Porous osteoinductive composites

Abstract

To provide an osteoinductive composite, in particular, an osteoinductive composite comprising osteoinductive granules embedded in a matrix which can be used in a medical treatment of bone loss or bone defect.SOLUTION: The invention is directed to a porous osteoinductive composite comprising osteoinductive granules embedded in a porous matrix, where more than 5% of the surface area of the osteoinductive granules is exposed from the matrix as determined by scanning electron microscopy (SEM) imaging. Such a composite can suitably be used in a medical treatment, e.g., in a medical treatment of connective tissue and / or bone loss or defect.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is directed to osteoinductive composites. In particular, the present invention relates to osteoinductive composites comprising osteoinductive granules embedded in a matrix that can be used to treat missing bone or bone defects. [Background technology]

[0002] Lost bone or bone defects can be treated with bone graft composites. Several of these composites are known in the art. For example, osteoconductive composites based on calcium phosphate and silk or collagen matrices are known, such as those sold under the trade names i-Factor™, Vitoss™, Formagraft™, and Mastergraft™. The drawback of these materials is that they are only osteoconductive, i.e., they lack the potential to stimulate new bone formation in sites where there is no natural bone to provide osteoblasts.

[0003] EP 2749301 describes a biocompatible, resorbable composite for bone fusion, comprising osteoconductive particles dispersed within a porous polymer matrix having a plurality of fluid passages exposing at least a portion of the osteoconductive particles to the exterior of the polymer matrix.

[0004] EP 2730295 describes certain calcium phosphate-collagen fiber composites that are capable of inducing bone replacement through bone remodeling.

[0005] US Patent Application Publication No. 2008 / 0138381 describes certain bone implant composites that include a collagen matrix and calcium-based minerals.

[0006] U.S. Patent Application Publication No. 2017 / 0304502 describes a method for producing a bone implant, the method including applying mechanical force to an aqueous slurry of insoluble collagen fibers to entangle the insoluble collagen fibers and form a semi-solid mass of entangled insoluble collagen fibers, and freeze-drying the semi-solid mass of entangled collagen fibers to form the bone implant. A bone implant comprising entangled insoluble collagen fibers is also described.

[0007] US Pat. No. 5,338,772 describes an implant material based on a composite of calcium phosphate ceramic particles and a bioabsorbable polymer.

[0008] Composites that induce and stimulate new bone formation in the absence of natural bone (i.e., osteoinductive composites) are also known. Osteoinductive composites typically derive their osteoinductivity from osteoinductive granules present in the composite. Examples of synthetic osteoinductive granules include calcium phosphate (CaP)-based ceramics, such as those described in International Publication No. 2015 / 009154 (incorporated herein in its entirety). An example of a composite containing such granules is a putty composite in which these granules are combined with a polymer carrier, such as those described in International Publication No. 2016 / 144182 and commercially available as MagnetOs™. However, a drawback of this putty is that it is not porous, meaning that clinicians are unable or very limited in their ability to augment the putty-based construct with the patient's own tissue (e.g., blood, bone marrow aspirate, BMA) before placing the construct in the patient's body. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent No. 2749301 [Patent Document 2] European Patent No. 2730295 [Patent Document 3] U.S. Patent Application Publication No. 2008 / 0138381 [Patent Document 4] U.S. Patent Application Publication No. 2017 / 0304502 [Patent Document 5] U.S. Patent No. 5,338,772 [Patent Document 6] International Publication No. 2015 / 009154 [Patent Document 7] International Publication No. 2016 / 144182 [Non-patent literature]

