

Description

[Technical Field]

[0001] The present invention is in the field of medical implants, particularly implants comprising bioactive and biocompatible materials suitable for use in osteoinductive and / or osteogenic applications. The present invention is also in the field of 3D printable compositions, particularly compositions comprising bioactive and / or biocompatible organic polymers. [Background technology]

[0002] Medical knowledge regarding the management of trauma and other bone defects has progressed and improved significantly worldwide. However, autologous bone grafting is still considered the gold standard for repairing bone defects. Clinical benefits are not guaranteed, and donor site complications and morbidity are not uncommon. In some cases, one or more additional interventions are required, and graft materials are limited. To date, segmental bone defects caused by trauma, bone tumors, revision surgery, or infection remain a major challenge for trauma surgeons. Although several materials and various implant options have been developed or improved, a perfect solution, especially for filling large defects, remains elusive.

[0003] Tissue engineering is the artificial modification or reconstruction of human tissues, intentionally directing tissue growth through controlled molecular signals and / or directed physical / mechanical channeling. Currently, the fabrication of three-dimensional scaffolds is dominated by traditional manufacturing techniques, such as phase separation, solvent casting, membrane coating, electrospinning, molding, and foaming. However, all of these methods share the major drawback of being unable to fully control the structure of the scaffold, its pore network, and pore size, resulting in inconsistent and less-than-ideal scaffolds.

[0004] 3D printing technology is becoming increasingly important in tissue engineering because it offers the ability to integrate biomedical device designs into actual manufacturing processes, thereby providing the opportunity to produce objects with controlled macro- and microstructures. Compared to traditional material processing techniques for tissue engineering of highly complex tissues like bone, 3D printing not only offers much more precise and reproducible detail, but also a more systematic approach for further research and development. In real-time conditions, this methodology offers the opportunity to design and print customized scaffolds during the surgical procedure based on real-time assessment and imaging of the required implant, thereby assisting the surgeon with high reproducibility of the machined results and minimizing potential errors in the surgical process. Summary of the Invention [Problem to be solved by the invention]

[0005] In addition to being printable and osteoinductive / osteoconductive, materials suitable for tissue engineering, especially bone regeneration applications, must also be biodegradable, antibacterial, endotoxin-free, nontoxic, and mechanically stable. Many materials have been reported for bone scaffolds, including collagen, hyaluronic acid, hydroxyapatite, bioglass, titanium, polylactic acid, PMMA, and carbon nanotubes. However, it has become clear that all of these materials, while osteoconductive, lack osteoinductive properties and therefore do not meet the osteoinductive / osteoconductive requirements of implants (Baldwin et al., 2019, J Orthopaed Trauma 33:203; D'Souza et al., 2019, Biomedics 7:1). Therefore, there is an urgent need to develop 3D-printable osteoinductive and osteoconductive biomaterials to meet the needs of orthopedic applications in the near future. [Means for solving the problem]

[0006] The present invention aims to overcome the above-mentioned deficiencies and drawbacks of the prior art. It is an object of the present disclosure to provide tissue scaffolds, compositions and methods for fabricating such scaffolds, in which bioregenerative properties, particularly those characterized by consistent osteoinductive / osteoconductive properties, are combined with a printable solution, particularly 3D printing. A key feature is the incorporation of unconventional chitin-based materials into biocompatible and / or biodegradable osteoconductive polymers to form composites suitable for use in additive manufacturing methods, e.g., based on material extrusion.

[0007] Thus, in one aspect, an implantable tissue scaffold is provided that comprises a mixture of a biocompatible organic polymer and chitin that is physically embedded within the biocompatible organic polymer.

[0008] The chitin used in the scaffolds and compositions described herein is a partially deacetylated chitin-based material with a controlled distribution of glucosamine moieties in the polymer chain. The chitin-based material can be provided as discrete particles dispersed within a biocompatible organic polymer. The implantable tissue scaffolds can be 3D printed using conventional 3D printing techniques.

[0009] The implantable tissue scaffold is adapted for implantation into the human or animal body. Upon implantation, the chitin embedded within the scaffold is slowly released and degraded by natural processes to form desired short-chain chitooligosaccharides (COS). The COS thus released promotes healing and natural tissue growth at the implant site.

[0010] Another embodiment relates to a composition for 3D printing, comprising at least one biocompatible organic polymer in the range of about 75% to 99.95% by weight and chitin in the range of about 0.05% to 20% by weight. The chitin is preferably embedded in the biocompatible organic polymer in the composition in the form of discrete particles. The chitin may be partially deacetylated, as described in more detail herein.

[0011] Therefore, implantable tissue scaffolds can be formed by a process of 3D printing the composition.Accordingly, another embodiment relates to a method for making tissue scaffolds, comprising 3D printing the composition described herein.3D printing can be performed based on three-dimensional modeling of the tissue defect to be repaired and / or healed, such as bone defects and fractures.Such modeling can be based on the analysis of one or more imaging methods, including computed tomography (CT), magnetic resonance imaging (MRI), radiological bone scan, ultrasound imaging, and radionucleotide bone imaging.

[0012] Another aspect relates to a method of promoting tissue formation, comprising implanting a tissue scaffold described herein into a human or animal body at a site requiring regenerative tissue formation. In certain embodiments, the target tissue is bone tissue. In certain embodiments, the method relates to a human body. In certain embodiments, the method relates to an animal body.

[0013] Another aspect relates to a method for treating a bone tissue defect comprising implanting a tissue scaffold described herein into the human or animal body at a site requiring regenerative bone tissue formation.

[0014] Yet another aspect relates to an implantable tissue scaffold as described herein for use in treating a tissue defect in the human or animal body. The tissue defect may preferably be a bone tissue defect, such as a fracture or a missing bone.

[0015] The above features, along with additional details of the invention, are further illustrated in the following examples, which are intended to further illustrate the invention and are not intended to limit the scope of the invention in any way.

