A COLLAGEN SCAFFOLD

Abstract

A collagen scaffold for the delivery of bioactive agents such as antimicrobials comprising a first collagen matrix layer and a second collagen matrix layer in which the first collagen matrix layer comprises a first bioactive agent physically entrapped in the first collagen matrix layer and the second collagen matrix layer comprises a second bioactive agent chemically attached to the second collagen matrix layer for an initial high concentration elution of antimicrobial from the first collagen matrix layer followed by a sustained release from the second collagen matrix layer to prevent re-infection.

Claims

1. A collagen scaffold comprising: a first collagen matrix layer and a second collagen matrix layer wherein the first collagen matrix layer comprises a first bioactive agent physically entrapped in the first collagen matrix layer and the second collagen matrix layer comprises a second bioactive agent chemically attached to the second collagen matrix layer.

2. A collagen scaffold as claimed in claim 1 wherein the first bioactive agent is releasable in a burst from the first collagen matrix layer and the second bioactive agent is releasable in a sustained release from the second collagen matrix layer.

3. A collagen scaffold as claimed in claim 2 wherein the second bioactive agent is releasable in response to the presence of microbes.

4. A collagen scaffold as claimed in claim 2 or claim 3 wherein the second bioactive agent is releasable in response to a microbial enzyme.

5. A collagen scaffold as claimed in claim 4 wherein the microbial enzyme is a protease.

6. A collagen scaffold as claimed in claim 5 wherein the protease is a collagenase.

7. A collagen scaffold as claimed in any of claims 1 to 6 wherein the second bioactive agent is crosslinked to the second collagen matrix layer.

8. A collagen matrix as claimed in claim 7 wherein the second bioactive agent is covalently crosslinked to the second collagen matrix layer.

9. A collagen scaffold as claimed in any of claims 1 to 8 wherein the first and second bioactive agents can be the same or different.

10. A collagen scaffold as claimed in any of claims 1 to 9 wherein the first and/or second bioactive agents comprise an antimicrobial.

11. A collagen scaffold as claimed in claim 10 wherein the antimicrobial comprises an antibiotic or an antifungal.

12. A collagen scaffold as claimed in claim 11 wherein the antibiotic comprises free amine and/or carboxylic groups for chemical attachment to the second collagen matrix layer.

13. A collagen scaffold as claimed in claim 12 wherein the antibiotic is chemically attached to the second collagen matrix layer at an amide bond.

14. A collagen scaffold as claimed in any of claims 11 to 13 wherein the antibiotic is selected from the group consisting of vancomycin, gentamycin and teicoplanin.

15. A collagen scaffold as claimed in any of claims 1 to 14 wherein the first collagen matrix layer and/or the second collagen matrix layer comprises hydroxyapatite to form a collagen-hydroxyapatite scaffold.

16. A collagen scaffold as claimed in any of claims 1 to 15 wherein the first collagen matrix layer and/or the second collagen matrix layer comprises glycosaminoglycans to form a collagen-glycosaminoglycans scaffold.

17. A collagen scaffold as claimed in any of claims 1 to 16 wherein the first collagen matrix layer is integrated with the second collagen matrix layer.

18. A collagen scaffold as claimed in any of claims 1 to 17 wherein the first and/or second collagen matrix layers comprise lyophilized collagen matrix layers.

19. A collagen scaffold as claimed in claim 18 wherein the second collagen matrix layer is lyophilized to the first collagen matrix layer.

20. A process for producing a multilayer collagen scaffold comprising: forming a first collagen matrix layer having a chemically attached first bioactive agent; and forming a second collagen scaffold matrix layer having a physically entrapped second bioactive agent on the first collagen matrix layer to produce the multilayer collagen scaffold.

21. A process for producing a multilayer collagen scaffold as claimed in claim 20 wherein first collagen matrix layer is formed by physically entrapping the first bioactive agent in the first collagen matrix layer and chemically attaching the physically entrapped first bioactive agent to the first collagen matrix layer.

