1. FIELD OF THE INVENTION

The present invention relates to a bovine bone grafting material and process for preparing the same.

2. BACKGROUND TO THE INVENTION

Vital bone tissues exhibit a certain degree of natural healing, however large defects due to bony injury following trauma, cancer, dental disease or osseous disease, can require surgical intervention and bone replacement. First-line treatments for such defects are bone grafting procedures. Bone grafts provide an osteoconductive scaffold to support osseous ingrowth and may also contain osteoinductive substrates for osteoprogenitor chemotaxis and mitogenesis, which promote wound healing.

Osteogenesis is the process of bone development and maturation. This involves osteoblasts which are the cuboidal rows of cells that form new bone. Osteoclasts are large multinucleate cells that break down bone tissues. Bone is a dynamic tissue that continuously undergoes remodelling and requires communication between osteoblasts and osteoclasts.

Graft materials can be osteoinductive and/or osteoconductive. When osteoinductive, they stimulate immature and multipotent stem cells to become preosteoblastic (immature osteoblasts) and then osteoblasts; that is they induce new bone forming cells and activate osteogenesis. Osteoconduction is an attribute of a material that permits new bone to grow on its surface. An osteoconductive material thus supports tissue ingrowth on its surface and the development of new bone formation. (Agrawal S., 2020)

In addition to being osteoconductive and ideally osteoinductive, the ideal bone grafting material should be biocompatible, non-immunogenic, cost effective, possess adequate mechanical strength, and should eventually be replaced by the host's own bone.

Autografts are bone grafts retrieved from the patient undergoing treatment. They are considered the gold standard bone replacement graft due to their high clinical success and good regenerative outcomes. However, autograft harvesting procedures are expensive and time-consuming, while also accompanied by significant donor site morbidity and pain. An alternative source of bone is xenografts derived from cattle bone. Use of bovine bone materials is widely recognized and has been proven to be safe for a number of human applications. Such materials have advantages over autografts because they can be mass-produced at a reasonable cost, are abundant in supply, and do not require additional surgery for sample retrieval. Furthermore, cancellous bovine bone has similar morphological structure and physicochemical properties to human cancellous bone, making it a favourable human bone substitute material.

Bone is a composite tissue comprising an organic matrix and inorganic minerals. The organic matrix is composed mainly of collagen whereas the inorganic phase consists of calcium, phosphorous, and oxygen, combined to form hydroxyapatite (HA). Since bovine bone materials are sourced from non-human species, the collagen and other protein constituents of bone present a risk of cross-species immunogenicity, necessitating a protein removal step (deproteinization). Generally, a sterilization step follows, in order to obtain regulatory approval for human applications.

A common approach for deproteinization involves thermal processing. Typically, commercially available bone grafting materials are heated to 300 -1400 °C, to remove organic materials and reduce the risk of immunogenic responses and infection. However, thermal processing has a direct effect on the physicochemical and mechanical characteristics of the material, such as porosity, surface roughness, crystallinity, and mechanical strength. Such changes in the physiochemical characteristics of bone graft materials may influence their handling properties, in vivo resorption, and their osteoconductive attributes.

Tadic and Epple (2004) compared 14 commercially available bone grafting materials and found that increased manufacturing temperatures between 300 - 1200 °C enhanced material crystallinity (Tadic & Epple, 2004). Crystallinity and temperature exposure while manufacturing is inversely proportional to the rate of resorption (Lu, 2002). Resorption of a graft material over time allows the patient's own bone to replace the graft material. This is a desirable attribute as it reduces the chance of an immune response and allows natural strength and remodelling.

Therefore, one way of decreasing the resorption rate of a bovine bone material is to heat-treat the bone source at a high temperature. BioOss® (BO, Geistlich Pharma AG, Wolhusen, Switzerland) is a widely-used, commercially available bovine bone material provided in granular form. BioOss® is obtained by chemically treating degreased bovine bone followed by heat treatment at 350 °C (Heinz Lussi, 1988).

However, the higher crystallinity and lower resorption rate, resulting from thermal processing has also been associated with resistance to biodegradation, lack of degradation by osteoclasts, and limited osteoconductive activity (Scalera, Gervaso, Sanosh, Sannino, & Licciulli, 2013).

The success of bone grafting when combined with dental implant placement greatly depends on osteointegration - the formation of a direct structural and functional connection between living bone and the surface of the implant. Current approaches suffer from clinical disadvantages associated with connective tissue encapsulation and a slow rate of resorption, which can interfere with both new bone formation and osteointegration (Lindhe, Cecchinato, Donati, Tomasi, & Liljenberg, 2014) (Stavropoulos, Kostopoulos, Mardas, Nyengaard, & Karring, 2001) (Donos, et al., 2004) (Slotte & Lundgren, 1999) (Orsini, et al., 2007)

Poor resorption of the bone graft results in long term, non-viable graft material at the graft site and may present an increased risk of infection and poor remodelling for optimal physicomechanical properties.

On the other hand, too rapid resorption may result in the graft material failing to provide an adequate physical scaffold, with an increased risk of graft failure.

Accordingly, there is a need for a safe bone grafting material which has an optimal resorption rate and good osteogenic properties, or that at least overcomes some of the technical issues in the field and/or that at least provides the public with a useful choice.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

3. SUMMARY OF THE INVENTION

In one aspect the invention relates to a process for preparing a bone graft material comprising heating tissue-free, cancellous bovine bone immersed in water for at least one hour at about 100 °C to about 220 °C in a closed vessel.

In another aspect the invention relates to a bovine bone graft material comprising cancellous bovine bone that has been immersed in water for at least one hour at about 100 °C to about 220 °C in a closed vessel.

In one embodiment the volume of water relative to the volume of the closed vessel is about 1: 1 to about 0.05: 1, preferably about 0.2: 1 to about 0.8: 1, more preferably about 0.5: 1. In one embodiment, the bone graft material is bleached and/or sterilised.

In one embodiment, the bone graft material is loaded with silver nanoparticles (AgNP), preferably alpha lipoic acid capped AgNP.

In one embodiment the bone graft material is loaded with alpha lipoic acid capped AgNP at a concentration of 75 pg or greater, per gram of bone. In one embodiment the bone graft material is loaded with alpha lipoic acid capped AgNP at a concentration of 75 - 150 pg, per gram of bone; preferably 75 - 100 pg, per gram of bone.

In one aspect the invention provides the use of a bovine bone graft material of the invention in a bone xenograft procedure.

4. BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described by way of example only and with reference to the drawings in which:

Figure 1 is a pair of graphs showing the osteoblast quantities at 7 and 21 days on bone scaffold specimens using a picogreen DNA assay (A), and cell counting of DAPI stained nuclei using confocal laser scanning microscopy and Fiji: image J analysis (B), as described in Example 3, N=3. Results expressed as mean ± SD. Dotted lines: 7-day negative control (black) and 21-day negative control. Significant values *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2 provides confocal laser scanning microscopy (CLSM) of DAPI stained osteoblast nuclei (blue) at 7 (left) and 21 days (right) on bone scaffold specimens processed at 100 °C (A), 130 °C (B), 160 °C (C), 190 °C (D), 220 °C (E), BioOss® (F), as described in Example 3. Scale bar= 200 pm. Representative images of N=3.

Figure 3 is a graph showing alkaline phosphatase production determined from osteoblasts cultured on bone scaffold specimens at 7 days, as described in Example 3, N=3. Results expressed as mean ± SD. Dotted line: 7-day negative control. Significant values *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

Figure 4 provides confocal laser scanning microscopy (CLSM) of osteoblast nuclei (blue) and osteocalcin (green) cultured in osteogenic media for 21 days on bone scaffolds heat- treated at 100 °C (A), 130 °C (B), 160 °C (C), 190 °C (D), 220 °C (E), BioOss® (F) as described in Example 3. Scale bar = 200 pm.