[0010] [Non-Patent Document 1] Kumar et al., Fibers 6 (2018) 45, (doi:10.3390 / fib6030045) [Non-patent document 2] Ligon et al., Chemical Reviews, 117(2017) pp. 10212-10290, (10.1021 / acs.chemrev.7b00074) [Non-patent document 3] Dill and Morgelin, Int Wound J., 17(2020) pp. 618-630 [Non-patent document 4] Rahaman et al., Acta Biomateralia, 2011(6) pp. 2355-2373 [Non-patent document 5] Schneider, CA, Rasband, WS, Eliceiri, KW, "NIH Image to ImageJ: 25 years of image analysis" Nature Methods 9, pp. 671-675, 2012 [Non-patent document 6] ISO 527-2 -Plastics - Determination of Tensile Properties - Part 2: Test conditions for molding and extrusion plastics Summary of the Invention [Problem to be solved by the invention]

[0011] It would therefore be desirable to provide an osteoinductive composite that does not suffer from the drawbacks of known synthetic bone graft composites, is preferably porous and / or resorbable, and allows for the composite to be augmented with autologous tissue and / or other materials. [Means for solving the problem]

[0012] The inventors have surprisingly discovered that there is a correlation between the osteoinductive properties of the granules in the composite and the surface exposure, and that good osteoinductive properties in porous structures can be achieved if the surface of the granules is sufficiently exposed and not covered by a carrier material or matrix that binds to the granules in the composite.

[0013] That is, the present invention is directed to a porous osteoinductive composite comprising osteoinductive granules contained in a porous matrix, preferably wherein greater than 5% of the surface area of ​​the osteoinductive granules is exposed from said matrix as determined by scanning electron microscope (SEM) imaging. Sufficient exposure of the granule surface can alternatively or additionally be described and / or achieved in other ways as described herein. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a photograph of a preferred embodiment according to the present invention. [Figure 2] 1 is a photograph of a preferred embodiment according to the present invention. [Figure 3] 1 shows an SEM image of a preferred embodiment according to the present invention. [Figure 4] 1 shows an SEM image of a preferred embodiment according to the present invention. [Figure 5] 1 shows an SEM image of a comparative example. [Figure 6] 1 shows an SEM image of a comparative example. [Figure 7] 1 shows an SEM image of a comparative example. [Figure 8] 1 shows an SEM image of a comparative example. [Figure 9] 1 shows an SEM image of a comparative example. [Figure 10] FIG. 1 shows the results of surface exposure. [Figure 11] FIG. 1 shows the results of fiber content. DETAILED DESCRIPTION OF THE INVENTION

[0015] The granules according to the present invention are contained in a matrix, meaning that the granules are at least partially embedded, i.e., embedded, fixed, and / or surrounded by the matrix. The matrix can be seen as providing a support or carrier structure for the granules. However, in contrast to the polymeric materials in the putties described in WO 2016 / 144182, for example, the matrix does not cover the entire surface of the granules.

[0016] Without wishing to be bound by theory, the inventors believe that by providing surface exposure, for example, macrophages, osteoclasts, mesenchymal stem cells (MSc), osteoblasts and / or osteoprogenitor cells can use a matrix that remains structurally intact under physiological conditions for a sufficiently long period of time to infiltrate the composite, promoting pro-healing mechanisms and / or bone regeneration, without interfering with the differentiation of the cells to form new bone.

[0017] As used herein, a surface exposed from the matrix means that the surface is not directly covered by the matrix, so that the surface is unrestricted and readily accessible to macrophages, osteoclasts, osteoblasts, and osteoprogenitor cells. From SEM images, one skilled in the art can visually distinguish between the matrix and the granules and determine whether the surface area of ​​a granule is exposed or covered by the matrix material.

[0018] Therefore, the surface exposure of granules in a composite can be determined by SEM as follows: For several representative granules (e.g., from at least three granules) on the SEM image, the surface of the granules is analyzed and the amount of the surface that is covered and exposed is measured. This measurement can be assisted by software packages such as ImageJ.

[0019] The inventors have discovered that greater surface exposure of the granules leads to greater bone formation after implantation of the composite. Thus, preferably, more than 10%, more preferably more than 20%, even more preferably more than 30%, and most preferably more than 40% of the surface area of ​​the osteoinductive granules is exposed from the matrix, as determined by scanning electron microscope (SEM) imaging.

