3D printed bioactive scaffold

Abstract

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

Description

Technical Field

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

[0002] Medical knowledge on the management of traumatic and other bone defects has greatly advanced and improved around the world. However, the gold standard for repairing bone defects is still considered to be autologous bone transplantation. Clinical benefit is not guaranteed, and donor site complications and morbidity are not uncommon. Sometimes, one or more additional interventions are required, and the graft material is limited. To date, segmental bone defects caused by trauma, bone tumors, revision surgery or infection remain a huge challenge for trauma surgeons to overcome. Although a variety of materials and various implant options have been developed or improved, the perfect solution, especially the filling of critical size defects, remains to be explored.

[0003] Tissue engineering is the remodeling or reconstruction of human tissues through artificial manipulation, intentionally directing tissue growth through controlled molecular signals and / or specified physical / mechanical pathways. Currently, the preparation of three-dimensional scaffolds is dominated by conventional manufacturing techniques, which include phase separation, solvent casting, membrane coating, electrospinning, molding, and foaming. However, all of these methods have a major drawback, that is, they cannot fully control the scaffold's architecture, its pore network and pore size, resulting in inconsistent and less than ideal scaffolds.

[0004] 3D printing techniques are becoming increasingly critical in tissue engineering because of their ability to integrate biomedical device design into the actual manufacturing process, providing the opportunity to fabricate objects with both controlled macro- and micro-architectural structures. Compared to conventional material processing techniques for tissue engineering of highly complex tissues like bone, 3D printing techniques provide more precise, reproducible fine details and offer a more systematic approach for further research and development. The high reproducibility of the instrumentation process under real-time conditions will help surgeons and minimize potential errors during the surgical procedure, as the method provides the opportunity to design and print customized scaffolds based on real-time assessment and imaging of the desired implant during the surgical procedure.

[0005] In addition to being printable and osteoinductive / osteoconductive, materials suitable for tissue engineering, especially bone regeneration applications, should also be biodegradable, antimicrobial, endotoxin-free, non-toxic and mechanically stable. Many materials for bone scaffolds have been reported, including collagen, hyaluronic acid, hydroxyapatite, bioglass, titanium, polylactic acid, PMMA, carbon nanotubes, etc. However, it was found that all these materials are osteoconductive, but lack osteoinductive properties, and therefore do not meet the osteoinductive / osteoconductive requirements required for implants (Baldwin et al., 2019, J Orthopaed Trauma [Orthopedic Trauma] 33: 203; D'Souza et al., 2019, Biomedicines [Biomedicine] 7: 1). Therefore, in this context, it is urgent to develop 3D printable osteoinductive and osteoconductive biomaterials to meet the needs of orthopedic applications in the near future. Summary of the invention

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

[0007] Thus, in one aspect, an implantable tissue scaffold is provided comprising a mixture of a biocompatible organic polymer and chitosan, wherein the chitosan is physically embedded in the biocompatible organic polymer.

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

[0009] The implantable tissue scaffold is suitable for implantation in humans or animals. After implantation, the chitin embedded in the scaffold will be slowly released and degraded by natural processes, resulting in the formation of desired short-chain chitosan oligosaccharides (COS). The COS thus released will promote the healing of the implant site and the formation of natural tissue growth.

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

[0011] Thus, an implantable tissue scaffold can be formed by a process of 3D printing a composition. Therefore, another aspect relates to a method for preparing a tissue scaffold, the method comprising 3D printing a composition as described herein. 3D printing can be performed based on three-dimensional modeling of a tissue defect (such as a bone defect, a fracture, etc.) to be repaired and / or healed. This modeling can be based on analysis of one or more imaging methods, including computed tomography (CT), magnetic resonance imaging (MRI), radiological bone scanning, ultrasound imaging, radionuclide bone imaging.

[0012] Another aspect relates to a method for promoting tissue formation, the method comprising implanting a tissue scaffold as described herein into a site in a human or animal body where regenerative tissue formation is desired. In certain embodiments, the tissue in question is bone tissue. In certain embodiments, the method relates to the human body. In certain embodiments, the method relates to an animal body.

[0013] Another aspect relates to a method for treating bone tissue defects, the method comprising implanting a tissue scaffold as described herein into a site in a human or animal body where regenerative bone tissue formation is desired.

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

[0015] The above features and additional details of the present invention will be further described in the following examples, which are intended to further illustrate the present invention but are not intended to limit the scope thereof in any way. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 1 Shown are images of partially deacetylated chitin (PDC) used to prepare tissue scaffolds obtained by scanning electron microscopy (SEM).

[0018] Figure 2 Shown is a SEM image of a tissue scaffold, where PDC particles (shown as circles) and larger CaP particles (shown as rectangular boxes) are observed.

[0019] Figure 3 Images obtained by micro-CT are shown showing a homogeneous distribution of CaP particles within the filaments, with a cross-sectional view in (a) and a longitudinal view in (b) for different CaP / PDC compositions (see Table 2).

[0020] Figure 4 Shown are the results of a stability study of partially deacetylated chitin at 200 °C during 3D printing, as determined by X-ray diffraction (XRD); (a) stability over time; (b) crystallinity and crystal size at different treatment times.

[0021] Figure 5 Different types of unit cells for 3D printing PDC / PLA scaffolds are shown, including straight stacked beams (SSB, A), porous sodalite crystals (PSC, B), and porous hexagonal prisms (PHP, C). For each scaffold type, the porosity varies between 25%, 50%, and 70% (from left to right for each scaffold shown).

[0022] Figure 6 SEM images of the 3D printed scaffolds at the macro and micro levels are shown. For each unit cell type, the macro view is shown on the right and the micro view is shown on the left.

[0023] Figure 7 Compressive strength determination results of 3D printed PDC / PLA scaffolds containing 0.25%, 0.5%, and 1.5% PDC are shown.

[0024] Figure 8 The compressive strength of 3D printed PDC / PLA scaffolds containing varying amounts of PDC for two types of structures: straight stacked beams (SSB) and porous sodalite crystals (PSC).

[0025] Fig. 9 The conceptual framework according to the present invention in the context of bone regeneration applications is illustrated.

