CN117098567A - Porous osteoinductive composite material - Google Patents

Abstract

The present invention relates to a porous osteoinductive composite material comprising osteoinductive particles embedded in a porous matrix, wherein more than 5% of the surface area of the osteoinductive particles is exposed from said matrix as determined by Scanning Electron Microscope (SEM) imaging. Such composites may be suitably used in medical treatment, for example of connective tissue and/or bone loss or bone defects.

Description

Porous osteoinductive composite material

The present invention relates to osteoinductive composite materials. In particular, the present invention relates to osteoinductive composites comprising osteoinductive particles embedded in a matrix useful for treating bone loss or bone defects.

Bone loss or bone defects may be treated by bone graft composites. Several of these composites are known in the art. For example, a bone-conducting composite based on calcium phosphate and silk or collagen matrix, e.g. according to the trade name i-Factor ^TM 、Vitoss ^TM 、Formagraft ^TM And Mastergraft ^TM Those that are commercially available are known. A disadvantage of these materials is that they are only osteoconductive, which means that they lack the possibility of stimulating new bone formation at sites of natural bone where osteoblasts are not provided.

EP2749301 describes a biocompatible, resorbable composite for bone synthesis comprising bone-conducting particles dispersed within a porous polymer matrix having a plurality of fluid channels exposing at least a portion of the plurality of bone-conducting particles to the exterior of the polymer matrix.

EP2730295 describes a specific calcium phosphate-collagen fiber composite material which can induce bone replacement by bone remodeling.

US 2008/0138881 describes a specific bone implant composite material comprising a collagen matrix and a calcium-based mineral.

US2017/0304502 describes a method of manufacturing a bone implant, the method comprising applying mechanical force to an aqueous slurry of insoluble collagen fibers to entangle the insoluble collagen fibers so as to form a semi-solid mass of entangled insoluble collagen fibers; and freeze-drying the semi-solid mass of entangled collagen fibers to form a bone implant. A bone implant comprising entangled insoluble collagen fibers is also described.

US 5338772 describes an implant material based on a composite of calcium phosphate ceramic particles and a bioabsorbable polymer.

Composites that induce and stimulate new bone formation in the absence of natural bone (i.e., osteoinductive composites) are also known. Osteoinductive composites generally acquire their osteoinductive properties from osteoinductive particles present in the composite. Examples of synthetic osteoinductive particles include the calcium phosphate (CaP) based ceramics described in WO 2015/009154 (which is incorporated herein in its entirety). An example of a composite material comprising such particles is a putty (putty) composite material, wherein the particles are combined with a polymer carrier as described in WO 2016/144182 and may be used as magnetOs ^TM Commercially available. However, a disadvantage of this putty is that it is not porous and does not allow or very limited to allow a clinical practitioner to augment a putty-based construct with the patient's autologous tissue (e.g., blood, bone marrow aspirate, BMA) prior to placement of the construct in the patient.

It is therefore desirable to provide an osteoinductive composite that does not suffer from the drawbacks of known synthetic bone graft composites, and that is preferably porous and/or absorptive, and that allows the composite to be reinforced with autologous tissue and/or other materials.

The inventors have unexpectedly found a correlation between the osteoinductive properties of particles in a composite material and surface exposure, and that good osteoinductive properties can be achieved in a porous structure if the surfaces of the particles are sufficiently exposed and not covered by a carrier material or matrix to which the particles in the composite material are bound.

Accordingly, the present invention relates to a porous osteoinductive composite material comprising osteoinductive particles comprised in a porous matrix, wherein preferably more than 5% of the surface area of the osteoinductive particles is exposed from said matrix, as determined by Scanning Electron Microscope (SEM) imaging. The sufficient exposure of the particle surface may alternatively or additionally be described and/or achieved in other ways described herein.

Figures 1 and 2 are photographs of a preferred embodiment according to the present invention.

Fig. 3 and 4 show SEM images according to a preferred embodiment of the present invention.

Fig. 5-8 show SEM images of the comparative examples.

The particles according to the invention are contained in a matrix, which means that they are at least partially embedded, e.g. incorporated, immobilized and/or surrounded, by the matrix. The matrix may be considered to provide a support or carrier structure for the particles. However, the matrix does not cover the entire surface of the particles, as opposed to the case of polymeric materials in putties such as described in WO 2016/144182.