22. A process for producing a multilayer collagen scaffold as claimed in claim 21 wherein the first bioactive agent is chemically attached to the first collagen matrix layer with a crosslinker.

23. A process for producing a multilayer collagen scaffold as claimed in claim 22 wherein the first bioactive agent is covalently attached to the first collagen matrix layer.

24. A process for producing a multilayer collagen scaffold as claimed in claim 23 wherein the first bioactive agent is covalently attached to the first collagen matrix layer at an amide bond.

25. A process for producing a multilayer collagen scaffold as claimed in any of claims 22 to 24 wherein the crosslinker comprises 1-Ethyl-3-(3-Dimethlamniopropyl)-carbodiimide (EDAC).

26. A process for producing a multilayer collagen scaffold as claimed in any of claims 21 to 25 wherein the first and second collagen matrix layers are formed by lyophilization.

27. A process for producing a multilayer collagen scaffold as claimed in any of claims 21 to 26 wherein the second collagen matrix layer is formed on the first collagen matrix layer by freeze drying the second collagen matrix layer on the first collagen matrix layer.

28. A process for producing a multilayer collagen scaffold as claimed in any of claims 21 to 27 wherein the first collagen matrix layer and/or the second collagen matrix layer comprises hydroxyapatite to form a collagen-hydroxyapatite scaffold.

29. A process for producing a multilayer collagen scaffold as claimed in any of claims 21 to 28 wherein: the first collagen matrix layer and/or the second collagen matrix layer comprises glycosaminoglycans to form a collagen-glycosaminoglycans scaffold; and/or the first and second bioactive agents can be the same or different; and/or the first and/or second bioactive agents comprise an antimicrobial; and/or the antimicrobial comprises an antibiotic or an antifungal; and/or the antibiotic comprises free amine and/or carboxylic groups; and/or the antibiotic is selected from the group consisting of vancomycin, gentamycin and teicoplanin.

30. A method of treating a subject comprising: implanting a collagen scaffold in the subject wherein the collagen scaffold comprises a first collagen matrix layer and a second collagen matrix layer wherein the first collagen matrix layer comprises a first bioactive agent physically entrapped in the first collagen matrix layer and the second collagen matrix layer comprises a second bioactive agent chemically attached to the second collagen matrix layer releasing the first active agent in a burst from the first collagen matrix layer and releasing the second active agent in a sustained release from the second collagen matrix layer.

31. A method of treating a subject as claimed in claim 30 wherein the second bioactive agent is released in response to the presence of microbes.

32. A method of treating a subject as claimed in claim 31 wherein the second bioactive agent is released in response to a microbial enzyme.

33. A method of treating a subject as claimed in claim 32 wherein the microbial enzyme is a protease.

34. A method of treating a subject as claimed wherein the protease is a collagenase.

35. A method of treating a subject as claimed in any of claims 30 to 34 wherein the first and second bioactive agents can be the same or different.

36. A method of treating a subject as claimed in any of claims 30 to 35 wherein the first and/or second bioactive agents comprise an antimicrobial.

37. A method of treating a subject as claimed in claim 36 wherein the antimicrobial comprises an antibiotic or an antifungal.

38. A method of treating a subject as claimed in claim 37 wherein the antibiotic comprises free amine and/or carboxylic groups for chemical attachment to the second collagen matrix layer.

39. A method of treating a subject as claimed in claim 37 or claim 38 wherein the antibiotic is chemically attached to the second collagen matrix layer at an amide bond.