[0026] Fig.10 Shown are CT images of surgical sites in rat femurs receiving a control implant (A) and an implant containing 0.25% chitosan (B).

[0027] Fig.11 Shown are the results of bone formation determined from CT images of animals included in the rat study.

[0028] Fig.12Shown are (a) a highly swollen partially deacetylated (about 50%) chitosan material; and (b) a transparent clear solution formed when the chitosan material is dissolved at 100% solubility. DETAILED DESCRIPTION

[0029] The present disclosure provides a conceptual framework for 3D printable biopolymer composites with bioregenerative properties. Such materials are suitable for, for example, the production of custom 3D printed scaffolds and material extrusion (fused deposition molding, FDM). In these composites, chitin is incorporated into biodegradable polymers to provide biopolymer composites comprising biodegradable polymers and chitin materials. Biodegradable polymers provide mechanical stability and osteoconductive properties, while chitin is believed to provide osteoinductive properties to the resulting composite in addition to the well-known antimicrobial and hemostatic properties of chitosan.

[0030] Biopolymer composites can be 3D printed to generate scaffolds with any desired structure and porosity. Thus, designed scaffolds can be designed and printed for in situ use, where the scaffold provides mechanical stability at the implant site. Over time, the biodegradable polymer is replaced by naturally occurring bone tissue, which is aided by the osteoinductive properties of the chitin material in the polymer. The new bone tissue migrates into the porous scaffold, where it is formed, stimulated by the osteoconductive properties of the scaffold and the osteoinductive properties of the chitin material.

[0031] Chitin is a linear polysaccharide composed of N-acetylglucosamine (GlcNAc) monosaccharides linked by 1-4β linkages to form a linear biopolymer. Chitin is an essential component of the exoskeletons of crustaceans and insects and some specific organs of molluscs (such as the pen of squid and cuttlefish).

[0032] Chitosan materials can be partially or fully deacetylated, with 0% deacetylated chitosan being a homopolymer of N-acetylglucosamine subunits linked by covalent β-(1→4)-linkages and 100% deacetylated chitosan being a homopolymer of glucosamine (GlcN) subunits linked by covalent β-(1→4)-linkages. Chitosan is a deacetylated form of chitosan that contains a mixture of GlcN and GlcNAc.

[0033] There are 3 different types of chitosan, namely, α, β and γ chitosan. All of these chitosans have different crystalline states. This is mainly due to the orientation or packing of the polymer chains in the matrix. Due to this difference, for deacetylated chitosans with the same % DD, the physicochemical properties can vary greatly depending on the crystalline state.

[0034] On the other hand, there are at least 3 different types of deacetylation methods, including a) solid or heterogeneous deacetylation at high temperature (referred to herein as SST), b) liquid or homogeneous deacetylation at low temperature (referred to herein as LSL), and c) solid or heterogeneous deacetylation at low temperature (referred to herein as SSL). The SST deacetylation method is currently widely used in the chitin industry. Comparing the distribution of 50% DD glucosamine deacetylation, SST deacetylation will produce a large number of block-shaped or clustered glucosamine in the polymer chain; for the LSL method, the sequence of glucosamine and n-acetyl-D-glucosamine is usually arranged in an alternating manner, that is, one monomer is next to another monomer; and for SSL, it is between the SST and LSL forms, or in a random form. Due to this distribution pattern of glucosamine or N-acetyl-D-glucosamine, for similar deacetylation degrees, the physicochemical properties of deacetylated materials can have completely different properties, for example, for the same % DD materials, one material may have a high swelling property after contact with water, while the SST material may not swell; or one may be completely soluble in acid and the other may show only little dissolution. Upon degradation in vivo, the differences in the oligomeric patterns obtained by these 3 deacetylations will be even more significant, leading to different in vivo effects on tissue regeneration.

[0035] Degree of deacetylation (%DD) and mean molecular weight are the two most important properties considered in the current chitin industry. However, this refers to chitin with a %DD of 75% or more. For chitin with 75% DD and lower, more characterization is needed to understand its properties, especially the distribution of the glucosamine part in the polymer chain and the crystalline state of the material. Partially deacetylated chitin (chitosan) or chitosan has more complex physicochemical properties, making it to some extent or highly crystalline, or alternatively, partially deacetylated chitin can have an amorphous structure. These properties are highly dependent on the manufacturing process, and therefore depend on the molecular composition of the partially deacetylated chitin. Further complicating factors are due to the different types of chitin, i.e. α, β and γ forms, which all have their unique properties.

[0036] Therefore, for chitin with similar deacetylation degree, different distribution of glucosamine moieties will have a great influence on all its physical properties, including toughness or mechanical properties, swelling, dissolution and thermal properties of the material (Aiba, S. Int J Biol Macromol [International Journal of Biomacromolecules], 1991, 13 (1), 40-44; Sannan, T. et al., Makromol Chemie [Polymer Chemistry], 1976, 177 (12), 3589-3600). As described, the current manufacturing process of the chitosan industry is dominated by less controlled high temperature processes in its production, resulting in inferior chitosan, which can only be applied to industries with lower requirements and will not meet the needs of medicine or bone subjects. The material used in this article is the result of carefully controlling the distribution of glucosamine to achieve its desired arrangement to optimize the physicochemical properties and produce the desired oligosaccharide (COS) pattern after degradation in vivo, thereby effectively initiating tissue regeneration. The resulting material preferably has randomly distributed glucosamine moieties (and therefore also randomly distributed N-acetylglucosamine) moieties within the polymer chain.

[0037] Chitosan materials are preferably manufactured by specific processes to meet specific properties, especially a) controlled distribution of glucosamine moieties in the chitosan matrix and within the polymer chain; b) low amorphous state to ensure a smooth and desired degradation pattern. These two parameters are critical to a certain extent for chitosans with similar deacetylation degrees, and different distributions of glucosamine moieties will have a great influence on the physical properties of different chitosan polymers, including the toughness or mechanical properties of the material, swelling, dissolution, thermal properties, and resistance to precipitation when dissolved.