Figure 5 provides high magnification scanning electron microscopy images of osteoclasts cultured on bone scaffold specimens heat-treated at 100 °C (A & B) 130 °C (C &D) and BioOss® (E & F), as described in Example 4. Scale bar = 100 pm (left) and 10 pm (right). N=4.

Figure 6 is a graph showing the elastic modulus of the heat-treated bovine bone processed at 100 °C, 160 °C, 220 °C and BioOss®, as described in Example 5. Mean ± SD. * * * P < 0.0005.

Figure 7 is a graph showing the percentage weight loss of the water content, organic components and crystal transformation calculated for heat-treated bovine bone and BioOss® from an average of two replicates, as described in Example 5.

Figure 8 is a graph of the crystallinity of the heat-treated bone and BioOss®, as described in Example 5. Mean ± SD. * P < 0.05, ** P < 0.0085, *** P < 0.0008.

Figure 9 is a pair of graphs showing remaining organic content. TGA(A) and FTIR (B) on heat treated 160°C bone; 160°C + Bl, heat treatment to 160°C with bleaching, 160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2); and and BioOss®. Mean ± SD. *** P < 0.001. **** P < 0.0001.

Figure 10 is a series of photographs of the inhibition zones of E. coli as a response to increasing AgNP concentration on the bovine bone graft material of the invention (bleached and gamma irradiated), as described in Example 8. Inhibitory zones were evident at AgNP concentration > 75 pg/g of bone.

Figure 11 is a graph showing osteoblast viability using Prestoblue at day 7 for bone at increasing processing temperatures (all bleached and gamma irradiated) and increasing AgNP concentrations, as described in Example 9. Bl + Ga = bleaching and gamma irradiated (Example 2). Mean ± SD.

Figure 12 is a graph showing osteoclast viability using Prestoblue at 7 days for bone at increasing processing temperatures (all bleached and gamma irradiated) and increasing AgNP concentrations, as described in Example 10. Bl + Ga = bleaching and gamma irradiated (Example 2). Mean ± SD.

Figure 13 is a set of images of all Group 1 mid-slice sites of the CT scans of rabbit cranial defects at 16 weeks used for analysis, as described in Example 11. Empty defect; BioOss®; and heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2).

Figure 14 is a pair of graphs showing quantification of Group 1 graft material (Graft - square black), connective tissue (CT - circular blue) and new bone (New Bone - triangular red) within the circular defect (5.5 mm circle), mid location within the rabbit cranial bone defect, as described in Example 11. No graft material (Control); BioOss®, (BO); 160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2). Mean ± SD. * P <0 .05, ** P < 0.01, *** P < 0.001. Tukey's adjusted ANOVA - unpaired.

Figure 15 is a set of images of all Group 2 mid-slice sites of the CT scans of rabbit cranial defects at 16 weeks used for analysis, as described in Example 11. Empty defect; heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2); and heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2), with the addition of AgNP (Example 7, 8).

Figure 16 is a pair of graphs showing the quantification of graft material (Graft - square black), connective tissue (CT - circular blue) and new bone (New Bone - red triangle) within the circular defect (5.5 mm circle), mid location within the rabbit cranial bone defect, as described in Example 11. No graft material (Control); 160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2); AgNP-160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2), with the addition of AgNP (Example 7, 8). Mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Tukey's adjusted ANOVA - unpaired.

Figure 17 is a set of representative resin embedded images of the rabbit cranial defects at week 4. Empty; BioOss®; 160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2); AgNP-160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation (Example 1, 2), with the addition of AgNP (Example 7, 8). NB = new bone, RG = residual graft, CT = connective tissue. Magnification xlO.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Definitions and abbreviations

The term "comprising" means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

The term "about" as used herein means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be construed as including a deviation of+/- 5% of the value.

The term "cancellous" as used herein with reference to bone means the spongy or trabecular bone characterized by its porous, honeycomb-like structure.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to

10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and/or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

5.2 The bovine bone graft material of the invention and preparation process

The inventors have surprisingly found that the process of deproteinating bovine bone by heating in water under pressure, provides a bone graft material with advantageous properties.

In one aspect the invention relates to a process for preparing a bone graft material comprising heating tissue-free, cancellous bovine bone immersed in water for at least one hour at about 100 °C to about 220 °C in a closed vessel.

In another aspect the invention relates to a bovine bone graft material comprising cancellous bovine bone that has been immersed in water for at least one hour at about 100 °C to about 220 °C in a closed vessel.

In one embodiment the tissue-free, cancellous bovine bone is immersed in water at about 130 °C or greater in a closed vessel. In one embodiment the pressure in the closed vessel is at least about 2.69 bar.

In one embodiment the tissue-free, cancellous bovine bone is immersed in water at about 160 °C or greater in a closed vessel. In one embodiment the pressure in the closed vessel is at least about 6.22 bar.

In one embodiment the tissue-free, cancellous bovine bone is immersed in water at about 190 °C or greater in a closed vessel. In one embodiment the pressure in the closed vessel is at least about 12.93 bar.

In one embodiment the tissue-free, cancellous bovine bone is immersed in water at about 220 °C or in a closed vessel. In one embodiment the pressure in the closed vessel is about 24.58 bar. In one embodiment the tissue-free, cancellous bovine bone is immersed in water at about 130 °C to about 160 °C in a closed vessel. In one embodiment the pressure in the closed vessel is at least about 2.69 bar.

In one embodiment the tissue-free, cancellous bovine bone is heated for up to about 5 hours, preferably up to about 3 hours, more preferably, about 2 hours.

Tissue-free, cancellous bovine bone for use in the process of the invention can be obtained by any method known in the art. Generally, the tissue-free, cancellous bovine bone will be in the form of pieces, such as cubes and/or slices. For example, pieces of cancellous bovine bone may be boiled and then centrifuged, to remove tissue particles, such as bone marrow and fat. Alternatively, tissue could be removed by suction or pressure, following heating to about 80 °C in a solvent such as water.

In one embodiment the cancellous bovine bone for use in the process of the invention is sourced from the lower femur of a cattle beast. The cancellous bovine bone obtained from this region may be any suitable length but will generally not exceed about 30 mm width.

In one embodiment the tissue-free, cancellous bovine bone immersed in water comprises pieces of up to 30 mm length in any dimension.

In one embodiment the pieces of tissue-free, cancellous bovine bone immersed in water are cubes. In one embodiment, the cubes are about 4 to about 12 mm ³ , preferably about 6 to about 10 mm ³ , more preferably about 8 mm ³ .

In one embodiment the pieces of tissue-free cancellous bovine bone immersed in water are slices. In one embodiment the slices are about 30 x 60 x 30 to 10 x 10 x 3 mm. In one embodiment the slices are about 25 x 25 x 2 mm.

In one embodiment the pieces of tissue-free cancellous bovine bone immersed in water are a combination of cubes and slices.

The process is carried out in a device comprising a sealable heating chamber that is able to withstand increased pressure (closed vessel). The closed vessel may be any suitable device known in the art.

The amount of water relative to the size of the vessel interior is also important, to ensure that the interior of the vessel reaches the pressure necessary to deproteinate the bone pieces.

In one embodiment the volume of water relative to the volume of the closed vessel is about 1: 1 to about 0.05: 1 preferably about 0.2: 1 to about 0.8: 1, more preferably about 0.5: 1. The bone must be covered with water. The bone graft material prepared by the process of the invention may undergo further processing, depending on the application for which it is intended.

