Tissue substitute material with biologically active coating

Abstract

The present invention relates to a tissue substitute material for implantation, comprising (a) a substrate to be implanted covered with (b) a controlled release coating containing (c) at least one biologically substance that decreases bacterial growth, wherein the (b) controlled release coating is a bioavailable, biocompatible polymer material and wherein the (c) at least one biologically active substance that decreases bacterial growth. The present invention also relates to a method to prepare the tissue substitute material, as wells the uses thereof.

Claims

1. A tissue substitute material for implantation, comprising (a) a substrate to be implanted covered with (b) a controlled release coating containing (c) at least one biologically active substance that decreases bacterial growth, wherein the (b) controlled release coating is a bioavailable, biocompatible polymer material comprising alginate and pectin; wherein the (c) at least one biologically active substance that decreases bacterial growth is selected from the group consisting of antibiotics and a mixture thereof, and wherein at least one water soluble part of the polymer material has been selectively converted into water insoluble form.

2. The tissue substitute material according to claim 1, wherein the polymer material contains water insoluble Ca-alginate.

3. The tissue substitute material according to claim 2, wherein the substrate is bone allograft.

4. The tissue substitute material for implantation according to claim 1, which is a bone substitute material for implantation, wherein the substrate is bone allograft, and wherein the (b) controlled release coating is a bioavailable, biocompatible polymer material consisting of alginic acid and pectin.

5. The tissue substitute material according to claim 1, wherein the substrate is a known tissue substitute selected from the group consisting of implants made from metal, plastic, and standalone polymer material suitable for the preparation of the coating.

6. The tissue substitute material according to claim 1, wherein the (b) controlled release coating is alginic acid pectin copolymer.

7. The tissue substitute material according claim 1, wherein the (b) controlled release coating essentially consists of alginic acid within the range of 70 to 90% and pectin within the range of 10 to 30%.

8. The tissue substitute material according to claim 1, wherein the (c) at least one biologically active substance that decreases bacterial growth is selected from the group consisting of gentamicin, ciprofloxacin, vancomycin, amoxicillin.

9. The tissue substitute material according to claim 1, further comprising other biologically active ingredients to enhance cell migration, adhesion and growth.

10. The tissue substitute material according to claim 1, wherein the biologically active ingredient to enhance cell migration, adhesion and growth is selected from the group consisting of growth factors including PDGF, TGF-/315 vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and epidermal growth factor; albumin, platelet rich plasma (PRP), platelet pure plasma (PPP) and platelet rich fibrin (PRF) and blood separation products that contain cell growth and/or cell migration enhancing agents.

11. A method for preparing a tissue substitute material for implantation according to claim 1, comprising (a) preparing a homogenous coating on a substrate to be implanted from at least one biologically active substance that decreases bacterial growth selected from the group consisting of antibiotics and a mixture thereof; (b) preparing a film coating from the water-soluble monomers of a biocompatible polymer material of alginic acid in combination with pectin; (c) drying the water-soluble film coating; (d) converting the water soluble film coating into water insoluble film coating; (e) drying the water-insoluble film coating.

12. The method according to claim 11, wherein the tissue is a tissue from the musculoskeletal system.

13. The method according to claim 12, wherein the substrate is bone allograft.

14. The method according to claim 11, wherein the substrate is alginate beads.

15. The method according to claim 11, wherein the antibiotic coating is prepared in step (a) by freeze drying, solvent evaporation or vacuum evaporation.

16. The method according to claim 11, wherein the film coating is prepared in step (a) spraying or casting.

17. The method according to claim 11, wherein the drying in step (c) and/or (e) is accomplished in a drying chamber or exsiccator, or by using moderate heating and vacuum.

18. The method according to claim 11, wherein the biocompatible polymer material selected is alginic acid pectin copolymer, wherein the alginic acid within the range of 70 to 90% and pectin within the range of 10 to 30%, and wherein in step (d), the conversion of the soluble film coating into water insoluble film coating is accomplished by using a Ca.sup.2+ ion containing solution.