[0038] The different physicochemical properties of chitin materials also lead to different biological responses to the material. Therefore, for the biological effects produced, different glucosamine distributions will affect the enzyme cleavage sites during degradation in vivo, thereby producing different degradation oligomeric products. Since it is believed that these degradation products support biological activity by binding to chitinase-like proteins (CLPs) or other receptors in humans or animals, the properties and distribution of these products (short-chain oligosaccharides) will lead to different biological responses and therefore have significantly different effects on tissue regeneration.

[0039] The percentage of deacetylation degree (%DD) is the fraction of GlcN in a copolymer consisting of GlcNAc and GlcN. The %DD value of a chitin / chitosan sample is one of the key factors in evaluating its properties. Chitin with a %DD value less than 35% is insoluble in weak acids, such as 1% acetic acid. However, chitin / chitosan with a %DD greater than 75% is soluble in weak acids. The solubility of partially deacetylated chitin in weak acids is greatly affected by its processing method. Chitin materials with randomly distributed glucosamine and a %DD of about 50% are soluble in weak acids (such as acetic acid). Therefore, such materials are expected to have higher bioavailability than poorly soluble chitin materials.

[0040] Chitosan that has been partially deacetylated (i.e., chitosan with a degree of deacetylation>0%) is sometimes also referred to as chitosan. Therefore, chitosan can generally include any desired degree of deacetylation. In addition, chitosan can be randomly deacetylated, i.e., the deacetylation can be at a random position in the polymer chain, or chitosan can be block-deacetylated, i.e., the deacetylation pattern is accompanied by clustered or aggregated glucosamine in the polymer chain. As described herein, the term "chitosan" refers to a chitosan material that can have any degree of deacetylation, i.e., the degree of deacetylation can be any value from 0% to 100%. As described herein, the term "chitosan" refers to a partially (i.e., >0%) deacetylated chitosan material. Therefore, the terms "chitosan" and "chitosan" can be interchangeably used for partially deacetylated chitosan (PDC) materials.

[0041] Compared to fully acetylated chitin, partially deacetylated chitosan oligosaccharides (oligosaccharides) are smaller and water-soluble, and are prepared by deacetylation of chitin to produce chitosan. Chitin or chitosan can be hydrolyzed to produce chitosan oligosaccharides with different degrees of acetylation. Short-chain chitosan oligosaccharides are more soluble in water than polymeric chitin; in particular, significantly deacetylated chitosan oligosaccharides are highly water-soluble.

[0042] Swelling index and solubility are two simple methods to evaluate the glucosamine distribution of chitin with 75% DD and lower. The more random the distribution of glucosamine in the polymer chain, the more the chitin swells and the better the solubility. This is especially pronounced in 50% DD chitin. For example, the swelling index of chitin films coated with randomly deacetylated 50% DD chitin can reach 6-10 times its dry weight and generally have high solubility. Moreover, for chitin with more randomly distributed glucosamine, the chitin will have a relatively low crystalline state. In addition, the prepared chitin solution will have greater resistance to precipitation than chitin solutions with less random distribution.

[0043] Bone graft materials are typically divided into 3 types, namely osteoconductivity, osteoinductivity and osteogenicity. Osteoconduction refers to a graft material used as a scaffold for new bone growth, which is continuously grown by native bone. The idea is to allow new bone to passively grow into the scaffold or channel itself (e.g., grow into a hole, channel or conduit) to reach a specified size. Osteoinduction is a process in which, for example, bone formation is induced by biomaterials. Osteoinduction involves cell recruitment and activation of these cells to develop into pre-osteoblasts. It is a normal phenomenon seen in bone healing. In the case of fractures, most of the bone is healed by an osteoinductive process. Osteogenesis refers to a process in which osteoblasts that exist directly (as osteocytes, osteoblasts or chondroblasts) or that are indirectly derived from previously undifferentiated stem cells produce new bone by laying osteoid or via endochondral ossification of cartilage.

[0044] Recent studies conducted by the applicant have shown that chitosan has osteoinductive properties, which makes it an ideal candidate as a component in a material for bone regeneration (Kjalarsdóttir et al., 2019, Regen Biomater [Regenerative Biomaterials] 6:231). In addition, the degree of deacetylation of chitosan and the method of preparing the material play a decisive role in the osteoinductive properties. Thus, it has been shown that, with this deacetylation method, chitin with a degree of deacetylation in the range of about 50% to 70% leads to particularly high levels of tissue regeneration than highly deacetylated chitosan.

[0045] Chitosan has been shown to provide many advantages in biomedical applications, including biocompatibility and biodegradability and the regulation of inflammatory response in a controlled manner. In addition, due to the bioabsorbability, antimicrobial, nontoxic and polycationic properties of chitosan and the great potential in tissue regeneration, it has a wide range of applications in medicine / medical fields, such as: antimicrobial agents, controlled drug delivery, blood anticoagulants, wound dressings and tissue engineering, including bone and nerve regeneration. Chitosan has been extensively studied in bone tissue engineering. Chitosan can work alone, or it can be blended with other polymers, natural and synthetic materials, which is generally considered to be an effective way to develop tissue engineering, chitosan-based materials (such as 3D freeze-dried scaffolds, hydrogels, films and other scaffolds).

[0046] Chitosan has demonstrated its osteoinductive / osteoconductive properties in many studies (Tan et al. 2014, Biomaterials 35:7828; Geffre et al. 2010, Future Sci OA 4:FSO225). In vivo studies have shown that chitosan alone is sufficient to stimulate osteogenesis [Pang et al. 2017, Oncotargt 8:35583; Ho et al. 2015, Int J Nanomedicine 10:5941]. More importantly, both in vitro and in vivo studies revealed that the degree of deacetylation (DD) of chitosan materials has a decisive influence on bone formation (Lieder et al. 2012, J Biomed Mater Res Part A, 100A:3392; Kjalarsdottir et al. 2019, Regen Biomater 6:231). However, chitosan itself lacks sufficient mechanical strength to be suitable for load-bearing applications.

[0047] To overcome this deficiency of chitosan and provide a mechanically stable solution, the incorporation of chitosan, particularly partially deacetylated chitosan, into a suitable biocompatible and / or biodegradable polymer is provided in the tissue scaffolds and compositions described herein.