Following the heat treatment, the resulting bone graft material may be rinsed with water and dried using any suitable process, such as sterile air drying at room temperature or low temperature (eg, 55 °C).

In one embodiment, the bone graft material is bleached. Bleaching removes residual organic material. Any suitable bleaching agent, for example sodium hypochlorite, hydrogen peroxide, ethylenediamine, hydrazine, proteases. In one embodiment the bone graft material is rinsed, before bleaching with 0.5- 2% sodium hypochlorite, preferably about 0.5 to 1% sodium hypochlorite, more preferably about 1% sodium hypochlorite.

In one embodiment, the bone graft material is sterilised. Multiple methods of sterilisation of xenograft bone materials are known in the art.

In one embodiment, the bone graft material is steam sterilised at about 120 °C to about 134 °C for about 19 minutes, preferably about 134 °C for about 19 minutes, with about a 30 minute drying cycle in the autoclave.

In one embodiment, the bone graft material is gamma irradiated. Preferably the bone graft material is gamma irradiated at a dose of 15-35 kGy.

In one embodiment, the bone graft material is loaded with silver nanoparticles (AgNP). AgNP can be made in many different ways and their antibacterial attributes (Bruna, Maldonado-Bravo, Jara, & Caro, 2021), make them suitable for loading on to the bovine bone graft material of the invention.

In one embodiment the AgNP are alpha lipoic acid capped AgNP.

In one embodiment the bone graft material is loaded with alpha lipoic acid capped AgNP at a concentration of 75 pg or greater, per gram of bone. In one embodiment the bone graft material is loaded with alpha lipoic acid capped AgNP at a concentration of 75 - 150 pg or greater, per gram of bone; preferably 75 - 100 pg or greater, per gram of bone.

The invention also relates to a bovine bone graft material prepared in accordance with the process above.

5.3 The properties of the bovine bone graft material of the invention

The process of the invention provides a new bovine bone graft material with many advantageous properties, as discussed below. For example, the bovine bone graft material of the invention is better at encouraging the growth of osteoblasts than commercially available BioOss®. The bone graft material of the invention undergoes gradual resorption compared to BioOss® and thus can be replaced by native bone giving it a significant advantage over other products. Osteoblasts (bone forming cells) bound to bovine bone graft material of the invention heated at 130 and 160 °C proliferated better than and laid down a more mature bone matrix than BioOss®. This is particularly important for encouraging the growth of native bone into a defect. Bovine bone graft material of the invention loaded with silver nanoparticles has antimicrobial properties giving it a distinct attribute superior to BioOss®. The antimicrobial activity seen with the alpha lipoic acid capped-AgNPs was without the cytotoxic cell effects observed with other AgNPs and in fact enhances bone cell proliferation. (Xie, Wang, & Wu, 2019) No adverse immune response was observed in the rabbit trial for the invention.

Osteogenic properties

Osteoblast cell binding and proliferation on bovine bone graft material produced at different temperatures showed a marked and surprising change with increased temperature (see Example 3). Bovine bone immersed in water heated to 100 °C, 130 °C and 160 °C in a closed vessel all bound osteoblasts which then proliferated on the surface (Figure 1). BioOss® was slower at day 7 than bovine bone graft material of the invention prepared at 160 °C (160 °C bone graft material) (Figure 1). At day 21 BioOss® showed more variability than other samples. Without being bound by theory, it is thought that this may be due to the heterogeneity seen in binding with SEM (Figure 2) where 30% of BioOss® particles did not bind cells.

An important question is whether the cells on the different bone materials were behaving as maturing osteoblasts (ie laying down new bone). ALP quantification (a marker of bone maturation) was conducted at Day 7 and results showed significantly more ALP from osteoblasts on 160 °C bone graft material of the invention than when on BioOss® (Figure 3), suggesting that this temperature produces bovine bone graft material that results in the best production of new bone. Consistent with this, confocal imagining (Figure 2) shows very few osteoblasts on 190 °C bone graft material of the invention and BioOss®, while 160 °C bone graft material of the invention had a high density of cells (Figure 2C). This was further validated at day 21 with osteocalcin, a specific marker of osteoblast function, which correlates with the bone formation rate. Osteocalcin staining was highly evident for 130 °C and 160 °C bone graft material of the invention (Figure 4). Osteogenesis had proceeded to such an extent on the 130 °C and 160 °C bone graft materials that the particles were welded to the plastic with new bone. BioOss® induced little osteocalcin production by the osteoblasts and the graft particles were highly mobile (Figure 4). The culture of osteoclasts on the bone graft material of the invention (Example 4) prepared at 100 °C (Figures 5A & B) 130 °C (Figures 5C &D) and BioOss® (5E & F) indicated BioOss® bound osteoclasts but that the cells never produce a resorptive phenotype (ie flattening with a resorption pit), thus suggesting they remain inactive.

These results consistently point to relatively small changes in heat and pressures having a marked effect on osteogenic cell behaviours with 160 °C bone graft material of the invention producing the optimal osteogenic graft material.

Physico-chemical properties

The elasticity of the bovine bone graft material of the invention was compared to BioOss® in Example 5. Bovine bone heated between 100-220 °C in a closed vessel gave a bovine bone graft material that was significantly more flexible than BioOss® which was stiffer (Figure 6). This suggests that the bovine bone graft material of the invention has different properties compared to BioOss® and that cells might like these properties and adhere to the material.

The residual organic content in the bovine bone graft material of the invention was higher than for BioOss® (Figure 7) which may also make it more attractive for osteogenesis. The organic content may be reduced significantly in the 160 °C bone graft material by subsequent bleaching and gramma irradiation, both of which are known to reduce organic content in bone. The crystallinity of the bone was also measured and the 160 °C bone graft material of the invention was not different from BioOss® (Figure 8). These parameters show that in some attributes heat treatment makes a distinctly different graft product to BioOss®.

Processing of 160 °C bone graft material (Example 1) with bleaching and gamma irradiation (Example 2) was conducted and the organic loss assessed. A reduction in organic material may be desirable to reduce any chance of an adverse immunogenic response. The reduction in organic material was measured with Fourier Transform Infrared Spectroscopy (FTIR) and thermogravimetric analysis (TGA) methodology (Example 6). FTIR reports a loss of organic material based on molecular bonds and TGA measures the burn of organic material. Both methods showed a marked reduction in organic material with bleaching and gamma irradiation. By TGA, 160 °C bone graft material had 14.42% organic material with significant reduction to 10.44% with bleaching. Gamma irradiation of the 160 °C bleached bone graft material of the invention caused further significant reduction to 5.95% (Figure 9), which was slightly higher than BioOss® at 2.11%, and consistent with other products that have met approval (MoaBone® (Molteno Opthalmic Ltd) - a natural hydroxyapatite xenograft comprising 7.57% organic material).

Antimicrobial properties

An antimicrobial (non-antibiotic) graft material for intra-oral bone regeneration would provide significant clinical advantage. The addition of alpha lipoic acid capped-AgNPs to the bovine bone graft material of the invention gives an antimicrobial efflux (Figure 10). An AgNP concentration dependent antimicrobial effect was detected, as described in Examples 7 and 8. An inhibitory zone was evident at the boundary of the 75 pg/g AgNP bone disc and was 0.5 mm- 1 mm wide for & 100 pg/g AgNP-bone discs. This shows that AgNP can be used with graft materials, for antimicrobial effect, when present in a concentration above 75 pg/g of bone.

