WO2021124108A1 - Biocomposite, methods of production and uses thereof - Google Patents

Abstract

The present disclosure relates to a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules ("HEPHAPC granules), a method of obtaining a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules ("HEPHAPC granules), and uses thereof, preferably a heparinized biocomposite comprising nanohydroxyapatite/collagen granules ("HEPHAPC granules"). The new heparinized biocomposite comprising nanohydroxyapatite/collagen granules ("HEPHAPC granules) is able to release high concentrations of antibiotic improving the treatment of bone infection and osteomyelitis and thereafter induce new bone regeneration capability.

Description

BIOCOMPOSITE, METHODS OF PRODUCTION AND USES THEREOF TECHNICAL FIELD The present disclosure relates a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules”), a method of obtaining a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules”), and uses thereof; preferably a heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules”), which is able to release high concentrations of antibiotic and subsequently improve bone regeneration capability. BACKGROUND Osteomyelitis is a chronic bone infection caused by bacteria which may result in bone necrosis and amputation, with serious risk of deadly septicaemia. This bone infection is often associated with diabetic foot ulcers, joint replacement surgeries and open fractures. Osteomyelitis affects around 4 million people worldwide every year, of which 0.5 million is in the US and the EU. Several micro-organisms may be the cause of osteomyelitis, Staphylococcus aureus (S. aureus) being the most common pathogen. Current treatment involves debridement of necrotic and infected regions of the bone and soft tissues, followed by intravenous administration of antibiotics for 4 – 6 weeks and oral therapy that can last an additional 8-10 weeks. This infection is difficult to control, particularly when caused by drug resistant strains of S. aureus, such as Methicillin-resistant S. aureus (MRSA) [1]. The conventional treatment presents several limitations such as the need for a minimum of two surgeries, one surgery with tissue debridement and antibiotic administration (this can be repeated several times until complete eradication) and a second surgery to promote bone reconstruction at the defect site. Since the infected regions are poorly irrigated, high doses of antibiotics are needed to obtain locally acceptable concentrations. Finally, systemic intravenous administration of antibiotics requires long-term hospitalization, results in pain, impaired mobility, impaired long-term bone regeneration, major collateral effects, extensive labour time lost and high associated costs. Considering all these limitations, there is a substantial need to improve therapeutic interventions for osteomyelitis. To treat orthopaedic infections, poly(methyl methacrylate) (PMMA) has been the gold standard biomaterial used for local antibiotic delivery. However, it is still fraught with limitations such as the high rates of re-infection and the need for subsequent surgical interventions to remove the PMMA. Such subsequent surgical intervention exposes the patient to new risks of infection and thus increased morbidity. These issues could be overcome by using novel bioactive materials. Recently, bioactive materials have been developed and evaluated for local delivery of antibiotics in the treatment of osteomyelitis. These bioactive implants could provide local antibiotics concentrations in the tissue for a duration long enough to eradicate the infection. Example of these materials are bioactive glass, hydroxyapatite, ceramic based cement and, injectable gelling biodegradable polymer [2]. However, such implantable materials exhibiting bioactivity and capacity to promote bone regeneration cannot simultaneously allow for localised and controlled drug release Further, some of these materials lack the porosity necessary to promote growth of new bone as their compact structures does not mimic bone extracellular matrix and hence, they are unable to promote rapid osteoblast proliferation, bone repair and bone regeneration. Thus, the ideal strategy would include the provision of a biomaterial allowing localized and controlled antibiotic release while being capable of promoting bone regeneration at the defective site. Biocomposite materials seem to be a promising choice for local antibiotic delivery yet they have been comparatively less researched as compared to ceramics and polymers. Particularly, there is growing interest in using nanohydroxyapatite/collagen biocomposites as a drug delivery carrier for bone as they not only are capable of mimicking the composition and structure of bone tissue [3], but also promote bone regeneration and have the capacity to carry and to release different biomolecules locally. Heparin has already been immobilized on different biomaterials such as nanohydroxyapatite/collagen composites to improve their performance as drug delivery systems. Heparin improves the binding capability of the biocomposite material without compromising the structure, the releasing capacity and the bioactivity of several other relevant biomolecules such as antibiotics [4] and growth factors [5]. Given that osteomyelitis treatment requires local antibiotic delivery followed by bone regeneration, this biocomposite can be a promising treatment strategy. However, handling properties are extremely important for the commercial success of these materials as they must be functional and easy to manipulate by surgeons during the implantation process. Document WO2015/162561 relates to a composition for treatment and/or prevention of infections, namely bone diseases, in particular osteomyelitis, via a controlled release of antibiotics, subsequently inducing regeneration of bone tissue that often undergoes necrosis due to infection. The document relates in particular to a pharmaceutical composition comprising one or more granules containing calcium phosphate, collagen and one or more polymers of heparin, and an antibiotic in an effective therapeutic amount, wherein the antibiotic is bound to the heparin polymers. These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION The present disclosure relates to a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules), a method of obtaining a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules) and uses thereof, preferably a heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules”). The new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules) is able to release high concentrations of antibiotic and improve the induction of bone regeneration. The biocomposite material of the present subject-matter is different from the one disclosed in the document WO2015/162561 because the new material is subjected to a different second heat-treatment cycle which uses a different temperature for the second plateau (sintering temperature) during the production of the bioceramic material of the present disclosure. The new second heat-treatment cycle associated to the use of a different, highly resilient foam induces an increased porosity in the material and a different morphology. Altogether, these factors influence most of the biocomposite´s characteristics such as structure, grain size, actual surface area. The characteristics of the biocomposite allows a high antibiotic concentration to be released and improves the induction of bone regeneration and treatment of bone infections. In an embodiment, nanohydroxyapatite/collagen granules contain vancomycin, a glycopeptide antibiotic known to be very effective against Gram positive bacteria such as MRSA. The antibacterial activity of the granules containing vancomycin was evaluated against MRSA in terms of suspension and adherent bacteria. Since this biocomposite material is intended to be used as a medical device, its cytotoxicity towards L929 fibroblasts was also evaluated according to ISO 10993-5 for medical devices certification. This cytotoxicity analysis is complemented with live/dead cell staining assessment. An aspect of this disclosure relates to heparinized nanoHA/collagen biocomposite with interconnected macroporous granules (hereinafter “HEPHAPC granules”). This macroporous structure induced a suitable biological response in terms of bone regeneration, an aspect to take