[0020] Granule surface exposure can also be expressed as the relative number of granules that exhibit at least a partially exposed surface. This can also be determined by SEM. For several representative granules (e.g., at least three granules) on the SEM image, the granule surfaces are analyzed, and the amount of granules that exhibit at least a partially exposed surface visible in the SEM image is divided by the total amount of detectable (either covered or at least partially exposed) granules. In a preferred embodiment, more than 20%, preferably more than 50%, and most preferably more than 75% of the granules exhibit at least a partially exposed surface area as determined by scanning electron microscope (SEM) imaging.

[0021] Another factor that may affect osteoinductive performance is the morphological structure of the matrix at the micrometer scale (i.e., 1-1000 μm scale). The morphological structure of a matrix can be described by the shape or form of its structural elements. Such elements may have, for example, a bulky shape or the shape of a sheet or fiber on the micrometer scale. A bulky shape refers to a shape that extends substantially evenly in all three dimensions on the micrometer scale. A sheet refers to a shape that extends substantially evenly in two dimensions on the micrometer scale. A fiber refers to a shape that extends more in one dimension than the other two dimensions on the micrometer scale. Thus, one skilled in the art can describe the structural elements of a matrix on the micrometer scale from SEM images. Unless otherwise specified, the morphology of the matrix and its structural elements will be described herein on the micrometer scale.

[0022] For good osteoinductivity, it is preferred that the matrix primarily contain fibers. The presence of sheets is less preferred, and bulky features are even less preferred, as these elements may obstruct both the surface of the granules and the passage into the interior of the composite. Thus, in preferred embodiments, the matrix contains fibers and sheets in a ratio of greater than 1:1, preferably greater than 3:1, more preferably greater than 4:1, and most preferably greater than 8:1, where fibers are defined as structures with a thickness and width of less than 50 μm, and sheets are defined as structures with a thickness and / or width greater than 50 μm. Thus, it is even more preferred that the matrix is ​​free of bulky features (defined for this purpose as features having a size greater than 50 μm in all three dimensions). Thus, the morphology of a matrix can also be described in terms of the relative amount of sheet and fiber structures to all structural elements of the matrix. Thus, in preferred embodiments, the matrix contains greater than 50%, preferably greater than 70%, more preferably greater than 90% fibers and / or less than 50%, preferably less than 30%, more preferably less than 10% sheets.

[0023] In a preferred embodiment of the present invention, the matrix is ​​fibrous, meaning that it contains fibers. Preferably, the fibers have an average diameter of less than 50 μm, preferably less than 30 μm. The length of the fibers may be longer, for example, much longer than 100 μm, or even longer, more than 500 μm. The diameter of the fibers can be measured using SEM images, optionally assisted by software packages such as ImageJ.

[0024] To allow sufficient penetration and access of arterial macrophages, osteoclasts, osteoblasts, and osteoprogenitor cells into the composite, it preferably exhibits a porosity in the range of 60-95%, preferably 70-90%. Herein, porosity is expressed as the void volume relative to the total volume of the composite, including the voids, and can be determined by taking the volume of a sample, measuring the mass of the components therein, and then calculating the volume of these components based on their known densities. The mass and amount of the composite components are typically known from their manufacturing process. Alternatively, they can be determined as described herein below to determine the mass of the matrix and granules.

[0025] The porous nature of the composites according to the present invention advantageously allows for the uptake of fluids, such as autologous tissue fluids. To facilitate fluid uptake, the composite preferably exhibits capillary or wicking properties. More preferably, the composite exhibits wicking of greater than 50%, preferably greater than 100%. Wicking can be determined by immersing the composite in a liquid for a certain period of time (i.e., 20 seconds) and measuring the difference in mass before and after immersion.