Safety was considered in vitro with autophagic encapsulation of AgNPs by osteoblasts and osteoclasts observed at high dose AgNPs. This process of the membrane encapsulation into lysosomes is a cell survival mechanism. This can lead to apoptosis which is a natural process of programmed cell death, rather than necrosis which is associated with a marked inflammatory reaction. Osteoclasts (RAW 264.7 cells) were more susceptible to AgNP than osteoblast (Saos-2 cells) thus favouring bone formation. The IC50 of AgNP on osteoblasts at 4 h was 88.5 pg/mL and at 96 h was 13.5 pg/mL (Porter, et al., 2021).

In addition, bovine bone graft materials of the invention were bound with AgNPs at different concentrations and tested on Saos-2 osteoblasts, as described in Example 9 (Figure 11). 160 °C Bone graft material treated both with and without AgNP consistently had more alive cells than BioOss® with no AgNP. 160 °C bone graft material also had more cells than bone subjected to other heat treatments, and this was sustained with 150 pg/ml AgNP present, suggesting that the AgNP-treated bone graft material enhanced osteoblast survival.

Osteoclast viability was not affected by increasing the concentration of AgNPs on the bovine bone graft material of the invention, as described in Example 10 and shown in Figure 12. BioOss® with no silver was however found to greatly increase osteoclast viability. The inventors tested if these osteoclasts had morphology consistent with being active: that is, large, flattened cells with a ruffled boarder that would resorb bone and leave a bone resorption pit. SEM was conducted (Example 4, Figures 5E & F) to address this question. It was clear that although the osteoclasts bind to BioOss® they were not found to be active, which is consistent with the lack of BioOss® graft resorption seen in patients. These results suggest that the AgNP-loaded bovine bone graft material of the invention is an effective antimicrobial adjunct to bovine bone with a safe profile and a recommended dose between 75-150 pg/g of bone (Example 8-10, Figures 10-12).

Otago bone tested in an animal model to assess its ability to regenerate bone

A rabbit cranial model with 6 mm defects (4 per animal) was used to test bone ingrowth in response different graft materials (Example 11). The 160 °C bone graft material of the invention with subsequent bleaching and gramma irradiation (160°C + Bl + Ga) was found to result in more new bone than the control empty defects (Figure 13 & 14). The lower amount of 160°C + Bl + Ga graft material used (Figure 13 & 14), and the lighter colour of some of the particles may be evidence of its remodelling which when compared to BioOss® was a solid white infill material. The presence of more connective tissue with 160°C + Bl + Ga as compared to BioOss® is also an indicator that more new bone can be produced into this space (Figure 13). In vivo the 160°C + Bl + Ga was better than nothing and equivalent or slightly better (more connective tissue) than BioOss®.

Animal testing of bovine bone graft material of the invention loaded with AgNPs at 100 pg/g (160 °C heat treated then bleached and gamma irradiated) was conducted in a bone repair model (Example 11). This concentration of AgNPs was well within the antimicrobial dose in vitro (Figure 10). The addition of AgNPs to the bone graft material resulted in no adverse effects on wound healing and osteogenesis (Figures 15 and 16). This is very encouraging and points to a AgNP construct of 160 °C, bleached and gamma irradiated bone graft material offering efficacy for osteogenesis while offering control of antimicrobial ingress.

There was no evidence of an inflammatory reaction to the 160°C + Bl + Ga graft material without and with AgNP in vivo within the rabbit cranial model supporting its safety (Figure 17).

5.4 Uses of the bovine bone graft materials of the invention

The results discussed above indicate that the bovine bone graft material of the invention is suitable for use in a range of xenografting applications.

Bone xenograft materials are commonly prepared as a particulate consisting of either small (0.25 to 1mm ) or large (1.0 to 2.0mm) particles I bone granules. They are also available in a block form that can be screwed onto jawbone, and as a fine powder incorporated into a putty or paste.

The range of possible products was recently reviewed by (Zhao, et al., 2021) and their development and use by (Duncan & Coates, 2021). A person skilled in the art would know how to treat the bovine bone graft material of the invention to convert it to the particular form needed for the application under contemplation. For example, a powdered bovine bone graft material can be obtained by grinding the treated bone pieces.

Examples of oral applications for the bovine bone graft material of the invention include:

1. Guided tissue regeneration: This involves regenerating bone and periodontal ligament by surgically placing a particulate graft into an intra-bony wound adjacent to a tooth surface; typically this is then covered by a resorbable membrane (a thin sheet eg: made of collagen). The most common cause of intra-bony wounds is periodontal disease, but other causes include surgical endodontics (root canal treatment).

2. Guided bone regeneration: This involves regeneration of only bone, by surgically placing a particulate graft into an intra-bony wound. Often this is covered by a resorbable membrane. Typical applications include (a) into the tooth socket after tooth extraction (b) prior to, or during placement of titanium dental implants (c) into the maxillary antrum ("sinus-lift surgery") to create bone support for dental implants (d) for regeneration of bone around previously-healthy titanium dental implants that have developed a chronic infection ("peri-implantitis").

3. Onlay bone grafts: This involves screwing a block of bone onto the external surface of the jaw bone, most commonly for augmentation prior to the placement of titanium dental implants.

4. Repair following maxillofacial trauma, cancer, or cleft lip and palate: This may require a combination of bone blocks, bone particles, pastes, and reservable membranes.

In one aspect the invention provides the use of a bovine bone graft material of the invention in a bone xenograft procedure.

6. EXAMPLES

EXAMPLE 1: Manufacture of bovine bone graft material of the invention

Tissue was removed from prion free NZ bovine bone blocks (25 x 25 x 25 mm) by boiling, rinsing with 80 (+/- 5) deg Celcius water and centrifugation (Molteno Ophthalmic Ltd (batch no. 1810/SBE2) Dunedin, New Zealand). The blocks were then cut into smaller 8 x 8 x 8 mm cubes and 25 x 25 x 2 mm bone slices using a Struers Accutom-50 cutting machine (Struers, Denmark). Thermal processing was conducted in a custom- made cylindrical stainless-steel vessel with external diameter of 120 mm and internal diameter of 70 mm and height of 105 mm fitted with a pressure gauge to record the increase in pressure commensurate with the increase in temperature. Three bone cubes (25 x 25 x 25 mm) and 21 slices (25 x 25 x 2 mm) were added and covered with distilled water then heated at a rate of 4-6 °C/min and held for 2 h at either 100 °C, 130 °C, 160 °C, 190 °C or 220 °C producing 5 sample groups of heat-treated bone graft material of the invention. Treatment was followed by a 5 minute cooling period. The pressure recorded at each temperature is presented in Table 1. The pressure was read directly from the attached pressure gauge and validated using the Clausius-Clapeyron relation. Finally, the bone samples were rinsed with distilled water, and air dried for 24 h in a sterile environment at room temperature (RT).

Prepared bone scaffold specimens are referred to as the respective temperature groups of 100 °C, 130 °C, 160 °C, 190 °C, 220 °C.

Table 1. Pressure recorded at each thermal bone-processing temperature within the stainless-steel vessel.

EXAMPLE 2: Bleaching and sterilization of the bovine bone graft material

Bovine bone graft material prepared in accordance with Example 1 was bleached with 1% sodium hypochlorite solution. The heat-treated bone graft material was dried in a sterile environment at room temperature and then immersed in a container of sodium hypochlorite solution, which was placed in a vacuum pot. The air was expelled for about 10 minutes to create a vacuum. The containers were left in a UV-free environment at ambient temperature (15-25°C) for 24 hours. The sodium hypochlorite solution was drained and the bone graft material centrifuged at 92 g. The bleaching process was repeated four times. Following the final treatment, the bone graft material was rinsed with clean water then reverse osmosis water and placed on perforated trays in an ambient temperature (15-25°C) drying cabinet until dry.

