1. FIELD OF THE INVENTION

The invention relates to an anti-microbial gelatin-methacryloyl (GelMA) hydrogel nanocomposite comprising silver nanoparticles (AgNP). The nanocomposite can be used in a variety of dental and surgical applications.

2. BACKGROUND TO THE INVENTION

Multifunctional hydrogels are important components and agents for engineering and treating damaged tissues. Hydrogels are polymeric material which retain liquid. They can be made from petrochemical-based or naturally-occurring materials. Natural protein hydrogels can be tailored for biocompatibility, cell attachment, hydrophilic nature and enzymatic degradation and stiffness.

In recent years, nano-engineered hydrogel constructs have expanded the range of medicinal applications for which hydrogels can be used. These hydrated polymer networks include nanomaterials embedded in their matrix.

GelMA macromer is a photocross-linkable version of gelatin (denatured collagen) with amine and carboxyl groups that are partially conjugated with methacrylate or methacrylamide groups. GelMA macromer has attracted the widespread interest of researchers because of its excellent biocompatibility, biodegradability, and moldability. GelMA macromer contains natural Arg-Gly-Asp (RGD) sequences, which can facilitate biological interaction between cells and scaffolds. Various structures have been constructed from GelMA macromer, including 3D scaffold, injectable gel, bio-printed scaffold, and electrospun fibrous membrane via precise fabrication methods such as light-induced crosslinking, extrusion 3D printing, electrospinning, or micro fluidics.

A GelMA hydrogel can be prepared by cross-linking the GelMA macromer polymer chains dispersed in an aqueous medium. Cross-linking can be via numerous mechanisms including physical gelation, ionic interactions and chemical cross-linking. The latter is preferred because it is more precise and controllable, as well as irreversible.

The mechanical properties of GelMA hydrogel such as elasticity, compressibility and hardness can be tuned by altering the concentration of GelMA macromer, crosslinking conditions and reagents; and by introducing nanomaterials into the hydrogel matrix.

GelMA nanocomposites have been prepared with a wide range of nanomaterials including carbon-based materials such as graphene, carbon nanotubules, reduced graphene oxide and nanodiamonds; inorganic materials such as nanosilicates, nanohydroxyapatite, bioactive glasses and mesoporous silica; and metallics such as silver and gold nanoparticles.

Some GelMA nanocomposites have been explored for use in tissue repair and construction. Tissue replacement is vulnerable to rejection from the immune system and is prone to infections.

GelMA nanocomposites containing silver are thought to have good potential in tissue repair and construction applications due to the known antimicrobial activity of silver ions. Silver has historically been used as an antimicrobial and the synthesis of silver nanoparticles (AgNP) with a size less than 100 nm has opened up new avenues for biomedical research (Lara, Garza-Trevino, Ixtepan-Turrent, & Singh, 2011; Xu et al., 2020). AgNP have been investigated extensively due to their superior physical, chemical, and biological and antimicrobial characteristics compared to bulk silver forms. They have broad antimicrobial activity which includes antibacterial, antifungal, antiviral, as well as being sporicidal (Tehri, Vashishth, Gahlaut, & Hooda, 2022).

For example, Jahan et al. suggests a possible incorporation of AgNP into GelMA for wound healing of soft tissues (Jahan, George, Saxena, & Sen, 2019) and Cao et al (Cao et al., 2021) investigated a AgNP/GelMA construct as a wound dressing. However, there are questions regarding how well the AgNP would be retained in the GelMA hydrogels during clinical applications.

While all tissue regeneration poses challenges, bone regeneration is particularly difficult due to its internal application and the need to induce the patient's own cells to regenerate replacement bone that can withstand considerable physical force.

Bone is a composite tissue comprising an organic matrix and inorganic minerals. The organic matrix is composed mainly of collagen which makes up approximately 90% of the matrix proteins present. The inorganic phase consists of calcium, phosphorous and oxygen, combined to form hydroxyapatite (HA) which provides strength and also some rigidity. New bone formation is referred to as the process of osteogenesis. Osteoblasts (bone producing cells) regenerate bone and they interact with osteoclasts (bone removing cells), which results in a dynamic bone material capable of remodelling. When graft materials are applied to a bone defect, the material can be osteoinductive or osteoconductive. When osteoinductive it stimulates immature and multipotent stem cells to become preosteoblasts (immature osteoblasts) and then osteoblasts; that is, it induces new bone forming cells and activates 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.

Bone grafting materials are commonly used to replace missing, damaged or diseased bone throughout the entire skeleton (orthopaedic applications) including around teeth, the jawbones and facial bones (oral and maxillofacial applications). Oral applications include the treatment of periodontal (gum) diseases, which cause loss of bone and periodontal ligament around teeth resulting in the loss of the tooth or teeth (known as guided tissue regeneration, GTR); supporting bone healing or regenerating lost bone in tooth sockets after tooth extraction (known as alveolar ridge preservation, ARP); regeneration of sufficient bone in the jaws to support installation of dental implants (screws made of titanium, zirconia or related metal alloys) to replace missing teeth, and also regeneration of bone around dental implants that has been destroyed by subsequent infectious peri-implant disease (peri-implantitis). These latter two applications of bone grafting are known as guided bone regeneration, GBR. Other oral applications may include GBR after bone loss due to dental disease (endodontic lesions) or after amputation of tooth roots (apicoectomy or root resection). Maxillofacial applications include GBR of bone lost due to trauma, infection, birth defects and cancer.

Oral grafting must deal with the risk of infection, as the mouth has high levels of bacteria present, including disease-causing (pathogenic) bacteria. Microbial infection is a common complication of bone grafting in oral sites including around teeth and dental implants. These infections can be difficult to treat using systemic (blood-borne) antibiotics due to poor blood supply in the regenerating bone and surrounding tissues. Surgical revision is often required, which adds further morbidity for the patient. The emergence of antibiotic-resistant bacteria in recent years has further complicated the treatment of bacterial infections. Prevention of infection concomitant with placing the bone graft, using locally-released antimicrobial agents, is the preferred option.

Although there are a range of grafting materials available, many have drawbacks limiting their acceptability and use. Pore size, pore interconnectivity, and porosity can modulate degradability, vascularization, and bone tissue ingrowth. Autografts are sourced from elsewhere in the body of the individual receiving the graft and are considered the gold standard, being the safest and most biocompatible. However they require a secondary surgical site, which is associated with pain and risk of infection, making them less desirable for patients. Allografts are sourced from human patients other than the individual receiving the graft, typically from banked bone or cadaveric bone. They are highly biocompatible but the harvesting of bone from cadavers is less acceptable to some patients and can be associated with the risk of disease transmission. Alloplasts are laboratory-manufactured artificial constructs which may include tricalcium phosphate, hydroxyapatite or resorbable polymers such as polylactic acid, polyglycolic acid or polycaprolactone. Xenografts (animal origin grafts) are commonly used and come in the form of decellularized bone. Common source species include cattle (bovine bone) pigs (porcine bone) or horses (equine bone). Generally, a porosity of more than 50% by volume and pore sizes between 200 and 800 pm is recommended as optimal for the development of bone tissue. Bone particle sizes range from 0.5 mm to 2 mm; smaller sizes are preferred for periodontal GTR, peri-implant GBR and socket ARP; larger sizes are preferred for maxillofacial and orthopaedic applications.

There are a number of xenograft options available. BioOss® (BO, Geistlich Pharma AG, Wolhusen, Switzerland) is a widely-used commercially available bovine bone material provided in granular form. Its major drawbacks are high crystallinity and a lower resorption rate, resulting from thermal processing which has then been associated with resistance to biodegradation, and lack of remodelling by osteoclasts.

Compositions comprising polymer-based hydrogels may be able to overcome some of these drawbacks.

It is therefore an object of the invention to provide GelMA nanocomposites that overcome at least some of the disadvantages associated with existing tissue repair and regeneration materials and/or that will at least provide the public with a useful choice.

3. SUMMARY OF THE INVENTION

The invention provides a dual osteogenic/non-antibiotic antimicrobial hydrogel for use in tissue healing. The hydrogel of the invention is a nanocomposite material comprising capped silver nanoparticles (AgNP) dispersed within the matrix of gelatin- methacryloyl (GelMA) hydrogel. The nanocomposite hydrogel can be combined with bone graft material to form a bone graft construct that enhances osteointegration in bone regeneration applications.

In one aspect the invention provides a nanocomposite hydrogel comprising capped AgNP dispersed within the matrix of GelMA hydrogel wherein the hydrogel is prepared by photocrosslinking GelMA macromer with visible light in the presence of (a) capped AgNP and (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt. In one aspect the invention provides a GelMA hydrogel comprising (a) capped AgNP, (b) a ruthenium(II) compound and (c) a persulfate salt, wherein the capped AgNP are dispersed within the matrix of the GelMA hydrogel.