[0048] Typically, the amount of chitosan in the tissue scaffold and / or composition can be in the range of about 0% to 20%, in the range of about 0% to 10%, in the range of about 0% to 5%, in the range of about 0.05% to 20%, in the range of about 0.05% to 15%, in the range of about 0.05% to 10%, in the range of about 0.05% to 5%, in the range of about 0.05% to 3%, in the range of about 0.1% to 20%, in the range of about 0.2 to 20%, in the range of about 0.5% to 20%, in the range of about 0.7% to 20%, or in the range of about 1%-20%. The lower limit 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%.

[0049] The amount of chitosan material used in the scaffold and / or composition can depend on its degree of deacetylation (%DD). Thus, a PDC material having a relatively low %DD (e.g., less than 75%) can be present in the scaffold in a lower amount than a PDC material having a relatively high %DD (e.g., greater than 75%). For example, for chitosan having a %DD greater than 75%, the amount of chitosan in the scaffold can be in the range of 0.1% to 20%, in the range of 0.1% to 10%, or in the range of 0.1% to 5%.

[0050] The chitosan material can be partially or completely deacetylated. For example, the deacetylation degree of the chitosan material can be in the range of 0% to 100%, in the range of about 10% to about 90% (w / w), in the range of about 10% to about 70% (w / w), in the range of about 20% to about 70% (w / w), in the range of about 30% to about 70% (w / w), in the range of 35% to about 65%, in the range of about 35% to 60%, in the range of about 40% to about 60% (w / w), in the range of about 40% to about 55%, or in the range of about 45% to about 55% (w / w).

[0051] The lower limit of suitable deacetylation degree range can 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 deacetylation degree can be about 70%, about 75%, about 80%, about 85%, about 90% or about 99%.

[0052] In certain applications, the degree of deacetylation of the chitosan material is 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).

[0053] The weight average molecular weight (MW) of the chitosan material may be in the range of 200 Da-2000 kDa, such as in the range of 1 kDa-1000 kDa, in the range of 5 kDa-500 kDa, in the range of 10 kDa-300 kDa, or in the range of 20 kDa-1500 kDa.

[0054] Preferably, chitosan material can include amorphous partially deacetylated chitosan (PDC). This material can be produced by a controlled process with a specific glucosamine distribution, thereby producing a material soluble in acidic aqueous solution and having a high swelling capacity. Such chitosan is preferably about 45% to about 55% deacetylated (such as about 50%), and is completely dissolved in acidic solution (that is, 100% solubility). Chitosan can have a weight average molecular weight within the range of 100kDa-400kDa, such as about 300kDa. Chitosan can absorb as much as 10 times, 15 times or about 20 times or more of its weight of water.

[0055] In some embodiments, chitosan is completely soluble in weakly acidic solutions, such as soluble in acetic acid solutions. In other words, chitosan is 100% or nearly 100% (such as greater than 99%) soluble in acidic solutions. The chitosan material dissolves instantly or nearly instantly (within a few minutes, such as within 10 minutes or within 5 minutes) when acidified. This is different from chitosan known in the art, which typically dissolves very slowly or does not dissolve at all in acidic solutions.

[0056] Solubility can be assessed by passing a solution containing PDC through a filter (eg, a 0.45 micron filter), where the absence of insoluble particles that fail to pass through the filter is a measure of complete solubility.

[0057] Chitosan materials have a high swelling capacity and form a gel when in contact with water. The ability to form a gel can be reflected by the ability to absorb (in a dry state) 10 times or more, 15 times or more, or 20 times or more of water. For chitosan with a high molecular weight (such as 150 kDa or more, 200 kDa or more, or 250 kDa or more molecular weight), the ability to form a gel that swells 10 times or more is particularly significant.

[0058] Chitosan material can be preferably in the form of microparticles. The average particle size of microparticles can be 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, in the range of 5 to 20 μm, or in the range of about 5 μm to about 15 μm. The lower limit of the range can be about 1 μm, about 2 μm, about 3 μm, about 4 μm or about 5 μm. The upper limit of the range can be about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm or about 50 μm.

[0059] The microparticles are preferably physically embedded and dispersed within a biocompatible organic polymer. Thus, the microparticles can be visualized as distinct 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 issues of chitin are eliminated. Thus, the physical embedding of chitin within the polymer is applicable to all chitins, independent of their degree of deacetylation.

[0060] The microparticles may preferably contain only chitosan material, ie the particles consist essentially only of chitosan material.Thus, the implantable stent comprises a biocompatible and / or biodegradable organic polymer and microparticles consisting essentially of chitosan material.

[0061] Any suitable organic polymer that is biocompatible and / or biodegradable can be used in the applications described herein. For example, the organic polymer can be selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

[0062] The organic polymer may include or consist of a thermoplastic, such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), polyethylene, or polypropylene, or a mixture thereof.

[0063] As an example, polylactic acid (PLA) is a widely used biomaterial that has proven its value in various medical applications. PLA is a biodegradable and bioactive thermoplastic aliphatic polyester derived from nature. The source of PLA is corn starch, cassava root, potato chips or starch, or sugar cane. PLA is biodegradable under nature and physiological conditions, forming harmless and nontoxic compounds through simple hydrolysis of the ester backbone. The advantages of PLA in bone engineering are its biocompatibility, thermoplasticity and mechanical properties. PLA has mechanical properties that make the material suitable for transient load-bearing applications and is easily processed by 3DFDM printing technology. PLA and its copolymers have been widely used in different fields, such as polymer engineering, tissue engineering, drug delivery systems and various vital medical implants.

[0064] PLA can generally be present as poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), or a mixture of PLLA and PDLA, known as poly(DL-lactic acid) (PDLLA). The mixture can conveniently be racemic, i.e. a 1:1 mixture of PLLA and PDLA. PDLLA tends to be in a more amorphous and less crystalline form than PLLA and PDLA.

[0065] Compared with other biodegradable polymers, PLA has attracted considerable attention as a bone engineering material in the past two decades due to its good processability and characteristics. PLA with high surface energy is easy to print, which makes it widely used in 3D printing. PLA has a glass transition temperature of 60°C and a melting temperature of 190°C (Total CorbionPLA). The basic mechanical properties of PLA are comparable to those of polystyrene and PET. However, the flexural modulus of PLA is higher than that of polystyrene, and PLA has good heat sealing properties. PLA compatible with the use according to the present invention can be any suitable PLA material, including, for example, poly-L-lactide type (PLLA) and poly-D-lactide (PDLA), including any combination and mixture of PLLA and PDLA. In some embodiments, PLA is PLLA.