[0026] The matrix material may be biocompatible and biodegradable. Suitable materials may be selected based on material properties such as strength, biodegradability, and the ability to form a porous matrix. As described above, the matrix preferably remains structurally intact in vivo and in vitro under physiological conditions for a period long enough to mitigate migration of the composite within the body. The presence of the matrix material may contribute to this mitigation. Therefore, the matrix preferably biodegrades to the extent that it retains its matrix material in vivo for at least 24 hours, a time during which the composite is particularly susceptible to migration. More preferably, the matrix is ​​resistant to biodegradation to the extent that the composite retains its matrix material in vivo for at least one week, preferably at least three weeks. In certain embodiments, the matrix biodegrades to the extent that the composite loses its matrix material in vivo after about six weeks or more.

[0027] The biodegradability of a composite can also be expressed as the composite's ability to remain structurally intact. In a preferred embodiment, the composite exhibits structural integrity in phosphate buffered saline at 37°C for at least 5 days, preferably at least 12 days. As used herein, structural integrity means that the composite has a solid shape and its dimensions can be accurately measured.

[0028] Several polymers and fibrillation techniques are known from other or similar tissue regeneration technologies. Examples of fiber formation methods include electrospinning, solution blowing, additive manufacturing, freeze-drying, etc. See, for example, Kumar et al., Fibers 6 (2018) 45 (doi:10.3390 / fib6030045) and Ligon et al., Chemical Reviews, 117 (2017) 10212-10290 (10.1021 / acs.chemrev.7b00074).

[0029] Examples of materials suitable for the matrix include natural polymers, semi-synthetic polymers, and synthetic polymers. Thus, in a preferred embodiment, the matrix comprises: - one or more natural polymers selected from the group consisting of collagen, gelatin, fibrin, hyaluronic acid, silk fibroin, chitosan, alginate, cellulose, lignin, hydrogels derived from decellularized tissue, and other ECM-derived or ECM-mimicking natural polymers; - one or more semi-synthetic polymers such as gelatin methacryloyl (gelMA), hyaluronic acid methacrylate (HAMA); one or more synthetic polymers selected from the group consisting of polyethylene glycol (PEG), poloxamers, polylactic acids (PLA) such as poly(L-lactic acid) (PLLA), poly(ethylene glycol-co-lactic acid) (PELA), poly(poloxamer-co-lactic acid) (POLA), polyglycolic acid (PGA), poly(lactic acid-co-glycolic acid) (PGLA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyamide (PA), polyacrylonitrile (PAN), combinations and copolymers thereof; or combinations thereof. See, for example, Kumar et al., Fibers 6 (2018) p. 45 (doi:10.3390 / fib6030045) and references therein.

[0030] Collagen has been found to be particularly suitable and preferred, as it has been found to provide a desirable balance between hydrophilicity, strength, biodegradability, flexibility, and morphology options. Type I and type II collagen are particularly preferred. The collagen may be of porcine, bovine, equine, fish, etc. Most preferably, the matrix comprises bovine type I collagen. The collagen may be non-natural (e.g., chemically cross-linked) or natural. Preferably, the matrix comprises natural collagen, as natural collagen has been found to provide the most preferred morphology, as described herein.

[0031] Native collagen has been found to be particularly fibrous. Those skilled in the art can visually distinguish native collagen from non-native collagen, for example, using SEM images. For example, the diameter of collagen fibrils in isolated native collagen is the same as or at least similar to the diameter of fibrils in dermal tissue (e.g., about 90 nm). See, for example, Dill and Morgelin, Int Wound J., 17 (2020) pp. 618-630. Furthermore, native collagen is typically more fibrous than non-native collagen.

[0032] The granules used in the composites of the present invention are osteoinductive. This means that the biocompatible composite comprises one or more materials that are osteoinductive. The osteoinductive properties of the biocompatible composite can be achieved, for example, by adding bone morphogenetic proteins (BMPs) and / or other growth factors. In an even more preferred embodiment, the granular synthetic material is inherently osteoinductive. This means that the granular synthetic material itself stimulates new bone even in a non-bone environment (e.g., osteoinductive ceramics, demineralized bone matrix - DBM).