In one aspect the invention provides a composition comprising (a) GelMA hydrogel, (b) capped AgNP, (c) a ruthenium(II) compound and (d) a persulfate salt.

In another aspect the invention provides a process for preparing a AgNP/GelMA nanocomposite hydrogel comprising crosslinking GelMA macromer with visible light in the presence of (a) capped AgNP and (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt.

In another aspect the invention provides a bone graft construct that comprises granules of bovine bone graft material dispersed within the matrix of a GelMA hydrogel, wherein the hydrogel is prepared by photocrosslinking GelMA macromer with visible light in the presence of (a) capped AgNP, (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt and (c) granules of bovine bone graft material.

In another aspect the invention provides a bone graft construct comprising (a) GelMA hydrogel comprising (a) capped AgNP, (b) a ruthenium(II) compound, (c) a persulfate salt and (d) granules of bovine bone graft material, wherein the capped AgNP and bovine graft material are dispersed within the matrix of the GelMA hydrogel.

In another aspect the invention provides a process for preparing a bone graft construct comprising crosslinking GelMA macromer with visible light in the presence of (a) capped AgNP, (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt and (c) bone graft granules.

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

In one embodiment the persulfate salt is selected from sodium, ammonium and potassium persulfate. Preferably, the persulfate salt is sodium persulfate.

In another aspect the invention provides a method for treating of inflamed dental pulp comprising applying to the inflamed dental pulp, a nanocomposite hydrogel of the invention.

In another aspect the invention provides a method for treating infected dental root canals comprising applying to the infected dental root canals, a nanocomposite hydrogel of the invention.

In another aspect the invention provides a method for treating infected and inflamed intro-oral mucosal ulceration, the method comprising applying to the intro-oral mucosal ulceration, a nanocomposite hydrogel of the invention. In another aspect the invention provides a method for supporting bone and fibrous connective tissue healing in periodontal pockets, the method comprising applying to the periodontal pockets a nanocomposite hydrogel of the invention.

In another aspect the invention provides a method for supporting bone healing in peri-implant pockets, the method comprising applying to the peri-implant pockets a nanocomposite hydrogel of the invention.

In another aspect the invention provides a method for supporting soft tissue healing and providing antimicrobial action in damaged or diseased tissues, the method comprising applying to the tissue a nanocomposite hydrogel of the invention.

In another aspect the invention provides a method for supporting bone healing and providing antimicrobial action in damaged or diseased bone with or without the presence of a implanted device, the method comprising applying to bone a nanocomposite hydrogel of the invention.

In another aspect the invention provides a use of the bone graft construct of the invention for intra-oral bone grafting.

In one embodiment, the bone grafting is applied to tooth sockets after tooth extraction. In one embodiment, the bone grafting is of bone defects around dental implants. In one embodiment, the bone grafting is to develop new bone prior to implant treatment. In one embodiment, the bone grafting is of periodontal intra-bony defects.

In another aspect the invention provides a method for supporting bone healing and providing antimicrobial action in damaged or diseased bone with or without the presence of an implanted device, the method comprising applying to the bone, a bone graft construct of the invention.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein that have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

4. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and with reference to the drawings in which: Figure 1 shows a transmission electron microscope image of alpha-lipoic acid capped AgNP (A), and a histogram of the size distribution of the AgNP prepared in Example 1 (B).

Figure 2 is a set of photographs of the two hydrogel systems; (A) HyStem-C, (B) HyStem-C with AgNP, (C) GelMA-Ru/SPS, (D) GelMA-Ru/SPS with AgNP.

Figure 3 is a graph showing the metabolic activity of human gingival fibroblasts encapsulated with 200 pg/ml AgNP (toxic concentration), and controls for 48 h; mean ± standard deviation, **** < 0.0001 (A); and confocal images of the live/dead cells with percentage live calculations in (B) stiff HyStem-C and (C) 5% GelMA-Ru/SPS; live cells - blue, dead cells - red.

Figure 4 is a set of graphs showing (A) the metabolic activity of human gingival fibroblasts after 48 h in GelMA-Ru/SPS with AgNP, * P<0.0001; and (B) the release of AgNP at 48 h frodm GelMA-Ru/SPS with and without human gingival fibroblast incorporation; mean ± standard deviation.

Figure 5A, B, C is a set of scanning electron micrographs showing the areas where scanning electron microscopy with energy dispersive spectroscopy was conducted in background gel areas (B) and white areas which could be Ag ions (W); (IB and W) HyStem-C, (2B and W) HyStem-C-AgNP, (3B and W) GelMA-Ru/SPS, (4B and W) GelMA-Ru/SPS-AgNP, (5B and W) GelMA-LAP, (6B and W) GelMA-LAP-AgNP, (7B and W) GelMA-12959, (8B and W) GelMA-I2959-AgNP, (9B and W) PVA-LAP, (10B and W) PVA-LAP-AgNP, (11B and W) PVA-I2959, (12B and W) PVA-I2959-AgNP.

Figure 6 is the Fourier Transform Infrared - Attenuated Total Reflectance (FTIR-ATR) of spectra of HyStem-C (A) and GelMA-Ru/SPS (B) without and with 100 pg/ml AgNP.

Figure 7 shows scanning electron microscopy energy dispersive X-ray spectroscopy on GelMA-Ru/SPS-AgNP with white triangulated spots positive for silver ions.

Figure 8 shows TEM images of constructs without uranyl acetate and lead citrate staining of a GelMA-Ru/SPS construct (A) and a GelMA-Ru/SPS-AgNP construct crosslinked with visible light (B).

Figure 9 is a pair of graphs showing the bacterial viability by Prestoblue® at 24h of E. coli (A) and S. aureus (B) when different concentrations of AgNP were encapsulated within GelMA-Ru/SPS-AgNP constructs; mean ± standard deviation.

Figure 10 is a set of graphs and images of the inhibition zones of E. coli (A) and S. aureus (B) when different concentrations of AgNP were encapsulated within GelMA- Ru/SPS-AgNP constructs; mean ± standard deviation. Figure 11 shows the typical antibacterial clear zone of the GelMA-Ru/SPS-AgNP (100 pg/ml of AgNP) 18-month post fabrication on E. coli (a) and S. aureus (b).

Figure 12 are representative 3D-reconstructions of CT scans of sites at 4- and 16- week timepoints from rabbit cranial defects. Empty Defect, BioOss®; optimized bone (OB); AgNP functionalized optimized bone (AgNP-OB); AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB).

Figure 13 contains representative images of the graft material within the circular defect (6 mm circle) 4- and 16-weeks post implantation from rabbit cranial defects. No graft material (Empty defect); BioOss®; optimized bone (OB); AgNP functionalized optimized bone (AgNP-OB); AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB). Circle shows 6 mm defect.

Figure 14 comprises images of all mid-slice sites from the CT scans of defects at 4 weeks used for analysis from rabbit cranial defects. No graft material (Empty defect); BioOss®; optimized bone (OB); AgNP functionalized optimized bone (AgNP-OB); AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB).

Figure 15 comprises images of all mid-slice sites of the CT scans of defects at 16 weeks used for analysis from rabbit cranial defects. No graft material (Empty defect); BioOss®; optimized bone (OB); AgNP functionalized optimized bone (AgNP-OB); AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB).

Figure 16 is a pair of graphs showing the quantification of residual graft material (RG - circle), connective tissue (CT - square) and new bone (NB - triangle) within the circular defect (6 mm circle), mid location within the cranial bone from rabbit cranial defects. No graft material (Control); BioOss® (BO); optimized bone (OB); AgNP functionalized optimized bone (AgNP-OB); AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB); mean ± standard deviation; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001; Tukey's adjusted ANOVA - unpaired.

Figure 17 provides representative resin embedded images of the rabbit cranial defects at week 16 (Magnification xlO); (A) No graft material (Control), (B) BioOss®, (C) optimized bone, (D) AgNP functionalized optimized bone (AgNP-OB); (E) AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB); NB - new bone, RG - residual graft, CT - connective tissue. 5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Definitions and abbreviations

As used herein 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 "treating" and "treatment" as used herein refer to dealing with a disease or condition in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The terms "treating" and "treatment" also include "prophylaxis" unless otherwise indicated.

The term "gel" means a substantially dilute cross-linked system which exhibits reduced flow when in the steady state.

The term "hydrogel" means a gel comprising a network of polymer chains that are hydrophilic. Hydrogels are highly absorbent natural or synthetic polymeric networks.

The term "photoinitiating system" means a compound or combination of compounds that produces free radicals when exposed to light.

The term "AgNP" refers to silver nanoparticles, which are particles of silver between 1 nm and 100 nm in size. The term "capped AgNP" refers to silver nanoparticles bound to a capping agent.