into consideration in cases of osteomyelitis where infected bone tissue undergoes necrosis and generates defects above a critical size therefore not being able to self-heal and thus requires bone regeneration. The granules sintered at 1050 °C presented higher mechanical strength, making them more stable and adequate to be clinically applied due to improved handling properties. This biocomposite material also allowed a vancomycin releasing profile with an intense initial burst followed by sustained release for 19 days during which the vancomycin concentrations were always above MIC for MRSA thus resulting in inhibition of MRSA, in some cases total inhibition of MRSA, as well as inhibition of MRSA biofilm formation. Furthermore, the materials were not cytotoxic to fibroblasts according to ISO 10993-5:2009 standard (Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity) and live/dead bacterial viability assay. The HEPHAPC granules containing vancomycin appear to be a promising material for osteomyelitis treatment. Many devices have been develop showing potential to help regenerate bone in critical defect areas, similar to the property of HEPHAPC in the absence of the antibiotic. The major benefits associated with HEPHAPC include improved patient wellbeing (reduced pain and morbidity, reduced amputation risk, quicker recovery), and the possibility of cost reductions (single surgery with definitive closure thus avoiding the need for subsequent surgery, reduced hospitalization periods, ambulatory treatment and recovery, and consequently increased comfort for the patients, reduced absence from labour, and major treatment costs reduction). This biocomposite allows for the provision of a platform product, as a vehicle for other antibiotics or other active molecules such as proteins, specific peptides or growth factors. In fact, it presents the advantage of allowing the drug selection decision to be made by the surgeon who could include the drug into the composite prior to the surgery as appropriate. Such a product would provide a broader market potential than a single drug with a fixed dose composition in a composite. In an embodiment, the morphology and collagen distribution of the HEPHAPC granules were analysed using scanning electron microscopy (SEM). X-ray energy dispersive spectroscopy (EDS) was also carried out for elemental analysis. In an embodiment, Fourier transformed infrared spectroscopy (FTIR) was used to evaluate the presence of functional chemical groups on the HEPHAPC granules. The analysis was performed using a Bruker Tensor 27 FTIR spectrometer in the wavelength ranging from 400 cm^-1 to 4000 cm^-1, with 4 cm^-1 resolution.128 scans were accumulated for each sample. In an embodiment, the nanohydroxyapatite surface topography and roughness of the granules (sintered at different temperatures) were assessed using Atomic Force Microscopy (AFM) in tapping mode (for example Veeco Metrology Multimode/Nanoscope IVA) with a silicon tip (for example RTESPA-V2 Bruker). For each sample, three images were acquired with areas of 1 µm x 1 µm in which the roughness parameters were evaluated, namely average roughness (Ra) and root- mean-square (Rq). 3D topographic images of the granules surface were obtained using the apparatus software (for example NanoScope, Digital Instruments/Veeco). In an embodiment, X-ray diffraction (XRD) was carried out using a diffractometer (for example PANanalytical Empyrean diffractometer) using CU-Kα radiation (Kα = 1.5460 Å) for 2θ range of values from 20˚ to 40˚ to evaluate the phase composition and crystallinity of HEPHAPC granules. To determine the crystalline structure of HEPHAPC granules, distances between peaks were compared to JCPSD 04-016-2958 from International Center of Diffraction Data. In an embodiment, X-ray micro-computed tomography (micro-CT) analysis was used to analyse 3D structure with a focus on the macroporosity of HEPHAPC_830 and HEPHAPC_1050 granules. SkyScan 1272 System (SkyScan) was used. The granules were scanned under high resolution mode of 0.9 μm, using a pixel size of 8.79 μm. The X-ray source was set at 60 kV of energy and 166 μA of current. 300 slices were used to create a binary image using a dynamic grey thresholding from 40 to 255 to distinguish ceramic material from porosity. The sliced 2D images were processed and the quantitative morphological parameters were quantified by CT-analyser (for example CTAn, v1.5.1.5., SkyScan). In addition, 3D virtual models of the granules were created using CT-Vox (for example SkyScan). In an embodiment, the porosity can be measured using many methods, in the present disclosure mercury porosimetry was used to complement the porosity evaluation (microporosity and submicron porosity evaluation), as mercury porosimetry possesses higher sensitivity for smaller scale features like micropores and nanopores. Parameters such as total surface area, average pore diameter and apparent density of HEPHAPC granules (in particular HEPHAPC_830 and HEPHAPC_1050) were determined. Mercury intrusion porosimetry was performed with high (up to 230000 kPa) and low pressure (350 kPa). In an embodiment a mercury intrusion porosimeter was used and the presence of open pores were detected in dimensions between 5.5 nm and 360 nm (minimum and maximum detection limit, respectively). In an embodiment, compression tests were performed to compare the mechanical strength of HEPHAPC_830 and HEPHAPC_1050. All tests were carried out using a texture analyzer (for example Stable Micro Systems, TA-XT2i), with the load applied vertically using a load cell of 49 N covering a distance of 3 mm at a displacement rate of 0.1 mm/s until the materials were fractured. The compression tests were performed on fifteen samples of each material and the force profile curves were used to estimate the associated compression strength (maximum force). In an embodiment, Vancomycin (for example Vancomycin from HIKMA Farmacêutica, SA) was adsorbed onto the HEPHAPC granules (HEPHAPC_830 and HEPHAPC_1050) for 2 h at 37 ˚C under 120 rpm in the orbital shaker (KS 4000 IC, IKA).20 mg of granules were submerged in microcentrifuge tubes containing 1 mL of vancomycin solution with a concentration of 50 mg/mL. Thereafter, the vancomycin solution was removed and the granules were transferred to new microcentrifuge tubes containing 1 mL of PBS. In an embodiment, previously prepared microcentrifuge tubes were placed at 37 ˚C under 120 rpm in orbital shaker to perform the vancomycin release tests. Every 24 hours for 21 days, 200 µL of the solution were removed and replaced with fresh PBS to determine the concentration of vancomycin released from the granules. The 200 ^L of solution which was removed from the microcentrifuge tube was then centrifuged for 5 minutes and 14000 rpm to sediment the particles to obtain a clear suspension. Vancomycin concentration was determined by molecular absorption spectrometry at 280 nm using a UV-Vis Spectrometer (for example NanoDrop ND – 1000). For this experiment the detection and quantification limits for vancomycin were also determined. All tests were performed in triplicate for each type of granules. In an embodiment, to assess the antimicrobial activity of both types of HEPHAPC granules, MRSA ATCC 33591 were cultivated in sterile tryptic soy broth (TSB) for 3 hours at 37 ˚C. Bacterial suspension was used on exponential phase bacteria and was adjusted to obtain the density of 10⁸ cell/mL (OD_{640 nm} =0.2) as determined by UV-Vis spectrophotometry. Thereafter, 20 mg of HEPHAPC_830 and HEPHAPC_1050 granules with antibiotic adsorbed as described above (five replicates) were placed in the wells of 48 well-plates each well containing 400 µL of bacterial suspension. After the granules were added to each well, the 48 well-plates were incubated at 37 ˚C under 120 rpm, for 24 h. HEPHAPC_830 and HEPHAPC_1050 granules without antibiotic adsorbed were used as a control. All tests were performed in triplicate. In an embodiment, the amount of planktonic and sessile bacteria in terms of colony- forming units (CFU) per mililiter was determined. For planktonic bacterial quantification, after 24 hours of incubation with the HEPHAPC granules, the suspension was removed and serially diluted in 0.9 % sodium chloride (NaCl). The dilutions were inoculated in agar and the plates were incubated at 37 ˚C for 18 hours. Results were expressed