40. A method of treating a subject as claimed in any of claims 37 to 39 wherein the antibiotic is selected from the group consisting of vancomycin, gentamycin and teicoplanin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The invention will now be described, by way of example only, with reference to the accompanying drawings and Examples in which:

[0064] FIG. 1 is a schematic representation of a multi-layer scaffold in the form of a dual-layer collagen scaffold for the delivery of antibiotics made up of a sustained release layer (formed by crosslinking the antibiotic to the collagen scaffold resulting in chemical attachment) and a burst release layer (formed by the direct incorporation of antibiotic within the collagen scaffold resulting in physically entrapment) with a scanning electron microscopy (SEM) image of the scaffold showing the integrated sustained release and burst release layers;

[0065] FIG. 2 is a schematic representation of the dual-layer collagen scaffold and an antibiotic release profile for the scaffold illustrating burst release for the directly incorporated antibiotic and sustained release for the crosslinked antibiotic;

[0066] FIG. 3(A) is an SEM image of a dual-layer antibiotic (teicoplanin) eluting CHA scaffold;

[0067] FIG. 3(B) are antibiotic release profiles from dual-layer scaffolds showing burst release via direct antibiotic incorporation and sustained release via crosslinking;

[0068] FIG. 3(C) is an image of an agar plate seeded with Staphylococcus aureus showing the zone of clearance achieved by CHA scaffolds with antibiotics directly incorporated;

[0069] FIG. 3(D) is a fluorescence micrograph of DAPI-stained osteoblasts seeded on antibiotic-eluting CHA scaffolds and infected with S. aureus in which the osteoblasts attach, remain viable, and migrate towards the scaffold center;

[0070] FIG. 4(A) is a release profile of a crosslinked antibiotic-eluting scaffold with and without supplementation with microbial collagenase demonstrating a responsive increase in antibiotic release in the presence of microbial collagenase;

[0071] FIG. 4(B) is a graphic illustration of agar diffusion tests performed with the dual layer scaffold of the invention against S. aureus compared with directly incorporated and crosslinked single layer scaffolds in which the dual-layer scaffold demonstrates enhanced bacterial clearance;

[0072] FIG. 5(A) illustrates the results of a pre-clinical trial in a murine model of osteomyelitis in which treatment with the dual-layer scaffold resulted in lower levels of bacteria through the treatment period. Results are presented as mean±standard error from mean. Significance; #p<0.1, * p<0.05;

[0073] FIG. 5(B) illustrates the results of the pre-clinical trial in which bacteriological analysis of the animals following euthanasia demonstrated fewer infected animals in the dual-layer scaffold group. Data points represent CFU counts from individual animals. Line represents mean value of each group. Significance; #p<0.1;

[0074] FIG. 5(C) is a representative micro-CT image analysis of a treated bone demonstrating evidence of mineralization within the defects treated with the dual-layer scaffold of the invention. Significance; #p<0.1, * p<0.05;

[0075] FIG. 6(A) is graph of the bacteriological analysis after 4 weeks inoculation period in a pre-clinical trial in a rabbit model of chronic osteomyelitis. Data points represent CFU counts from individual animals. Line represents mean value of each group;

[0076] FIG. 6(B) is a graph of the bacteriological analysis of the animals after the 8 week treatment period showing that infection was completely eradicated in the Gent dual-layer scaffold group. Data points represent CFU counts from individual animals. Line represents mean value of each group. Significance; * p<0.05;

[0077] FIG. 7 is a graph of high performance liquid chromatography results demonstrating the percentage of Teicoplanin antibiotic released with increasing concentrations of crosslinker with optimal crosslinking being observed through drug release where the retention of the antibiotic cannot be further increased;

[0078] FIG. 8(A) is a graph of the DNA content of control (antibiotic-free) dual-layer, vancomycin dual-layer and gentamicin dual-layer scaffolds seeded with MC3T3 cells from day 0 to day 21;

[0079] FIG. 8(B) is a graph of the alkaline phosphatase (ALP) activity at day 7 and day 21 of cell-seeded scaffolds demonstrating similar levels of osteogenesis in each scaffold group;

[0080] FIG. 8(C) are images of hematoxylin and eosin stained scaffolds at day 21 with the dashed line indicating the interface between the crosslinked and non-crosslinked layers of the scaffolds. Scale bar is 500 μm Significance; p<0.05, a vs Day 0, b vs. Day 2, c vs. Day 7, d vs. Vanc dual-layer, e vs. Gent dual-layer;