[0033] WO 2015 / 009154 (incorporated herein in its entirety) describes granules based on calcium phosphate (CaP) that are osteoinductive. The biodegradable polymeric material of the present invention has been found to be suitable for maintaining the osteoinductive properties of the granules as described in WO 2015 / 009154.

[0034] Granular synthetic materials according to the present invention may comprise calcium phosphate, bioactive glass, etc. Rahaman et al., Acta Biomateralia 2011(6) pp. 2355-2373, provide an overview of various synthetic materials for tissue engineering suitable for the present invention. The granules may be ceramic granules. Preferably, the granules comprise calcium phosphate. Such granules have proven to be particularly suitable for tissue regeneration.

[0035] The osteoinductive granules preferably have a size in the range of 100 to 2500 μm, preferably 250 to 1000 μm.

[0036] The osteoinductive properties of a particular granular synthetic material are generally due to its specific micro- and submicrosurface structure. Water can affect this structure, thereby affecting the osteoinductive properties of the granular synthetic material. Furthermore, other properties of the granular synthetic material may also be affected by the presence of water, for example, because the synthetic material may be partially dissolved by water. Furthermore, it is known that water can also affect the osteoinductive performance of other drugs, namely BMP and DBM. Therefore, to prevent or limit the loss of osteoinductive properties, the composite is preferably anhydrous (i.e., contains less than 2% water by weight, relative to the total weight of the composite). Anhydrous in the present invention therefore means that the granular synthetic material in the environment is sufficiently anhydrous to limit the loss of biological activity of the granular synthetic material so that the granular synthetic material sufficiently retains its chemical and structural properties and the biocompatible composite remains effective and therefore applicable.

[0037] The amount of osteoinductive granules in the composite is preferably greater than 50% by weight, more preferably greater than 75% by weight, and most preferably greater than 90% by weight, based on the weight of the composite, and / or the amount of matrix in the composite is preferably less than 50% by weight, more preferably less than 25% by weight, and most preferably less than 10% by weight. The masses and amounts of the composite components are typically known from their manufacturing process. Alternatively, the masses and amounts of the composite components can, in appropriate embodiments, be determined by measuring the ash content of the composite. That is, the ash content reflects the granular inorganic content, and the burned and vaporized organic content reflects the matrix.

[0038] The osteoinductive composite according to the present invention can have the form of a sheet, strip, block, rod, or stick, depending on the intended implantation site. See FIG. 1 for an exemplary preferred strip shape. Preferably, it is pliable and / or malleable, allowing it to be cut at 15°C (i.e., the temperature normally found in an operating room) for ease of placement by the clinician (see FIG. 2). For good handling, it is further preferred that the composite exhibit a tensile strength of at least 0.1 MPa, preferably between 0.1 MPa and 5 MPa, and / or a modulus of elasticity in the range of 2 to 300 MPa.

[0039] Another aspect of the present invention is a method for preparing a porous osteoinductive composite according to any of the preceding claims, the method comprising mixing osteoinductive granules with a solution containing a matrix, followed by freeze-drying the mixture.

[0040] The osteoinductive composites according to the present invention can be used in therapeutic methods, such as in the treatment of connective tissue and / or bone loss or bone defects.

[0041] The osteoinductive composite can induce and direct three-dimensional bone regeneration at the implanted defect site. When placed next to viable host bone, new bone is deposited on the implant's surface. The composite is resorbed and replaced by bone during the natural process of bone remodeling.

[0042] In a preferred embodiment, the osteoinductive composite is gamma sterilized.

[0043] The osteoinductive composite according to the present invention can be used as a bone void filler for voids and gaps that are not essential for the stability of bone structure. The osteoinductive composite can be used to treat surgically created bone defects or bone defects resulting from bone trauma. The osteoinductive composite can be used to fill bone voids or gaps in the skeletal system (i.e., limbs, spine, skull, mandible, maxilla, and pelvis), and can be combined with autologous bone, blood, platelet-rich plasma (PRP), and / or bone marrow.