as the decimal logarithm of the colony-forming units (CFU) per mililiter of suspension. All tests were performed in triplicate. For sessile bacteria quantification, the HEPHAPC granules were removed and carefully rinsed three times with 0.9 % NaCl to remove non-adherent or loosely adherent bacteria. Thereafter, the HEPHAPC granules were transferred to a new 48 well-plate with NaCl and sonicated for 15 minutes in an ultrasonic bath to release the sessile bacteria. Negative controls were obtained by incubating granules in TSB without adding bacteria. Finally, the sonicated solutions were used to obtain serial dilutions that were then placed in TSA agar and incubated at 37 ˚C for 18 hours. The number of colonies was counted and the CFU´s /mL were determined. All experiments were performed in triplicate. In an embodiment, HEPHAPC granules with and without vancomycin were observed by SEM to assess the sessile bacteria. After incubation with bacterial suspension, the materials were fixed in 1.5 % (v/v) glutaraldehyde in cacodylate buffer (0.14 M) for 10 minutes and dehydrated in a gradient series of ethanol solutions (50, 70, 90 and 100 %) for 10 minutes each step. The samples were dried overnight at room temperature and prepared for SEM visualization. In an embodiment, cell viability assays were assessed according to “ISO 10993:2009 – Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity”. L929 fibroblasts derived from subcutaneous connective tissue were cultivated in alpha minimum essential medium (α – MEM) (Sigma- Aldrich) supplemented with 1 % penicillin-streptomycin (P/S) (Gibco) and 10 % fetal bovine serum (FBS). Cells were seeded at a density of 1 x 10⁶cells/mL in 96-well plates and incubated in a humified environment for 24 hours at 37 ˚C with 5 % of CO2. In an embodiment, extracts were prepared according to ISO 10993:2012 – Biological evaluation of medical devices - Part 12: Sample preparation and reference materials. Firstly, vancomycin was adsorbed onto HEPHAPC granules, as described above, thereafter these extracts were immersed in cell culture medium and placed inside an incubator for 24 hours at 37 ˚C, in humidified 5 % CO₂ atmosphere. The granules mass to volume ratio of the medium was 0.2 grams per mL. In an embodiment, to evaluate cell viability, MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromid) was performed according to the guidelines presented in ISO 10993 – 5:2009 standard [26]. After cell seeding and incubation for 24 hours, the culture medium was discarded and 100 µL of each extract were added to each well. The same volume of extract was used for negative control (cell culture medium alone) and for cytotoxic positive control (1 % Triton X-100). After 24 hours of cell incubation with extracts or controls, 10 µL of MTT (5 mg/mL) were added to each well and incubated for another 3 hours at 37 ˚C. Afterwards, the solution was removed and 100 µL of dimethyl sulfoxide (DMSO) were added to each well. Thereafter, the absorbance was measured at a wavelength of 570 nm using a microplate reader. In accordance with ISO 10993 – 5:2009 standard, samples with 50 % extract were also prepared and cell viability for this extract should be equal or higher than 100 % extract in order to validate the assay. For each experiment, five replicates were used per group and, data were normalized with respect to the negative control. In an embodiment, cell viability was also evaluated using live/dead cell staining. Viable and dead cells are stained in different fluorescent colours. Calcein-AM stains viable cells green, while propidium iodide (PI) stains dead cells red. As performed in the MTT assay, cells were incubated with sample extracts for 24 hours, using 1 % Triton X-100 as a positive control and supplemented cell culture medium as negative control. Sample extracts and controls were then removed and cells were washed with PBS. Additionally, 50 µL of Calcein AM diluted in 2 µL/mL of α- MEM without phenol red and 50 µL PI solution were added to each well and then the cells were incubated in the dark for 45 minutes, at 37 ˚C. Once stained, cells were observed under an inverted fluorescence microscope with green filters at 488 nm and red filters at 598 nm for fluorescence visualization. The images were processed by an image analysis software. In an embodiment, statistical analysis of the results was performed using the SPSS statistical software (Statistical Package for the Social Sciences Inc., USA). One-way analysis of variance (One- way ANOVA) was performed followed by Tukey HSD post hoc test. Levels of p ≤ 0.05 were considered statistically significant. Triplicate experiments were performed and 5 replicates were used per experiment. The results were expressed as the arithmetic mean ± standard deviation. In an embodiment, the present disclosure relates to a method of obtaining a heparinized biocomposite comprising the following steps: obtaining a bioceramic by subjecting a hydroxyapatite infused scaffold to a first heat- treatment cycle and a second heat-treatment cycle, wherein the first heat-treatment cycle is at a temperature from 400 °C to 700 °C, including a 1-hour temperature plateau, and the second heat-treatment cycle is at a temperature from 1000 °C - 1300 °C, including a 1-hour temperature plateau; adding collagen to the bioceramic obtained in the previous step to obtain a collagen film on the surface of said bioceramic to obtain a coated bioceramic; adding to obtain a heparinized biocomposite; binding a suitable antibiotic to the heparinized biocomposite. In an embodiment, the heating rate of the first heat-treatment cycle is 1 °C/min and the heating rate of the second heat-treatment cycle is 4 °C/min. In an embodiment, the first heat-treatment cycle temperature is 600 °C and the second heat-treatment cycle temperature is 1050 °C (sintering temperature). In an embodiment, the method further comprises a step of breaking the bioceramic into granules. In an embodiment, the heparin is associated to the collagen so that heparin integrates the disrupted film, that is added during collagen crosslinking. Preferably the crosslinker is N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide and N-hydroxysuccinimide. In an embodiment, a heparinized biocomposite is obtainable according to the method described above, wherein the heparinized biocomposite comprises: calcium phosphate coated granules, wherein the granules are coated with a collagen film, and heparin and an antibiotic in an effective therapeutic amount, wherein the antibiotic is bound to the heparin, and wherein the antibiotic release from the heparinized biocomposite is sustained for up to 20 days, preferably for 19 days. In an embodiment, the heparinized biocomposite´s antibiotic release is sustained at a concentration of at least 2 µg/mL, preferably a concentration of at least 2.7 ug/mL. In an embodiment, the compression strength of the heparinized biocomposite is from 1 x 10 ^-2 MPa to 30 x 10 ^-2 MPa, preferably 2 x 10 ^-2 MPa to 28 x 10 ^-2 MPa, wherein the pore size ranges between 50 nm – 600 µm. In an embodiment, the heparinized biocomposite´s porosity is from 60 % to 80 %, preferably from 65 % to 78 %. In an embodiment, the heparinized biocomposite´s pore size distribution is as follows: pores having a size from 50 nm to 500 nm, preferably 90 to 300 nm; pores having a size from pore size is from 0.8 µm to 6 µm, preferably 0.9 to 5 µm; and pores having a size from 250 µm to 600 µm, preferably from 300 µm to 400 µm. The pore size characterization was obtained by crossing three techniques: Scanning Electron Microscopy, mercury porosimetry and Micro-CT. In an embodiment, the wall thickness between the pores is from 14 µm to 35 µm, preferable from 15 µm to 33 µm. In an embodiment, the collagen is collagen type I. In an embodiment, the collagen film is a disrupted film. In an embodiment, the antibiotic is selected from the following list: vancomycin, amoxicillin, gentamicin, piperacillin-tazobactam, derivatives of piperacillin-tazobactam, amoxicillin or any mixtures thereof. In an embodiment, the antibiotic is preferably vancomycin or gentamicin. In an embodiment, the heparinized biocomposite density is from 0.9 g/mL to 1.5 g/mL, preferably 0.95 g/mL to 1.27 g/mL. In an embodiment, the total surface area of the heparinized biocomposite is from 3 m²/g to 30 m²/g, preferably from 4 m²/g to 30 m²/g. In an embodiment, the heparinized biocomposite is for use in inducing regeneration of bone. In an embodiment, the heparinized biocomposite is for prevention or treatment of bone infections. In an embodiment, the heparinized biocomposite is for treatment of osteomyelitis. In an embodiment, a pharmaceutical composition comprising the heparinized biocomposite is obtained. In an embodiment, the pharmaceutical composition is for in situ administration. In an embodiment, the pharmaceutical composition is an injectable composition. BRIEF DESCRIPTION OF THE DRAWINGS The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention. Figure 1A shows SEM images of nanoHA_830 (1-3), nanoHA_1050 granules (5-7) and, AFM topographic images of nanoHA_830 and nanoHA_1050 granules (4, 8). Figure 1B shows SEM images of HEPHAPC granules. Figure 1C shows the results of EDS analysis of the HEPHAPC_830 granules collagen region (1) and nanoHA region (2). Figure 2A shows the FTIR spectra of HEPHAPC_830 and HEPHAPC_1050 granules. Figure 2B shows XRD diffractograms of HEPHAPC_830 and HEPHAPC_1050. Figure 3 shows the Micro-CT results for HEPHAPC granules. 