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 nanocomposite hydrogel of the invention

The invention provides a bifunctional nanocomposite hydrogel with adventitious properties that make it highly suitable for tissue regeneration. The nanocomposite hydrogel provides an antimicrobial matrix that induces cellular regeneration while controlling bacterial loading. The nanocomposite hydrogel can also be combined with bone grafting granules of bovine bone graft material to produce an antimicrobial bone graft construct with excellent bone regeneration properties.

The nanocomposite hydrogel of the invention comprises capped AgNP dispersed within the matrix of GelMA hydrogel wherein the hydrogel is prepared by photocrosslinking GelMA macromer with visible light in the presence of (a) capped AgNP and (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt.

GelMA macromer is a photocrosslinkable version of gelatin (denatured collagen) with amine and carboxyl groups that are partially conjugated with methacrylate or methacrylamide groups. GelMA macromer has attracted the widespread interest of researchers because of its excellent biocompatibility, biodegradability, and moldability. GelMA macromer contains natural Arg-Gly-Asp (RGD) sequences, which can facilitate biological interaction between cells and scaffolds. Various structures have been constructed from GelMA macromer, including 3D scaffold, injectable gel, bio-printed scaffold, and electrospun fibrous membrane via precise fabrication methods such as light-induced crosslinking, extrusion 3D printing, electrospinning, or microfluidics.

A GelMA hydrogel can be prepared by cross-linking the GelMA macromer polymer chains dispersed in an aqueous medium. Cross-linking can be via numerous mechanisms including physical gelation, ionic interactions and chemical cross-linking. The latter is preferred because it is more precise and controllable, as well as irreversible.

In one aspect the invention provides a nanocomposite hydrogel comprising capped AgNP dispersed within the matrix of GelMA hydrogel wherein the hydrogel is prepared by photocrosslinking GelMA macromer with visible light in the presence of (a) capped AgNP and (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt. In another aspect the invention provides a GelMA hydrogel comprising (a) capped AgNP, (b) a ruthenium(II) compound and (c) a persulfate salt, wherein the capped AgNP are dispersed within the matrix of the GelMA hydrogel.

In another aspect the invention provides a process for preparing a AgNP/GelMA nanocomposite hydrogel comprising crosslinking GelMA macromer with visible light in the presence of (a) capped AgNP and (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt.

For the above aspects of the invention:

In one embodiment the concentration of GelMA in the hydrogel is about 2.5 to 10% wt/v, ideally 5% wt/v. In one embodiment the GelMA macromer has a degree of methacrylation of about 5-40%.

The AgNP used in present in the nanocomposite hydrogel of the invention are bound to a capping agent. Capping agents control the shape and size of the AgNP, preventing agglomeration usually by steric hindrance and/or electrostatic repulsion.

Capping agents such as alpha-lipoic acid covalently bind to the AgNP not only altering the size and physico-chemical properties, but also conferring functionality. Capping agents can include surfactants, small ligands, polymers, dendrimers, cyclodextrins, and polysaccharides.

In one embodiment the AgNP are those with a terminal carboxylic acid in the capping agent including but not limited to lipoic acid, oleic acid, citric acid and gallic acid.

In one embodiment the AgNP are those with a similar charge in the capping agent.

In one embodiment the capped AgNP are alpha-lipoic acid capped AgNP. Alpha-lipoic acid capped AgNP are known to be relatively small and have anti-oxidant and antiinflammatory functionality.

In one embodiment the concentration of capped AgNP is about 50 to about 100, 200, 300, 400 500 or 1000 pg/ml of GelMA hydrogel. Preferably the concentration of capped AgNP is about 50 to about 200 pg/ml.

In one embodiment the ratio of ruthenium(II) compound to persulfate salt is about 1: 10.

In one embodiment the concentration of ruthenium(II) compound is about 0.5 mM in the final GelMA hydrogel. In one embodiment the concentration of persulfate salt is about 5 mM in the final GelMA hydrogel.

The GelMA macromer is photocrosslinked with visible light using a standard techniques in the art. In one embodiment the GelMA macromer and photoinitiator system are exposed to visible light for about 2 to 15 minutes.

In one embodiment the visible light (wavelength 400-450 nm) has an intensity of about 10 to 50 mW/cm ² .

In one embodiment the ruthenium(II) compound is tris(2,2-bipyridyl)- dichlororuthenium(II) hexahydrate.

In one embodiment the capped AgNP have an average diameter of between about 1 to about 100 nm, preferably about 1 to about 50 nm, more preferably about 1 to about 15 nm and most preferably, about 1 to about 12 nm.

Alpha-lipoic acid capped silver nanoparticles with a diameter of between 1-12 nm can produced as per Example 1.

The resulting nanocomposite hydrogel demonstrates properties that make it highly suitable for tissue regeneration.

Examples 2 and 3 describe a comparison of the nanocomposite hydrogel of the invention GelMA-Ru/SPS-AgNP compared with HyStem-C loaded with alpha-lipoic acid capped AgNP (HyStem-C-AgNP). HyStem-C comprises thiol-modified hyaluronan (Glycosil), thiol-reactive crosslinker, PEGDA (Extralink) and thiol-modified denatured collagen (Gelin-S®).

Both nanocomposite hydrogels were prepared as set out in Example 2. As can be seen in Figure 2 the GelMA-Ru/SPS-AgNP (5% GelMA) gave an even incorporation of the silver nanoparticles while the HyStem-C-AgNP resulted in dark condensations within the gel. This suggests that the GelMA-Ru/SPS interacted with the AgNP to maintain an even distribution and retention of the nanoparticles in a way that was distinctly better than HyStem-C.

In one embodiment the capped AgNP are reasonably evenly distributed throughout the GelMA hydrogel matrix. A reasonably even distribution of capped AgNP can be assumed if the capped AgNP cannot be detected visually, such as in Figure 2D. Where clumping can be seen, such as in Figure 2B, the capped AgNP are not reasonably evenly distributed. Retention of the AgNP within the gel was tested in Example 3 using primary human gingival fibroblasts; with the AgNP at a toxic concentration of 200 pg/ml. Good viability was seen with cells in GelMA-Ru/SPS alone, however addition of AgNP resulted in cell death suggesting high retention of the Ag ions. Cell survival in HyStem-C was not affected by the addition of AgNP suggesting the Ag ions are rapidly lost from the construct. Examination of low AgNP concentrations, down to 0.5 pg/ml, in the GelMA-Ru/SPS-AgNP constructs, was found to retain the AgNP activity (Figure 4). The release profile of AgNP from the GelMA-Ru/SPS-AgNP was markedly influenced by the presence of cells possibly indicating that cellular protein may also bind the AgNP and allow sustained release around the construct. The release of < 60% when examining 100 pg/ml AgNP in GelMA-Ru/SPS over 48 h suggests that the GelMA- Ru/SPS-AgNP would provide good antimicrobial protection over the initial healing period when there is a higher risk of microbial ingress. The nanocomposite hydrogel of the invention would also retain longer term antimicrobial activity for additional protection of the regenerative tissues.

The nanocomposite hydrogel of the invention was compared against a number of hydrogel systems in Example 4. These systems included HyStem-C (extra-link crosslinking). GelMA with Ru/SPS or 0.05% LAP (Lithium phenyl-2,4,6- trimethylbenzoylphosphinate) where crosslinking was with visible light (400-450nm) or 0.05% 12959 (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) with ultraviolet crosslinking. PVA (polyvinyl alcohol-methacrylate) was also investigated with 0.05% LAP with visible light crosslinking or 0.05% 12959 with ultraviolet light crosslinking.

Surprisingly only GelMA-Ru/SPS-AgNP gels with light crosslinking were able to bind the AgNP and hold them as visible nanoparticles (Figures 5 and 7). Scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) showed that all the other gels with AgNP had Ag ions dispersed throughout the matrix while only GelMA- Ru/SPS-AgNP gels had visible discrete AgNP with other areas being negative for Ag ions (Figure 5, Table 1). This suggests the GelMA-Ru/SPS-AgNP hydrogel is surprisingly unique in its ability to retain AgNP compared to other crosslinking and hydrogel systems investigated.

To investigate if bond changes occurred with GelMA-Ru/SPS with and without AgNP and for HyStem-C with and without AgNP, the inventors used Fourier Transform Infrared-Attenuated Total Reflection (FTIR-ATR). With HyStem-C no new peaks or significant shifts in wave numbers were observed with the addition of AgNP thus suggesting there may be no active binding of AgNP to the HyStem-C components. Addition of AgNP to GelMA-Ru/SPS however resulted in the shifting of peaks around the 3273 cm ^-1 region (corresponding to the -OH groups), in addition to amide II and amide I shifting to lower wave lengths of -1529 cm ^-1 , 1629 cm ^-1 , respectively. This indicates that the AgNP are actively interacting with the GelMA-Ru/SPS system which then supports the finding of the uniqueness of the GelMA-Ru/SPS-AgNP hydrogel (Example 5, Figure 6).