[0016] Those skilled in the art will appreciate that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows an image obtained by scanning electron microscopy (SEM) of partially deacetylated chitin (PDC) used to create tissue scaffolds. [Figure 2] Shown is an SEM image of a tissue scaffold visualizing PDC particles (shown as circles) and larger CaP particles (shown as rectangular boxes). [Figure 3] Images obtained by micro-CT showing the homogeneous distribution of CaP particles within the filaments are shown for different compositions of CaP / PDC (see Table 2), with a cross-sectional view in (a) and a longitudinal view in (b). [Figure 4] Figure 1 shows the results of a stability study of partially deacetylated chitin during 3D printing at 200 °C as determined by X-ray diffraction (XRD): (a) stability over time; (b) crystallization rate and crystal size at various processing times. [Figure 5] Figure 1 shows the various types of unit cells used in 3D-printed PDC / PLA scaffolds, including straight laminated beams (SSB, A), porous sodalite crystals (PSC, B), and porous hexagonal pillars (PHP, C). For each scaffold type, the porosity is varied between 25%, 50%, and 70% (from left to right for each scaffold shown). [Figure 6] SEM images of the 3D printed scaffolds are shown at both the macro and micro levels, with macro images on the right and micro images on the left for each cell type. [Figure 7] 1 shows the compressive strength measurements of 3D printed PDC / PLA scaffolds containing 0.25%, 0.5%, and 1.5% PDC. [Figure 8] Figure 1 shows the compressive strength of 3D printed PDC / PLA scaffolds containing different amounts of PDC for two types of structures: straight laminated beam (SSB) and porous sodalite crystal (PSC). [Figure 9] 1 illustrates a conceptual framework according to the present invention for bone regeneration applications. [Figure 10] CT images of the surgical site of a rat femur containing a control implant (A) and an implant containing 0.25% chitosan (B) are shown. [Figure 11] The results of bone formation determined from CT images of the animals used in the rat test are shown. [Figure 12] (a) shows a highly swollen, partially deacetylated (approximately 50%) chitin-based material, and (b) shows the clear solution formed when the chitin-based material is dissolved with 100% solubility. DETAILED DESCRIPTION OF THE INVENTION

[0018] This disclosure provides a conceptual framework for 3D-printable biopolymer composites with bioregenerative properties. Such materials are suitable for use, for example, in the fabrication of customized 3D-printed scaffolds and material extrusion (fused deposition modeling, FDM). In these composites, chitin is incorporated into a biodegradable polymer, providing a biopolymer composite comprising a biodegradable polymer and a chitin-based material. The biodegradable polymer provides mechanical stability and osteoconductive properties, while chitin is believed to impart osteoinductive properties to the resulting composite, in addition to the well-known antibacterial and hemostatic properties of chitosan.

[0019] Biopolymer composites can be 3D printed to produce scaffolds with any desired structure and porosity. This allows engineered scaffolds to be designed and printed for in situ use, providing mechanical stability at the implant site. Over time, the biodegradable polymer is replaced by naturally formed bone tissue, which is facilitated by the osteoinductive properties of the chitin-based material within the polymer. The new bone tissue migrates into the porous scaffold, where it is stimulated by the osteoconductive properties of the scaffold and the osteoinductive properties of the chitin-based material to form new bone tissue.

[0020] Chitin is a linear polysaccharide composed of N-acetylglucosamine (GlcNAc) monosaccharides linked by 1-4β bonds to form a linear biopolymer. Chitin is a basic component of the exoskeletons of crustaceans and insects and certain organs of mollusks, such as the shells of squid and cuttlefish.

[0021] Chitin-based materials can be partially or fully deacetylated; 0% deacetylated chitin is a homopolymer of N-acetylglucosamine subunits linked by β-(1→4) covalent bonds, while 100% deacetylated chitin is a homopolymer of glucosamine (GlcN) subunits linked by β-(1→4) covalent bonds. Chitosan is the deacetylated form of chitin and contains a mixture of GlcN and GlcNAc.

[0022] There are three different types of chitin: α-, β-, and γ-chitin. These chitins all have different crystalline states, which are primarily due to the orientation or packing of the polymer chains within the matrix. Because of these differences, deacetylated chitins with the same %DD can have significantly different physicochemical properties depending on their crystalline state.

[0023] On the other hand, there are at least three different types of deacetylation methods: a) high-temperature solid-state or heterogeneous deacetylation (hereinafter referred to as SST), b) low-temperature liquid-state or homogeneous deacetylation (hereinafter referred to as LSL), and c) low-temperature solid-state or heterogeneous deacetylation (hereinafter referred to as SSL). Currently, the SST deacetylation method is commonly adopted in the chitin industry. Comparing the distribution of glucosamine deacetylation at 50% DD, SST deacetylation produces a large amount of block or clustered glucosamine within the polymer chain. In the LSL method, the arrangement of glucosamine and N-acetyl-D-glucosamine is generally alternating, i.e., one monomer is arranged next to the other, while in SSL, the arrangement is random, which is intermediate between SST and LSL. Because of this distribution pattern of glucosamine or N-acetyl-D-glucosamine, the physicochemical properties of deacetylated materials can have completely different characteristics even when the degree of deacetylation is the same. For example, some may have the property of swelling significantly in contact with water, whereas SST materials with the same %DD may not swell, or some may be completely soluble in acid, whereas others may show little solubility. Upon degradation in the body, the differences in the oligomer patterns obtained by these three types of deacetylation become even more pronounced, leading to differences in their in vivo effects on tissue regeneration.

[0024] The degree of deacetylation (%DD) and average molecular weight are the two most important characteristics considered in the current chitin industry. However, this applies to chitin with a %DD of 75% or greater. For chitin below 75% DD, more characterization is needed to understand its properties, particularly the distribution of glucosamine moieties within the polymer chain and the crystalline state of the material. Partially deacetylated chitin (chitosan) or chitosan has more complex physicochemical properties; therefore, it may be somewhat or highly crystalline, or partially deacetylated chitin may have an amorphous structure. These characteristics are highly dependent on the manufacturing process and, therefore, on the molecular composition of the partially deacetylated chitin. Further complicating factors include the unique properties of different types of chitin, i.e., α, β, and γ forms.

[0025] Therefore, for chitins with similar deacetylation degrees, differences in the distribution of glucosamine moieties significantly affect all physical properties of the material, including toughness or mechanical properties, swelling, dissolution, and thermal properties (Aiba, S. Int J Biol Macromol, 1991, 13(1), 40-44; Sannan, T., et al., Makromol Chemie, 1976, 177(12), 3589-3600). As mentioned above, the current chitosan industry's manufacturing process lacks control over the high-temperature process during production, resulting in low-quality chitosan that can only be applied to low-demand industries and does not meet the demand for medical or orthopedic purposes. The material used herein is the result of optimizing its physicochemical properties and carefully controlling the distribution of glucosamine to a desired arrangement so that, after in vivo degradation, it will have a desirable oligosaccharide (COS) pattern that effectively initiates tissue regeneration. The resulting material preferably has a random distribution of glucosamine moieties (and thereby a random distribution of N-acetylglucosamine) within the polymer chains.