19. The method according to claim 11, further comprising the inclusion of other biologically active ingredients to enhance cell migration, adhesion and growth into the biocompatible polymer coating material, selected from the group consisting of growth factors including PDGF, TGF-/315 vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and epidermal growth factor; albumin, platelet rich plasma (PRP), platelet pure plasma (PPP) and platelet rich fibrin (PRF) and other blood separation products that contain cell growth and/or cell migration enhancing agents.

20. A method for treating or inhibiting bone infection, said method comprising implanting the tissue substitute material of claim 1 in the form of a graft in a patient in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Coating methods. As a first step, bone allografts were incubated in an antibiotic solution for 24 hours. Subsequently, the soaked graft was removed from the solution and freezed at −80° C. followed by lyophilization for 24 hours using a Labconco Freezone 2.5′ freeze-dryer (soaked preparation). In order to maximize the drug content of the graft an alternative approach was also performed when the grafts were frozen while still submerged in the antibiotic solution and the whole system was freeze-dried (saturated preparation). The Na-Alg film was prepared by adding Na-Alg solution on the antibiotic coated freeze-dried bone. Then the graft was dried in an oven. The process was repeated with the dried coated graft turned upside down, thus the double layer Na-Alg film was formed. Sodium alginate was then converted into calcium alginate by Ca ion containing aqueous solution.

(2) FIG. 2 shows the short-term release of the antibiotics over a 48 hour period. prepared by the soaked or the saturated method.

(3) FIG. 3 Release profile of amoxicillin, ciprofloxacin or vancomycin with sustained release Ca-Alg film coating (n=3). Although the coating method was the same in each case, the effective release term is different among the three drugs with amoxicillin lasting up to 8 days, ciprofloxacin up to 28 days while vancomycin reaches 50 days.

(4) FIG. 4. Calculated daily release of antibiotics from one implanted femoral head graft. Values are expressed as percentages of the daily recommended iv dose (amoxicillin 2000 mg/day, ciprofloxacin 1600 mg/day, vancomycin 1000 mg/day). Please note that values are calculated from in vitro data, not actual in vivo implantations, which may vary significantly due to the type of grafting and the individual difference among patients. Nonetheless, the data indicate that the antibiotics implanted with the bone grafts may significantly increase the daily systemic dose only for the first day. Therefore, in case of large volumes of antibiotic bone grafts are implanted the systemic dosing must be carefully monitored during the first few postop days.

(5) The present invention is further illustrated by the experimental examples described below; however, the scope of the invention will by no means be limited to the specific embodiments described in the examples.

(6) Materials and Methods

(7) Antibiotic Solution

(8) All chemicals were purchased from Sigma except from vancomycin, which was purchased from Hangzhou APIChem Technology Co., Ltd., China. The bone blocks were a generous gifts from the West-Hungarian Regional Tissue Bank Freeze-dried femoral head blocks were cut to 0.05±0.01 g cube-shaped pieces.

(9) The antibiotic containing solution was prepared by adding one or more antibacterial agent(s) (e.g. amoxicillin, ciprofloxacin, etc.) to an aqueous solution. The aqueous solution may also contain a suspension that is the growth factor enriched phase of a blood product. The antibacterial agent content is typically between 0.1 mg/ml to 100 mg/ml, we used 10 mg/ml concentration. We can use the mixture when a homogenous solution or suspension is formed this can be achieved with using a magnetic or overhead stirrer. We can also use this technique when the applied antibiotic is lipophilic, but in this case we need to use organic solvents.

(10) Microspheres

(11) The antibiotic content may also be present in the form of microspheres. These microspheres are water insoluble spheres with encapsulated antibiotic content. The production of these spheres is based on converting water soluble alginate solution, which contains antibiotic, into water insoluble alginate solution. This can be done by spraying Na-Alg into a Ca ion containing solution, and so the water insoluble spheres are formed. The spheres can be filtered off, dried and used later as antibiotic bone coating. The spheres may also be formed from chitosan, or any biocompatible polymer that is suitable for the purpose.