This finding of unique retention of AgNP in GelMA-Ru/SPS was further investigated with images from SEM-EDS and TEM. In both cases discrete particles of approximately 10 nm were visible (Example 6, Figures 7 and 8). The findings of the physical characterization of GelMA-Ru/SPS-AgNP thus supports this nanocomposite hydrogel being unique in its ability to bind and retain AgNP.

In Example 7 the antimicrobial activity of f. coli and S. aureus encapsulated in 10 pl of 5% GelMA-Ru/SPS with different concentrations of AgNP was investigated. At AgNP concentrations of > 5 pg/ml significant cytotoxicity was observed with broth cultures indicating complete killing of the bacteria (Figure 9). This defined the fate of bacteria within the GelMA-Ru/SPS-AgNP hydrogels. To investigate the diffusion of AgNP to control E. coli and S. aureus around the gels, bacterial inhibitory zones were quantified. AgNP in GelMA-Ru/SPS-AgNP produced a zone of inhibition at 100 pg/ml or greater (Figure 10).

Interestingly one of the problems with AgNP can be their drop in effectiveness over time with a typical shelf life of 4-6 weeks. GelMA-Ru/SPS-AgNP constructs were made, freeze-dried and then tested against E. coli and S. aureus 18 months later. An antimicrobial clearance zone was clearly evident (Figure 11). The hydrogel of the invention thus clearly has a good shelf life with AgNP incorporation recommended at 100 pg/ml to obtain both intra and external antimicrobial activity. This surprising result of the AgNP antimicrobial activity over long periods of time is another strong attribute supporting the uniqueness of the nanocomposite hydrogels of the invention.

The hydrogel of the invention can be used for external and internal tissue repair and regeneration with the advantage of non-antibiotic antimicrobial activity. In addition, it provides an antimicrobial formulation for implants and other medical devices. The hydrogel of the invention can be used as in a freeze dried, mouldable, trimmable construct either reconstructed or dry; in addition it can be used as an emulsion, gel, spray or ointment and photocrosslinked in situ. Other excipients include, without intended limitation, fillers, binders, lubricants, emulsifiers, wetting agents, buffers, preservatives, antimicrobials and/or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantage for pharmaceutical use.

The nanocomposite hydrogels of the invention can also be combined with bone graft material to make a highly effective bone graft construct. The bone graft material adds a further osteoconductive scaffold for bone regeneration within the nanocomposite hydrogel while retaining the advantageous antimicrobial properties associated with the GelMA-Ru/SPS-AgNP hydrogel. Bone particles can be made from cattle, pigs and horse and can include cortical (compact bone of in the external layers of bones) or cancellous bone (spongy bone with trabecular rods and plates surrounded by spaces). Particle sizes typically range from 0.5 mm to 2 mm; smaller sizes are preferred for periodontal, peri-implant and socket grafting; larger sizes are preferred for maxillofacial and orthopaedic applications.

In another aspect the invention provides a bone graft construct that comprises granules of bovine bone graft material dispersed within the matrix of a GelMA hydrogel, wherein the hydrogel is prepared by photocrosslinking GelMA macromer with visible light in the presence of (a) capped AgNP, (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt and (c) granules of bovine bone graft material.

In another aspect the invention provides a bone graft construct comprising (a) GelMA hydrogel comprising (a) capped AgNP, (b) a ruthenium(II) compound, (c) a persulfate salt and (d) granules of bovine bone graft material, wherein the capped AgNP and bovine graft material are dispersed within the matrix of the GelMA hydrogel.

In another aspect the invention provides a process for preparing a bone graft construct comprising crosslinking GelMA macromer with visible light in the presence of (a) capped AgNP, (b) a photoinitiating system comprising a ruthenium(II) compound and a persulfate salt and (c) bone graft granules.

The embodiments set out above also apply to the bone graft aspects of the invention.

To prepare the bone graft construct, GelMA macromer is crosslinked with capped AgNP using a visible light photoinitiating system comprising a ruthenium(II) compound and a persulfate salt in the presence of bone graft granules.

In one embodiment the bone graft granules comprise about 5 to about 80 wt% of the bone graft construct, preferably about 10 to about 60 wt%, more preferably about 20 wt%.

The bone graft granules may be distributed relatively evenly throughout the GelMA hydrogel matrix. Alternatively, an amount of AgNP/GelMA-Ru/SPS hydrogel could be produced first as a base, onto which a mixture of GelMA macromer, AgNP and bone graft granules is positioned and then crosslinked using the photoinitiating system.

In some applications it may be advantageous to include the bone graft granules in only a portion of the construct, for example, in the portion that will be next to the bone but not in the epithelial facing portion.

In Example 8, an in vivo rabbit cranial defect model was conducted with GelMA- Ru/SPS-AgNP containing AgNP at a concentration of 100 pg per ml of hydrogel and the addition of previously produced bone graft granules 0.5 mm - 1.2 mm, with larger or smaller granules possible (Example 8). The AgNP-GelMA-OB construct had considerably less graft material than other test constructs (Figure 12). AgNP-GelMA-OB supported the regeneration of native bone/regenerative bone with significantly less of the defect containing xenografting material compare to the BioOss®, optimized bone (OB), or the AgNP functionalized optimized bone (AgNP-OB).

The GelMA-Ru/SPS-AgNP hydrogel of the invention provided a matrix for regeneration of native/natural bone rather than the graft material being entrapped in the bone with limited resorption. BioOss® sites at 16 weeks had significantly more graft material. The presence of BioOss® years after placement of the graft means that the patient has xenograft present that may trigger an immune response. This is greatly reduced in the AgNP-GelMA-OB graft site (Figure 16).

The appearance of the wound site containing AgNP-GelMA-OB was different from other constructs and it is unclear if the darker colour is due to more blood vessels or more connective tissue. It is unlikely due to the presence of AgNP as the OB group was also dark in appearance (Figure 13). There is no evidence from the qualitative or quantitative analysis that the present of AgNP adversely affected healing (Figures 14- 17). This is a critical finding as others have suggested that the presence of Ag can adversely affect tissue healing. The amount of new bone in the AgNP-GelMA-OB group was similar to the other test groups and significantly greater than empty control sites (Figure 16). It would also be expected that the connective tissue areas would transform to bone, with time, perhaps providing an advantage over the other test materials.

6. EXAMPLES

Example 1: Production of alpha-lipoic capped silver nanoparticles

Methods: Alpha-lipoic acid capped AgNP were prepared by the following process. To prepare the microemulsions (pEms), 40 ml of an AOT (docusate sodium 5 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. ICN 10289425, 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 AgNP. This mixture was stirred 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, centrifugated 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 with anhydrous ammonia. The resulting yellow- brown colloidal suspension was centrifuged twice at 16,000 g for 45 min, with the final supernatant collected and retained for characterization (Cotton et al., 2019).

The AgNP concentration was quantified using inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7500ce instrument, Agilent Technologies; California, USA), by adding 4 ml of concentrated HNO3 in a Teflon digestion vessel (Cat. No. 010- 500-264, SPS Science, Quebec, Canada), followed by gentle heating. The samples were digested at 95°C for 1 h. During the digestion process, the sample's volume was reduced to ~0.2 ml, which was made up again to 3 ml using deionized water. Transmission electron microscopy (TEM) images were obtained using a Philips CM100 BioTWIN TEM (Philips/FEI Corporation; Eindhoven, Holland) equipped with a LaB6 emitter fitted with a MegaView III Olympus digital camera. The samples were prepared by depositing 10 pl of alpha-lipoic acid capped AgNP onto plasma glowed, carbon-coated (400 mesh) copper grids. After 60 s, the excess was blotted with filter paper and the sample was left to air dry before viewing. Particle size diameter was measured manually for 100 particles using ImageJ software, U. S. National Institutes of Health, Bethesda, Maryland, USA.

Results: After synthesis of the colloidal suspension of AgNP their appearance was dark yellow/brown, which indicated stability, as well as good dispersion of the nanoparticles. The hydrodynamic size of AgNP batches used in these experiments were determined to be 1-12 nm in size with a concentration ranging between 1800 to 2900 pg/ml and were used within 28 days of being produced. TEM images showed spherical particles with an average diameter of 6 nm (Figure 1). Example 2: Encapsulation of AgNP into a HyStem-C hydrogel compared with a GelMA-Ru/SPS hydrogel

Methods: HyStem-C-AgNP reconstitution and sample preparation

HyStem-C (Cat. No. HYSC020-1KT, Sigma-Aldrich, Auckland, NZ), was used as per the manufacturer instructions. Briefly, the hydrogel components were brought to room temperature. Gylcosil and Gelin were reconstituted with 1 ml of degassed water (DG- H2O). HyStem-C was prepared by reconstituting the 0.50 ml of Extralink with 0.25 ml DG-H2O and mixed by gently inverting several times to give a 'stiff' gel, which had previously been determined to give better retention of AgNP. Gelin and Glycosil were incubated for 30 mins at 37°C, 5% CO2, then placed horizontally on a shaker for 2 h at 60 cycles per min, until fully dissolved. Within 2 h of reconstituting the solutions with the degassed water, Glycosil and Gelin were mixed in a ratio of 1: 1, and slowly pipetted up and down to avoid aeration. The Glycosil/Gelin mix was placed on a shaker for further 30 mins. The cross-linker was then added and the constructs pipetted directly in a well-plate and left undisturbed for 20 min to allow for gelation. AgNP were added at a concentration of 0.1 to 200 pg/ml, or control carrier deionized water pH 10, to maintain a constant final volume.