3D Micro-CT images of HEPHAPC_830 (A-C) and HEPHAPC_1050 (D-F) are shown in different orientations as well as a cross- section view of the granules (C and F). Figure 4 shows the concentration of vancomycin release from HEPHAPC granules at different time points. The values correspond to the vancomycin concentration present in solution for each day. * Represents a statistically significant difference between HEPHAPC_830 and HEPHAPC_1050. Figure 5 shows the antibacterial activity of HEPHAPC_830 and HEPHAPC_1050 with and without vancomycin after 24 h of incubation with MRSA. Figure 5A shows the quantification of planktonic and sessile bacteria expressed in log10 (CFU/mL). * represents a statistically significant difference compared to HEPHAPC granules without vancomycin (830 and 1050) (p < 0.05). Figure 5B SEM images of adherent MRSA on HEPHAPC granules. Figure 6 shows the cell viability of L929 cells after 1 day of incubation with the extracts. Figure 6A shows the results of MTT cytotoxicity assay performed in accordance with ISO 10993- 5:2009. * represents a statistically significant result when compared to the negative control (p < 0.05). Figure 6B shows the results of the Live/Dead viability assay where viable cells are stained green and dead cells red. DETAILED DESCRIPTION The present disclosure relates a new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules), a method of obtaining an new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules), and uses thereof; preferably a heparinized biocomposite comprising nanohydroxyapatite-collagen granules (“HEPHAPC granules”). The new heparinized biocomposite comprising nanohydroxyapatite/collagen granules (“HEPHAPC granules) is able to release high concentrations of antibiotic locally and induce bone regeneration. In an embodiment, the nanohydroxyapatite microaggregates (nanoHA) granules´ morphology, microstructure, porosity and collagen distribution was assessed by SEM. In Figure 1A, the presence of interconnected macro-porosity is evident for granules sintered at both temperatures. Also, the presence of micro-porosity is visible on the granules sintered at both temperatures. In terms of microstructure, the nanohydroxyapatite microaggregates maintained the initial morphology of spherical agglomerates for both heat-treatment cycle sintering temperatures (Figure 1A 2 and 6). In contrast, the submicron is clearly visible for granules sintered at 830 °C as opposed to the ones sintered at 1050 °C where there is a partial decrease of the nano- porosity (Figure 1A 3 and 7). Figure 1B shows the collagen distribution in a fiber-like/network heterogeneous structure for both materials. With SEM analysis, it was also possible to observe that granules sintered at 1050 °C resulted in an increased grain size. Atomic Force Microscopy (AFM) was also performed to evaluate the 3D topography and the images obtained for nanoHA granules are represented in Figure 1A 4 and 8, each showing different surface topographies. For granules sintered at 1050 °C, in particular, an increase in grain size was observed but both revealed morphologic features characteristics of nanoHA structures. Roughness analysis over 1x1 µm² area showed that the amplitude parameters (Ra and Rq) revealed significant statistical differences between the nanoHA granules sintered at the two different temperatures. The results indicate that the granules sintered at 1050 °C had higher Ra and Rq values (Ra = 43.9 ± 1.4 nm and Rq = 54.2 ± 1.8 nm) as compared to those sintered at 830 °C (Ra = 15.9 ± 2.7 nm and Rq = 21.2 ± 3.5 nm). X-ray energy dispersive spectroscopy (EDS) analysis of HEPHAPC granules was performed on two distinct regions for collagen and nanoHA. Samples sintered at both temperatures presented similar results (only HEPHAPC_830 is presented here). The EDS performed on collagen region presented the expected elements of collagen (C, O and N) (Figure 1C 1), and for the nanoHA region the presence of calcium (Ca) and phosphorus (P) was detected (Figure 1C 2). The results of EDS analysis for HEPHAPC_1050 were similar to the ones referred above and thus they are not presented. Figure 1A shows the SEM images of nanoHA_830 (1-3), nanoHA_1050 granules (5-7) and, AFM topographic images of nanoHA_830 and nanoHA_1050 granules (4, 8). Figure 1B shows SEM images of HEPHAPC granules. Figure 1C shows the results of the EDS analysis of the HEPHAPC_830 granules collagen region (1) and nanoHA region (2). In an embodiment, Fourier-transformed infrared spectroscopy (FTIR) was performed. Figure 2A shows the FTIR spectra for HEPHAPC_830 and for HEPHAPC_1050. The IR transmission of both spectra exhibits similar peaks and bands. The spectra revealed the presence of phosphate ion (PO₄ ^3-) groups (474, 569, 602, 962, 1045 and 1091 cm^-1) and OH- (633 and 3572 cm^-1) corresponding to HA characteristics peaks. The bands at approximately 3497 cm^-1 and 1638 cm^-1 indicate the presence of lattice water in the material, these bands were less intense for granules sintered at 1050 °C. For the granules sintered at 830 °C, the presence of carbonate ion (CO₃ ^2-) was detected by the peak at 1411 cm^-1. The broad band observed at 2000-2500 cm^-1 may be assigned to the hydrogen phosphate ion (HPO₄ ^2-) groups. Similar results were obtained for nanoHA_830 and nanoHA_1050 granules (data not shown). Collagen amides and heparin were included in the nanoHA granules in low amount and hence their peaks were not detected as their presence was below the equipment’s detection limit. In an embodiment, X-ray Diffractography was performed. The XRD diffractograms of granules sintered at both temperatures are shown in Figure 2B. For both types of granules, XRD analyses revealed the phase purity for hydroxyapatite without detection of further phases. However, for the granules sintered at 1050 ˚C, the width of these peaks is sharper thus indicating increased crystallinity. XRD also showed the peak positions characteristics of HA with hexagonal crystal phase, similar to data obtained from International Center of Diffraction Data. The results of XRD analysis for nanoHA granules were similar to those of HEPHAPC granules (data not shown). Figure 2A shows the FTIR spectra of HEPHAPC_830 and HEPHAPC_1050 granules while Figure 2B shows the XRD diffractograms of HEPHAPC_830 and HEPHAPC_1050. In an embodiment, structural analysis was performed using micro-CT in order to visualize the 3D structure of the HEPHAPC granules and to determine porosity, mean macropore size and the pore walls´ thickness for HEPHAPC_830 and HEPHAPC_1050 granules (Table 1 below). Figure 3 shows the irregular morphology of HEPHAPC granules and confirms the presence of interconnected macro-porosity. In an embodiment, mercury porosimetry analysis was performed to allow quantitative analysis of total surface area, average pore diameter and bulk density of HEPHAPC_830 and HEPHAPC_1050. The results presented in Table 1 show that HEPHAPC_830 has a higher surface area as compared with HEPHAPC_1050. HEPHAPC_830 has an average pore diameter considerably smaller, in the nanometric range, as compared to HEPHAPC_1050 which is in the micrometric range. Table 1 - Parameters obtained with SEM, mercury porosimetry and Micro-CT assay for HEPHAPC granules.