Sterilization of bovine bone was conducted by gamma irradiated at a dose of 25-32 kGy (MSD Animal Health, Gamma Department, New Zealand).

EXAMPLE 3: Bone temperature treatment and testing with osteoblasts and comparison to BioOss®.

Saos-2 osteoblast proliferation growth on bovine bone treated to different temperatures

Method: The human osteosarcoma cell line (Saos-2 (ATCC® HTB-85™)) (p=15) was grown in standard cell culture conditions (37 °C, 5% CO2) in cell culture medium containing McCoy's (Cat. No. 36600021; ThermoFisher, MA, USA) I 15% FBS, 50 pg/mL of gentamicin and 5 mL of antibiotic-antimycotic.

Osteogenic assays were conducted on standardized bone granules (1 mm x 2 mm x 2 mm) as per Example 1. Prior to cell assays, the granules were sterilized with 70% ethanol. The methodology involved pre-treating each bone group with media, seeding cells at high density onto the granules and then transferring the granules for each group into replicate wells, thus ensure only cell/granule interactions were being assayed rather than cell/plastic. Enough granules for each group (100 °C, 130 °C, 160 °C, 190 °C, 220 °C, and BioOss®), were placed into one well each of a 48 well plate and were preincubated overnight with McCoys/15% FBS. Extra granules were included for each group to allow for granule loss during processing, therefore each collated group contained ~ 30 granules. Saos-2 cells were seeded at 160,000 cells per well (400 pL) onto the bone particles and were incubated for 16 h. The granules were aseptically transferred to four wells per group (N=4) each containing 5 granules per well in a new 48 well plate containing 500 pL of McCoys/15% FBS supplemented with 100 pM L-ascorbic acid-2- phosphatase, 10 nM dexamethasone, and 5 mM g-glycerophosphate (osteogenic media). The cell bound granules were incubated under standard cell culture conditions over 21 days. Experimental assays were performed on day 7 and day 21.

At 7 days a triplex assay was performed which included confocal microscopy of NucBlue® Live reagent (Hoechst 33342) and propidium iodide (PI) stained cells (ReadyProbes™ Cell Viability Imaging Kit; Thermofisher), analysis of ALP activity, and measurement of DNA content using a Picogreen DNA assay. Subsequently at 21 days, a duplex assay was performed using confocal microscopy of DAPI & PI stained samples, and analysis of DNA content using a picogreen assay. A separate set of bone particles (n=5) were used for DAPI/osteocalcin immunolabelling and imaged with confocal microscopy. Picogreen DNA quantification

A low and high-range concentration calibration for DNA content in trypsin was performed using the picogreen assay kit, as per manufacturer instructions (Pl 1496, Quant-iT™ PicoGreen™ dsDNA Assay Kit, Invitrogen). The bone granules were aseptically transferred from wells into separate 1.5 ml tubes and trypsin-EDTA (0.25% v/v, 200 pL) added and incubated at RT for 4 min. The bone granules were agitated to lift cells from the bone surface and the samples were divided to allow use in the picogreen assay and the ALP assay (100 pL for each). A working solution of IX tris-HCI EDTA (TE; 10 mM Tris-HCI, 1 mM EDTA, pH 7.5) was made and the Quant-iT™PicoGreen reagent was diluted 200-fold in 1 X TE and stored protected from light. An equal volume of picogreen (0.1 ml) was added to the bone granule derived cell samples in a 96 well plate and incubated for 5 min at RT, protected from light. Fluorescence was measured at ex/em: 480 nm/ 520 nm using a Synergy 2 Plate Reader and Gen 5 software.

Alkaline phosphatase fluorometric assay

Immediately after trypsination of bone granule samples, 100 pL of resulting supernatant was centrifuged at 1000 x g for 4 min to pellet cells and bone granules. The trypsin was removed and discarded. Ice cold PBS (200 pl) was placed onto the bone-cell pellet and then centrifuged at 1000 x g for 4 min. PBS was removed from the bone-cell pellet and replaced with the assay buffer (100 pl), which was then pipetted up and down rapidly (ab83371 Alkaline Phosphatase Assay Kit Fluorometric, Abeam). The samples were centrifuged at 13,000 x g, 4 °C, for 3 min and the supernatant was collected and kept at -80 °C until required. The supernatant was allowed to equilibrate at room temperature prior to further analysis. Bone granules without cells that had been processed through the trypsin and the centrifugation procedure were used as a background control. Assay buffer (10 pL) was added to samples (100 pL) and 4-methylumbelliferyl phosphate disodium salt (MUP; 20 pL), added to the test/control samples and background control assay buffer. A stop solution was added to designated test background controls. Samples were incubated for 30 min at 25 °C, protected for light. The stop solution was then added to the samples, calibration standards, and background wells. The well plate was gentle shaken, and fluorescence was measured using Ex/Em = 360/440 nm. A calibration of ALP enzyme concentration was conducted as directed by manufacturer instructions during the assay.

Live/dead staining of cells on bone scaffolds

The culture media was removed from each well containing bone granules and each was washed 3 times with PBS. PBS (400 pl) was placed on the specimens and Nunc blue and PI (15 pl for each) were added to each well. The samples were incubated in the dark at RT for 30 min. Samples were subsequently washed using PBS and were maintained in 100 pl of PBS during confocal laser scanning microscopy. Image acquisition of live/dead stain was performed a on Nikon A1+ inverted confocal laser scanning microscope (Kurobane Nikon Co., Japan). Confocal images were analysed for cell counts using Fiji software.

Bone maturation on cells bound to the bone scaffolds

Immunohistochemistry was performed using an osteocalcin (Cat. No. abl3421; Abeam) mAb antibody. The culture media was removed from the bone granules/Saos-2 cells after 21 days of culture and samples were gently washed with PBS and fixed using methanol (100 pL) for 5 min. They were then washed again with PBS (500 pL) and incubated in tween-20 (1% in PBS) for 10 min. PBS washes prior to blocking with 20% goat serum (G9023; Sigma)/PBS (500 pL) were conducted and the osteocalcin antibody (2.5 pg/ml in 5% goat serum/PBS (300 pl)) was applied to each sample and was left to incubate at 4°C overnight. The samples were washed 3 times with 1% skimmed milk powder/PBS (500 pL) for 15 min each with gentle rotation. Secondary antibody (goat anti-mouse IgG secondary Dylight 488; Cat. No. NBP1-72872) (0.2 mg/ml) was incubated in the dark and then samples were washed three times in PBS. Granules were further stained for 5 min with DAPI (300 nM, 300 pL) and washed with PBS three times before imaging on a Nikon A1+ inverted confocal laser scanning microscope.

Results:

Osteoblast cell (Saos-2) number on materials as assessed by picogreen and DAPI on Days 7 and 21.

The two methodologies give very similar results. Osteoblast cell number as determined from DNA quantitation (Picogreen assay), and DAPI counts, on heated bone scaffolds at 7 and 21 days and are shown in Figure 1. 190 °C and 220 °C bone graft material exhibited no or few cells, with values consistent with media only control wells. At 7 and 21 days, 100 °C, 130 °C, and 160 °C bone graft material demonstrated the highest number of cells amongst tested groups, along with BioOss®. The BioOss® specimens demonstrated a noticeable increase in cell number between 7 and 21 days but had high variability between replicates. By Picogreen staining 160 °C bovine bone contained a similar number of cells to BioOss® at both days. Using DAPI analysis at day 7, 160 °C bone graft material had significantly more cells to than BioOss® at day 7, and at day 21 the BioOss® show high variability. Images of osteoblasts (Saos-2) on bone by confocal microscopy

Osteoblasts were more evident on 160 °C bone graft material (Figure 2C) one than any other bone group examined (Figure 2). BioOss® (Figure 2F) had low/moderate numbers of cells.