[0044] In certain embodiments, the osteoinductive composites can be used in therapeutic methods for replacing or supplementing autologous and / or allogeneic cancellous corpus corpora, for example, in filling and bridging bone defects in the skeleton, including the spine, plastic reconstruction of damaged or resected bone areas, filling intervertebral implants, etc.

[0045] In certain embodiments, the osteoinductive composites can be used in therapeutic procedures to fill or reconstruct multi-walled (artificial or deformed) bone defects, such as defects after bone cyst removal, augmentation of atrophied alveolar ridges, sinus lift or sinus floor elevation, filling alveolar defects after tooth extraction to preserve the alveolar ridge, filling tooth extraction defects to create an implant bed, filling bi- or multi-walled bone pockets as well as dental bifurcation and trifurcation, defects after surgical removal of retained teeth or orthodontic osteotomy, and other multi-walled bone defects of the alveolar process and facial skull.

[0046] Thus, another aspect of the present invention is a method for treating connective tissue and / or bone loss or defects in a patient, comprising the steps of providing a porous osteoinductive composite according to the present invention, optionally augmenting said composite with autologous tissue such as BMA, blood or PRP of said patient, and implanting the composite into the patient.

[0047] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The term "and / or" includes any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprises" and / or "comprising" specify the presence of stated features but do not exclude the presence or addition of one or more other features.

[0048] Although, for purposes of clarity and conciseness of description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the present invention may include embodiments having all or any combination of the described features. [Example]

[0049] The invention can be illustrated by the following non-limiting examples.

[0050] Preparation and analysis of composite strips The composites were prepared by mixing MagnetOs™ granules ranging in size from 250 to 1000 μm with various aqueous collagen solutions at a ratio of 0.7 ml of granules per 1.0 ml of sample material. The mixtures were then poured into stainless steel molds, frozen, lyophilized, and sterilized using gamma irradiation prior to physicochemical characterization and in vivo evaluation.

[0051] Table 1 shows the physical and chemical properties of the obtained composite strips. SEM images are shown in Figures 3 to 9.

[0052] For Example 1, natural bovine type 1 collagen was used, provided by MedSkin Solutions Dr. Suwelack AG, Germany. SEM images of Example 1 are shown in Figures 3 and 4.

[0053] For Comparative Examples 1 to 10, bovine type I collagen supplied and processed by SouthernLights Biomaterials was used. FIG. 5 shows an SEM image of Comparative Example 1. 6 and 7 show SEM images of Comparative Example 2. 8 and 9 show SEM images of Comparative Example 10. For comparison, i-Factor™, Formagraft™, Vitoss™, and Mastergraft™ constructs are also included.

[0054] Analysis method Granule exposure The surface exposure of granules in the composites was determined by SEM as follows: SEM images were recorded from the top surface of the composite. The granule surfaces were then analyzed using ImageJ (Schneider, CA, Rasband, WS, Eliceiri, KW, "NIH Image to ImageJ: 25 years of image analysis," Nature Methods 9, pp. 671-675, 2012) for at least three representative granules in the SEM images to determine the surface coverage and exposure.

[0055] Granules can be viewed as 3D volumes with angular lines and circular pores representing irregularly shaped porous granules. A granule's surface was considered covered by matrix material if it was amorphous, smooth, shiny, homogeneous, and / or unstructured, possibly due to the presence of cubic salts resulting from product processing. A granule's surface was considered exposed if its microsurface structure was visible on the surface.

[0056] porosity Porosity is expressed as the void volume relative to the total volume of the composite including the voids and was determined using the following formula: Porosity%=l-(B d / P d ) In the formula, B d is the bulk density of the composite, which is determined by measuring the mass and volume, and P d is the known particle density of the component expressed as a ratio proportional to the matrix:granule mass % of the composite.