GelMA-Ru/SPS-AgNP preparation and sample preparation

GelMA macromer was synthesized as previously described by Lim et al (Lim et al., 2019). Briefly, gelatin (porcine skin, type A, 300 g Bloom strength, Cat. No. G1890, Sigma Aldrich, USA) was dissolved in Phosphate Buffered Saline (PBS) at 10 wt%, with 0.6 g of methacrylic anhydride (Cat. No. 276685, Sigma Aldrich, USA) per gram of gelatin added to the solution and left to react for 1 h at 50°C under constant stirring. The obtained solution was then filtered through a 0.22 pm sterile filter, lyophilized under sterile conditions and stored until used. Prior to use, a 10% stock solution was prepared by dissolving the lyophilized GelMA-Ru/SPS in PBS and incubating at 37°C overnight.

Different final GelMA macromer concentrations of 2.5%, 5% and 10% were investigated. Stock solutions of 50 mM Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate ruthenium (Ru; Cat. No. 544981, Sigma-Aldrich, Auckland, NZ) and 500 mM sodium persulphate (SPS; Cat. No. 216232, Sigma-Aldrich, Auckland) were made in PBS. All solutions were sterilized with 0.22 pm filter prior to use. Nanocomposite hydrogels were prepared by adding 50% of GelMA stock solution, 48% PBS, 1% of 50 mM Ru and 1% of 500 mM SPS. Final GelMA-Ru/SPS constructs comprised 5% wt/v GelMA with 0.5 mM Ru and 5 mM SPS. Within the GelMA- Ru/SPS-AgNP constructs, the volume of AgNP solution to be used was deducted from the total amount of PBS added. Constructs were pipetted into well-plates and light crosslinked for 3 mins using visible light (Wavelength = 400-450 nm) at a distance of 30 mW/cm ² .

Results: Encapsulation of AgNP in gel constructs

At the macroscopic level, aggregates of AgNP were immediately observed upon addition to the hydrogel components of HyStem-C (Figure 2 A, B), unlike GelMA- Ru/SPS where the AgNP appeared evenly dispersed (Figure 2 C, D). As soon as the AgNP were added to the GelMA- Ru/SPS matrix, the original yellow matrix changed to brown and then after cross linking with light for 3 mins reversed again to yellow, which might indicate a chemical interaction.

Example 3: Retention of AgNP within hydrogel constructs as measured by primary human gingival toxicity and the AgNP release profile

Methods: Cell encapsulation in HyStem-C and GelMA-Ru/SPS constructs

Primary human gingival fibroblasts (HGF) were obtained with consent from a female patient undergoing routine crown-lengthening surgery at the Faculty of Dentistry, University of Otago. All experiments were performed in accordance with the guidelines of the National Ethics Advisory Committee, New Zealand, and approved by the Human Ethics Committee, University of Otago, reference number H17/112. The HGFs were grown in Dulbecco's Modified Eagle Medium (DMEM; Cat. No. 10569010, Life Technologies New Zealand limited, Auckland, NZ) supplemented with 10% Fetal Bovine Serum (FBS; Cat. No. 10091148, Life Technologies New Zealand Limited, Auckland, NZ), 1% Antibiotic-Antimycotic (100X) (Anti-Anti; Cat. No. 15240062, Life Technologies New Zealand Limited, Auckland, NZ), 0.5% Gentamicin (Cat. No. 15710064, Life Technologies New Zealand Limited, Auckland, NZ). Cells were used at 5 million cells/ml of hydrogel. Encapsulation of HGF cells within the HyStem-C constructs were conducted by resuspending the cell pellet at a ratio of 4: 1 Glycosil/Gelin mix to Extralink (cross-linker). Constructs of 10 pl were pipetted into a 96 well-plate and incubated at 37°C, 5% CO2 for 20 mins to allow for gelation before addition of the culture media. After gelation, 400 pl of media was gently added at the side of the well to avoid disturbing the construct (n=3). 5% GelMA with HGF constructs were produced by resuspending the cell pellet in GelMA/PBS and mixing gently, followed by the addition of the crosslinkers Ru and SPS. Constructs of 10 pl in size were pipetted into a 96 well plate and light crosslinked for 3 mins using visible light (Wavelength = 400-450 nm) at 30 mW/cm ² (n=3). After cross-linking, 400 pl of media was added gently at the side of the well and the constructs incubated at 37°C, 5% CO2. Three cell-free controls of each condition were also prepared. AgNP were added as described in Examples 1 and 2. Metabolic activity of human gingival fibroblast cells within the gel constructs with AgNP

The retention of AgNP within the hydrogels and their effects on the metabolic activity of the encapsulated HGFs in HyStem-C and 5% GelMA-Ru/SPS was determined. AgNP were incorporated at a toxic dose of 200 pg/ml with 5 million HGF cells/ml within 10 pl constructs. The samples were incubated at 37°C, 5% CO2, for 44 h then 10% Prestoblue® cell viability regent (Cat. No. G8080, Promega, In Vitro Technologies, USA) added to each well (n=3). The well-plates were incubated again at 37°C, 5% CO2 and fluorescent measurements recorded at the 48 h time point. Metabolic activity was measured by transferring 50 pl of solution, from each well plate to a 96 well-plate and fluorescence measurement undertaken using a Synergy 2 Multi-mode microplate reader with an excitation/emission of 535/615 nm.

Cell viability with AgNP as assessed by live/dead staining

Following the metabolic activity experiments, the same constructs were labelled using a cell viability imaging kit (Cat. No. R37610, Life Technologies, New Zealand limited, Auckland, NZ). Constructs were washed with PBS (x3), and 15 pl of both NucBlue® and propidium iodide added to each well-plate in 400 pl of fresh DMEM/10% FBS. The well-plate was incubated for 45 mins and then washed (x3) with PBS, prior to being observed under inverted confocal microscopy. Representative images were used for counting live/dead cells.

The effect of AgNP on human gingival fibroblast cells in GelMA-Ru/SPS-AgNP constructs

Only in GelMA-Ru/SPS-AgNP did entrapped AgNP cause cell death at 200 pg/ml. To determine the cytotoxicity of the 5% GelMA-Ru/SPS-AgNP constructs (10 pl) were prepared with and without 5 million HGF cells/ml. AgNP were incorporated at 0, 0.1, 0.5, 1, 5, 10, 50, and 100 pg/ml (n=3) and metabolic activity measured at 48 h with Prestoblue®.

Silver nanoparticle release from GelMA-Ru/SPS

ICP-MS was used to determine the concentration of AgNP released from the GelMA- Ru/SPS-AgNP constructs after 48 h of in vitro incubation in 400 pl of DMEM/10% FBS. Constructs of GelMA-Ru/SPS (10 wt%) at 10 pl in size were prepared with and without 5 million HGF cells/ml. AgNP were incorporated at the following concentrations 0, 0.1, 0.5, 1, 5 ,10, 12.5, 25, 50, 100 and 200 pg/ml (n=3). After 48h incubation at 37°C, 5% CO2, 250 pl of the media supernatant was transferred to a Teflon digestion vessel and ICP-MS conducted. A standard curve was generated by preparing media containing AgNP at 0, 0.0002, 0.002, 0.02, 0.2 and 2 pg/ml. Results: Metabolic activity and live/dead staining of HGFs in gel constructs with AgNP

The 5% GelMA-Ru/SPS-AgNP constructs (10 pl) were used as the preferred model due to cell compatibility, even AgNP distribution, and suitable handling properties. There was a correlation between the cytotoxicity of the AgNP and their distribution. Even distribution of AgNP in GelMA-Ru/SPS-AgNP (Figure 2D) induced a significant decrease in cell viability (Figure 3A), meanwhile AgNP aggregates in HyStem-C-AgNP did not result in cytotoxicity at the same toxic concentration of 200 pg/ml (Figure 3A). This suggested that the AgNP in HyStem-C-AgNP are less active than when retained in GelMA-Ru/SPS.