[0081] FIG. 9(A) is a graph of the bone volume per total volume (BV/TV) of radial defects in a rabbit model of chronic osteomyelitis either left empty or treated with a Vanc dual-layer scaffold or a Gent dual-layer scaffold;

[0082] FIG. 9(B) is a graph of the bone density of defects at weeks 4, 8, 10 and 12, and

[0083] FIG. 9(C) are CT reconstructions of radial defects at weeks 4, 8 10 and 12 showing bone healing in all groups over time. Scale bar is 3 mm. Significance: p<0.05; a vs Week 4, b vs. Week 8, c vs. Week 10, d vs. Vanc dual-layer, e vs. Gent dual-layer.

DETAILED DESCRIPTION OF THE INVENTION

Bioactive Agents—Antimicrobials

[0084] In the following Examples, teicoplanin, vancomycin and gentamycin are shown to be suitable antibiotics for incorporation into the dual-layer collagen scaffolds as they are the clinical drugs of choice for the treatment of chronic infection caused by bacteria. However, the dual-layer collagen scaffolds of the invention allow the controlled delivery of other antibiotics with free amine and/or carboxylic groups that can be covalently cross-linked to the collagen. In addition, other effective and cost saving bioactive agents including antimicrobials such as antibiotics and antifungals for the treatment of fungal, gram positive and negative bacterial infections can also be employed in the dual-layer collagen scaffolds of the invention.

Example 1—Synthesis

[0085] Porous three-dimensional collagen-based scaffolds were fabricated following a well-established freeze-drying method (O'Brien et al., 2005). Briefly, for the typical fabrication of collagen scaffolds, the collagen was suspended in an acidic solution. If required, the collagen slurry solution can contain glycosaminoglycans for soft tissue regeneration (a CG scaffold), HA for bone regeneration (a CHA scaffold) or any other natural material that has suitable side chains for attachment of the antimicrobials.

[0086] The obtained slurry was then heavily degassed (to eliminate air bubbles that may generate non-controlled porosity) prior to adding the antimicrobial that possessed suitable attachment sites.

[0087] The antimicrobial was added to the collagen slurry using a syringe (direct incorporation) and injected through a needle (18G 1½″) into the slurry, with gently mixing without introducing air bubbles. The collagen solution with the antimicrobial was then lyophilised by a freeze-drying process. In this process, the slurry was first frozen where both the final freezing temperature and the rate of freezing (ramp) were controlled to tune the resulting pore size. Next, the pressure was lowered to vacuum allowing the sublimation of ice crystals from the slurry to vapour resulting in the creation of a highly porous antibiotic-eluting collagen scaffold with an interconnected pore structure and homogenous pore size (O'Brien et al., 2005).

[0088] Freeze dried antibiotic-eluting collagen scaffolds were then crosslinked using 1-Ethyl-3-(3-Dimethlamniopropyl)-carbodimide hydrochloride (EDAC) and N-Hydroxysuccinimide (NHS) as a catalyst, resulting in the chemical attachment of the antibiotic to the collagen via covalent bonds. Briefly, collagen antibiotic scaffolds were immersed in a solution of EDAC/NHS, at increasing concentrations, to crosslink the antibiotic to the collagen. The EDAC was utilized at ranges from 8× to 32× the standard operating concentration (48 mM-192 mM per gram of collagen). The crosslinked antibiotic-collagen scaffolds were then washed from all EDAC residues. Increasing the concentrations of the EDAC crosslinker resulted in the occupation of all the binding sites available between the antibiotic and the collagen based scaffold. As shown in FIG. 7, this ultimately can slow the release of the antibiotic, achieving a controlled release system.

[0089] The dual-layered antibiotic-eluting collagen scaffold shown in FIG. 1 was formed by placing a crosslinked antibiotic-eluting collagen scaffold (described above) on a stainless steel plate and pouring additional collagen slurry containing an antibiotic (directly incorporated) onto the top. The freeze drying process was then repeated to provide an inter connecting dual layered scaffold that contains antimicrobials to achieve a final antibiotic-eluting scaffold with a dual release profile.