Figure 3 shows the Micro-CT results for HEPHAPC granules. 3D Micro-CT images of HEPHAPC_830 (A-C) and HEPHAPC_1050 (D-F) shown in different orientations and a cross-section view of the granules (C and F). In an embodiment, compression tests were performed in order to compare the mechanical strength of both materials, one sintered at 830 °C and the other at 1050 °C. Cylindrical specimens were produced with length to diameter ratio of 2:1 to minimize the end effect imposed by compressive load. The compression strength was determined from the stress-strain curves and the results show a higher value of compression strength for materials sintered at 1050 °C (Table2). Table 2 – Compression strength results for nanoHA granules sintered at the two temperatures.

In an embodiment, vancomycin release profile was analysed. The vancomycin release profile of HEPHAPC granules is shown in Figure 4. For both materials, the vancomycin release profile obtained had a high initial burst followed by 19 days of sustained release at concentrations always above the minimum inhibitory concentration (MIC) for MRSA (MIC = 2 µg/mL). For the first 17 days, there is a higher statistically significant amount of antibiotic being released by HEPHAPC_830 as compared to HEPHAPC_1050. After 20 days, the vancomycin concentration present did not reach the quantification limit (concentration values bellow ≤ 9.11 µg / mL) and on the 21st day, the concentration did not reach the detection limit (2.73 µg / mL). Figure 4 shows the quantity of vancomycin released from HEPHAPC granules versus time. The values correspond to the vancomycin concentration present in solution for each day. * Represents a statistically significant difference between HEPHAPC_830 and HEPHAPC_1050. In an embodiment, to assess the antibacterial activity of the HEPHAPC granules, sessile and planktonic bacteria were quantified and expressed in Log₁₀ (CFU/mL) (Figure 5A). After 24 hours of MRSA incubation, HEPHAPC granules with vancomycin were able to kill the vast majority of, preferably eradicate, the bacteria as opposed to granules without antibiotic. This result was observed for both heat-treatment cycle sintering temperatures, 830 °C and 1050 °C. For HEPHAPC granules without vancomycin, no statistically significant differences were observed for both temperatures in terms of bacterial growth. With regard to adherent bacteria, Figure 5B shows that the vancomycin adsorbed on granules inhibited bacterial adhesion onto granules. SEM also showed the typical spherical morphology of Staphylococcus aureus and the bacteria clusters formation on granules without antibiotic. Figure 5 shows the antibacterial activity of HEPHAPC_830 and HEPHAPC_1050 with and without vancomycin after 24 hours of incubation with MRSA. Figure 5A Quantification of planktonic and sessile bacteria expressed in log₁₀ (CFU/mL). * represents a statistically significant difference compared to HEPHAPC granules without vancomycin (830 and 1050) (p < 0.05). Figure 5B SEM images of adherent MRSA on HEPHAPC granules. Figure 6A shows the results of the MTT assay used to assess L929 cell viability. After 24 hours incubation with the extracts, cell viability was above 70 % for all sample extracts with the exception of HEPHAPC_830 V. All other materials were considered non-cytotoxic according to ISO 10993-5 standard. In Figure 6B, shows the results of live/dead cell viability, live cells were stained green and dead cells in red. The controls used performed as expected since the cells treated with positive control (1 % Triton X-100) are represented in red and the negative control has most of cells viable and stained in green. In an embodiment, for HEPHAPC_830, HEPHAPC_1050 and HEPHAPC_1050 V, Figure 6B shows that these materials were similar to the negative control, with most of the cells viable and labelled in green as opposed to HEPHAPC_830 V where the number of living cells is visibly reduced and the number of dead cells visibly high. Figure 6 shows the viability of L929 cells after 1 day of incubation with the extracts. Figure 6A shows the results of MTT cytotoxicity assay performed in accordance to ISO 10993-5:2009. * represents a statistically significant result when compared to the negative control (p < 0.05). Figure 6B shows the results of the Live/Dead viability assay with viable cells stained in green and dead cells in red. In an aspect of this disclosure, a heparinized nanoHA/collagen biocomposite was produced using two heat-treatment cycle sintering temperatures and their effect on granules properties were studied. Their performance as a drug delivery system to treat osteomyelitis was also evaluated. In an embodiment, the morphological, chemical and mechanical characterization of the heparinized nanoHA/collagen biocomposite was performed. In addition, vancomycin release kinetics was assessed and the antibacterial activity was evaluated using MRSA. Finally, the effect of the heparinized nanoHA/collagen biocomposite on L929 cell viability was also studied. In an embodiment, SEM results showed the granular morphology of the biocomposites. The granular morphology of the biocomposites facilitates filling of irregular and specific bone defects. The presence of interconnected macro-porosity was also visible. The macro-porosity is bigger for the granules sintered at 1050 °C than for the ones sintered at 830 °C as observed in Figure 1. This structure plays an important role in bone regeneration since it facilitates cell adhesion, proliferation, differentiation, as well as vascularization and extracellular matrix production. The interconnected porosity is a very important factor because it increases bonding between the granules and the host tissue, allowing for cell migration, neovascularization, bone tissue growth, as well as nutrient and protein flux. Micro-porosity was present in the granules sintered at both temperatures. The micro-porosity provides a high surface area that induces protein adsorption, ion exchange, formation of apatite and creates anchorage points for the cells. With regard to nanoporosity, it was clearly present in the granules sintered at 830 °C, but its presence is reduced for granules sintered at 1050 °C. The increase in the heat-treatment cycle sintering temperature favoured the increase in grain size, as expected and as observed by other authors [6]. This explains the reduction of nano-porosity for granules sintered at 1050 °C; this is confirmed by mercury porosimetry. In an embodiment, the results obtained by AFM showed that the granules sintered at 1050 °C have a bigger grain size than those sintered at 830 °C (Figure 1A, 4 and 8). The increase in heat- treatment cycle sintering temperature caused an increase in grain size [34, 38] which supports the microstructure differences observed by SEM. According to the analysed area, the granules sintered at 1050 °C showed statistically significantly higher Ra and Rq values as compared to granules sintered at 830 °C. At this nanometric scale, the increased heat-treatment cycle sintering temperature resulted in a significant increase in roughness as measured by