Without wishing to be bound by theory, the inventors believe that by providing surface exposure, a matrix that remains structurally intact under physiological conditions for a sufficient period of time may be used, for example, to promote the invasion of macrophages, osteoclasts, mesenchymal stem cells (MSc's), osteoblasts and/or osteoprogenitor cells into the composite material that promote the healing mechanism and/or bone regeneration, while not preventing the differentiation of the cells into new bone.

By a surface exposed from the matrix is meant herein that the surface is not directly covered by the matrix such that the surface is free and accessible to macrophages, osteoclasts, osteoblasts and osteoprogenitor cells to contact the surface. From SEM images, one skilled in the art can visually distinguish between the matrix and the particulate material and can determine whether the surface area of the particles is exposed or covered by the matrix material.

Thus, the surface exposure of the particles in the composite material can be determined by SEM as follows. For many representative particles (e.g., from at least three particles) on the SEM image, the surface of the particles is analyzed and the amount of coverage and exposure of the surface is measured. Such measurements may be aided by a software package such as ImageJ.

The inventors found that more surface exposure of the particles resulted in more bone formation after implantation of the composite material. Thus, preferably more than 10%, more preferably more than 20%, even more preferably more than 30%, most preferably more than 40% of the surface area of the osteoinductive particles is exposed from the matrix, as determined by Scanning Electron Microscope (SEM) imaging.

The exposure of the particle surface may also be expressed as the relative number of particles exhibiting an at least partially exposed surface. This can also be determined by SEM. For many representative particles (e.g., from at least three particles) on the SEM image, the surface of the particles is analyzed, and the amount of particles exhibiting at least a partially exposed surface is divided by the total amount of detectable particles (covered or at least partially exposed) visible in the SEM image. In a preferred embodiment, more than 20%, preferably more than 50%, most preferably more than 75% of the particles exhibit at least partially exposed surface area as determined by Scanning Electron Microscope (SEM) imaging.

Another factor that can affect osteoinductive capacity is the morphological structure of the matrix on the micrometer scale (i.e., on the scale of 1-1000 μm). The morphological structure of a matrix can be described by the shape or morphology of its structural elements. Such elements may for example have a bulk shape (bulk shape), or a sheet or fibre shape on the micrometer scale. By bulk shape is meant a shape that extends substantially equally in all three dimensions on the micrometer scale. By flakes is meant shapes that extend substantially equally in two dimensions on a micrometer scale. Fiber means a shape that extends more in one dimension than in the other two dimensions on the micrometer scale. From SEM images, the skilled person can describe the structural elements of the matrix correspondingly on the micrometer scale. The morphology of the matrix and its structural elements is described herein on the micrometer scale unless explicitly indicated otherwise.

For good osteoinductive properties, it is preferred that the matrix comprises mainly fibres. The presence of flakes is less preferred and a bulk shape is less preferred as these elements will block the surface of the particles and the channels into the interior of the composite. Thus, in a preferred embodiment, the matrix comprises fibers and flakes in a ratio of more than 1:1, preferably more than 3:1, more preferably more than 4:1, most preferably more than 8:1, wherein fibers are defined as structures having a thickness and width of less than 50 μm and flakes are defined as structures having a thickness and/or width of more than 50 μm. Thus, it is more preferred that the matrix is free of bulk shapes (for this purpose, defined as shapes having dimensions exceeding 50 μm in all three dimensions). Thus, the morphology of the matrix can also be described based on the relative amounts of the sheet and fibrous structure with respect to all structural elements of the matrix. Thus, in a preferred embodiment, the matrix comprises more than 50%, preferably more than 70%, more preferably more than 90% fibres and/or less than 50%, preferably less than 30%, more preferably less than 10% flakes.

In a preferred embodiment of the invention, the matrix is fibrous, which means that it comprises fibers. Preferably, the fibres have an average diameter of less than 50 μm, preferably less than 30 μm. The length of the fibers may be much longer, for example over 100 μm, or even longer, such as over 500 μm. The diameter of the fibers can be measured using SEM images, optionally aided by a software package such as ImageJ.