[0066] Other suitable biomaterials that may also or alternatively be used in the scaffolds and compositions described herein include poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyhydroxyalkanoates (PHA), polyethylene, or polypropylene, and any mixtures or combinations thereof.

[0067] The chitosan material may be uniformly mixed in the polymer material. The chitosan may exist as particles physically dispersed and embedded in the polymer material, that is, the chitosan exists as small different particles in the polymer material.

[0068] The chitosan material can typically be in the form of microscopic particles 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, or in the range of about 3-10 μm.

[0069] It may be appropriate that the tissue scaffold is at least partially porous, ie at least part / portion of the scaffold has a porous structure. By making the scaffold porous, cell recruitment and cell growth into the site of the scaffold is promoted, thereby enhancing and / or accelerating the tissue regeneration process at the implant site.

[0070] The porosity of the scaffold can generally be 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%. In some embodiments, the scaffold is about 20% porous, about 25% porous, about 30% porous, about 35% porous, about 40% porous, about 45% porous, about 50% porous, about 55% porous, about 60% porous, about 65% porous, about 70% porous, or about 75% porous. In this context, the term "porous" refers to the open, i.e., polymer-free volume of the three-dimensional scaffold structure.

[0071] The porosity and / or shape of the pores of the implant may vary as desired, taking into account factors such as the mechanical strength required of the scaffold, the nature of the tissue to be regenerated, and the amount of chitin material incorporated into the scaffold. Typically, the scaffold may have a porosity in the range of about 10% to about 90%.

[0072] The scaffold will have a mechanical strength that can be changed by changing the porosity of the 3D printed structure. 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.

[0073] The tissue scaffold may preferably lack non-biodegradable materials, ie the tissue scaffold is completely biodegradable, with the result that after implantation, the tissue scaffold slowly degrades in situ over time. Thus, the tissue scaffold may preferably contain no metal or alloy components or other non-biodegradable components.

[0074] Without intending to be bound by theory, it is believed that the chitosan material implanted in the tissue scaffold degrades through a natural degradation process in vivo catalyzed by natural chitinase, lysozyme, or through other natural degradation processes. The degradation process releases short-chain chitosan oligosaccharides, especially with a controlled distribution of glucosamine, which are believed to represent a bioactive form of chitosan material that has osteoinductive properties, as further described herein.

[0075] The resulting chitosan oligosaccharides trigger tissue regeneration processes at the implant site, such as recruiting bone-forming cells around the 3D-printed implant inserted into the bone defect, while blood cells form around the implant site, leading to osteogenesis and bone tissue regeneration. Over time, the implant, along with the incorporated chitosan material, will completely degrade, but will have completed its role in promoting healthy tissue formation. In the case of bone regeneration, this includes guided ossification, leading to tissue remodeling and the formation of natural bone tissue.

[0076] In the context of bone regeneration processes, the conceptual framework according to the present invention can be used as Fig. 9 As shown in the figure. Therefore, chitin materials (exemplified as partially deacetylated chitin or chitosan) have osteoinductive properties in addition to known antimicrobial and hemostatic properties. Chitin materials can be incorporated into biocompatible polymers (exemplified here as PLA), thereby producing filaments suitable for 3D printing applications. Subsequently, under the guidance of images obtained by, for example, CT or MRI, 3D printing is performed on the filaments to produce a scaffold with bioinductive (e.g., osteoinductive / osteoconductive) properties. The scaffold thus produced can then be implanted into a site where tissue regeneration is required, such as a site of a bone defect (e.g., missing bone or bone deformation), including a fracture. Once implanted, the chitin in the scaffold degrades to release bioactive chitosan oligosaccharides that promote tissue regeneration at the implant site, which is illustrated here by the initiation of osteogenesis and subsequent bone tissue regeneration. Over time, the guided ossification process is dominant, wherein the scaffold initially implanted has been degraded by a natural process and replaced by remodeled natural bone tissue.

[0077] The biocompatible polymer can be any suitable polymer material that provides the required mechanical stability and chemical integrity during 3D printing. Preferably, the biocompatible polymer is biodegradable. Exemplary suitable biocompatible polymer materials include polylactic acid (PLA) and polyhydroxyalkanoate (PHA).

[0078] Except chitin, it may be advantageous to mix calcium phosphate in biopolymer composite.Therefore, the gained support will comprise biopolymer / calcium phosphate composite, and chitin material is embedded therein.Calcium phosphate is the well-known biological activity and biodegradable transplant material known for bone cement application.This material can be in the form of crystal, and wherein its crystallinity (crystal size, crystal integrity, grain size) can change.

[0079] The amount of calcium phosphate can be in the range of 0.2%-20% (w / w), such as in the range of about 0.5%-15% (w / w), in the range of about 0.5%-10% (w / w), in the range of about 1%-10% (w / w), in the range of about 2%-10% (w / w), or in the range of about 2%-8% (w / w).

[0080] The calcium phosphate can be in the form of microparticles having an average diameter in the range of about 1 to 100 μm, in the range of about 5 to 70 μm, in the range of about 5 to 60 μm, in the range of about 10 to 70 μm, in the range of about 10 to 60 μm, in the range of about 10 to 50 μm, or in the range of about 10 to 40 μm, wherein the microparticles are dispersed in a biocompatible organic polymer.

[0081] In some embodiments, the calcium phosphate particles have a diameter of less than 80 μm, less than 70 μm, less than 60 μm, or less than 50 μm. In some embodiments, the calcium phosphate particles have a diameter of greater than 2 μm, greater than 5 μm, or greater than 10 μm.

[0082] Incorporating calcium phosphate into the described composite material can enhance the benefits of chitosan, thereby providing a biopolymer composite material with optimal properties (mechanical strength and bioactivity). Therefore, with regard to mechanical stability and bioactivity, by changing the composition (type of biodegradable polymer and amount of chitosan and optional added calcium phosphate), the mechanical strength and bioactivity of the resulting composite material can be adapted to the desired properties in vivo.