the difference in topography between highest picks and lowest valleys. The results obtained by AFM show that there is a greater variation of the surface topography for lower heat-treatment cycle sintering temperature, that is, the oscillations observed are smaller in amplitude but are in much greater numbers. At higher temperature, greater topographic oscillations are produced, causing higher peaks to valleys differences and consequently higher roughness values even though the surfaces are "smoother". In an embodiment, FTIR spectra revealed that there are no differences in the HA characteristics peaks of the two heat-treatment cycle sintering temperatures. The intensity of the peak corresponding to the presence of H₂O is higher for samples sintered at 830 °C than those sintered at 1050 °C as some crystallinity water appears to be lost due to the increase in the temperature of the heat-treatment. In addition, the presence of CO₃ ^2- ions at residual levels for the materials sintered at 830 ° C was also detected, probably due to the fixation of the CO₂ from the atmosphere during the preparation of the nanoHA. When the heat-treatment cycle sintering temperature increased, the CO₃ ^2- ions were no longer detected. As expected, similar EDS peaks for each element were also obtained for both materials. In an embodiment, XRD analyses showed pure hexagonal crystal phase for hydroxyapatite on both materials, with none unexpected phases being present. This result is in agreement with data from International Centre for Diffraction Data. Sharper peaks were observed for granules sintered at 1050 °C, indicating a more crystalline structure. In an embodiment, Micro-CT analysis revealed the presence of interconnected macro- porosity for both granules and similar values of mean macroporosity and mean macropore size. This result shows that the interconnected macroporosity in the biocomposite is at least adequate for bone regeneration since high porosity (above 50 %) and a minimum recommended pore size of 100 μm (typically above 200 μm) are essential requirements for osteoconduction. In an embodiment, mercury porosimetry results complemented the results from Micro-CT, as expected. Higher heat-treatment cycle sintering temperature resulted in a decrease of nanoporosity and consequently lower surface area [6]. In fact, average micrometric pore diameters for HEPHAPC_1050 were found, in opposition to HEPHAPC_830 that presented average pore size below 100 nm. Moreover, the granules have a higher apparent density and a higher average pore diameter compared to HEPHAPC_830. Increasing temperature during heat-treatment favoured a stronger binding between crystallite grains. Furthermore, the compressive strength is inversely dependent on porosity. Although a highly porous scaffold is preferred as it favours bone cell adhesion to the scaffold and regeneration, this is achieved at the expense of the mechanical strength which yields insufficient mechanical properties. In an embodiment, the bioceramic sintered at 1050 °C have higher maximum compression stress values, indicating superior mechanical strength. This superior mechanical strength was also observed during handling of the various materials. In fact, handling is a very important feature for ease of handling of these materials during surgery, for the ease of manipulation and implantation [34]. This increase in mechanical strength was due to higher heat-treatment cycle sintering temperature which provided a greater number of contact points in heat-treatment cycle sintering and consequently a stronger grain bonding and a higher compressive strength. As a general trend, the compression strength of a scaffold may depend on its macro-porosity and macropore size, density and pore wall thickness. In this study, the macro-porosity and macropore size were very similar for both materials, so it seems that these two factors did not play any important role on the effect on mechanical strength. However, a higher mean value of pore wall thickness was obtained for granules obtained with a second heat-treatment cycle sintering temperature of 1050 °C (mean of the pore wall thickness is between 20 µm and 32 µm) as compared to the ones obtained with a second heat-treatment cycle sintering temperature of 830 °C (mean of the pore wall thickness is between 13 µm and 17 µm). This observation justifies the compression test results since thicker pore walls led to higher mechanical strength. Additionally, nanoHA_1050 also presented higher density than nanoHA_830 and consequently a higher compressive strength. This behaviour is similar to another previously reported. In an embodiment, the mechanical strength of HEPHAPC_1050 (material obtained with a second heat-treatment cycle sintering temperature of 1050 °C) is similar [6] or higher than those reported in the literature and makes these materials adequate to be used clinically for non-load bearing application sites. In an embodiment, the heat-treatment cycle sintering temperature of nanoHA also influenced the vancomycin release kinetics from HEPHAPC granules. This also explains why HEPHAPC_830 showed a higher antibiotic release for the first 17 days as compared to HEPHAPC_1050. This difference can be explained by the fact that the materials sintered at 830 °C present a much larger surface area, thus having many more binding sites and therefore increasing vancomycin adsorption and further release. In an embodiment, for biocomposites sintered at both temperatures, the obtained release profiles always displayed high initial bust followed by 19 days of sustained release where concentration of vancomycin is always above MIC for MRSA (MIC = 2 μg / mL). This release profile is recommended if the objective is to have a complete eradication of the pathogen and treat bone infections. Higher concentrations of antibiotic may be required for eradication of a biofilm caused by resistant bacteria. Particularly, concentrations in order of a 1000-fold higher than conventional therapeutic concentrations may be required [2]. Therefore, HEPHAPC granules are effective since the amount of vancomycin released in the first days is about 1000-fold higher than the MIC. In this study, the vancomycin adsorption time was reduced to 2 hours and the vancomycin solution concentration was doubled in comparison with a previous work [4]. This resulted in a shorter and therefore more adequate time for material preparation in terms of antibiotic loading prior to surgery. This also results in a considerable increase in the amount of vancomycin released from granules and an extension of further five days for the drug to be fully released into the defective site. In an embodiment, HEPHAPC granules with adsorbed vancomycin were able to kill the vast majority, preferably eradicate, the planktonic and sessile bacteria. Although granules sintered at 1050 °C released lower amounts of vancomycin, the concentration released from this material was enough to eradicate at least a substantial amount of MRSA. For granules without vancomycin, no differences between the granules sintered at both temperatures were seen in