In order to allow the macrophages, osteoclasts, osteoblasts and osteoprogenitors to penetrate and penetrate sufficiently into the composite material, it preferably exhibits a porosity in the range of 60% -95%, preferably in the range of 70% -90%. Porosity is expressed herein as the relative void volume relative to the total volume of the composite material including voids, and can be determined by taking into account the volume of the sample, measuring the weight of the components therein, and calculating the volume of the components accordingly based on the known densities of the components. The weight and amount of the composite material is generally known from the production process thereof. Alternatively, it may be determined as described below for determining the weight of the substrate and particles.

The porous nature of the composite material according to the invention advantageously allows the absorption of fluids, such as autologous tissue fluids. For ease of fluid absorption, the composite preferably exhibits capillary or wicking properties. More preferably, the composite exhibits more than 50%, preferably more than 100% wicking. The wicking effect can be determined by immersing the composite material in the fluid for a set point in time (i.e., 20 seconds) and measuring the weight difference before and after immersion.

The materials used for the matrix are biocompatible and may be biodegradable. Suitable materials may be appropriately selected by considering material characteristics such as strength, biodegradability and ability to form a porous matrix. As described above, the matrix preferably remains structurally intact for a period of time sufficient under physiological conditions, both in vivo and in vitro, to mitigate migration of the complex in vivo. The presence of matrix material may assist in this mitigation. Thus, the matrix is preferably biodegradable to such an extent that the composite retains the matrix material in the body for at least 24 hours, during which time the composite may be particularly susceptible to migration. More preferably, the matrix is preferably resistant to biodegradation to such an extent that the composite retains the matrix material in the body for at least 1 week, preferably at least 3 weeks. In certain embodiments, the matrix is biodegradable to the extent that the composite loses its matrix material in vivo after about 6 weeks or more.

The biodegradability of a composite material may also be expressed as the ability of the composite material to maintain structural integrity. In a preferred embodiment, the composite exhibits structural integrity for at least 5 days, preferably at least 12 days, under phosphate buffered saline solution at 37 ℃. In this context, structurally complete means that the composite material has a solid shape and its dimensions can be precisely measured.

From other or similar tissue regeneration techniques, several polymer and fibrillation techniques are known. Examples of fiber forming methods include electrospinning, solution-spraying (solution-blowing), additive manufacturing, freeze drying, and the like. See, for example, kumar et al, fibers 6 (2018) 45 (doi: 10.3390/fib 6030045) and Ligon et al, chemical Reviews,117 (2017) 10212-10290 (10.1021/acs. Chemrev.7b00074).

Examples of suitable materials for the matrix include natural polymers, semisynthetic 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, and other ECM-derived or ECM-mimicking natural polymers derived from decellularized tissue;

-one or more semisynthetic polymers, such as methacryloylated gelatin (gelMA), methacryloylated hyaluronic acid (HAMA);

-one or more synthetic polymers selected from: polyethylene glycols (PEG), poloxamers (polyoxamers), polylactic acids (PLA) such as poly (L-lactic acid) (PLLA), poly (ethylene glycol-lactic acid copolymer) (PELA), poly (poloxamer-lactic acid copolymer) (POLA), polyglycolic acid (PGA), poly (lactic acid-glycolic acid copolymer) (PGLA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyamide (PA), polyacrylonitrile (PAN), combinations and copolymers of the above;

or a combination of the above. See, e.g., kumar et al, fibers 6 (2018) 45 (doi: 10.3390/fib 6030045) and references therein.

Collagen was found to be particularly suitable and preferred as it was found to provide the desired balance between hydrophilicity, strength, biodegradability, flexibility and morphological options. Type I and type II collagen are particularly preferred. The collagen source can be pig, cow, horse, fish, etc. Most preferably, the matrix comprises bovine type I collagen. Collagen may be non-native (e.g., chemically cross-linked) or native. Preferably, the matrix comprises native collagen, as this was found to provide the most advantageous morphology as described herein.

Natural collagen has been found to be particularly fibrous. The skilled artisan can visually distinguish between native collagen and non-native collagen using, for example, SEM images. For example, the collagen fibril diameter of isolated native collagen is related to the fibril diameter of dermal tissueAt or at least near (e.g., about 90 nm). See, e.g., dill andint Wound j.17 (2020) 618-630. Moreover, natural collagen is generally more fibrous than non-natural collagen.