[0026] Chitin-based materials are preferably manufactured by specific processes to meet certain characteristics, in particular a) a controlled distribution of glucosamine moieties within the chitin matrix and polymer chains, and b) a low amorphous state to ensure a smooth, desirable degradation pattern. These two parameters are important to the extent that, for chitins of similar deacetylation degree, differences in the distribution of glucosamine moieties significantly affect the physical properties of different chitin polymers, such as the toughness or mechanical properties of the material, swelling, dissolution, and thermal properties, and resistance to precipitation after dissolution.

[0027] Differences in the physicochemical properties of chitin-based materials also lead to differences in biological responses to the materials. Therefore, with regard to the biological effects that occur during degradation in the body, differences in the distribution of glucosamine affect the enzymatic cleavage sites, resulting in different degradation oligomer products. Because these degradation products are thought to exert their biological activity by binding to chitinase-like proteins (CLPs) or other receptors in the human or animal body, the nature and distribution of these products (short-chain oligosaccharides) can lead to different biological responses, resulting in significantly different effects on tissue regeneration.

[0028] The percent degree of deacetylation (%DD) is the proportion of GlcN in a copolymer composed of GlcNAc and GlcN. The %DD value of a chitin / chitosan sample is an important factor in evaluating its properties. Chitin with a %DD value of less than 35% is insoluble in weak acids, such as 1% acetic acid. However, chitin / chitosan with a %DD value of more than 75% is soluble in weak acids. The solubility of partially deacetylated chitin in weak acids is significantly affected by its processing method. Chitin-based materials with randomly distributed glucosamine and a %DD of approximately 50% are soluble in weak acids, such as acetic acid. Therefore, such materials are expected to have higher bioavailability than less soluble chitin-based materials.

[0029] Partially deacetylated chitin, i.e., chitin with a degree of deacetylation greater than 0%, is sometimes referred to as chitosan. Thus, chitosan can generally be constructed with any desired degree of deacetylation. Furthermore, chitosan can be randomly deacetylated, i.e., deacetylation can occur at random positions along the polymer chain. Alternatively, chitosan can be block deacetylated, i.e., the deacetylation pattern has glucosamines clustered or aggregated within the polymer chain. The term "chitin" as used herein refers to chitin-based materials that can have any degree of acetylation. That is, the degree of deacetylation can range anywhere from 0 to 100%. The term "chitosan" as used herein refers to chitin-based materials that are partially (i.e., greater than 0%) deacetylated. Thus, the terms "chitin" and "chitosan" can be used interchangeably to refer to partially deacetylated chitin (PDC)-based materials.

[0030] Compared to fully acetylated chitin, partially deacetylated chitooligosaccharides (oligosaccharides) are smaller and more water-soluble and are prepared by deacetylating chitin to produce chitosan. Either chitin or chitosan can be hydrolyzed to produce chitooligosaccharides with various degrees of acetylation. Short-chain chitooligosaccharides are more water-soluble than polymeric chitin, and chitooligosaccharides with a high degree of deacetylation are particularly highly water-soluble.

[0031] Swelling index and solubility are two simple methods for assessing the distribution of glucosamine in chitin of 75% DD or less. The more randomly the glucosamine is distributed in the polymer chain, the more swellable and soluble the chitin is. This is particularly evident in 50% DD chitin. For example, the swelling index of a chitin film coated with randomly deacetylated 50% DD chitin can reach 6–10 times its dry weight, and it generally has high solubility. In chitin with more randomly distributed glucosamine, the chitin also has a relatively low crystalline state. In addition, in prepared chitin solutions, more randomly distributed chitin is more resistant to precipitation than less randomly distributed chitin.

[0032] Bone graft materials are typically classified into three types: osteoconductive, osteoinductive, and osteogenic. Osteoconductive refers to the implantation of a material that functions as a scaffold for new bone growth that is permanently maintained by natural bone. The goal is for new bone to passively grow within the scaffold or channel itself to achieve specified dimensions (e.g., pores, channels, or pipes). Osteoinduction, for example, is the process by which bone formation is induced by a biomaterial. Osteoinduction involves the recruitment of cells and their activation to develop into preosteoblasts, a normal phenomenon observed during bone healing. In the setting of a fracture, the majority of bone heals through the osteoinductive process. Osteogenesis refers to the process by which bone-generating cells, either directly present (as osteocytes, osteoblasts, or chondroblasts) or indirectly present from previously undifferentiated stem cells, form new bone by depositing osteoid or by endochondral ossification of cartilage.

[0033] Recent research conducted by the present applicant has shown that chitosan has osteoinductive properties, making it an ideal candidate as a component of bone regeneration materials (Kjalarsdottir, et al., 2019, Regen Biomater 6:231). Furthermore, the degree of deacetylation of chitosan and the method of preparation of the material play a crucial role in its osteoinductive properties. Thus, chitin with a deacetylation degree ranging from approximately 50% to 70% using this deacetylation method has been shown to result in significantly higher levels of tissue regeneration than highly deacetylated chitosan.

[0034] Chitosan has been shown to offer many advantages in biomedical applications, including controlled biocompatibility and biodegradability and modulation of inflammatory responses. Furthermore, chitosan is bioabsorbable, antibacterial, nontoxic, polycationic, and has great potential in tissue regeneration, making it suitable for a wide range of pharmaceutical and medical applications, including as an antibacterial agent, controlled drug delivery, blood anticoagulant, wound dressing, and tissue engineering, including bone and nerve regeneration. Chitosan has been extensively studied in bone tissue engineering. Chitosan can function alone or be blended with other polymers and natural and synthetic materials, which is generally considered an effective method for developing chitosan-based tissue engineering materials, such as 3D freeze-dried scaffolds, hydrogels, films, and other scaffolds.

[0035] Chitosan has been shown to have osteoinductive / osteoconductive properties in numerous studies (Tan et al. 2014, Biomaterials 35:7828; Geffre et al. 2010, Future Sci OA4:FSO225). In vivo studies suggest that chitosan alone is sufficient to stimulate bone formation (Pang et al. 2017, Oncotargt 8:35583; Ho et al. 2015, Int J Nanomedicine 10:5941). More importantly, the degree of deacetylation (DD) of chitosan-based materials has been shown to have a decisive influence on bone formation both in vitro and in vivo (Lieder et al. 2012, J Biomed Mater Res Part A,100A:3392; Kjalarsdottir et al. 2019, Regen Biomater 6:231). However, chitosan itself does not possess sufficient mechanical strength for load-bearing applications.