terms of bacterial adhesion. Therefore, in this case, surface changes did not affect bacterial adhesion, as observed in other studies. In an embodiment, cytotoxicity evaluation was carried out. Cell viability assay was carried out following ISO 10993-5 standard for medical device certification. According to this ISO standard, samples with cell viability lower than 70 % are considered cytotoxic. Except for HEPHAPC_830 V granules, cell viability was above 70 % thus indicating that these materials were not to be considered cytotoxic. This difference may be related to lower mechanical resistance of HEPHAPC 830 granules, since these materials degrade faster, forming higher amounts of fragments and debris that may be toxic to cells. This was also observed in live/dead cell staining, as HEPHAPC_830 with or without vancomycin presented higher number of dead cells and also a reduced number of adhered cells, in comparison with the control. This was also observed by other authors that used lower heat-treatment cycle sintering temperatures (e.g.600 and 800 °C). Biocomposite materials sintered at lower temperatures have greater toxic effect on cells as compared to those sintered at temperatures higher than 1000 °C [7]. Therefore, HEPHAPC_1050 proved to be more effective in terms of not inducing any toxicity to cells as opposed to HEPHAPC_830. In an embodiment, to obtain porous nanoHA granules, scaffolds were produced using the polymer sponge replication method as described elsewhere [4]. Briefly, a high resilience polyurethane sponge (density of 42 Kg/m³ and hardness of 3.4 kPa, SupraCell AR 4234 BR) was impregnated with a nanoHA slurry (nanoXIM·HAp202, Fluidinova SA, Portugal). The ceramic slurry was prepared using the ratio of 5:4.5:0.2 for nanoHA powder (g), distilled water (mL) and dispersive agent Dolapix CE64 (mL), respectively. After soaking into the slurry, the sponges were dried at 37 °C in the oven for 1 h and then subjected to two different heat-treatment cycles in a sintering furnace (Thermolab): heating rate of 1 °C /min with 1 h plateau at 600 °C to burn out the polyurethane foam, followed by a heating rate of 4 °C /min with 1 h plateau at 830 °C and alternatively the other cycle with a plateau at 1050 °C. These two different sintering temperatures were used to compare their effect on the properties of the material. In an embodiment, 90 days sub-chronic toxicity study of the HEPHAPC_1050 granules after intra-osseous implantation in rabbits (GLP study) was performed. In an embodiment, in vivo local and systemic toxicology evaluations of the HEPHAPC_1050 granules (with and without vancomycin) were performed, under GLP tests, for sub-chronic conditions (90 days) in animal model (15 male rabbits). The results indicated that no clinical signs were recorded in any animal of the two studied situations. All animals from both treated groups (HEPHAPC_1050 granules without vancomycin and HEPHAPC_1050 with vancomycin) and control group showed no tissue reaction at the implantation sites during the whole observation period. All individual body weight values were within normal range in all groups and was stable or increased during the observation period. The body temperature of all animals was recorded in the range of 38.4 °C - 39.6 °C, which corresponds to the physiologic values on the rabbit. Food consumption was well-balanced in all animals. No changes in the haematology (red blood cells count, white blood cells count and coagulation) and clinical chemistry parameters (Glu, Na, K, Cl, Alb/Glo concentrations, LDH, ALT, GGT activities) were observed during the study that could be connected with the administration of the HEPHAPC_1050 granules. The highest values of protein, bilirubin and urobilinogen urine concentration were observed, at the end of the study period, in the group of rabbits treated with HEPHAPC_1050 granules with Vancomycin. The association between the results and the implantation of the HEPHAPC_1050 granules could not be excluded. No changes in other urine analysis parameters were observed during the study that could be associated with the implantation of the HEPHAPC_1050 granules. Organs weight analysis (e.g. liver, kidneys, spleen, brain, heart, thymus, adrenals, thyroid gland, testes, prostate, epididym) did not show any treatment related changes. Both HEPHAPC_1050 granules without vancomycin and HEPHAPC_1050 granules with vancomycin did not cause gross or histopathological changes in the test rabbits’ liver and kidneys which would be indicative of a toxic effect. All bone defects after intra-osseous implantation of HEPHAPC_1050 granules were completely healed with minimal to marked periosteal fibrosis similar to the control group. On the contrary, mild to severe amount of foreign body granulomas was observed in the bone marrow around the granules of implanted material. These granulomas were in most cases lined by a layer of newly formed bone. Implantation of HEPHAPC_1050 granules with vancomycin showed similar result without considerable differences. The irritation index, according to ISO 100993-10, which evaluates local tissue reaction was 7.08 for the group implanted with HEPHAPC_1050 granules without vancomycin and 5.74 for the group implanted with HEPHAPC_1050 granules with Vancomycin, as compared to the control group. The irritation index observed correspond to the category of slight irritation. The results of the tests indicate that both HEPHAPC_1050 granules without vancomycin and HEPHAPC_1050 granules with vancomycin did not induce toxic effects on the animals during the 90 days of post implantation study. The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The embodiments described above are combinable. References 1.. Jiang J-L, Li Y-F, Fang T-L, Zhou J, Li X-L, Wang Y-C, et al. Vancomycin-loaded nano-hydroxyapatite pellets to treat MRSA-induced chronic osteomyelitis with bone defect in rabbits. Inflammation research.2012;61(3):207-15. 2.. Ferguson J, Diefenbeck M, McNally M. Ceramic Biocomposites as Biodegradable Antibiotic Carriers in the Treatment of Bone Infections. Journal of Bone and Joint Infection.2017;2(1):38. 3. Sun F, Zhou H, Lee J. Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites for bone regeneration. Acta biomaterialia.2011;7(11):3813-28. 4. Coelho CC, Sousa SR, Monteiro FJ. Heparinized nanohydroxyapatite/collagen granules for controlled release of vancomycin. Journal of Biomedical Materials Research Part A.2015;103(10):3128-38. 5. Teixeira S, Yang L, Dijkstra PJ, Ferraz M, Monteiro F. Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering. Journal of Materials Science: Materials in Medicine.2010;21(8):2385-92. 6. Gervaso F, Scalera F, Kunjalukkal Padmanabhan S, Sannino A, Licciulli A. High‐performance hydroxyapatite scaffolds for bone tissue engineering applications. International Journal of Applied Ceramic Technology.2012;9(3):507- 16. 7. Wang C, Duan Y, Markovic B, Barbara J, Howlett CR, Zhang X, et al. Proliferation and bone-related gene expression of osteoblasts grown on hydroxyapatite ceramics sintered at different temperature. Biomaterials. 2004;25(15):2949-56.