[0057] Mass% matrix / granules The collagen to granule mass ratio was determined by calcining the organic material (i.e., collagen) using a furnace. After measuring the mass of the sample, the sample was placed in a crucible and placed in a furnace under the following conditions: The temperature rises from room temperature to 150°C in 2 hours. The temperature rises from 150°C to 500°C in 7 hours. The temperature rose from 500°C to 600°C in 3 hours and 20 minutes. Hold at 600°C for 3 hours and 20 minutes. The furnace is shut down and allowed to cool slowly to room temperature overnight.

[0058] After cooling, the remaining inorganic material was weighed and the difference in mass of the material before and after being placed in the furnace was calculated using the following formula: Ash content %=M_f / M_i×100 where Mi is the initial material mass before entering the furnace and Mf is the final material mass after removal from the furnace.

[0059] morphology SEM images were recorded from the top surface of the composite. The dimensions and content of the fibers and sheets were then measured and averaged using ImageJ. For this purpose, fibers were defined as structures with thickness and width less than 50 μm, and sheets were defined as structures with thickness and / or width greater than 50 μm.

[0060] Average fiber dimensions were determined using ImageJ by measuring a minimum of 100 fibers and / or sheets per SEM image taken at a magnification of 250x or less. The average was determined based on measurements from three representative SEM images.

[0061] In vitro testing The moisture loss was determined by calculating the difference in mass percent of the sample after immersion for 20 seconds in phosphate buffered saline preheated to 37°C, where moisture loss is defined as the change in mass recorded between the dry mass and the hydrated mass.

[0062] Structural integrity after 12 days was determined by immersing samples in phosphate-buffered saline preheated to 37°C for up to 12 days and determining whether the samples remained structurally intact enough to allow accurate dimensional measurements.

[0063] Mechanical testing Tensile strength and modulus were determined according to "ISO 527-2 -Plastics - Determination of Tensile Properties - Part 2: Test conditions for molding and extrusion plastics".

[0064] [Table 1]

[0065] In vivo studies and sample evaluation Four male beagle dogs (12 months old) were used and underwent surgery under general aseptic conditions and anesthesia. MagnetOs granules (control) and most of the composites in Table 1 were implanted intramuscularly into the dorsal muscle (1 ml per sample), while the composite from Example 1 was also implanted into the condyle (φ6 × 10 mm) for collagen resorption analysis. Surgical procedures were performed to obtain explants at various time points. Finally, the animals were sacrificed, and samples were collected along with the surrounding tissue. Routine undecalcified histology was performed, and sections (10–20 μm) were stained with methylene blue / basic fuchsin to observe bone or Van Gieson stain to observe collagen. Histomorphometry was performed using the histological overview, and the area percentage of the target (e.g., collagen residue or bone) within the available space was calculated as target area × 100 / (target area − calcium phosphate (CaP) material).

[0066] One-way analysis of variance (ANOVA) with Tukey's post-test multiple comparisons and two-way ANOVA with Bonferroni's post-test multiple comparisons were performed.

[0067] The in vivo stability of the complex was found to be as follows: Collagen % for intramuscular implantation: Week 0 = 10.2 ± 1.7% Week 3 = 3 ± 0.4% Week 6 = 0.2 ± 0.3% Week 12 = 0% Collagen % for femoral condyle implant: Week 0 = 10.2 ± 1.7% Week 3 = 1.4 ± 1.7% Week 6 = 0% Week 12 = 0%

[0068] The bone growth results are shown in Table 2.