Osteoblast alkaline phosphatase production

ALP production levels from osteoblasts on heated bone specimens at 7 days are shown in Figure 3. ALP production was significantly increased when bone was heat treated at 160 °C compared to 100 °C (p = 0.0064). Cells cultured on 130 °C bone graft material also demonstrated higher mean quantities of ALP but greater viability between samples. No ALP production was recorded from 190 °C and 220 °C bone graft materials, which was consistent with the lack of osteoblast growth observed on these bone specimens. ALP concentrations recorded from cells on 100 °C bone graft material were significantly higher than those recorded from 190 °C and 220 °C bone graft materials but were not significantly different from BioOss®. Cells on 160 °C bone graft material produced significantly more ALP than BioOss®. This suggests that 160 °C bone graft material was associated with more osteogenic activity.

Osteoblast osteocalcin secretion as a marker of maturing bone

Osteocalcin, a small non-collagenous protein produced exclusively by osteoblasts, is generally regarded as a marker of bone formation. The presence of osteocalcin on the heat-treated bone specimens is shown in Figure 4 and can be distinguished as green fluorescence. Ossteocalcin was found to be beneath cells (DAPI stained nuclei). Osteocalcin presence was more distinct in areas where cells were particularly dense and overlapped as might be expected. Cells attached to 130 °C and 160 °C bone graft materials had proliferated further than the margins of the bone granules and had connected the granule to the bottom of the well; every nuclei observed was associated to an area of osteocalcin production. 190 °C bone graft material had residual osteocalcin evident in small quantities on the bone, however cells did not attach. 220 °C bone graft material showed no presence of osteocalcin. BioOss® possessed the least osteocalcin associated with cells amongst the specimens. It was interesting to observe that some groups of bone granules had adhered to the culture well, due to cell sheeting from granule to the well surface, in particularly 100% of granules for 160 °C bone graft material were immobile, 60% for 130 °C, 40% for 100 °C, and full mobility was seen for 190 °C and 220 °C bone graft materials, and BioOss® within wells. This suggest that 130 °C and 160 °C bone graft materials were associated with osteoblasts producing mature bone matrix and consistent with high levels of osteogenesis (bone formation). EXAMPLE 4: Scanning electron microscopy of osteoclasts (RAW 264.7) on BioOss®

Method: Mouse alveolar macrophage cells (RAW 264 .7 (ATCC® TIB-71™)) (passage 5) were grown in standard cell culture conditions (37 °C, 5% CO2) in cell culture medium containing DMEM (Cat. No. 10569010; ThermoFisher, MA, USA) / 10% FBS (Cat. No. F8067; Merck, NJ, USA), 50 pg/mL of gentamicin (Cat. No. 15710064; Life Technologies) and 5 mL of antibiotic- antimycotic (Cat. No. 15240062; Life Technologies Ltd).

Bone discs made as per Example 1 were prepared from the 25 mm x 25 mm x 2 mm bone slices, using a 5.2 mm circular soft tissue punch (Ref 32Z2002 Nobel Biocare, Kloten, Switzerland), producing 5.2 mm x 2 mm discs. Prior to cell seeding, bone discs (N=4) were sterilized by soaking in 90% EtOH (10 min, x3), phosphate buffered saline (PBS) washed (10 min, x3), and a final wash performed in DMEM/10% FBS for (10 min, x3). The bone discs were then placed onto sterile parafilm and air-dried in a sterile environment. BioOss® granules were also sterilized using the same methodology. Bone discs or granules were then placed into a 96 well plate in DMEM/10% FBS (100 pL) overnight at 37 °C, 5% CO2. After 16 h, RAW 264.7 cells were seeded at 2000 cells per sample well (100 pL) onto the overnight-incubated bone discs containing the 100 pL of pre-incubation media to give a final volume of 200 pL. Following overnight incubation, each bone disc was aseptically moved to a 48 well plate well containing oMEM (500 pL) (Cat. No. 32571036; ThermoFisher, MA, USA) supplemented with 10% FBS, RANK-L (50 ng/ml) and colony stimulating factor (25 ng/ml CSF). The discs were then incubated at 37 °C in 5% CO2 for a duration of 7 days. An additional 500 pL of supplemented oMEM 10% FBS was added to the existing media after 48 h. After 96 h, 500 pL of media was removed and replaced with fresh supplemented oMEM 10% FBS. On day 7, the specimens were analysed via SEM.

To conduct the SEM culture media was removed from the specimens and this was replaced with 2.5% glutaraldehyde in sodium cacodylate buffer (0.19 M, pH 8.4). The plates were then placed on an orbital mixer at RT for 60 min. Cells were then washed three times for 5 min each in sodium cacodylate buffer (0.1 M) and stained using 1% osmium tetroxide (OSO4) in sodium cacodylate for 1 h. Post staining; the cells were washed three times for 5 min each with cacodylate buffer. Cell bound-bone discs were then dehydrated using a graded ethanol series: 30%, 50%, 70%, 80%, 95% and 100% for 5 min each and transferred to safe cell specimen holders, ensuring the discs and holders remained submerged in 100% ethanol. The samples were then dried using a critical point dryer with liquefied carbon dioxide as the transitional fluid. Specimens were then mounted on aluminum stubs with carbon tape and were sputter coated with a goldpalladium mix using a Peltier-cooled high-resolution sputter coater (Emitech K575X, EM Technologies Ltd; Kent, England). Specimens were examined using a JEOL FE-SEM 6700 (Joel Ltd; Tokyo, Japan).

Results: Osteoclast growth on BioOss® was variable, cells grew evenly across ~ 70% of examined granules, however ~ 30% granules exhibited no osteoclast presence. This perhaps reflects different bone types in BioOss®. Osteoclast morphology on 100 °C and 130 °C bone graft materials had a bimodal population of small, rounded cells and flattened elongated cells (Figure 5 A-D). Osteoclasts that adhered to BioOss® were typically very small and rounded (Figure 5 E). There was no evidence of pits on the BioOss®, which indicates the cells were not able to actively resorb the BioOss®.

EXAMPLE 5: Physico-chemical characteristics of heated bone blocks at different temperatures.

Method: Elastic modulus calculated using atomic force microscopy (AFM)

Bone graft material of the invention (non-bleached and non-gamma irritated) as per Example 1 was prepared at three different temperatures (low = 100 °C, medium = 160 °C, high = 220 °C) and compared against BioOss®. Bone slices measuring 2 mm in thickness (n=3) were prepared from heat-treated bone 25 mm ³ cubes. The heated bone slices and BioOss® granules were affixed to a standard glass microscope slide (Thermofisher, Massachusetts, USA). AFM measurements were conducted using a Bioscope catalyst atomic force microscope (Bruker, Massachusetts, USA). The imaging was performed using a silicon probe (RTESPA-150, Bruker) with a spring constant of 5 N/m and resonant frequency of 150 kHz. Nanoscope software 9.4 (Bruker, USA) was used to calculate the elastic modulus.

Results: The elastic modulus is a measure of the resistance to elastic deformation in the bone from an applied stress. The lower the elastic modulus, the more rubber-like; the higher the elastic modulus, the stiffer it is. The elastic modulus measured using AFM showed that bovine bone treated to 100 - 220 °C was significantly more elastic than BioOss® (Figure 6).