The particles used in the composite material of the present invention are osteoinductive. This means that the biocompatible composite material comprises one or more materials that are osteoinductive. The osteoinductive properties of the biocompatible composite material may be achieved, for example, by the addition of Bone Morphogenic Proteins (BMP) and/or other growth factors. In an even more preferred embodiment, the particulate synthetic material is osteoinductive in nature. This means that the particulate composite material itself can stimulate new bone (e.g., osteoinductive ceramics, demineralized bone matrix-DBM) even in non-bone environments.

WO 2015/009154 (which is incorporated herein in its entirety) describes calcium phosphate CaP-based particles with osteoinductive properties. As described in WO 2015/009154, the biodegradable polymeric materials of the present invention were found to be suitable for maintaining the osteoinductive properties of the particles.

The particulate synthetic material according to the present invention may comprise calcium phosphate, bioactive glass, and the like. Rahman et al acta biomaterialia 2011 (6) 2355-2373 reviewed various synthetic materials suitable for use in the present invention for tissue engineering. The particles may be ceramic particles. Preferably, the particles comprise calcium phosphate. Such particles have proven to be particularly suitable for tissue regeneration.

The osteoinductive particles preferably have a size in the range of 100-2500 μm, preferably 250-1000 μm.

The osteoinductive properties of certain particulate synthetic materials are generally attributed to their specific microscopic and sub-microscopic surface structures. Water can affect this structure and thus the osteoinductive properties of the particulate composite material. Other properties of the particulate composite material may also be affected by the presence of water, as the composite material may be partially dissolved, for example, by water. Furthermore, it is known that water can also affect the osteoinductive potential of other agents, namely BMP and DBM. Thus, the composite is preferably anhydrous (i.e., contains less than 2wt% water based on the total weight of the composite) to prevent or limit loss of osteoinductive properties. Thus, anhydrous in the present invention means that the environmental particulate composite material is sufficiently anhydrous for the particulate composite material to retain chemical and structural properties sufficiently to limit the decrease in biological activity of the particulate composite material so that the biocompatible composite material remains effective and thus can be used.

The amount of osteoinductive particles in the composite is preferably more than 50wt%, more preferably more than 75wt%, most preferably more than 90wt%, and/or the amount of matrix in the composite is preferably less than 50wt%, more preferably less than 25wt%, most preferably less than 10wt%, based on the weight of the composite. The weight and amount of the composite components are generally known from the production process thereof. Alternatively, in suitable embodiments, the weight and amount of the composite constituents may be determined by measuring the ash content of the composite. Thus, ash content reflects particulate inorganic content, while the burning and evaporating organic content reflects the matrix.

The osteoinductive composite material according to the invention may have the form of a sheet, strip, block, rod or stick, depending on the intended site for implantation. Exemplary and preferred strips are shown in fig. 1. Preferably, it is formable, such as pliable and/or flexible, and can be cut at 15 ℃ (i.e. typical temperatures in an operating room) as this facilitates placement by a clinical practitioner (see fig. 2). For good handling it is further preferred that the composite material exhibits a tensile strength of at least 0.1MPa, preferably 0.1MPa-5MPa and/or an elastic modulus in the range of 2-300 MPa.

Another aspect of the invention is a method of preparing a porous osteoinductive composite material according to any preceding claim, the method comprising mixing osteoinductive particles with a solution comprising a matrix, and then freeze drying the mixture.

The osteoinductive composite material according to the invention can be used for medical treatment, such as of connective tissue and/or bone loss or bone defects.

The osteoinductive composite material can induce and guide three-dimensional regeneration of bone at the site of its implantation defect. When placed beside a viable host bone, new bone will be deposited on the surface of the implant. In the natural process of bone remodeling, the composite absorbs and is replaced by bone.

In a preferred embodiment, the osteoinductive composite material is gamma sterilized.

The osteoinductive composite material according to the invention can be used as bone void filler for voids and gaps which are not inherent in the stability of the bone structure. The osteoinductive composite material can be used to treat bone defects resulting from surgery or bone defects resulting from bone trauma. The osteoinductive composite material may fill in bone voids or interstices of the skeletal system (i.e., limbs, vertebrae, skull, mandible, maxilla, and pelvis) and may be combined with autologous bone, blood, platelet Rich Plasma (PRP), and/or bone marrow.