[0036] To overcome this drawback of chitosan and provide a mechanically stable solution, the tissue scaffolds and compositions described herein provide for the incorporation of chitin, particularly partially deacetylated chitin, into a suitable biocompatible and / or biodegradable polymer.

[0037] Typically, the amount of chitin in the tissue scaffold and / or composition can be in the range of about 0% to 20%, about 0% to 10%, about 0% to 5%, about 0.05% to 20%, about 0.05 to 15%, about 0.05% to 10%, about 0.05% to 5%, about 0.05 to 3%, about 0.1 to 20%, about 0.2 to 20%, about 0.5 to 20%, about 0.7 to 20%, or about 1 to 20%. The lower end of the range can be about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0%. The upper limit of the range can be about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4%, about 4.5%, about 5%, about 7%, about 8%, about 10%, about 12%, about 14%, about 15%, about 16%, about 18%, or about 20%.

[0038] The amount of chitin-based material used in the scaffold and / or composition can depend on its degree of deacetylation (%DD). Thus, a PDC material with a relatively low %DD (e.g., less than 75%) can be present in a lower amount in the scaffold than a PDC material with a relatively higher %DD (e.g., greater than 75%). For example, for a %DD greater than 75% chitin, the amount of chitin in the scaffold can be in the range of 0.1-20%, 0.1-10%, or 0.1-5%.

[0039] The chitin-based material can be partially or completely deacetylated. For example, the chitin-based material can have a degree of deacetylation in the range of 0 to 100%, about 10% to about 90% (w / w), about 10% to about 70% (w / w), about 20% to about 70% (w / w), about 30% to about 70% (w / w), 35 to about 65%, about 35 to about 60%, about 40% to about 60% (w / w), about 40 to about 55%, or about 45% to about 55% (w / w).

[0040] The lower limit of the range of suitable degrees of deacetylation may be 0%, about 5%, about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.The upper limit of suitable degrees of deacetylation may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 99%.

[0041] In certain applications, the chitin-based material has a degree of deacetylation in the range of about 45% to about 95% (w / w), in the range of about 50% to about 90% (w / w), in the range of about 50% to about 80% (w / w), or in the range of about 50% to about 70% (w / w).

[0042] The chitin-based material may have a weight average molecular weight (MW) in the range of 200 Da to 2000 kDa, for example in the range of 1 kDa to 1000 kDa, in the range of 5 kDa to 500 kDa, in the range of 10 kDa to 300 kDa, or in the range of 20 kDa to 1500 kDa.

[0043] The chitin-based material may preferably comprise amorphous partially deacetylated chitosan (PDC). This material may be produced by a controlled process with a specific glucosamine distribution, resulting in a material that is soluble in aqueous acidic solutions and has a high swelling capacity. Such chitosan is preferably about 45% to about 55% deacetylated (e.g., about 50%) and completely soluble in acidic solutions (i.e., 100% solubility). The chitosan may have a weight-average molecular weight ranging from 100 kDa to 400 kDa, such as about 300 kDa. Chitosan can absorb 10, 15, or about 20 times its weight in water, or more.

[0044] In some embodiments, chitin can be completely dissolved in a weakly acidic solution, such as an acetic acid solution. In other words, chitin is 100% or nearly 100% (e.g., greater than 99%) soluble in an acidic solution. Chitin-based materials dissolve instantly or nearly instantly (within minutes, e.g., within 10 minutes or within 5 minutes) upon acidification. This differs from chitosan, known in the art, which typically dissolves very slowly or not at all in acidic solutions.

[0045] Solubility can be assessed by passing a solution containing PDC through a filter, such as a 0.45 micrometer filter, with the absence of insoluble particles that do not pass through the filter being a measure of complete dissolution.

[0046] Chitin-based materials have a high swelling capacity and form gels when in contact with water. The ability to form gels can be expressed as the ability to absorb 10 or more, 15 or more, or 20 or more times its weight in water (in the dry state). The ability to form gels swollen 10 or more times is particularly pronounced for chitins with high molecular weights, such as those with molecular weights of 150 kDa or more, 200 kDa or more, or 250 kDa or more.

[0047] The chitin-based material may preferably be in the form of microparticles. The microparticles may have an average particle size in the range of about 0.1 μm to about 50 μm, about 1 μm to about 25 μm, about 1 to 15 μm, 5 to 20 μm, or about 5 μm to about 15 μm. The lower limit of the range may be about 1 μm, about 2 μm, about 3 μm, about 4 μm, or about 5 μm. The upper limit of the range may be about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, or about 50 μm.

[0048] The microparticles are preferably physically embedded and dispersed within a biocompatible organic polymer. Thus, the microparticles can be visualized as independent physical entities within the organic polymer. The particles can be uniformly dispersed within the organic polymer. By physically embedding the particles within the polymer, the solubility problem of chitin is eliminated. Therefore, physically embedding chitin within a polymer is suitable for all chitins, regardless of the degree of deacetylation.

[0049] The microparticles may preferably comprise only chitin-based material, i.e., the particles consist essentially of chitin-based material, such that the implantable scaffold comprises a biocompatible and / or biodegradable organic polymer and microparticles consisting essentially of chitin-based material.

[0050] Any suitable organic polymer that is biocompatible and / or biodegradable may be used for the applications described herein. For example, the organic polymer may be selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

[0051] The organic polymer may comprise or consist of a thermoplastic resin such as polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polyethylene, polypropylene or mixtures thereof.

[0052] For example, polylactic acid (PLA) is a widely used biomaterial that has proven its value in various medical applications. PLA is a naturally derived, biodegradable, and bioactive thermoplastic aliphatic polyester. PLA is produced from corn starch, cassava root, chips, or starch, or sugarcane. PLA can be biodegraded both in nature and under physiological conditions by simple hydrolysis of the ester backbone, forming harmless, non-toxic compounds. The advantages of PLA in bone engineering are its biocompatibility, thermoplasticity, and mechanical properties. PLA possesses mechanical properties that make it suitable for temporary load-bearing applications and can be easily processed using 3D FDM printing technology. PLA and its copolymers are widely used in diverse fields, including polymer engineering, tissue engineering, drug delivery systems, and various critical medical implants.

[0053] PLA is commonly found as a mixture of PLLA and PDLA, referred to as poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), or poly(DL-lactic acid) (PDLLA). The mixture may conveniently be racemic, i.e., a 1:1 mixture of PLLA and PDLA. PDLLA tends to be a more amorphous, less crystalline form than PLLA and PDLA.