(12) Chitosan Based Short-Term Release Coating

(13) Chitosan-based preparations were prepared by using 1 ml aqueous 2% chitosan solution to dissolve the antibiotic. The bone samples were placed in this solution and incubated at room temperature for 24 hours and frozen and lyophilized afterwards in a similar manner as the saturated preparations.

(14) Alginate Based Short-Term Release Coating

(15) Alginate-based preparations should be created in another way since this polymer has a basic pH and antibiotics precipitate in it. First, the bone grafts were coated by the saturated freeze-dried method as described above then a film coating of alginate was created on top of the antibiotic layer. The Na-Alg film was prepared by adding 1 ml 4% Na-Alg solution on the antibiotic coated freeze-dried bone. Then the graft was dried in an oven at 40° C. for 4 hours on teflon plates. The process was repeated with the dried coated graft turned upside down, thus the double layer Na-Alg film was formed. Sodium alginate was then converted into calcium alginate by CaCl2. The Na-alginate coated bone grafts were placed in the 10% CaCl2 solution for exactly 60 seconds then washed with distilled water and dried in an oven at 40° C. The methods for preparing the coatings are presented in FIG. 1.

(16) Antibiotic Release Measurements

(17) The chosen antibiotics (amoxicillin, ciprofloxacin and vancomycin) have characteristic absorbances in the UV range in aqueous solutions, allowing the use of UV spectroscopy to assess the concentrations with a spectrophotometer. The absorbance-concentration diagrams were plotted using all antibiotics and the linear phase of this diagram was used to calculate the concentration from the absorbances according to the Lambert-Beer law (Table 2).

(18) TABLE-US-00002 TABLE 2 UV measurement characteristics of the investigated antibiotics Characteristic Linear absorbance- absorbance (nm) concentration interval Amoxicillin 229 0.22-3.7 Ciprofloxacin 275 0.085-2.29 Vancomycin 280  0.06-2.00

(19) Measurements of release kinetics were performed by incubating each sample separately in 2 ml of water in a 24 well plate at room temperature. Concentration measurements were performed at regular intervals by removing the supernatant for spectroscopy and replenishing with fresh solvent. The frequency of solution changes and the length of the experiments were determined by preliminary experiments and set in a way that optimal kinetic curves can be constructed from the data. In a separate experiment with Ca-Alg coated amoxicillin grafts, the medium was pipetted back onto the graft after each measurement in order to evaluate the effect of drug accumulation in the medium on the release kinetics. Statistics were carried out using GraphPad Prism 5.0 software. All data were expressed as means±SEM (n=3).

(20) Freeze Drying

(21) Freeze drying was applied as a method to prepare antibiotic coating. In this method 1 cm.sup.3 or 2 cm.sup.3 of aqueous antibiotic solution is incubated with the bone for 10 hours. The mixture was then put in a refrigerator at −80° C. for 4 hours. The frozen samples were freeze dried at −50° C. and 2.1 Pa reduced pressure for 12 hours. After 12 hours of freeze drying, the bone was taken out from the freeze dried matrix, the drug formed a dense coating all over the surface of the bone.

(22) Film Casting

(23) The antibiotic coating was further modified with a controlled release film. This was achieved by adding 1 ml of the film coating material on the bone, which was placed in a teflon plate. The plate was put in an oven with the temperature set to 40° C. After 4 hours of drying, the plate was taken out, and the excess film coating was cut off the bone. The procedure with the bone after turning it upside down was repeated again. After drying it and cutting the excess film off again, the film and antibiotic coated bone was soaked in 10% Ca.sup.2+ containing solution for 60 seconds. The calcium ions convert the water soluble Na-Alg to water insoluble Ca-Alg, and so a controlled release coating can be formed. If we used more than one component to produce the film (composite film) the ratio of the materials can also change the rate of antibiotic release (e.g. 40% Na-Alg and 60% pectin). The modification with calcium only affects the alginate part, so the reduced amount of alginate enhances the release of the used antibiotic.