In particular embodiments, the osteoinductive composite material may be used in medical procedures to replace or supplement autologous and/or allogenic cancellous bone, for example, for filling and bridging plastic reconstruction of bone areas including bone defects, injuries or resections of the spine, filling of intervertebral implants.

In particular embodiments, the osteoinductive composite material may be used in medical procedures to fill or reconstruct multi-walled (artificial or degenerative) bone defects, such as defects after removal of bone cysts, enlargement of atrophic alveolar ridges, sinus augmentation or sinus floor lifting, filling alveolar defects after tooth extraction to protect the alveolar ridges, filling extraction defects used to form implant beds, filling double-walled or multi-walled bone bags and double and triple bifurcations of teeth, surgical removal of defects after retention teeth or corrective osteotomies, alveolar processes and other multi-walled bone defects of the facial skull.

Accordingly, another aspect of the present disclosure is a method of treating connective tissue and/or bone loss or bone defect in a patient, the method comprising providing a porous osteoinductive composite according to the invention, optionally reinforcing the composite with autologous tissue of the patient, such as BMA, blood or PRP, and implanting the composite into the patient.

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

For the purposes of clarity and brevity, features will be described herein as part of the same or separate embodiment, however, it is to be understood that the scope of the invention may include embodiments having a combination of all or some of the features described.

The invention may be illustrated by the following non-limiting examples.

Preparation and analysis of composite strips

By mixing magnetOs with a size of 250-1000 μm in a proportion of 0.7ml particles per 1.0ml sample material ^TM The particles are mixed with different aqueous collagen solutions to prepare a composite material. The mixture was then poured into stainless steel molds, frozen, freeze-dried, and sterilized using gamma irradiation prior to physicochemical characterization testing and in vivo evaluation.

Table 1 shows the physical and chemical properties of the obtained composite strips. SEM images are provided in fig. 3-9.

For example 1, native bovine type 1 collagen supplied by MedSkin Solutions dr. SEM images of example 1 are provided in fig. 3 and 4.

For comparative examples 1-10, bovine type I collagen provided and treated by SouthernLights Biomaterials was used.

Fig. 5 shows an SEM image of comparative example 1.

Fig. 6 and 7 show SEM images of comparative example 2.

Fig. 7 and 8 show SEM images of comparative example 10.

For comparison, also include i-Factor ^TM 、Formagraft ^TM 、Vitoss ^TM And Mastergraft ^TM Constructs。

Analysis method

Particle exposure

The surface exposure of the particles in the composite material was determined by SEM as follows. SEM images from top view of the composite were recorded. Then, for at least three representative particles on SEM images, the surface of the particles was analyzed and the amount of coverage and exposure of the surface was measured using ImageJ (Schneider, c.a., rasband, w.s., elibeiri, k.w. "NIH Image to ImageJ:25years of image analysis"Nature Methods 9,671-675,2012).

The particles may be considered as 3D volumes with angular lines and/or circular pores representing irregularly shaped porous particles. When the surface of the particles is amorphous, smooth, glossy, of uniform quality and/or non-structural, cubic salts may be present as a result of the processing of the product, the surface is considered to be covered by the matrix material. When the microscopic surface structure of the particle is visible on a surface, the surface is considered to be exposed.

Porosity of the porous body

Porosity is expressed as the relative void volume relative to the total volume of the composite material including the void and is determined using the following equation:

porosity% _d /P _d )

Wherein B is _d Is determined by measuring the bulk density of the composite material by mass and volume, and P _d Is the known particle density of the ingredients, which is proportional to the matrix of the composite material, the weight% of the particles.

Matrix/particle weight%

The weight ratio of collagen to particles is measured by burning off the organic material (i.e., collagen) using an oven. After measuring the mass of the sample, it was placed in a crucible and placed in a furnace under the following conditions:

2h to increase the heat from room temperature to 150℃

7h to increase the heat from 150℃to 500 DEG C

3h 20min to increase heat from 500℃to 600 DEG C

Maintaining at 600deg.C for 3h 20min

The furnace was turned off and slowly cooled back to room temperature overnight

Once cooled, the remaining inorganic material was weighed and the weight difference of the material before and after placement in the furnace was calculated using the following equation:

ash content% = m_f/m_i 100

Where Mi is the initial material weight before it is placed in the furnace and Mf is the final material weight after it is removed from the furnace.