[0054] PLA has attracted considerable attention as a bone engineering material over the past 20 years due to its superior processability and properties compared to other biodegradable polymers. Because of its high surface energy, PLA is easy to print and is therefore widely used in 3D printing. PLA's glass transition temperature is 60°C and its melting point is 190°C (Total Corbion PLA). Its basic mechanical properties are comparable to those of polystyrene and PET. However, PLA's flexural modulus is higher than that of polystyrene, and PLA has excellent heat-sealing properties. Suitable PLA for use in the present invention can be any suitable PLA-based material, including poly-L-lactide (PLLA) and poly-D-lactide (PDLA), as well as any combination or blend of PLLA and PDLA. In some embodiments, the PLA is PLLA.

[0055] Other suitable biomaterials that may also or instead be used in the scaffolds and compositions described herein include poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyhydroxyalkanoic acid (PHA), polyethylene or polypropylene, and any mixtures or combinations thereof.

[0056] The chitin-based material can be homogeneously mixed into the polymer-based material, or the chitin can be present as physically dispersed and embedded particles within the polymer-based material, i.e., the chitin is present as small, discrete particles within the polymer-based material.

[0057] Chitin-based materials can typically be in the form of fine particles having an average particle size in the range of about 0.1 μm to about 50 μm, about 1 μm to about 25 μm, about 1 to 15 μm, or less than 20 μm, or about 5 μm to about 15 μm, or about 3 to 10 μm.

[0058] It may be appropriate for the tissue scaffold to be at least partially porous, i.e., at least one or more portions of the scaffold have a porous structure, which facilitates cell recruitment and proliferation to the site of the scaffold, thereby enhancing and / or accelerating the tissue regeneration process at the implantation site.

[0059] The porosity of the scaffold can typically range from about 10% to about 90%, from about 20% to about 70%, from about 30% to about 70%, or from about 40% to about 60%. In some embodiments, the scaffold has a porosity of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%. In this context, the term "porosity" refers to the volume of the three-dimensional scaffold structure that is open, i.e., does not contain polymeric material.

[0060] The porosity and / or pore shape of the implant can be varied as needed, taking into consideration factors such as the required mechanical strength of the scaffold, the nature of the tissue requiring regeneration, and the amount of chitin-based material incorporated into the scaffold. Typically, the scaffold can have a porosity ranging from about 10% to about 90%.

[0061] By varying the porosity of the 3D printed structure, the scaffold will have variable mechanical strength. Thus, typically, the scaffold can have a mechanical strength with a yield point in the range of 1-50 MPa, 2-50 MPa, 5-50 MPa, or 10-40 MPa. In some cases, the scaffold has a porosity of 50% or less and a mechanical strength of at least 10 MPa.

[0062] The tissue scaffold may preferably be free of non-biodegradable materials, i.e., the tissue scaffold is entirely biodegradable, such that, once implanted, the tissue scaffold gradually degrades in situ over time. Thus, the tissue scaffold preferably does not include components that are metals or alloys or other components that are not biodegradable.

[0063] Without being bound by theory, it is believed that chitin-based materials embedded within tissue scaffolds are degraded by natural degradation processes in the body catalyzed by natural chitinases, lysozymes, or other natural degradation processes. The degradation process releases short-chain chitooligosaccharides, particularly with a controlled distribution of glucosamine, which are believed to be the bioactive form of chitin-based materials and exhibit osteoinductive properties as further described herein.

[0064] The chitooligosaccharides produced in this way induce tissue regeneration processes at the implantation site, such as the recruitment of bone-forming cells around a 3D-printed implant inserted into a bone defect, along with blood cell formation around the implantation site, resulting in activated bone formation and bone tissue regeneration. Over time, the implant, along with the incorporated chitin-based material, is completely degraded, but still promotes healthy tissue formation. In the case of bone regeneration, this includes the induction of ossification, which leads to tissue remodeling and the formation of natural bone tissue.

[0065] In relation to the bone regeneration process, the conceptual framework according to the present invention can be explained as shown in FIG. 9 . Thus, chitin-based materials (exemplified here as partially deacetylated chitin or chitosan) possess osteoinductive properties in addition to known antibacterial and hemostatic properties. Chitin-based materials can be incorporated into biocompatible polymers, exemplified here as PLA, from which filaments suitable for 3D printing applications are generated. The filaments are then 3D printed, guided by images obtained, for example, by CT or MRI, to generate bioinductive, e.g., osteoinductive / osteoconductive, scaffolds. The scaffolds thus generated can then be implanted at sites requiring tissue regeneration, such as bone defects (e.g., missing bone or bone deformity), including fractures. Upon implantation, the chitin within the scaffold is degraded to release bioactive chitooligosaccharides, which promote tissue regeneration at the implantation site, as demonstrated here by the initiation of bone formation and subsequent bone tissue regeneration. Over time, a guided ossification process occurs in which the originally implanted scaffold is degraded by natural processes and replaced by remodeled native bone tissue.

[0066] The biocompatible polymer can be any suitable polymer-based material that provides the necessary mechanical stability and chemical integrity during 3D printing. Preferably, the biocompatible polymer is biodegradable. Exemplary suitable biocompatible polymer-based materials include polylactic acid (PLA) and polyhydroxyalkanoic acid (PHA).

[0067] In addition to chitin, it may be advantageous to incorporate calcium phosphate into the biopolymer composite. The resulting scaffold would consist of a biopolymer / calcium phosphate composite with chitin-based material embedded in it. Calcium phosphate is a well-known bioactive and biodegradable implant material known for use in bone cement applications. This material can be in crystalline form, and its crystallinity (crystal size, crystal perfection, granularity) can be varied.

[0068] The amount of calcium phosphate may be in the range of 0.2% to 20% (w / w), for example in the range of about 0.5% to 15% (w / w), in the range of about 0.5% to 10% (w / w), in the range of about 1% to 10% (w / w), in the range of about 2% to 10% (w / w), or in the range of about 2% to 8% (w / w).

[0069] The calcium phosphate may be in the form of microparticles that may have an average diameter in the range of about 1 to 100 μm, about 5 to 70 μm, about 5 to 60 μm, about 10 to 70 μm, about 10 to 60 μm, about 10 to 50 μm, or about 10 to 40 μm, and the microparticles are dispersed in a biocompatible organic polymer.

[0070] In some embodiments, the calcium phosphate microparticles have a diameter of less than 80 μm, less than 70 μm, less than 60 μm, or less than 50 μm, hi some embodiments, the calcium phosphate microparticles have a diameter of more than 2 μm, more than 5 μm, or more than 10 μm.