[0090] The dual layered collagen scaffold can contain the same antimicrobial in each layer or two different antimicrobials as required.

[0091] The efficacy of the antibiotic-eluting dual-layer collagen scaffold was demonstrated in two pre-clinical trials.

Example 2—Pre-Clinical Trial 1

[0092] In pre-clinical trial 1, uni-cortical bone defects (Ø0.8 mm) in the tibiae of C57BL6 mice were inoculated with 2×10.sup.3 CFUs of bioluminescent S. aureus. After 2 weeks, the infected site was debrided (Ø1.2 mm) and either left empty or treated with a vancomycin-eluting dual-layer scaffold. Animals were sacrificed at day 11 post-treatment and tibiae were homogenized and counted for bacteria. Statistical comparisons were performed using t-tests. In vivo imaging during the treatment demonstrated lower levels of bioluminescent bacteria in scaffold treated animals compared to animals not treated with the scaffold (see FIG. 5(A)). Furthermore, bacteriological analyses of the animals at euthanasia demonstrated fewer infected animals in the scaffold treated group (see FIG. 5(B)). As shown in FIG. 5(C), micro-computed tomography (μCT) analysis also showed that the treated defects were beginning to fill with newly formed mineral.

Example 3—Pre-Clinical Trial 2

[0093] Pre-clinical trial 2 utilised an animal model representative of chronic osteomyelitis. Infections were established in the radii of New Zealand White rabbits using inoculations of 2×10 CFUs S. aureus over a period of 4 weeks. Following surgical debridement (6 mm), rabbits then underwent treatment for a period of 8 weeks until euthanasia. The treatment groups were: 1) empty, 2) commercially available gentamicin-eluting collagen fleece (Septocoll E (Trade Mark)), 3) vancomycin-eluting dual-layer collagen scaffold (Vanc dual-layer) and 4) gentamicin-eluting dual-layer collagen scaffold (Gent dual-layer). During the treatment period, all groups received systemic antibiotics (Cefazolin 25 mg/kg), administered subcutaneously, twice daily, for 4 weeks. Results showed that inoculation resulted in the development of a sequestrum containing S. aureus in all rabbit radii, demonstrating the successful establishment of OM (see FIG. 6(A)). After the 8 week treatment period, only 1/6 (one out of six) rabbits in the empty group were infection-free, indicating that systemic antibiotic administration following debridement was insufficient to treat the infection. Infections were cleared in 4/6 rabbits in both the Septocoll E (Trade Mark) group and the Vanc dual-layer collagen group. However, the most effective treatment was found to be the Gent dual-layer scaffold, which cleared the infection in 6/6 rabbits, a statistically significant improvement (p=0.0152) over the empty group (see FIG. 6(B)).

Characterisation

[0094] As shown in FIG. 3(A), in the dual-layer scaffold structure incorporating crosslinked and directly incorporated antibiotic produced as described above, the porous scaffold structure necessary for efficient tissue regeneration was maintained. In addition, the porosity (>98%) and mean pore size, essential for optimal proliferation and differentiation of osteoblasts during bone regeneration, were not compromised after the loading of the drug into the collagen structure. Depending on the incorporation method, different release kinetics of the antibiotic from the materials were obtained. FIG. 3(B) shows the different delivery profiles obtained from different antibiotic-loaded materials for 60 days. At day 10, approximately 90% of the teicoplanin was released when it was directly loaded into the slurry during the CHA scaffold fabrication.

[0095] The chemical attachment of antibiotic onto the scaffold, achieved by the chemical crosslinkers that generated a stable amide bond between the drug and the collagen component of the scaffold, resulted in the retention of the drug within the scaffold. This ultimately resulted in a sustained release of the antibiotic, with approximately 11% teicoplanin released at day 60.