Claims

C L A I M S 1. A method of obtaining a heparinized biocomposite comprising the following steps: obtaining a bioceramic by subjecting a hydroxyapatite infused scaffold to a first heat- treatment cycle and a second heat-treatment cycle, wherein the first heat-treatment cycle is at a temperature from 400 °C to 700 °C, including a 1-hour temperature plateau, and the second heat-treatment cycle is at a temperature from 1000 °C - 1300 °C, including a 1-hour temperature plateau; adding collagen to the bioceramic obtained in the previous step to obtain a collagen film on the surface of said bioceramic to obtain a coated bioceramic; adding heparin to obtain a heparinized biocomposite; binding a suitable antibiotic to the heparinized biocomposite.

2. The method according to the previous claim wherein heating rate of the first heat-treatment cycle is 1 °C/min and the heating rate of the second heat-treatment cycle is 4 °C/min.

3. The method according to any of the previous claims wherein the first heat-treatment cycle temperature is 600 °C and the second heat-treatment cycle temperature is 1050 °C.

4. The method according to any of the previous claims further comprising a step of breaking the bioceramic into granules.

5. The method according to any of the previous claims wherein the heparin binds to the collagen during a collagen crosslink, preferably wherein the crosslinker is N-(3-dimethylaminopropyl)- N’-ethylcarbodiimide and N-hydroxysuccinimide.

6. A heparinized biocomposite obtainable according the method described in any of the previous claims wherein the heparinized biocomposite comprises: calcium phosphate coated granules, wherein the granules are coated with a collagen film, and heparin and an antibiotic in an effective therapeutic amount, wherein the antibiotic is bound to the heparin, and wherein the antibiotic release from the biocomposite is sustained for up to 20 days, preferably for 19 days.

7. The biocomposite according to the previous claim wherein antibiotic release is sustained at a concentration of at least 2 µg/mL, preferably a concentration of at least 2.7 ug/mL.

8. The biocomposite obtainable by the method described in the previous claims 1-5 wherein the compression strength of the biocomposite is from 1 x 10 ^-2 MPa to 30 x 10 ^-2 MPa and, wherein the pore size ranges between 50 nm – 600 µm.

9. The biocomposite according to any of the previous claims 6-8 wherein the compression strength of the biocomposite is from 2 x 10 ^-2 MPa to 28 x 10 ^-2 MPa.

10. The biocomposite according to any of the previous claims 6-9 wherein the porosity is from 60 % to 80 %.

11. The biocomposite according to any of the previous claims 6-10 wherein the porosity is from 65 % to 78 %.

12. The biocomposite according to any of the previous claims 6 - 11 comprising pores having a size from 50 nm to 500 nm, preferably 90 to 300 nm; pores having a size from pore size is from 0.8 µm to 6 µm, preferably 0.9 to 5 µm; and pores having a size from 250 µm to 600 µm, preferably from 300 µm to 400 µm.

13. The biocomposite according to any of the previous claims 6 - 12 wherein the wall thickness between the pores is from 14 µm to 35 µm, preferable from 15 µm to 33 µm.

14. The biocomposite according to any of the previous claims 6 - 13 wherein the collagen is collagen type I.

15. The biocomposite according to any of the previous claims 6 - 14 wherein the collagen film is a disrupted film.

16. The biocomposite according to any of the previous claims 6 – 15 wherein the antibiotic is selected from the following list: vancomycin, amoxicillin, gentamicin, piperacillin-tazobactam, derivatives of piperacillin-tazobactam, amoxicillin or any mixtures thereof.

17. The biocomposite according to the previous claim wherein the antibiotic is vancomycin or gentamicin.

18. The biocomposite according to any of the previous claims 6 - 17 wherein the biocomposite density is from 0.9 g/mL to 1.5 g/mL.

19. The biocomposite according the previous claim wherein the biocomposite density is from 0.95 g/mL to 1.27 g/mL.

20. The biocomposite according to any of the previous claims 6 - 19 wherein the total surface area is from 3 m²/g to 30 m²/g.

21. The biocomposite according the previous claim wherein the total surface area is from 4 m²/g to 30 m²/g.

22. A biocomposite according to any of the previous claims 6 - 21 for use in inducing regeneration of bone.

23. A biocomposite according to any of the previous claims 6 - 21 for prevention or treatment of bone infections.

24. The biocomposite according to any of the previous claims 6 - 21 for treatment of osteomyelitis.

25. A pharmaceutical composition comprising the biocomposite described in any of the previous claims 6 - 21.

26. The pharmaceutical composition according to the previous claim wherein the composition is for in situ administration.

27. The pharmaceutical composition according to any of the previous claims 25 -26 wherein the composition is an injectable composition.