[0071] The incorporation of calcium phosphate into the described composites can enhance the advantages of chitosan, resulting in biopolymer composites with optimal properties (mechanical strength and bioactivity). Thus, by varying the composition (type of biodegradable polymer and amount of chitosan, optional addition of calcium phosphate), the mechanical strength and bioactivity of the resulting composites can be tailored to the in vivo required properties in terms of mechanical stability and bioactivity.

[0072] Computer images, such as those obtained by computed tomography (CT) and magnetic resonance imaging (MRI), can be used to design protocols for 3D printing appropriate porous implant scaffolds, such as those to replace missing and / or fractured bones or portions of bone. This enables personalized applications in which images of patients in need of bone regeneration therapy are used to design and manufacture customized 3D-printed scaffolds that promote the natural formation of healthy, normal bone tissue at the implantation site. The bone or portion of bone to be treated or repaired using the implantable scaffold can be any suitable human or animal bone. For example, the bone can be a bone of the human hand, a human jaw or skull, a bone of the human face (such as the nose or temporal bone), a human scapula, a human patella, a human sternum, a human rib, a bone of the human foot, or any human weight-bearing bone (such as, but not limited to, the tibia, fibula, femur, sacrum, sternum, or vertebra).

[0073] Printing resolution can be very high, i.e., resolutions of up to ±0.5 mm, ±0.4 mm, ±0.3 mm, ±0.2 mm, or ±0.1 mm are possible. Printing is also very fast, i.e., on the order of minutes to hours at most, meaning that an implant can be designed, printed, and introduced in a single medical procedure. Mechanically, this technology can be used to design or replace virtually any bone or portion of bone in the human or animal body, thereby providing a unique method for replacing and regenerating broken or damaged bone tissue.

[0074] The present invention can be illustrated by the following exemplary, non-limiting aspects and embodiments.

[0075] In one aspect, the present invention provides an implantable tissue scaffold comprising a mixture of a biocompatible organic polymer and chitin embedded within the biocompatible organic polymer.

[0076] In some embodiments, the chitin is partially or fully deacetylated chitin (PDC).

[0077] In some embodiments, the tissue scaffold comprises chitin in the range of about 0.05% to 20% (w / w).

[0078] In some embodiments, the tissue scaffold comprises chitin in the range of about 0.05%-10% (w / w), 0.05%-5% (w / w), 0.05%-2% (w / w), 0.5%-2% (w / w), or 1%-2% (w / w).

[0079] In some embodiments, the chitin has a degree of deacetylation in the range of about 2% to about 99%, in the range of about 6 to about 90%, in the range of about 10% to about 70%, in the range of about 20% to about 70%, in the range of about 30% to about 70%, in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

[0080] In some embodiments, the chitin has a degree of deacetylation in the range of about 35% to about 75%, in the range of about 35% to about 70%, in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

[0081] In some embodiments, the scaffold comprises chitin in the range of about 0.1% to about 1.5% with a degree of deacetylation in the range of about 45% to about 55%.

[0082] In some embodiments, chitin has a weight average molecular weight (MW) in the range of 200 Da to 2000 kDa, e.g., 1 kDa to 1000 kDa, 5 kDa to 500 kDa, 10 kDa to 300 kDa, or 20 kDa to 150 kDa. Chitin may preferably have a molecular weight in the range of about 100 kDa to about 400 kDa, about 200 kDa to about 400 kDa, or about 250 kDa to about 350 kDa, e.g., about 300 kDa.

[0083] In some embodiments, chitin can absorb at least 10 times, such as at least 15 times or at least 20 times its dry weight in water.

[0084] In some embodiments, the chitin-based material may be characterized by one or more of: (i) a degree of deacetylation in the range of 30-70%, (ii) a weight average molecular weight in the range of 40 kDa to 400 kDa, (iii) the ability to form a gel upon contact with water, and (iv) complete (100%) solubility in dilute acid.

[0085] In some embodiments, the chitin is in the form of particulates having an average particle size in the range of about 0.1 μm to about 50 μm, in the range of about 1 μm to about 25 μm, in the range of about 1 to 15 μm, or less than 20 μm, or in the range of about 5 μm to about 15 μm.

[0086] In some embodiments, the microparticles are physically embedded and dispersed within a biocompatible organic polymer, hi some embodiments, the biocompatible organic polymer is selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

[0087] In some embodiments, the biocompatible organic polymer comprises one or more heat-resistant organic polymers.

[0088] In some embodiments, the biocompatible organic polymer comprises at least one thermoplastic resin such as polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polyethylene, polypropylene, or a mixture thereof.

[0089] In some embodiments, the tissue scaffold has a porosity in the range of about 10% to about 90%, in the range of about 20% to about 70%, in the range of about 30% to about 70%, or in the range of about 40% to about 60%.

[0090] In some embodiments, the tissue scaffold further comprises calcium phosphate in an amount ranging from 0.2% to 20% (w / w), hi some embodiments, the calcium phosphate is in the form of microparticles having an average diameter ranging from about 1 to 100 μm, about 10 to 70 μm, or about 5 to 60 μm, which microparticles are dispersed within the biocompatible organic polymer.

[0091] In some embodiments, the tissue scaffold does not include a metal or alloy.

[0092] Another aspect relates to a composition for 3D printing comprising at least one biocompatible organic polymer in the range of about 75% to 99.95% by weight and chitin in the range of about 0.05% to 5% embedded within the biocompatible organic polymer. In one such embodiment, the chitin is partially or fully deacetylated chitin.

[0093] In some embodiments, the composition comprises chitin in the range of about 0.05% to 20%, chitin in the range of about 0.05% to 10%, chitin in the range of about 0.05% to 5%, chitin in the range of about 0.05% to 2%, or chitin in the range of about 1% to 2%.

[0094] In some embodiments, the chitin in the composition is partially deacetylated chitin having a degree of deacetylation in the range of about 0 to 75%, 6% to about 70%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 35% to about 65%, about 40% to about 70%, about 40% to about 60%, or about 45% to about 55%.

[0095] In some embodiments, the chitin in the composition is chitin having a degree of deacetylation in the range of about 35% to about 75%, in the range of about 35% to about 70%, in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

[0096] In some embodiments, the chitin in the composition has a weight average molecular weight (MW) in the range of 200 Da to 2000 kDa, such as in the range of 1 kDa to 1000 kDa, in the range of 5 kDa to 500 kDa, or in the range of 10 kDa to 300 kDa or 20 kDa to 150 kDa.