Live/dead staining, imaged with confocal microscopy, and quantified for the percentage of live cells in ImageJ confirmed 51.2% of HGFs were alive in HyStem-C- AgNP while only 3.7% of HGFs were alive in GelMA-Ru/SPS-AgNP constructs at 48 h with 200 pg/ml AgNP.

Metabolic activity of HGFs and release of AgNP from GelMA-Ru/SPS-AgNP Concentrations > 0.5 pg/ml of AgNP in GelMA-Ru/SPS-AgNP constructs encapsulated with HGFs showed significant cytotoxicity, with little to no metabolic activity recorded (Figure 4A). Only in 0.1 pg/ml AgNP did cells maintain their metabolic activity at levels comparable to the positive control.

Increasing AgNP release of 20 - 60 % w/w of AgNP was observed from 5% GelMA- Ru/SPS-AgNP (10 pl) constructs without cells, containing 0.1 - 1 pg/ml AgNP (Figure 4B). However, GelMA-Ru/SPS-AgNP constructs containing AgNP > 1 pg/ml demonstrated a plateaued AgNP release of ~50 % w/w, suggesting a maximum AgNP concentration within the hydrogel and constant release upon incorporation of increasing concentrations. Constructs containing HGFs had a consistently lower percentage release compared to the AgNP-free constructs.

Example 4: Unique distribution of AgNP in GelMA compared to other hydrogels

Method: Scanning electron microscopy with energy dispersive spectroscopy (SEM- EDS) was used to assess the distribution of silver in constructs and visualize whether different gels retained the silver as nanoparticles. Samples were prepared and stored overnight at - 80°C before freeze drying for 24 h. SEM-EDS analysis of carbon coated constructs (1-12 below) was performed using a JEOL JEM-7500F (Tokyo, Japan). EDS spectra were obtained from ten randomly chosen areas per sample. Five areas were selected from the electrodense (white) areas representing smaller white areas of either small NaCI crystals or AgNP, and five were selected from the grey/black areas representing the matrix.

The constructs were:

1. HyStem-C

2. HyStem-C-AgNP

To make HyStem-C the hydrogel components were brought to room temperature. Gylcosil and Gelin were both reconstituted with 1 ml of degassed water (DG-H2O). The stiff hydrogel consistency was prepared by reconstituting the Extralink with 0.25ml of DG-H2O. Gelin and Glycosil were incubated for 30 mins at 37°C, 5%CO2, then placed horizontally on a shaker for 2 h (60 cycles per min), until fully dissolved. Within 2 h of reconstituting the solutions with the DG-H2O, Glycosil and Gelin were mixed in a ratio of 1: 1, and slowly pipetted up and down to avoid aeration. The Glycosil/Gelin mix was placed on a shaker for further 30 mins. Extra-link was then added, and the constructs were pipetted into 6 mm silicone molds. The silicone mold was stored at -80°C overnight before freeze drying for 24 h.

To make the HyStem-C-AgNP constructs the AgNP were added at a concentration of 100 pg per ml of HyStem-C.

3. (Visible light) GelMA-Ru/SPS

4. (Visible light) GelMA-Ru/SPS-AgNP

GelMA-Ru/SPS constructs were prepared by adding 50% of GelMA stock solution (10% wt/v), 48% PBS, 1% Ruthenium, and 1% SPS. Ru (MW= 748.63 g/mol) at a concentration of 50 mM was prepared by dissolving 10 mg of Ru in 267 pl of PBS. SPS (MW=238.1 g/mol) at a concentration of 500 mM was prepared by dissolve 100 mg of SPS in 840 pl of PBS. Final GelMA-Ru/SPS constructs comprised of 5% w/v GelMA with 0.5 mM Ru and 5 mM SPS. All solutions were sterilized with 0.22 pm filter prior to use.

Constructs of 85 pl in size were pipetted into 6 mm silicone molds and light crosslinked for 3 mins using visible light (400-450nm) at 30 mW/cm ² . The silicone molds were stored at -80°C overnight before freeze drying for 24 h.

To make the GelMA-Ru/SPS-AgNP constructs, the amount of AgNP at 100 pg per ml of GelMA was calculated and deducted from the total amount of PBS added. 5. (Visible light) GelMA-LAP

6. (Visible light) GelMA-LAP-AgNP

GelMA-LAP constructs were prepared by adding 50% of GelMA stock solution (10%wt), 45% PBS, 5% LAP. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, MW = 294.1 g/mol) at a final concentration of 1% wt/vol was prepared by dissolving 1 mg of LAP in 100 pl of PBS. All solutions were sterilized with 0.22 pm filter prior to use.

Constructs of 85 pl in size were pipetted into 6 mm silicone molds and light crosslinked for 3 mins using visible light (400-450nm) at an intensity of 30 mW/cm ² . The silicone molds were stored at -80°C overnight before freeze drying for 24 h.

To make the GelMA-LAP-AgNP constructs, the amount of AgNP at 100 pg per ml of GelMA was calculated and deducted from the total amount of PBS added.

7. (UV) GelMA-12959

8. (UV) GelMA-I2959-AgNP

GelMA-12959 constructs were prepared by adding 50% of GelMA stock solution (10%wt), 45% PBS, 5% 12959. 2-Hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone (12959, MW = 224.3 g/mol) at a final concentration of 1% wt/vol was prepared by dissolving 1 mg of 12959 in 100 pl of PBS. All solutions were sterilized with 0.22 pm filter prior to use.

Constructs of 85 pl in size were pipetted into 6 mm silicone molds and crosslinked for 3 mins using ultraviolet light at an intensity of 30 mW/cm ² . The silicone molds were then stored at -80°C overnight before freeze drying for 24 h.

To make the GelMA-I2959-AgNP constructs, the amount of AgNP at 100 pg per ml of GelMA was calculated and deducted from the total amount of PBS added.

9. (Visible light) PVA-LAP

10. (Visible light) PVA-LAP-AgNP

PVA-LAP constructs were prepared by adding 50% of PVA-MA stock solution (20%wt), 45% PBS, 5% LAP. PVA-MA stock solution (20wt%) was prepared by dissolving 0.2g of PVA-MA in 0.8 ml of PBS at 60°C for 30 min. Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP, MW = 294.1 g/mol) at a final concentration of 1% wt/vol was prepared by dissolving 1 mg of LAP in 100 pl of PBS. All solutions were sterilized with 0.22 pm filter prior to use.

Constructs of 85 pl in size were pipetted into 6 mm silicone moulds and light crosslinked for 3 mins using visible light (400-450nm) at an intensity of 30 mW/cm ² . The silicone moulds were then stored at -80 freezer overnight before freeze drying for 24 h.

To make the PVA-LAP-AgNP constructs, the amount of AgNP at 100 pg per ml of PVA- MA was calculated and deducted from the total amount of PBS added.

11. (UV) Polyvinyl alcohol (PVA)-I2959

12. (UV) PVA-I2959-AgNP

PVA-I2959 constructs were prepared by adding 50% of PVA-MA (polyvinyl alcoholmethacrylic acid) stock solution (20%wt), 45% PBS, 5% 12959. PVA-MA stock solution (20wt%) was prepared by dissolving 0.2g of PVA-MA in 0.8 ml of PBS at 60°C for 30 min. 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (12959, MW = 224.3 g/mol) at a final concentration of 1% wt/vol was prepared by dissolving 1 mg of 12959 in 100 pl of PBS. All solutions were sterilized with 0.22 pm filter prior to use.

Constructs of 85 pl in size were pipetted into 6 mm silicone molds and light crosslinked for 3 mins using ultraviolet light (365 nm) at an intensity of 30 mW/cm ² . The gels within the silicone molds were stored at -80°C overnight before freeze drying for 24 h.

To make the PVA-I2959-AgNP constructs, the amount of AgNP at 100 pg per ml of PVA-MA was calculated and deducted from the total amount of PBS added.

Results: The selected areas for SEM-EDS collection are shown in shown in Figure 5 and a summation of the results in Table 1. It was expected that small white areas might contain AgNP. Larger white areas were rich in Na+ and Cl- consistent with being salt crystals. Only in GelMA-Ru/SPS-AgNP (Figure 5, Sample 4W) were small white spots of approximately 10 nm in size detected consistent with the presence of AgNP. They appeared as three triangulated spots which contained Ag ions by SEM- EDS (Table 1). All other black areas examined by SEM-EDS in the GelMA-Ru/SPS- AgNP constructs were negative for Ag-i- suggesting the retention of Ag-i- within the localized spots (Figure 5, Sample 4; Table 1). In all other constructs generated with AgNP (Table 1; Samples 2, 6, 8, 10, 12) no structures consistent with being AgNP were detected. In addition, in all other gels the Ag-i- were evident in all tested areas suggesting and the presence of disperse silver ions and the loss of nanoparticle structure which allows retention of antimicrobial activity within the gel (Table 1). Table 1: Summary of the scanning electron microscopy with energy dispersive spectroscopy showing which gels were positive (+) and negative (-) for silver ions

*boxed area shows absence of silver ions in gel regions of constructs.