[0083] Computer images (such as images obtained by computer tomography (CT) and magnetic resonance imaging (MRI)) can be used to design a solution for 3D printing suitable porous implant scaffolds (such as scaffolds that replace missing and / or broken bones or bone parts). Thus, personalized applications are possible, i.e., using the imaging of patients who need bone regeneration treatment to design and produce customized scaffolds by 3D printing, these customized scaffolds promote the natural formation of healthy normal bone tissue at the implant site. The bone or bone part to be healed or repaired using an implantable scaffold can be any suitable human or animal bone. For example, bone can be human hand bone, human jawbone or skull, human facial bone (such as nasal bone or temporal bone), human shoulder bone, human patella, human sternum, human ribs, human foot bone or any human load-bearing bone (such as but not limited to tibia, fibula, femur, sacrum, sternum, vertebrae).

[0084] Printing resolution can be quite high, i.e. resolutions up to ±0.5 mm, ±0.4 mm, ±0.3 mm, ±0.2 mm, or ±0.1 mm are possible. Printing is also quite fast, i.e. on the order of a few minutes or at most a few hours, which means that it is feasible to design, print, and introduce an implant in a single medical procedure. Mechanically, virtually any bone or bone part in the human or animal body can be designed or replaced using this technology, thereby providing a unique way to replace and regenerate broken or damaged bone tissue.

[0085] The invention can be represented by the following exemplary non-limiting aspects and embodiments:

[0086] In one aspect, the present invention provides an implantable tissue scaffold comprising a mixture of a biocompatible organic polymer and chitosan, wherein the chitosan is embedded in the biocompatible organic polymer.

[0087] In some embodiments, the chitosan is partially or fully deacetylated chitosan (PDC).

[0088] In some embodiments, the tissue scaffold comprises chitosan in the range of about 0.05% to 20% (w / w).

[0089] In some embodiments, the tissue scaffold comprises chitosan in the range of about 0.05% to 10% (w / w), 0.05% to 5% (w / w), 0.05% to 2% (w / w), 0.5% to 2% (w / w), or 1%-2% (w / w).

[0090] In some embodiments, the chitosan is a chitosan having a degree of deacetylation ranging from about 2% to about 99%, ranging from about 6 to 90%, ranging from about 10% to about 70%, ranging from about 20% to about 70%, ranging from about 30% to about 70%, ranging from about 40% to about 60%, or ranging from about 45% to about 55%.

[0091] In some embodiments, the chitosan is chitosan having 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%.

[0092] In some embodiments, the scaffold contains chitosan 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%.

[0093] In some embodiments, the weight average molecular weight (MW) of chitosan is in the range of 200Da-2000kDa, such as in the range of 1kDa-1000kDa, in the range of 5kDa-500kDa, in the range of 10kDa-300kDa or 20kDa-150kDa. The molecular weight of chitosan can preferably be in the range of about 100kDa to about 400kDa, in the range of about 200kDa to about 400kDa, in the range of about 250kDa to about 350kDa, such as about 300kDa.

[0094] In some embodiments, chitosan can absorb at least 10 times (eg, at least 15 times or at least 20 times) its dry weight in water.

[0095] In some embodiments, the chitosan material may be characterized by one or more of the following: (i) a degree of deacetylation in the range of 30%-70%, (ii) a weight average molecular weight in the range of 40 kDa-400 kDa, (iii) the ability to form a gel upon contact with water, and (iv) complete (100%) solubility in dilute acid.

[0096] In some embodiments, the chitosan is in the form of microparticles having an average particle size ranging from about 0.1 μm to about 50 μm, from about 1 μm to about 25 μm, from about 1 to 15 μm or less than 20 μm, or from about 5 μm to about 15 μm.

[0097] In some embodiments, the microparticles are physically embedded and dispersed within a biocompatible organic polymer. In 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.

[0098] In some embodiments, the biocompatible organic polymer comprises one or more heat-resistant organic polymers.

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

[0100] In some embodiments, the porosity of the tissue scaffold is in a range of about 10% to about 90%, in a range of about 20% to about 70%, in a range of about 30% to about 70%, or in a range of about 40% to about 60%.

[0101] In some embodiments, the tissue scaffold further comprises calcium phosphate in an amount in the range of 0.2%-20% (w / w). In some embodiments, 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, in the range of about 5 to 60 μm, wherein the microparticles are dispersed in a biocompatible organic polymer.

[0102] In some embodiments, the tissue scaffold is free of metals or alloys.

[0103] Another aspect relates to a composition for 3D printing, the composition comprising at least one biocompatible organic polymer in the range of about 75%-99.95% by weight and chitosan in the range of about 0.05%-5%, wherein the chitosan is embedded in the biocompatible organic polymer. In one such embodiment, the chitosan is partially or fully deacetylated chitosan.

[0104] In some embodiments, the composition comprises chitosan in a range of about 0.05% to 20%, chitosan in a range of about 0.05% to 10%, chitosan in a range of about 0.05% to 5%, chitosan in a range of about 0.05 to 2%, or chitosan in a range of about 1%-2%.

[0105] In some embodiments, the chitosan in the composition is partially deacetylated chitosan having a degree of deacetylation in the range of about 0%-75%, 6% to about 70%, 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 35% to about 65%, in the range of about 40% to about 70%, in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

[0106] In some embodiments, the chitosan in the composition is chitosan having a degree of deacetylation ranging from about 35% to about 75%, ranging from about 35% to about 70%, or ranging from about 40% to about 60%, or ranging from about 45% to about 55%.

[0107] In some embodiments, the weight average molecular weight (MW) of chitosan in the composition is in the range of 200 Da-2000 kDa, such as in the range of 1 kDa-1000 kDa, in the range of 5 kDa-500 kDa, or in the range of 10 kDa-300 kDa or 20 kDa-150 kDa.

[0108] In some embodiments, the chitosan 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, in the range of about 1 to 15 μm, or in the range of about 5 μm to about 15 μm. In some embodiments, the microparticles are dispersed in a biocompatible organic polymer.

[0109] 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.