[0097] In some embodiments, the chitin in the composition is in the form of microparticles having an average particle size in the range of about 1 μm to about 50 μm, preferably in the range of about 1 μm to about 25 μm, about 1 to 15 μm, or about 5 μm to about 15 μm. In some embodiments, the microparticles are dispersed in a biocompatible organic polymer.

[0098] In some embodiments, the biocompatible organic polymer in the composition is selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof. In some embodiments, the biocompatible organic polymer comprises one or more heat-resistant organic polymers.

[0099] In some embodiments, the biocompatible organic polymer comprises at least one thermoplastic resin such as polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polyethylene, polypropylene, or a mixture thereof.

[0100] In some embodiments, the composition further comprises calcium phosphate in an amount ranging from 0.2% to 20% (w / w), hi some embodiments, the calcium phosphate is in the form of microparticles having an average diameter ranging from about 1 to 100 μm, or about 10 to 70 μm, or about 5 to 60 μm, and the microparticles are dispersed within the biocompatible organic polymer.

[0101] In some embodiments, the density or amount of N-acetylglucosamine (NAG) in the composition ranges from 0.01 to 100 mg / g, 0.01 to 50 mg / g, 0.02 to 15 mg / g, 0.1 to 15 mg / g, or 0.05 to 10 mg / g, preferably 0.1 to 5 mg / g. The amount of NAG in the composition may preferably be calculated per dry weight of the material.

[0102] Another embodiment relates to a method of making a tissue scaffold comprising 3D printing a composition described herein.

[0103] Another aspect relates to a method of promoting tissue formation comprising implanting a tissue scaffold described herein at a site requiring regenerative bone tissue formation.

[0104] In some embodiments, the partially deacetylated chitin oligomers contained in the tissue scaffold are released in situ from the tissue scaffold after implantation, thereby promoting tissue formation.

[0105] In some embodiments, the tissue is bone tissue.

[0106] Another aspect relates to an implantable tissue scaffold as described herein for use in treating bone defects in the human or animal body.

[0107] As used herein, including embodiments, singular terms are to be construed as including plural forms, and vice versa, unless the context dictates otherwise. Thus, it should be noted that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0108] Throughout this specification and the embodiments, the terms "comprise," "include," "have," and "contain," as well as variations thereof, are to be understood to mean "including, but not limited to," and are not intended to exclude other elements.

[0109] The present invention also covers the exact terms, features, values, ranges, etc. when these terms, features, values, ranges, etc. are used in combination with terms such as about, approximately, generally, substantially, essentially, at least, etc. (i.e., "about 3" also covers exactly 3, and "substantially constant" also covers exactly constant).

[0110] The term "at least one" should be understood to mean "one or more," and thus includes both embodiments including one or more components. Furthermore, dependent embodiments that refer to an independent embodiment describing a feature with "at least one" have the same meaning whether the feature is referred to with "the" or "at least one."

[0111] It will be understood that modifications can be made to the above-described embodiments of the invention while still falling within the scope of the invention. Features disclosed herein, unless expressly stated otherwise, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature represents an example of a generic set of equivalent or similar features.

[0112] The use of exemplary terms such as "for example," "e.g.," "for example," etc. is intended merely to facilitate description of the invention and does not limit the scope of the invention unless embodied. Any steps described herein may be performed in any order or simultaneously unless the context clearly dictates otherwise.

[0113] All features and / or steps disclosed herein may be combined in any combination, except for combinations in which at least some features and / or steps are mutually exclusive. In particular, preferred features of the invention are applicable to all aspects of the invention and may be used in any combination.

[0114] The present invention is further illustrated by the following non-limiting examples. [Example]

[0115] Example 1 Partially deacetylated chitin (PDC, 50% DD, average MW approximately 300 kDa) used to form the biodegradable composite was analyzed by scanning electron microscopy (SEM). As can be seen in Figure 1, PDC has the appearance of roughly spherical particles with diameters ranging from approximately 2 to 10 µm.

[0116] Example 2 50 mg of PDC (50% DD, average MW approximately 300 kDa) was weighed onto a small glass plate and 3 g of deionized water was applied around it. The deionized water was then applied to the PDC with a spatula to initiate absorption. After 30 minutes, any untrapped water was removed with a piece of filter paper and the weight was recorded. The PDC changed from a powder to a gel-like form, weighing 1010 mg, or more than 20 times its original weight (Figure 12A). 50 mg of acetic acid was then added to the swollen gel, followed by stirring with a spatula. The gel transformed into a clear solution within seconds (Figure 12B).

[0117] This experiment shows that the PDC material can absorb more than 20 times its dry weight in water and that the material can be completely dissolved in an acidic environment.

[0118] Example 3 Filaments for 3D printing were produced using partially deacetylated chitin (PDC) and the biopolymer polylactic acid (PLA) at different concentrations of PDC in the material, as shown in Table 1 below.

[0119] [Table 1]

[0120] Filaments were also produced that contained calcium phosphate (CaP), as shown in Table 2 below.

[0121] [Table 2]

[0122] Filaments containing PDC and CaP were analyzed by SEM to visualize the incorporation of PDC. As shown in Figure 2, the filaments contain PDC particles (represented by circles) and larger CaP particles (represented by rectangular boxes).

[0123] Micro-CT images (Fig. 3) show a homogeneous distribution of CaP particles within the filaments, and cross-sectional views are shown in (a) and longitudinal views in (b) for different CaP / PDC compositions (see Table 2).

[0124] Example 4 The stability of PDC during 3D printing at 200 °C was determined by X-ray diffraction (XRD). The results are shown in Figure 4. As can be seen from (a), all the important peaks of PDC, including the reflections at (020), (110), and (130), remain intact even after treatment at 200 °C for 30 minutes, demonstrating the stability of PDC after heat treatment. (b) shows the crystallinity and crystal size, both of which slightly decrease with increasing treatment time.

[0125] Example 5 For 3D printing of PDC / PLA parts, three types of unit cells were designed, including a straight laminated beam (SSB, A), a porous sodalite crystal (PSC, B), and a porous hexagonal pillar (PHP, C), as shown in Figure 5. Furthermore, the porosity was varied between 25% (a), 50% (b), and 70% (c).

[0126] Scanning electron microscope (SEM) images of the printed parts are shown at both the macro and micro levels in Figure 6, with macro images of each cell type on the right and micro images (in the boxes shown) on the left.

[0127] Example 6 The compressive strength of the 3D printed PDC / PLA materials was determined, and the results are shown in Figure 7. It is observed that the compressive strength is highest at the lowest porosity (25%) and the effect decreases as the amount of PDC in the material increases from 0.25% to 1.5%.