Morphology of the product

SEM images from top view of the composite were recorded. The size and content of the fibers and flakes were then measured and averaged using ImageJ. For this purpose, a fiber is defined as a structure having a thickness and width of less than 50 μm, and a sheet is defined as a structure having a thickness and/or width of more than 50 μm.

The average fiber size was determined by measuring at least 100 fibers and/or flakes per SEM image taken at a magnification of 250x or less using ImageJ. The average was determined based on measurements from three representative SEM images.

In vitro testing

The wicking effect was determined by calculating the wt% difference of the samples after 20 seconds of immersion in phosphate buffered saline solution preheated to 37 ℃. In this case, wicking ability is defined as the weight change between the recorded dry weight and the hydrated weight.

The structural integrity after 12 days was determined by immersing the sample in a phosphate buffered saline solution preheated to 37 ℃ for up to 12 days and determining if the sample remained structurally intact until its dimensions could be accurately measured.

Mechanical testing

Tensile Strength and elastic modulus are measured according to ISO 527-2-Plastic-tensile Properties-part 2: the test conditions for molding and extrusion of the plastic are determined.

TABLE 1

¹ Decomposition of

² Weight% of the silk fiber matrix

In vivo studies and sample evaluation

Four beagle dogs (male, 12 months old) were used and the procedure was performed under general sterile conditions and under anesthesia. The MagnetOs particles (control) and most of the composites in table 1 were implanted intramuscularly into the back muscle (1 ml per sample), while the composites of example 1 were also implanted into the condyles (phi 6x10 mm) for collagen absorption analysis. Surgical procedures were performed at different time points to obtain explants. Finally, animals were sacrificed and samples with surrounding tissues were harvested. Conventional non-decalcification histological examination was performed, sections (10-20 μm) were stained with methylene blue/basic fuchsin to view bone, or van Gieson to view collagen. Histomorphometry was performed using a histological overview and the area percentage of the target (e.g. collagen residues and bones) in the available space was calculated as target area x 100/(target area-calcium phosphate (CaP) material).

One-way analysis of variance (ANOVA) using a basis (tukey) post-hoc multiple comparisons and two-way ANOVA using Bonferroni post-hoc multiple comparisons were performed.

The in vivo stability of the composite was found to be as follows.

Intramuscular implanted collagen%:

week 0 = 10.2 ± 1.7%

Week 3 = 3 ± 0.4%

Week 6 = 0.2 ± 0.3%

Week 12 = 0%

Collagen% of femoral condyle implant:

week 0 = 10.2 ± 1.7%

Week 3 = 1.4 ± 1.7%

Week 6 = 0%

Week 12 = 0%

The bone growth results are shown in table 2.

TABLE 2

Statistical analysis of the correlation between surface exposure (%), fiber content (%) and bone formation (%)

Historical data designs were constructed using Design Expert 13 (Build 13.0.1.0; stat-Ease inc.) using surface coverage (%) or fiber content (%) as a continuous numerical factor and bone formation (%) as a response. box-cox analysis revealed that when using surface coverage as an argument, no data conversion was required, lambda was kept at 1.00. A linear regression model was generated and ANOVA analysis found that the effect from surface exposure was significant (< 0.0001), with a square R value of 0.935. When using fiber content as an argument, box-cox analysis suggests square root conversion with a k value of 0.0135. The resulting linear regression model and ANOVA analysis found that the effect from fiber content was significant (0.0029) with a square R value of 0.7279.

The results of the surface exposure are provided in fig. 10.

The results of the fiber content are provided in fig. 11.

Claims (20)

1. A porous osteoinductive composite comprising osteoinductive particles contained in a porous matrix, wherein more than 5% of the surface area of the osteoinductive particles is exposed from the matrix as determined by Scanning Electron Microscope (SEM) imaging.

2. The porous osteoinductive composite of the preceding claim, wherein more than 10%, preferably more than 20%, even more preferably more than 30%, most preferably more than 40% of the surface area of the osteoinductive particles is exposed from the matrix as determined by Scanning Electron Microscope (SEM) imaging.