Method: Thermogravimetric analysis (TGA) to measure organic content

The residual organic content of all bone graft material prepared as per Example 1 and BioOss® were determined using a Q50 Thermogravimetric Analyzer (TA instruments, New Castle, USA). The samples were ground into a fine powder, and 20 to 25 mg was packed into the platinum tray. The thermal decomposition was carried out in air at room temperature up to 1000 °C with a heating rate of 20 °C/min. Each sample and control were repeated twice and presented as an average of both runs. Results: The water content and the crystal formation of the samples was similar. Organic content in heated bone samples decreased with increasing temperature and BioOss® had the lowest organic content (Figure 7).

Method: Crystallinity analysis by x-ray diffraction (XRD)

The crystallinity of bone specimens was determined using powdered XRD (X'Pert Pro MPD, Malvern Panalytical, United Kingdom). Samples (n=3) of heat-treated bone graft material of the invention; "initial cleaning" which is tissue-free bone, otherwise untreated; and BioOss®, were ground into fine powder using an agate mortar and pestle, packed into a specimen holder, and measured with the diffractometer at scan steps of 0.05°. The diffractometer operated at 40 kV and 30 mA, using Cu Ko _radiation with a scan range of 20°-80° 20. Crystallinity was calculated using a method was adapted from (Landi, Tampieri, Celotti, & Sprio, 2000) where the crystalline phase was evaluated by the relation: Xc « _1- (VI 12/300/1300). 1300 is the intensity of 300 reflection and V112/300 is the intensity of the hollow between 112 and 300 reflections.

Results: Crystallinity measurement suggest that the 160 °C bone graft material was similar to BioOss® but bone graft material of the invention prepared by heating under pressure at lower temperatures differed from that prepared at higher temperatures. This suggests that higher temperatures results in more ordered structure and increase in crystal size (Figure 8).

EXAMPLE 6: Organic content in 160 °C bone graft material with and without bleaching and gamma irradiation

Method: FTIR analysis of organic content

The chemical analysis was performed with an attenuated total reflectance FTIR spectrometer (Alpha II, Bruker, Germany). The spectra were recorded in the range 4000

- 400 cm ^-1 , at a resolution of 4 cm ^-1 , with a total of 24 scans per run. The baseline was corrected for all the specimens (n=3). The mineral content: organic matrix (M:M) was calculated as the phosphate to amide I ratio (Pienkowski, 1997) The M:M was determined by calculating the ratio of the peak area assigned to phosphate bands at 900

- 1200 cm ^-1 to the peak areas of 1585-1720 cm ^-1 assigned to Amide I.

Method: TGA analysis of organic content is outlined in Example 5.

160 °C bone with and without bleaching and gamma irradiation (Examples 1 and 2) was comparted to BioOss®. Results: The TGA and FTIR results are given in Figure 9A and 9B. TGA showed heat treated 160 °C bone graft material had 14.42% organic content, and bleaching reduced this to 10.44%, with gamma irradiation further reducing this to 5.95%. 160 °C bone graft material which had been bleached and gamma irradiated thus had an organic content was similar to BioOss® at 2.11% (Figure 9A). FITR showed that bleaching of 160 °C bone graft material was the process that resulted in the most marked loss of collagen bonds (Figure 9B) as measured by FTIR (Figure 9B).

EXAMPLE 7: Preparation of alpha lipoic acid capped AgNP for loading onto bovine bone graft material

Alpha lipoic acid capped-AgNPs were prepared by the following process. To prepare the microemulsions (pEms), 40 ml of an AOT (docusate sodium salt > 96%, Cat. No. 86140, Sigma Aldrich, Missouri, USA) solution in 0.33 M heptane (Cat. No. H350-1, Fischer Scientific, New Hampshire, USA) were placed in two separate flasks. To the first solution, an aqueous solution of silver nitrate (AgNCh; Cat. No. 10224350; Fisher Scientific, New Hampshire, USA) (1.6 ml, 0.13 M) was added dropwise with stirring, forming pEm 1. To the second solution, an aqueous solution of sodium borohydride (NaBH4 crystalline 98- 99%, Cat. No. ICN10289425, Fischer Scientific, New Hampshire, USA) (1.6 ml, 1.84 M) was added dropwise with stirring, forming pEm 2. The flasks were placed in separate ice baths, and pEm 1 was covered with aluminium foil then pEm 2 added dropwise with continuous stirring. Upon addition, a colour change from light yellow to dark yellow- brown was observed, suggesting the production of AgNPs. This mixture was allowed to stir in the dark for up to 24 h. Subsequently, alpha lipoic acid (0.08 mM, dissolved in 0.25 ml ethanol) was introduced and the microemulsion was stirred for an additional 2 min. Upon discontinuation of the stirring, a 1 : 1 methanol/acetone mixture was added at an equivalent half volume of the combined microemulsion (40 ml). Phase separation was clearly observed, and a dark-coloured interface formed between the two phases, where the particles resided. The nanoparticles at the interface were carefully collected. Subsequently, the particles were washed 3 times with ethanol, with centrifugation at 6000 g for 5 min, and resuspended in 1-6 ml of deionised (DI) H2O (depending on the [Ag] desired) which was previously pre-adjusted to pH 10 (±0.5) with anhydrous ammonia. The resulting yellow-brown colloidal suspension was centrifuged twice at 16000g for 45 min, with the final supernatant collected and retained for characterization. AgNP size is typically <10 nm in size, with a narrow PDI <0.3, with a high concentration of approximately 3000 pg/mL (Cotton, et al., 2019). EXAMPLE 8: Antimicrobial efflux of AgNP from bovine bone graft material of the invention (bleached and gamma irradiated)

Method: Discs (5.2 mm diameter) of bovine bone graft material prepared in accordance with Examples 1 and 2 (heated under pressure at 160 °C, bleached and gamma irradiated) were loaded with AgNP (as made in Example 7) at increasing concentrations (0, 12.5, 25, 50, 75, 100, 150, 200, 500 pg/g) using an equal loading volume (30 pL) of AgNP solution. AgNP loading was performed in a sterile environment on parafilm to ensure a hydrophobic surface that allowed all AgNPs to be adsorbed onto the bone surfaces. Disc specimens were allowed to dry at room temperature in the dark.

A 16 h bacterial culture of E.coli in tryptic soy broth was adjusted to an optical density value of 0.4 OD. An inoculate of the aforementioned bacterial broth (1 mL) was added to tryptic soy agar (14 mL) in a 15 mL falcon tube, and gently poured into a sterile agar plate and left to semi-set. Discs of AgNP-loaded bovine bone material prepared in accordance with Example 1, 2, and 7 were then placed onto the semi-set agar, with the top of the disc remaining uncovered by the agar. Once the agar was set, phosphate buffered saline (30 pL) was pipetted onto the top of the bone. Plates were inverted and incubated for 24 h at 37 °C.

Result: The discs of bone graft material loaded with AgNP showed a concentration dependent antimicrobial effect. As can be seen in Figure 10, an inhibitory zone was evident at the boundary of 75 pg/g AgNP bone disc and was evident at 0.5 mm- 1 mm for > 100 pg/g AgNP-bone discs.