Example 5: Bond change with addition of AgNP to GelMA-Ru/SPS but not HyStem-C

Method: Fourier transform Infrared-attenuated total reflection (FTIR-ATR)

HyStem-C and GelMA-Ru/SPS constructs without and with 100 pg/ml AgNP were prepared as previously outlined in Example 4 and stored overnight at -80°C before freeze drying for 24 h. Spectra of the constructs were obtained on an ATR-FTIR spectrometer (Alpha II, Bruker, Germany). For each condition (n=2), 100 scans were acquired in the 4000-400 cm ^-1 range, with a resolution of 4 cm ^-1 .

Results: FTIR-ATR spectra of the HyStem-C and GelMA-Ru/SPS with and without AgNP are presented in Figure 6. The HyStem-C spectra revealed typical stretching peaks of OH- groups appearing at 3315 cm ^-1 . Additionally, typical absorption peaks of amides appeared at 1646 cm-1 (amide I), 1548 cm-1 (amide II) and 1241 cm-1 (amide III) which are the characteristic absorption peaks of C=O, N-H, and C-N, respectively (Garside, Lahlil, & Wyeth, 2005). The peak appearing at 1409 cm-1 is attributed to C=O stretching of the carboxyl groups within the HA (Zhang et al., 2018). Other significant peaks at 1105 cm-1, 1082 cm-1 and 1039 cm-1 are assigned to C-0 ester association, C-C groups, C-OH groups, respectively (Pan, Pereira, da Silva, Vasconcelos, & Celligoi, 2017). No new peaks or significant shifts in wave numbers were observed with addition of AgNP to the HyStem-C.

Spectra derived from GelMA-Ru/SPS showed a broad peak at 3289 cm-1 attributed to the stretching of the hydrogen bonded hydroxyl groups. The peaks at 1237 cm- ¹ , 1537 cm- ¹ and 1630 cm- ¹ are associated with the C-N stretching of amide III, N-H stretch of the amide II, and C=O stretching of amide I, respectively. The spectrum also displayed the characteristic bands of N-H stretching at 3078 cm-1 (amide B), C-H stretching at 2937 cm ^-1 , and 2878 cm ^-1 (amide A), and C-H deformation were also detected at 1444 cm ^-1 . Other relevant peaks were identified at: 1161 cm ^-1 (symmetric C-O-C stretching), 1199 cm- ¹ (v (O-C-O), and 1336 cm- ¹ (CH2). The spectra of the GelMA-Ru/SPS-AgNP did not show any new peaks corresponding to the AgNP, but it presents a shift of certain peaks, especially around the 3273 cm ^-1 region (corresponding to the -OH groups); in addition amide II and amide I also shifted to lower wave lengths of -1529 cm ^-1 , 1629 cm ^-1 , respectively. This finding indicated bond changes when AgNP were added to GelMA-Ru/SPS not seen with the addition of AgNP to HyStem-C.

Example 6: Visualization of intact lipoic capped silver in GelMA

Method: Carbon coating of the GelMA-Ru/SPS-AgNP constructs and SEM-EDS imaging methodology is given in Example 4. TEM imaging of the GelMA-Ru/SPS and GelMA- Ru/SPS-AgNP was performed on 10 pl constructs containing 0 or 200 pg/ml AgNP. Prior to imaging, the media was removed from the wells and replaced with a 100 pl of blue food dye to ensure the gels were visible during processing. After 20 min, the food dye was removed, and the constructs were washed with PBS and then fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 1 h. Constructs were then washed twice for 5 min in 0.1 M sodium cacodylate followed by a third wash in double distilled water for 5 min. The gels were carefully removed from the wells and transferred to 1 ml of distilled water. Water was removed with a plastic transfer pipette, with a 150-mesh copper grid on the tip, to aid in retention of the specimens. The specimens were exposed to serial alcohol dehydration using ethanol at 70%, 80%, 95%, and 100%, 5 mins each and then infiltrated with a 50:50 mixture of low viscosity Spurr's resin and 100% ethanol for 30 min. This was followed by a second infiltration with 100% Spurr's resin for 1 h. The old resin was then replaced with fresh resin and allowed to infiltrate for another 30 min. Finally, the constructs were removed and placed into silicone molds, which were filled with resin, covered and cured for 48 h at 60°C. The cured resin blocks were removed from the silicone molds and semi-thin sections (3 pm thick) were prepared. The sections were stained with methyl blue to verify the presence of gel before an ultrathin section of 80 nm were prepared. The sections were mounted onto formvar-coated copper slot grids and viewed under JEOL JEM-2200FS TEM.

Results: Carbon coating of GelMA-Ru/SPS-AgNP constructs and imaging with SEM- EDS clearly showed Ag+ positive triangulated particles approximately 10 nm in diameter consistent with being AgNP (Figure 7), which were not evident in any other gels examined (Figure 5). TEM imaging of GelMA-Ru/SPS-AgNP constructs revealed similar nanoparticle structures (Figure 8B) not present in the GelMA-Ru/SPS gels without AgNP (Figure 8A). The structures were of a size consistent with retention of AgNP structure (Figure 8B) within the GelMA-Ru/SPS-AgNP gels.

Example 7: Antibacterial properties of GelMA-Ru/SPS-AgNP constructs

Methods: Bacterial culture

Escherichia coli DH5o (5. coli), and Staphylococcus aureus Oxford NCTC6571 (S. aureus) were obtained from the University of Otago culture collection, Dental School and sub cultured on Tryptic Soy agar plates (Cat. No. 1335, Fort Richard, Mt. Wellington, New Zealand) with incubation at 37°C/5% CO2 for 24 h. Sixteen hours prior to experiments, the species were inoculated into 10 ml of tryptic soy broth (TSB; Cat. No. 211825, Difco Laboratories, Detroit, MI, USA) and incubated at 37°C/5% CO2.

Bacterial encapsulation in GelMA-Ru/SPS and GelMA-Ru/SPS-AgNP and antibacterial effects

Prior to encapsulation of E. coli and S. aureus into GelMA, a bacterial optical density (OD) of 0.01 per ml of hydrogel was calculated. The amount of broth was subtracted from the total volume of PBS used in the GelMA construct. 10 pl constructs were then crosslinked at 450 nm for 3 min in a 48 well-plate and incubated at 37°C/5% CO2 for 20 h as previously described in Example 4.

GelMA-Ru/SPS-AgNP constructs with either E. coli and S. aureus were encapsulated (10 pl) with 0, 0.1, 0.5, 1, 5 ,10, 50, or 100 pg/ml of AgNP (n=3) and no bacterium as a GelMA-Ru/SPS control. The samples were pipetted into 48 well-plate, placed in a sealable plastic bag to maintain humidity, and incubated at 37°C/5% CO2. After 21h of incubation, 400 pl of broth with 40 pl of the Prestoblue® regent were added and the well-plates were incubated for another 3 h before recording at the 24 h time point.

Metabolic activity was measured by transferring 50 pl of solution, from each well plate to a 96 well-plate and fluorescence measurement undertaken using a Synergy 2 Multimode microplate reader with an excitation/emission of 535/615 nm. Antibacterial properties of GelMA-Ru/SPS-AgNP in disc diffusion assays

The bacterial OD of f. coli and S. aureus was measured and adjusted to 0.1 OD. One ml of the adjusted broth was added to 19 ml of TBS and mixed slowly up and down before pouring in an empty sterile petri dish, and then left to set. Using a biopsy punch, 3 holes measuring 5 mm in diameter were punched per plate. 10 pl of the standard consistency (5 wt%) un-crossed linked GelMA with the following concentrations of AgNP 0, 25, 50, 100, and 200 pg/ml, was pipetted directly inside the punched hole and cross linked for 3 mins at 450 nm. The plates were then incubated for 24 h at 37°C/5% CO2. Images were taken using a Canon camera and the inhibition zone diameters was measured using Image J software.

Results: Bacterial encapsulation and the effects of GelMA-Ru/SPS-AgNP The metabolic activity of f. coli and S. aureus encapsulated in 10 pl 5% GelMA- Ru/SPS with different doses of AgNP was investigated (Figure 9 A & B). At 24h, concentrations of AgNP > 0.1 pg/ml slightly decreased the bacterial viability for both tested organisms, however, at > 5 pg/ml significant antibacterial activity was observed. At 48h, the 10 pl plated broth did not show any bacterial growth at concentrations of 5 pg/ml or higher, indicating complete killing of the tested bacterium.