3. A porous osteoinductive composite according to any of the preceding claims, wherein more than 20%, preferably more than 50%, most preferably more than 75% of the particles exhibit an at least partially exposed surface area as determined by Scanning Electron Microscope (SEM) imaging.

4. A porous osteoinductive composite as claimed in any of the preceding claims, wherein the composite exhibits a porosity in the range of 60% -95%, preferably in the range of 70% -90%.

5. A porous osteoinductive composite according to any of the preceding claims, wherein the matrix comprises fibres and flakes in a ratio of more than 1:1, preferably more than 3:1, more preferably more than 4:1, most preferably more than 8:1, wherein fibres are defined as structures having a thickness and width of less than 50 μm and flakes are defined as structures having a thickness and/or width of more than 50 μm.

6. A porous osteoinductive composite according to any of the preceding claims, wherein the matrix comprises fibres having an average diameter of less than 50 μm, preferably less than 30 μm, more preferably less than 20 μm.

7. A porous osteoinductive composite according to any of the preceding claims, which exhibits a wicking effect of more than 50wt%, preferably more than 100 wt%.

8. The porous osteoinductive composite of any of the preceding claims, wherein the matrix comprises pores larger than mesenchymal stem cells and macrophages and openings to the pores.

9. A porous osteoinductive composite according to any of the preceding claims, wherein the composite exhibits structural integrity under phosphate buffered saline solution at 37 ℃ for at least 5 days, preferably at least 12 days.

10. The porous osteoinductive composite of any of the preceding claims, wherein 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, and other ECM-derived or ECM-mimicking natural polymers derived from decellularized tissue;

-one or more semisynthetic polymers, such as methacryloylated gelatin (gelMA), methacryloylated hyaluronic acid (HAMA);

-one or more synthetic polymers selected from: polyethylene glycol (PEG), poloxamers, polylactic acid (PLA) such as poly (L-lactic acid) (PLLA), poly (ethylene glycol-lactic acid copolymer) (PELA), poly (poloxamer-lactic acid copolymer) (POLA), polyglycolic acid (PGA), poly (lactic acid-glycolic acid copolymer) (PGLA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyamide (PA), polyacrylonitrile (PAN), combinations and copolymers of the above;

or a combination of the above.

11. A porous osteoinductive composite according to any of the preceding claims, wherein the matrix comprises collagen, preferably type I and/or type III collagen, more preferably bovine type I collagen.

12. The porous osteoinductive composite of any of the preceding claims, wherein the matrix comprises native collagen.

13. A porous osteoinductive composite according to any of the preceding claims, wherein the amount of osteoinductive particles in the composite is more than 55wt%, preferably more than 75wt%, most preferably more than 90wt%, based on the weight of the composite, and/or wherein the amount of matrix in the composite is less than 45wt%, preferably less than 25wt%, most preferably less than 10wt%.

14. The porous osteoinductive composite of any of the preceding claims, wherein the osteoinductive particles comprise calcium phosphate.

15. A porous osteoinductive composite as claimed in any of the preceding claims, wherein the composite comprises osteoinductive particles having a size in the range of 100-2500 μm, preferably 250-1000 μm.

16. A porous osteoinductive composite as claimed in any of the preceding claims, which exhibits a tensile strength in the range of 0.1MPa-5MPa and/or an elastic modulus in the range of 2-300 MPa.

17. A porous osteoinductive composite as claimed in any one of the preceding claims, which is formable at 15 ℃, such as in the form of a flexible and/or pliable sheet, strip, block, rod or stick.

18. A method of preparing a porous osteoinductive composite material according to any of the preceding claims, the method comprising mixing the osteoinductive particles with a solution comprising the matrix, and then freeze drying the mixture.

19. Use of the porous osteoinductive composite of any of claims 1 to 17 for medical treatment, preferably for medical treatment of connective tissue and/or bone loss or bone defects.

20. A method of treating connective tissue and/or bone loss or bone defect in a patient, the method comprising providing the porous osteoinductive composite of any of claims 1-17, optionally reinforcing the composite with autologous tissue of the patient, such as BMA, and implanting the composite into the patient.