EXAMPLE 9: Safety of AgNPs on Saos-2 osteoblasts on bone graft material of the invention

Method: Osteoblast (Saos-2 (ATCC® HTB-85™) cells were added to wells at 1.6 x 10 ⁵ cells per well and cultured on bone granules (1 mm x 2 mm x 2 mm) prepared in accordance with Example 1 and 2 with bleaching and gamma irradiation. Bone granules (23 per well, performed in triplicate wells) of different preparatory temperatures were loaded with increasing concentrations of AgNPs (0, 50, 100, 150 pg/g) as described in Examples 7 and 8. Once dry, the bone granules were placed into individual wells of a 48 well plate. Bone particles with no addition of cells, and with BioOss® served as controls. McCoy's media +15 % FBS (200 uL) supplemented with 100 pM L-ascorbic acid-2- phosphatase, 10 nM dexamethasone, and 5 mM g-glycerophosphate was added to wells for 16 h at 37 °C 5% CO2. Prestoblue® was added to each well at 7 days (40 uL) and the fluorescence measured. Results: Cell viability assays were carried out on osteoblasts at 7 days. The 160 °C bone consistently enhanced cell viability and performed better with increasing AgNP concentration (Figure 11). It also induced cell growth better than BioOss®. The presence of AgNP appears to enhance proliferation over the control with no AgNP for most constructs. At 150 pg/ml of AgNP more variability and decreased viability was observed on 100, 130 and 190 °C bone graft material surfaces.

EXAMPLE 10: Safety of AgNPs on RAW 264.7 osteoclasts on bone graft material of the invention

Method: Bone granules (5 per well, performed in triplicate) prepared in accordance with Example 1 and 2 with bleaching and gamma irradiation, and with different preparatory temperatures were loaded with increasing concentrations of AgNP (0, 50, 100, 150 pg/g) as described in Example 7 and 8. Once dry, the bone granules were placed into individual wells of a 48 well plate. Bone particles with no addition of cells, and BioOss® served as controls. oMEM + 10% FBS (200 pL) supplemented with 10% FBS, RANK-L (50 ng/ml) and colony stimulating factor (25 ng/ml CSF) was added to wells for 16 h at 37°C 5% CO2. Osteoclasts (RAW 264 .7 (ATCC® TIB-71™)) were added to wells at 5000 cells per well in the supplemented oMEM (200 pL). Prestoblue® was added to each well at 7 days (40 uL) and the fluorescence measured.

Results: Osteoclast viability was not altered by the presence increasing AgNP (Figure 12). BioOss® was better at binding osteoclasts in this assay.

EXAMPLE 11: In vivo analysis in a rabbit cranial model of bone regeneration

Method: Bone grafting materials

A comparison was made between the bovine bone graft material of the invention, empty sites, and BioOss® (Geistlich Pharma, Switzerland).

Method: Making the bovine bone graft material of the invention including treating of the bone, making particles, sieving, settings for gamma irradiation

Bovine bone blocks were treated as per Examples 1 and 2, being heated to 160 °C under pressure and then bleached. Particles were chiselled directly from the blocks and sieved with a small sieve (0.5/0.6 mm) and a larger sieve (1.2 mm) to obtain particles measuring approximately between 0.25 mm - 2 mm. Afterwards, the particles were packed in Eppendorf tubes (around 70 mg per tube) and gamma irradiated at 25-32 kGy (Example 2). This material was designated as "160°C + Bl + Ga, heat treatment to 160°C with bleaching and gamma irradiation". Bovine bone graft material of the invention was also loaded with alpha lipoic acid capped AgNP (AgNP) as made in Example 7. To generate the AgNP-160°C + Bl + Ga, the AgNPs was added to the bone particles (Example 8) inside the hood after the gamma radiation step. AgNP stock concentration was 2510 pg /ml and used within 4 weeks of production. The final concentration of the AgNP per construct was 100 pg per gram of 160°C + Bl + Ga graft material.

Method: Surgical approach. 12 New Zealand rabbits were used in these experiments. The experiment was conducted (Ethics number: 5.8.18-02996/2020; Approved from Malmo-Lund animal research board) at Lund University, Sweden. The animals were sedated with ketamine and further anaesthetised with an isofluran-NzO inhalation method. The skull hair was shaved, and the skin prepared with iodine and sterile drapes. Aseptic technique and sterile instruments were always used. Lidocaine with adrenaline were administered around the operative site. A linear incision was prepared running from the nasal bone to the mid sagittal crest. Soft tissues reflected, followed by periosteum dissection from the occipital, frontal, and parietal bones. Four x 6 mm osteotomy defects were drilled - two in frontal and two in parietal bone with a 6 mm trephine in a dental hand piece. The surgical area was irrigated with sterile saline solution to remove bone debris. To avoid any dural perforation, drilling was stopped before the inner table of the calvaria is fully penetrated. The defects were finalized carefully using a round bur and an elevator. Two experimental Groups included: Group 1 - Untreated defect, BioOss®, 160 °C bone graft material which was bleached and gamma irradiated (160°C + Bl + Ga). Group 2 - Untreated defect, 160°C + Bl + Ga, and 160 °C bone graft material which was bleached and gamma irradiated with 100 pg/ gm AgNP (AgNP-160°C + Bl + Ga). The treatments were rotated for each animal. The soft tissues were sutured after placement of test materials and Novalgin (50 mg/kg body weight) administered. The animals were kept in a purpose-designed room for experimental animals and fed a standard laboratory diet.

Four and sixteen weeks after surgery the rabbits were sacrificed to give two time points. Rabbits were sacrificed by an overdose of pentobarbital. Animals were perfusion fixed with 10% neutral buffered saline after a heparin flush, to maintain good tissue histology. The 4-week time point was chosen to study the early healing events, and the 16-week time point was chosen to evaluate bone graft substitute degradation. The experimental sites were excised, placed in 10% neutral, buffered formalin and prepared for analysis.

Method: Radiography and micro-computer tomography (CT scanning)

The samples to be radiographed were subjected to a sequential water substitution process as per the following; 48 h in 40% ethanol, 72 h in 70% ethanol (changed at 24 h). Using SKYSCAN high resolution in vivo x-ray microtomography at 60 kV the amount of newly formed bone and grafting materials were quantified using Image J software (NIH). The CT data was analysed mid-slice through the 6 mm cranial detect and the amount of bone, residual graft, new bone and connective tissue analysed using fixed thresholding. The region of interest was defined as a 5.5 mm circle within the cranial defect. Data was analysed in GraphPad PRISM7 (GraphPad Software, San Diego, CA, using Tukey's adjusted ANOVA - unpaired.

Results: CT analysis of 160 °C bone graft material, bleached and gamma irradiated

Quantitative analysis of mid-slice CT scans was conducted for each defect. All images were quantitatively analysed at both 4 and 16 weeks. Figure 13 shows the Group 1 midslice defects from CT scanning at week 16. The empty defects had evidence of only marginally more new bone. Significantly more new bone was measured for BioOss® and 160°C + Bl + Ga graft material than compared to empty sites. There was evidence of less graft material with 160°C + Bl + Ga bone, than BioOss®; equating to more connective tissue which would be transformed to new bone over time (Figure 14).

Results: CT analysis of 160 °C bone graft material, bleached and gamma irradiated with alpha lipoic acid capped AgNP

The addition of AgNP to the 160 °C bone graft material bleached and gamma irradiated (Examples 1, 2, 7, 8) was examined in the Group 2 experiment. Images at 16 weeks of healing (Figure 15) show no difference between 160°C + Bl + Ga and AgNP-160°C + Bl + Ga. There was no evidence of AgNP having and adverse effect on healing. Both 160°C + Bl + Ga and AgNP-160°C + Bl + Ga produced more new bone than the empty sockets alone (Figure 16).

Results: Histology of the rein-embedded tissue examined for any inflammatory reaction at week four of healing

Representative rein-embedded histological images are given in Figure 17. Evidence of new bone formation around 160°C + Bl + Ga and AgNP-160°C + Bl + Ga graft material was clearly evident. Within the connective tissue blood vessels were clear evidence of angiogenesis and healing/regeneration. No inflammatory infiltrate was evident in any of the sites.

Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto. 7. REFERENCES

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