[0128] The results for the PDC / CaP / PLA material are shown in Figure 8 for two types of structures, namely, straight laminated beam (SSB) and porous sodalite crystal (PSC). The results suggest that porosity is the most important factor determining the compressive strength of the scaffold.

[0129] Example 7 Clinical trials were conducted on 20 rats (Taconic, DK). The rats were 7 to 8 months old at the time of surgery. Cylindrical 3D-printed PLA implants (50% porosity) with an average size of 1 x 4 mm were inserted into the rat femurs under general anesthesia. The implants contained either no chitosan or 0.25% or 1.5% chitosan (50% DD, average molecular weight approximately 300,000 Da). All animals received implants in both femurs (left: L, right: R). The experimental protocol is shown in the table below.

[0130] [Table 3]

[0131] Postoperative monitoring showed that all animals were healthy and experienced minimal weight loss. Animals were sacrificed after 3 months.

[0132] Bone formation was assessed using computed tomography (CT) to determine the amount of bone formation.

[0133] In Figure 10, a representative image of bone formation in an animal with a control implant (a PLA implant without chitosan or calcium phosphate) is shown on the left (A), and an image showing bone formation in an animal with an implant containing 0.25% chitosan is shown on the right (B). As can be seen, extensive bone formation is observed in the animals with chitosan-containing implants, while bone formation is much more limited in the animals with the control implants.

[0134] A comparison of the treatment groups is shown in Figure 11. The 0.25% implants showed significantly greater new bone formation (mm ) compared to the control (shown as B). 3 Animals receiving implants containing 1.5% chitosan (D, E) show significantly increased bone formation compared to controls (B), although the effect of the higher chitosan content is small.

[0135] Overall, this rat study showed that even after three months, animals receiving chitosan-containing implants had a clear increase in bone formation compared to controls.

Claims

1. 1. An implantable tissue scaffold comprising a mixture of a biocompatible organic polymer and chitin embedded within said biocompatible organic polymer, wherein the implantable tissue scaffold comprises chitin in the range of about 0.05% to 20% (w / w).

2. 2. The implantable tissue scaffold of claim 1, wherein the chitin has a degree of deacetylation in the range of about 2% to about 99%, about 6 to 90%, about 6% to about 70%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, or about 45% to about 55%.

3. 3. The implantable tissue scaffold of claim 1 or 2, wherein the chitin has a random distribution of glucosamine.

4. 4. The implantable tissue scaffold of claim 1, wherein the chitin in dry form is amorphous chitin.

5. 5. The implantable tissue scaffold of any one of claims 1 to 4, wherein the chitin in dry form is capable of absorbing at least 10, at least 15, or at least 20 times its weight in water.

6. 6. The implantable tissue scaffold of claim 5, wherein the chitin forms a gel upon exposure to water and can then be dissolved by the addition of an acid.

7. 7. An implantable tissue scaffold according to any one of claims 1 to 6, wherein the chitin has a weight average molecular weight (MW) in the range of 200 Da to 2000 kDa, such as in the range of 1 kDa to 1000 kDa, in the range of 5 kDa to 500 kDa, in the range of 10 kDa to 400 kDa, in the range of 20 kDa to 400 kDa or in the range of 100 kDa to 400 kDa.

8. 8. The implantable tissue scaffold according to any one of claims 1 to 7, wherein the chitin is in the form of microparticles having an average particle size in the range of about 0.1 μm to about 50 μm, in the range of about 1 μm to about 25 μm, in the range of about 1 to 15 μm or less than 20 μm, or in the range of about 5 μm to about 15 μm.

9. 9. The implantable tissue scaffold of any one of claims 1 to 8, wherein the biocompatible organic polymer is selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

10. 10. The implantable tissue scaffold according to any one of claims 1 to 9, further comprising calcium phosphate in an amount ranging from 0.2% to 20% (w / w), said calcium phosphate being in the form of microparticles having an average diameter in the range of about 1 to 100 μm, in the range of about 10 to 70 μm, or in the range of about 5 to 60 μm, dispersed within said biocompatible organic polymer.

11. 1. A composition for 3D printing comprising at least one biocompatible organic polymer in the range of about 75% to 99.95% by weight and chitin in the range of about 0.05% to 20% by weight, wherein the chitin is embedded within the biocompatible organic polymer.

12. 12. The composition of claim 11, wherein the chitin has a degree of deacetylation in the range of about 35% to about 75%, in the range of about 35% to about 70%, or in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

13. 12. The composition of claim 11, containing N-acetylglucosamine (NAG) in the range of 0.01 to 25 mg / g, or 0.02 to 15 mg / g, or 0.1 to 15 mg / g, or 0.05 to 7.5 mg / g, preferably 0.1 to 5 mg / g.

14. The composition of any one of claims 11 to 13, wherein the chitin has a random distribution of glucosamine.

15. 15. The composition of any one of claims 11 to 14, wherein the chitin in dry form is amorphous chitin.

16. 16. The composition of any one of claims 11 to 15, wherein the chitin in dry form is capable of absorbing at least 10, 15 or 20 times its weight in water.

17. 17. The composition of claim 16, wherein the chitin forms a gel upon exposure to water and can then be dissolved by the addition of acid.

18. 18. The composition of any one of claims 11 to 17, wherein the chitin has a weight average molecular weight (MW) in the range of 200 Da to 2000 kDa, such as in the range of 1 kDa to 1000 kDa, such as in the range of 5 kDa to 500 kDa, or in the range of 10 kDa to 300 kDa, or in the range of 20 kDa to 300 kDa.

19. 19. The composition of any one of claims 11 to 18, wherein the chitin is in the form of microparticles having an average particle size in the range of about 1 μm to about 50 μm, preferably in the range of about 1 μm to about 25 μm, in the range of about 1-15 μm or in the range of about 5 μm to about 15 μm.

20. 20. The composition of any one of claims 11 to 19, wherein the biocompatible organic polymer is selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

21. 21. The composition of any one of claims 11 to 20, further comprising calcium phosphate in an amount ranging from 0.2% to 20% (w / w), wherein the calcium phosphate is in the form of microparticles having an average diameter in the range of about 1 to 100 μm, in the range of about 10 to 70 μm, or in the range of about 5 to 60 μm, dispersed within the biocompatible organic polymer.

22. 22. A method of making a tissue scaffold, comprising 3D printing a composition according to any one of claims 11 to 21.

23. An implantable tissue scaffold according to any one of claims 1 to 10 for use in treating bone defects in the human or animal body.

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