Antibacterial properties of GelMA-Ru/SPS-AgNP in disc diffusion assays

The antimicrobial efficiency of the GelMA-Ru/SPS-AgNP was tested using nutrient agar media in disc diffusion assays. Figures 10 A and B show the diameter of the clearance zone (inhibition zone) around the constructs after a 24 h incubation of the agar plate at 37°C. Diameters of inhibition zones varied with type of tested microorganism and silver concentration used in the constructs, and ranged between 3 to 7 mm. Inhibition zones and therefore antimicrobial activity increased with increasing concentration of AgNP encapsulated in the GelMA-Ru/SPS-AgNP constructs. GelMA-Ru/SPS-AgNP constructs were found to be effective against both E. coli and S. aureus at 50 and 100 pg/ml respectively.

18-month post fabrication GelMA-Ru/SPS-AgNP constructs showed an E. coli inhibition zone of 9.7 mm and S. aureus had and inhibition zone of 8.2 mm (Figure 11).

Example 8: Rabbit cranial model of bone regeneration with alpha-lipoic acid capped silver nanoparticles in GelMA-Ru/SPS

Methods: Constructs

The following materia Is/ constructs were tested in 6 mm rabbit cranial defects: control empty defects (control), 160°C optimized bone graft material which was bleached and gamma irradiated (OB), AgNP functionalized optimized bone (AgNP-OB), AgNP functionalized GelMA crosslinked with Ru/SPS with OB as a bio-composite (AgNP- GelMA-OB), and BioOss® as a positive control (BO, Geistlich Pharma, Switzerland).

Making of the optimized bone (OB)

Tissue was removed from prion free NZ bovine bone blocks (25 x 25 x 25 mm) by boiling, rinsing with 80 (+/- 5) deg Celsius water and centrifugation (Molteno Ophthalmic Ltd, 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 containing distilled water (80 mL) and covering the bone. Bone cubes were heated at a rate of 4-6°C/min and held for 2 h at 160°C under pressure (90.28 psi). Treatment was followed by a 5 min cooling period, by running cold water over the vessel. The pressure was read directly from the chamber 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. Blocks were bleached with 1% sodium hypochlorite solution. Dried bone blocks were immersed in a container of sodium hypochlorite solution, which was placed in a vacuum pot. The air was expelled for about 10 min to create a vacuum. The containers were left in a UV-free environment at ambient temperature (15-25°C) for 24 h. 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. Particles were chiseled directly from the 25 x 25 x 25 mm 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 0.5 mm - 1.2 mm. Afterwards, the particles were packed in Eppendorf tubes (around 70 mg per tube) and sterilization conducted by gamma irradiated at a dose of 25-32 kGy (MSD Animal Health, Gamma Department, New Zealand).

Preparation of the AgNP constructs

AgNP stock concentration at 2510 pg /ml was produced on 6th of December 2019 and used within 4 weeks of production. The final concentration of the AgNP per construct was 100 pg per ml of gel and per gram of OB.

To make the AgNP-OB, the AgNP were added to the bone particles inside the hood after the gamma radiation step and allowed to dry. For the AgNP-GelMA-OB the AgNP were incorporated into the GelMA immediately prior to crosslinking with Ru/SPS as per Example 2 with addition of OB. Silicone moulds (6 mm in diameter) were sterilized in ethanol for 20 mins and left to dry inside the hood. 42.4 pl of GelMA-AgNP were plated in each of silicone moulds and cross linked for 3 mins. Another 42.4 pl of the GelMA-AgNP were added on the top of the first GelMA- AgNP layer, followed by adding 17.5 mg (approximately 20% of construct) of OB particles to that layer prior to cross-linking. The whole constructs were cross-linked for another 3 mins. Afterwards, the silicone mould was covered by parafilm and stored in the -80°C freezer for 24 h then freeze dried overnight.

Surgical approach

24 female New Zealand rabbits were used in these experiments. The experiment was conducted with ethics number: 5.8.18-02996/2020 from Malmo-Lund animal research board, Lund University, Sweden. The animals were sedated with ketamine and further anaesthetized 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 in the surgery. 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 was fully penetrated. The defects were carefully finished using a round bur and an elevator. Experimental groups included in this project were: Untreated defect (Control), BioOss® (BO), 160°C bone graft material which was bleached and gamma irradiated (OB), 160°C bone graft material which was bleached and gamma irradiated with 100 pg/gm AgNP (AgNP-OB), AgNP functionalized GelMA crosslinked with Ru/SPS with optimized bone as a bio-composite (AgNP-GelMA-OB). The treatments were rotated for each animal. The soft tissues were sutured after placement of test materials and an analgesic (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 with an overdose of pentobarbital to give two time points. 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.

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). After undertaking SKYSCAN high resolution x-ray micro-computer tomography at 60 kV, the amount of newly formed bone and grafting materials were quantified using Image J software (NIH). The micro-CT data was analyzed mid-slice through the 6 mm cranial detect and the amount of bone, residual graft, new bone and connective tissue analyzed using fixed thresholding. The region of interest for analysis was defined as a 5.5 mm circle within the cranial defect. Data was analyzed in GraphPad PRISM7 (GraphPad Software, San Diego, CA, using Tukey's adjusted ANOVA - unpaired.

Resin embedding

After micro-CT, the samples will be further subjected to water substitution for 72 h in 96% ethanol and 72 h in 100% ethanol, then placed in xylene for 72 h. They were resin embedded by placing the samples in methyl methacrylate (MMA) for 72 h followed by 3 days in 100 ml MMA + 2g at 4°C. Next, samples will be embedded in 100 ml MMA + 3g dibenzoylperoxide + 10 ml plastoid N or dibutylphthalate and allowed to polymerize at 37°C in an airtight environment. 4.5 pm slices were sectioned from the middle of the defect and stained with Goldner Trichrome.

Results: Micro CT analysis

The rabbit surgery resulted in n=6 (one BioOss® site in 4 week imaging group excluded). Reconstruction of 3D micro-CT images (Figure 12) indicated that the AgNP- GelMA-OB construct had less evidence of residual bone grafting material at both 4- and 16-weeks post-surgery compared to other test sites. The sites also had a different appearance when viewed before embedding being darker and more even in appearance (Figure 13). All mid-slice micro-CT images are shown in Figures 14 and 15, for 4- and 16- weeks respectively. At both time points the empty sites showed some limited evidence of new bone formation but were dominated by black space likely to contain connective tissue. In the empty sites it appeared that the size of the defect might extend out further into the cranium with large areas of remodeling bone at 16 weeks. BioOss® was evident as bright white particles at both 4- and 16- weeks. New bone, evident as a light grey color, was found surrounding the particles. Darker connective tissue areas more centrally were often evident. OB and AgNP-OB were very similar and showed less graft material than BioOss® with the material ranging from white to grey in appearance. The sites were more variable in appearance and new bone was evident associated with the bone granules. AgNP-GelMA-OB had the least amount of graft material evident at 4- and 16- week timepoints. New bone was evident beyond the areas containing the OB. The gel was not visible via micro-CT imaging but newly formed bone was evident throughout the construct.

Quantitative analysis at both 4- and 16- weeks is shown in Figure 16. Of note was that AgNP-GelMA-OB had significantly more residual connective tissue than BioOss® at both time points, thus suggesting more of the matrix was capable of transformation to bone over time while BioOss® remained entrapped within the new bone. There was significantly more new bone present in BioOss®, OB, AgNP-OB, and AgNP-GelMA-OB than in the empty socket thus indicating enhanced hard tissue healing. There was no significant difference in new bone between BioOss® and AgNP-GelMA-OB suggesting at least equivalence. The AgNP-GelMA-OB had the added advantage of low amounts of graft material allowing more of the site to be replaced by the natural regenerative bone and less of a foreign graft remaining at 16 weeks. The presence of AgNP with their antimicrobial activity had no measurable negative impact on bone regeneration.

Histology of the resin-embedded tissue examined for any inflammatory reaction at week 16 of healing

Representative rein-embedded histological images are shown in Figure 17. No inflammatory infiltrate was evident associated with the grafting materials. New bone formation around OB, AgNP-OB and AgNP-GelMA-OB graft material was evident. Blood vessels were observed within the connective tissue indicating angiogenesis and healing/ regeneration.

The above results above show that the GelMA-Ru/SPS-AgNP hydrogel of the invention is surprisingly unique in its ability to retain AgNP compared to other crosslinking and hydrogel systems investigated. The inventors believe that the antimicrobial properties of the GelMA-Ru/SPS-AgNP hydrogel would also be present in the bone graft constructs of the invention.

The hydrogel of the invention binds cells and enhances their growth in a 3- dimensional wound environment by providing a scaffold for growth and regeneration. When bone graft material was added to the GelMA-Ru/SPS-AgNP hydrogel good bone regeneration was measured with less residual graft. Thus a hydrogel bone graft construct with retention of nanoparticule Ag for antimicrobial control was produced. 7. REFERENCES

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