[0069] [Table 2]

[0070] Statistical analysis of the correlation between surface exposure (%), fiber content (%), and bone formation (%) A historical data design was constructed using Design Expert 13 (Build 13.0.1.0; Stat-Ease Inc.) using either surface coverage (%) or fiber content (%) as continuous factors and bone formation (%) as the response. Box-Cox analysis revealed that when surface coverage was used as the independent variable, no data transformation was necessary and lambda was maintained at 1.00. A linear regression model was generated, and ANOVA analysis revealed a significant effect of surface exposure (<0.0001) with an R-squared value of 0.935. When fiber content was used as the independent variable, the Box-Cox analysis recommended a square-root transformation with a k-value of 0.0135. The resulting linear regression model and ANOVA analysis revealed a significant effect of fiber content (0.0029) with an R-squared value of 0.7279.

[0071] The results of the surface exposure are shown in FIG.

[0072] The fiber content results are shown in FIG.

Claims

1. A porous osteoinductive composite comprising osteoinductive granules contained in a porous matrix, wherein the osteoinductive granules comprise calcium phosphate and / or bioactive glass, the matrix comprises collagen, and the matrix comprises more than 50% fibers and less than 50% sheets, where, as determined by scanning electron microscopy (SEM) imaging, fibers are defined as structures having a thickness and width of less than 50 μm, and sheets are defined as structures having a thickness and / or width of more than 50 μm.

2. The porous osteoinductive composite according to claim 1, wherein, as determined by scanning electron microscopy (SEM) imaging, more than 5% of the surface area of ​​the osteoinductive granules is exposed from the matrix.

3. The porous osteoinductive composite according to claim 1, wherein, as determined by scanning electron microscopy (SEM) imaging, more than 10% of the surface area of ​​the osteoinductive granules is exposed from the matrix.

4. The porous bone-inducible composite according to any one of claims 1 to 3, wherein, as determined by scanning electron microscopy (SEM) imaging, more than 20% of the granules have at least a partially exposed surface area.

5. The porous bone-inducing composite according to any one of claims 1 to 4, wherein the composite exhibits a porosity in the range of 60 to 95%.

6. The porous bone-inducing composite according to any one of claims 1 to 5, wherein the matrix comprises fibers having an average diameter of less than 50 μm.

7. A porous bone-inducing composite according to any one of claims 1 to 6, exhibiting water evaporation of more than 50% by mass.

8. The porous bone-inducible composite according to any one of claims 1 to 7, wherein the matrix includes pores larger than those of mesenchymal stem cells and macrophages, and openings to such pores.

9. The porous bone-inducible composite according to any one of claims 1 to 8, wherein the composite exhibits structural integrity for at least 5 days under phosphate-buffered saline at 37°C.

10. The porous bone-inducing composite according to any one of claims 1 to 9, wherein the matrix comprises type I and / or type III collagen.

11. The porous bone-inducing composite according to any one of claims 1 to 10, wherein the matrix comprises bovine type I collagen.

12. The porous bone-inducing composite according to any one of claims 1 to 11, wherein the matrix contains natural collagen.

13. A porous osteoinductive composite according to any one of claims 1 to 12, wherein the amount of osteoinductive granules in the composite is greater than 55% by mass of the composite, and / or the amount of matrix in the composite is less than 45% by mass.

14. A porous osteoinductive composite according to any one of claims 1 to 13, wherein the osteoinductive granules contain calcium phosphate.

15. A porous osteoinductive composite according to any one of claims 1 to 14, wherein the composite comprises osteoinductive granules having a size in the range of 100 to 2500 μm.

16. A porous bone-inducing composite according to any one of claims 1 to 15, exhibiting a tensile strength in the range of 0.1 MPa to 5 MPa and / or an elastic modulus in the range of 2 to 300 MPa.

17. A porous bone-inducing composite according to any one of claims 1 to 16, which is in the form of a sheet, strip, block, rod, or stick, and is pliable and / or flexible at 15°C.

18. A method for preparing a porous osteoinductive composite according to any one of claims 1 to 17, comprising the steps of mixing osteoinductive granules with a solution containing a matrix, and subsequently freeze-drying the mixture.

19. A porous bone-inducing composite according to any one of claims 1 to 17, for use in the treatment of connective tissue and / or bone loss or defects.

Images