[0110] In some embodiments, the biocompatible organic polymer includes at least one thermoplastic, such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), polyethylene, or polypropylene, or a mixture thereof.

[0111] In some embodiments, the composition further comprises calcium phosphate in an amount in the range of 0.2%-20% (w / w). In some embodiments, the calcium phosphate is in the form of microparticles having an average diameter in the range of about 1 μm to 100 μm, or about 10 μm to 70 μm, or about 5 μm to 60 μm, wherein the microparticles are dispersed in a biocompatible organic polymer.

[0112] In some embodiments, the density or amount of N-acetylglucosamine (NAG) in the composition is in the range of 0.01-100 mg / g, 0.01-50 mg / g, 0.02-15 mg / g, 0.1-15 mg / g, or 0.05-10 mg / g, preferably 0.1-5 mg / g. The amount of NAG in the composition can preferably be calculated according to the dry weight of the material.

[0113] Another aspect relates to a method of preparing a tissue scaffold, the method comprising 3D printing a composition as described herein.

[0114] Another aspect relates to a method of promoting tissue formation, the method comprising implanting a tissue scaffold as described herein at a site in need of regenerative bone tissue formation.

[0115] In some embodiments, the partially deacetylated chitin oligomers contained in the tissue scaffold are released from the tissue scaffold in situ after implantation, thereby promoting tissue formation.

[0116] In some embodiments, the tissue is bone tissue.

[0117] Another aspect relates to an implantable tissue scaffold as described herein for use in treating bone defects in a human or animal body.

[0118] As used herein, including in the Examples, terms in the singular should be interpreted as also including the plural, and vice versa, unless the context indicates otherwise. Thus, it should be noted that the singular forms "a / an" and "the" as used herein include plural referents unless the context clearly indicates otherwise.

[0119] Throughout the specification and examples, the terms “comprising,” “including,” “having,” and “containing” and variations thereof should be understood to mean “including but not limited to,” and are not intended to exclude other components / components.

[0120] Where terms, features, values ​​and ranges, etc. are used in conjunction with terms such as about, approximately, usually, substantially, essentially, at least, etc., the present invention also covers these exact terms, features, values ​​and ranges, etc. (i.e., "about 3" should also cover exactly 3, or "substantially constant" should also cover completely constant).

[0121] The term "at least one" should be understood to mean "one or more", and therefore includes two embodiments including one or more components. In addition, when the feature "at least one" is referred to as both "the" and "the at least one", the dependent embodiments referring to the independent embodiment describing the feature have the same meaning.

[0122] It should be understood that the foregoing embodiments of the present invention may be varied while still falling within the scope of the present invention. Unless otherwise specified, the features disclosed in the specification may be replaced by alternative features for the same, equivalent or similar purposes. Therefore, unless otherwise specified, each feature disclosed represents an example of a general series of equivalent or similar features.

[0123] The use of exemplary language, such as "for instance", "such as" and "for example" etc. is only intended to better illustrate the present invention, and unless so implemented, it does not indicate a limitation on the scope of the present invention. Unless the context clearly indicates otherwise, any steps described in the specification can be performed in any order or simultaneously.

[0124] All features and / or steps disclosed in the specification may be combined in any combination, except combinations in which at least some features and / or steps are mutually exclusive. In particular, the preferred features of the present invention are applicable to all aspects of the present invention and may be used in any combination.

[0125] The present invention is further described by the following non-limiting examples.

[0126] Examples

[0127] Example 1

[0128] Partially deacetylated chitin (PDC, 50% DD, average MW about 300 kDa) used to form the biodegradable composite was analyzed by scanning electron microscopy (SEM). Figure 1 As can be seen in FIG. 1 , PDC has the appearance of generally spherical particles with diameters ranging from about 2 to 10 μm.

[0129] Example 2

[0130] 50 mg of PDC (50% DD, average MW about 300 kDa) was weighed onto a small glass plate and 3 g of DI water was applied around it. The DI water was then brought into contact with the PDC with a spatula to initiate absorption. After 30 minutes, all untrapped water was removed with a piece of filter paper and the weight was recorded. The PDC transformed from its powder form into a gel and weighed 1010 mg or more than 20 times its initial weight ( Fig.12 A). Then, 50 mg of acetic acid was added to the swollen gel and then stirred with a spatula, the gel turned into a clear solution within a few seconds ( Fig.12 B).

[0131] This experiment showed 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.

[0132] Example 3

[0133] Filaments for 3D printing were produced using partially deacetylated chitin (PDC) and the biopolymer polylactic acid (PLA) at a number of different concentrations of PDC in the material as shown in Table 1 below:

[0134] Table 1

[0135] Material PDC, g PLA, g abbreviation 0.25%PDC / PLA 0.25 100 0.25% CS / PLA 0.5%PDC / PLA 0.50 100 0.5% CS / PLA 1.5%PDC / PLA 1.50 100 1.5% CS / PLA

[0136] Filaments were produced that also contained calcium phosphate (CaP), as shown in Table 2 below:

[0137] Table 2

[0138] Material PDC, g CaPs, g PLA, g abbreviation 0.25%PDC / CaP / PLA 0.25 0.5 100 0.25% CS / PLA 0.5%PDC / CaP / PLA 0.50 1.0 100 0.5% CS / PLA 1.5%PDC / CaP / PLA 1.50 4.5 100 1.5% CS / PLA

[0139] The filaments containing PDC and CaP were analyzed by SEM to visualize the incorporation of PDC. Figure 2 As can be seen in FIG. 1 , the filaments contain PDC particles (shown as circles) and larger CaP particles (shown as rectangular boxes).

[0140] Micro-CT imaging ( Figure 3 ) show the uniform distribution of CaP particles within the filaments, where for different CaP / PDC compositions (see Table 2), the cross-sectional view is shown in (a) and the longitudinal view is shown in (b).

[0141] Example 4

[0142] The stability of PDC at 200°C during 3D printing was determined by X-ray diffraction (XRD). The results are shown in Figure 4 As can be seen in (a), all the necessary peaks of PDC, including reflections at (020), (110), and (130), are intact even after treatment at 200°C for 30 min. This illustrates the stability of PDC upon thermal treatment. In (b), the crystallinity and crystal size are shown, both of which decrease slightly with increasing treatment time.