[0096] Furthermore, as shown in FIG. 4(B) when crosslinked and direct incorporation scaffolds were layered to create a dual-layer scaffold, this dual-layer scaffold demonstrated synergistically enhanced bacterial clearance when compared to crosslinked or direct incorporation scaffolds alone.

[0097] Moreover, bacterial enzymes such as microbial collagenases were able to break the chemical bond between the collagen and the antibiotic, leading to the responsive increased release of the drug in the presence of S. aureus or S. epidermidis. This was demonstrated by the zone of clearance achieved after the culture of these bacteria (10.sup.8 colony forming units) in the presence of crosslinked scaffolds as shown in FIG. 3(C).

[0098] The responsive release of antibiotic-loaded crosslinked scaffolds was further characterized by loading the scaffolds with a fluorescently tagged vancomycin antibiotic. As shown in FIG. 4(A) when microbial collagenase was added to the antibiotic-eluting crosslinked scaffolds, an increase in antibiotic release rate was observed confirming that, once implanted into an infected environment, the scaffolds of the invention facilitate enhanced release of the antibiotic when it is most needed.

[0099] Accordingly, the dual-layer collagen scaffolds of the invention provide a microbially responsive release, whereby increased bacterial presence causes a responsive spike in the release of antibiotics from the scaffold leading to eradication of the infection.

[0100] Importantly, the antibiotic loaded scaffolds of the invention also demonstrate good cell compatibility. As shown in FIG. 3(D), results achieved from the in vitro assessment of osteoblasts infected and cultured with live S. aureus on the antibiotic-eluting scaffolds of the invention demonstrated that osteoblast cells attached, remained viable, and migrated towards the centre of the antibiotic-eluting scaffolds. In contrast, parallel tests carried out with non-eluting materials identified that cells do not survive on scaffolds post-infection.

[0101] In vitro data sets demonstrated that 1) it is possible to load and release high doses of antibiotics from collagen based scaffolds in a controlled fashion while keeping the structural properties of these materials, 2) the released antibiotic retains antibacterial activity 3) the fabricated materials are cytocompatible and allow osteoblast and stem cell survival, attachment and migration and 4) the antibiotic-loaded crosslinked scaffolds are responsive to microbial activity.

[0102] As shown particularly in FIG. 5(C), the scaffolds of the invention can also provide an antimicrobial platform in optimal tissue regeneration compared to commercially available collagen sponges/fleeces without compromising optimal scaffold physiochemical properties.

[0103] The release of antibiotics is controlled through either chemically crosslinking the antibiotic to the collagen backbone and/or allowing free diffusion by direct incorporation of the antibiotic i.e. a burst release. Different antimicrobial(s) can be chemically attached to the scaffold as required for a controlled sustained release with ˜11% release over 2 months.

[0104] Moreover, as shown in the SEM image of FIG. 1 (and in FIG. 8(C)), the multi-layer scaffold of the invention does not contain barriers between the layers but is made up of seamlessly integrated layers so that 1) cell migration, 2) antibiotic diffusion, and 3) nutrient exchange/waste removal can continue unhindered. As a result, the multi-layer collagen scaffold of the invention is a unitary barrier-free scaffold containing a dual release system.

[0105] As shown in FIG. 4(A), the scaffolds of the invention function as reservoirs of antibiotics with the ability to release more antibiotic in response to a relapsing infection. Each layer of the dual-layer scaffold can incorporate the same or different bioactive agents such as antimicrobials e.g. two or more different antibiotics can be included in a multilayer scaffold. Moreover it is possible to optimise dosage of therapeutic antimicrobials that does not harm host cells or tissue, while maintaining a high microbial activity. The scaffold structure can therefore be created as required as a multilayered scaffold which allows the ability to combine a burst and sustained release profile at the same time, with the ability to contain a choice of selected antimicrobial(s).

[0106] As indicated above, the scaffolds of the invention facilitate the control of the release kinetics of antimicrobials in such a way as to enable an initial burst release to eradicate deep seated infections, and a longer controlled release to prevent infection reoccurrence.

[0107] FIG. 8(A) shows a graph of the DNA content of a control (antibiotic-free) dual-layer, vancomycin dual-layer and gentamicin dual-layer scaffolds seeded with MC3T3 cells. The cell seeded scaffolds were cultured in expansion medium for 2 days before being switched into an osteogenic medium for the duration of the experiment (21 days total). As shown in the graphs, all scaffolds demonstrated the capacity to promote proliferation of MC3T3 cells. FIG. 8(B) shows a graph of the alkaline phosphatase (ALP) activity of the cell-seeded scaffolds demonstrating similar levels of osteogenesis in each scaffold group while FIG. 8(C) shows images of hematoxylin and eosin staining of the scaffolds at day 21. As shown in the images, the dashed line indicates the interface between the crosslinked and non-crosslinked layers of the scaffolds. The excellent integration of the separate layers with no evidence of delamination is clearly visible in the image. Accordingly, despite being separate, the first and second collagen matrix layers form a seamless collagen scaffold that maintains its structural integrity in use.

[0108] As shown in FIG. 6 described above, multi-layer scaffolds of the invention are extremely effective at eliminating infection which is the primary objective and function of the scaffolds. As indicated above, the vancomycin dual layer scaffold eliminated infection in 4/6 animals and the gentamicin dual layer scaffold eliminated infection in 6/6 animals. However, a secondary objective of the scaffolds of the invention can be to facilitate bone tissue healing unhindered and FIGS. 8(A) to 8(C) therefore demonstrate the capacity of the vancomycin and gentamicin scaffolds of the invention to facilitate mammalian cell attachment, proliferation, and osteogenic differentiation in vitro. Despite antibiotics being known to have the potential to be toxic to mammalian cells, the results demonstrate that the scaffolds of the present invention did not inhibit osteogenesis (as demonstrated by the ALP/DNA data in FIG. 8(B)) in either vancomycin dual layer or gentamicin dual layer scaffolds compared to antibiotic-free dual layer scaffolds. Furthermore, although gentamicin dual layer scaffolds demonstrated a lower capacity to facilitate initial cell attachment, cell proliferation was subsequently observed to occur over time.

[0109] FIGS. 9(A) to 9(C) also demonstrate that bone tissue regeneration was not hindered by the collagen scaffolds of the invention (i.e. bone healing was visible in all groups over time) with FIG. 9(A) being a graph of the bone volume per total volume (BV/TV) of radial defects in a rabbit model of chronic osteomyelitis either left empty or treated with a Vanc dual-layer scaffold or a Gent dual-layer scaffold, FIG. 9(B) being a graph of the bone density of defects at weeks 4, 8, 10 and 12, and FIG. 9(C) showing CT reconstructions of radial defects at weeks 4, 8 10 and 12.

[0110] As shown in the drawings, the bone healing observed between 4 and 12 weeks in the empty group demonstrates that the size of the defect in this particular model is sub-critical and will heal itself over time. Although lower levels of BV/TV were observed in the gentamicin dual-layer group at 12 weeks compared to the empty group at 12 weeks, the results demonstrate that the primary objective of infection elimination was achieved while bone healing was still also achieved over time albeit at a lower rate i.e. an increase in BV/TV is still observed over time in the gentamicin dual-layer which demonstrates that bone healing is occurring, albeit at a slightly reduced rate.

[0111] In summary, in the collagen scaffolds of the invention, the physically entrapped antimicrobial in the first collagen matrix layer is released in an initial burst in vivo to attack and eliminate infection. Where the initial burst fails to eradicate the infection, the antimicrobials attached to the collagen matrix in the second layer via a crosslinking method to generate a drug-collagen covalent bond that can be broken by microbial infection are released via an adaptive release kinetic technology whereby the level of antibiotic release from the second layer is controlled by the degree of infection. Accordingly, in a highly infected site the bacteria begins to induce release of enzymes such as proteases which can liberate the collagen-bound antibiotic reservoir, thus leading to eradication of the infection and allowing the micro-environment necessary for tissue regeneration.