[0143] Example 5

[0144] like Figure 5 As shown in Figure 3, three types of unit cells are designed for 3D printing of PDC / PLA parts, including straight stacked beams (SSB, A), porous sodalite crystals (PSC, B), and porous hexagonal prisms (PHP, C). In addition, the porosity varies between 25% (a), 50% (b), and 70% (c).

[0145] exist Figure 6, scanning electron microscope (SEM) images of the printed parts are shown at both the macro and micro levels, where for each unit cell type, the macro view is shown on the right and the micro view is shown on the left (within the indicated boxes).

[0146] Example 6

[0147] Determine the compressive strength of 3D printed PDC / PLA materials, where the results are as follows Figure 7 The compressive strength is highest for the lowest porosity (25%), with a smaller effect observed when the amount of PDC in the material increases from 0.25% to 1.5%.

[0148] The results for PDC / CaP / PLA materials are shown in Figure 2 for two types of structured straight stacked beams (SSBs) and porous sodalite crystals (PSCs). Figure 8 The results indicated that porosity was the biggest determinant of the compressive strength of the scaffold.

[0149] Example 7

[0150] A clinical trial was conducted on 20 rats (Taconic, Denmark (DK)). The rats were 7-8 months old at the time of surgery. A cylindrical 3D-printed PLA implant (50% porosity) with an average size of 1×4 mm was inserted into the femur of the rats under systemic analgesia. The implant contained no chitosan, or contained 0.25% or 1.5% chitosan (50% DD, average molecular weight about 300,000 Da). Both femurs (left side: L, and right side, R) of all animals received implants, and the experimental scheme is shown in Table 3 below.

[0151] Table 3

[0152]

[0153] CS = chitosan (by weight); BCCS = chitosan plus calcium phosphate, % refers to the amount of chitosan by weight

[0154] During postoperative monitoring, all animals were healthy with minimal weight loss. After 3 months, the animals were sacrificed.

[0155] Bone formation was assessed using computed tomography (CT), and the volume of bone formation was determined.

[0156] exist Fig.10 In the figure, representative images of bone formation in animals receiving control implants (PLA implants without chitosan or calcium phosphate) are shown on the left (A), while images of bone formation in animals receiving implants containing 0.25% chitosan are shown on the right (B). As can be seen, there is a large amount of bone formation in animals receiving implants containing chitosan, while there is much less bone formation in animals receiving control implants.

[0157] exist Fig.11 In the Figure 2, a comparison of the treatment groups is shown. New bone formation was significantly increased (in mm) in the implants containing 0.25% compared to the control (shown in Figure 3). 3 Measured). Although bone formation was significantly increased in animals receiving implants containing 1.5% chitosan (D, E) compared to controls (B), the effect of higher chitosan content was smaller.

[0158] Overall, this rat study showed that even after 3 months, bone formation was significantly increased in animals receiving chitosan-containing implants compared to controls.

Claims

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

2. The implantable tissue scaffold according to claim 1, wherein: The chitosan is a chitosan having a degree of deacetylation in the range of about 2% to about 99%, in the range of about 6% to 90%, in the range of about 6% to about 70%, 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 35% to about 65%, in the range of about 40% to about 60%, or in the range of about 45% to about 55%.

3. The implantable tissue scaffold according to claim 1 or claim 2, wherein: The chitosan has randomly distributed glucosamine.

4. The implantable tissue scaffold according to any one of claims 1 to 3, wherein: This chitosan in dry form is amorphous chitosan.

5. The implantable tissue scaffold according to any one of claims 1 to 4, wherein: The chitosan in dry form can absorb 10 times or more, 15 times or more, or 20 times or more of its weight in water.

6. The implantable tissue scaffold according to claim 5, wherein: The chitosan, after being exposed to water, forms a gel which can be dissolved by the addition of acid.

7. An implantable tissue scaffold as claimed in any one of the preceding claims, wherein: The weight average molecular weight (MW) of the chitosan is in the range of 200 Da-2000 kDa, such as in the range of 1 kDa-1000 kDa, in the range of 5 kDa-500 kDa, in the range of 10 kDa-400 kDa, in the range of 20 kDa-400 kDa or in the range of 100 kDa-400 kDa.

8. An implantable tissue scaffold as claimed in any one of the preceding claims, wherein: The chitosan is in the form of microparticles having an average particle size ranging from about 0.1 μm to about 50 μm, from about 1 μm to about 25 μm, from about 1 to 15 μm or less than 20 μm, or from about 5 μm to about 15 μm.

9. An implantable tissue scaffold as claimed in any one of the preceding claims, wherein: The biocompatible organic polymer is selected from the group consisting of polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and mixtures thereof.

10. An implantable tissue scaffold as claimed in any of the preceding claims, further comprising calcium phosphate in an amount in the range of 0.2%-20% (w / w), wherein the calcium phosphate is in the form of microparticles dispersed in the biocompatible organic polymer, the average diameter of the microparticles being 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.

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

12. The composition according to claim 11, wherein The chitosan is chitosan having 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. The composition of claim 11, wherein The composition contains N-acetylglucosamine (NAG) in the range of 0.01-25 mg / g, or 0.02-15 mg / g, or 0.1-15 mg / g, or 0.05-7.5 mg / g, preferably 0.1-5 mg / g.

14. The composition according to any one of claims 11 to 13, wherein The chitosan has randomly distributed glucosamine.

15. The composition according to any one of claims 11 to 14, wherein This chitosan in dry form is amorphous chitosan.

16. The composition according to any one of claims 11 to 15, wherein The chitosan in dry form can absorb 10 times or more, 15 times or more, or 20 times or more of its weight in water.

17. The composition of claim 16, wherein After chitosan is exposed to water to form a gel, it can be dissolved by adding acid.

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

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

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

21. A composition as described in any of the preceding claims 11-20, further comprising calcium phosphate in an amount in the range of 0.2%-20% (w / w), wherein the calcium phosphate is in the form of microparticles dispersed in the biocompatible organic polymer, the average diameter of the microparticles being 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.

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

23. The implantable tissue scaffold of any one of claims 1 to 10 for use in treating bone defects in humans or animals.

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