Example 1: Short Term Antibiotic Release

(24) The drugs were highly soluble in water and were suitable to be stored at room temperature without any decomposition thus all the drugs were successfully applied on the surface of the bone. The original concentration of the antibiotic solutions used for incubating the bone grafts correlated with the amount of antibiotics on the bone surface estimated by the released total amount of drugs. 10 mg/ml starting solution was used in the experiment.

(25) Simple freeze-drying of antibiotics on the surface of bone grafts did not result in a sustained release of the compounds. Although minor differences were observed among the three antibiotics, each one is completely released within 48 hours (FIG. 2). Maximizing the antibiotic loading on the grafts by freezing them in the solution (saturated method) before lyophilization did not improve the release kinetics only the overall amount of antibiotics on the graft (FIG. 2B). Using a chitosan additive with the antibiotics did not significantly prolong the release of the drugs from the surface.

Example 2: Long Term Antibiotic Release

(26) Using a Ca-Alg film layer it was possible to reach a long-term sustained-release antibiotic coating. Interestingly, the type of antibiotic significantly affected the rate of drug release from the same type of coating. Amoxicillin was completely released within 8 days, ciprofloxacin within 28 days, while vancomycin was the longest with 50 days (FIG. 3). The amount of active ingredient released on the first day was approximately the same as the amount from the antibiotic bones, which did not contain Ca-alginate (FIG. 2). The total quantity of dissolved antibiotics over the 8, 28, or 50 or day period depending on the respective antibiotic was approximately the same than those without alginate coating, indicating that the amount of total antibiotic content did not increase only the release rate has changed (FIG. 3).

(27) To summarize the long-term release experiment, altogether 0.64±0.07 mg amoxicillin was eluted from the surface of 50 mg bone allograft, with complete dissolution in 8 days. In case of ciprofloxacin, 1.08±0.11 mg was the total eluted amount within 28 days. Vancomycin had the longest elution time for over 50 days during which 1.66±0.31 mg antibiotic was released altogether.

Example 3

(28) 50 mg bone chips were freeze dried in an aqueous solution that contained 10 mg/ml of vancomycin. One milliliter of 4% Na-alginate (Na-Alg) was added to the freeze dried bone in a teflon plate and the water content was allowed to evaporate in an oven at 40° C. The coating with Na-Alg was repeated once again. After the coating dried we soaked the coated bone in 10% CaCl.sub.2 solution for 60 seconds, to form water insoluble Ca-alginate (Ca-Alg) and the bones were dried again. Finally the dried Ca-Alg coated bone was irradiated with UV light to be sterile.

Example 4

(29) 100 mg bone was freeze dried in 10 mg/ml vancomycin solution. 1 ml 4% Na-Alg added dropwise to the bone and dried in an oven at 40° C. to form a composite film. The film coated bone was covered again with the composite film and the excess film residues were cut off. After drying, the coated bone was soaked in 10% calcium ion containing solution

Example 5

(30) 50 mg bone was freeze dried in 10 mg/ml ciprofloxacin solution. 4 ml 4% Na-Alg was mixed together with 1 ml 4% pectin to form a composite viscous solution. This viscous solution was added dropwise to the bone and dried in an oven at 40° C. to form a composite film. The film coated bone was covered again with the composite film and the excess film residues were cut off. After drying, the coated bone was soaked in 10% calcium ion containing solution. This method enables to change the release mechanism.

Example 6

(31) 50 mg bone was freeze dried in 50 mg/ml amoxicillin solution. 2 ml 4% Na-Alg was mixed together with 2 ml 4% pectin to form a composite viscous solution. This viscous solution was added dropwise to the bone and dried in an oven at 40° C. to form a composite film. The film coated bone was covered again with the composite film and the excess film residues were cut off. After drying, the coated bone was soaked in 10% calcium ion containing solution. This method enables to change the release mechanism and reduce the drug release time.

Example 7

(32) 1 ml aqueous 2% chitosan solution was used dissolve 10 mg gentamicin. The 50 mg bone sample was placed in this solution and incubated at room temperature for 24 hours. After the incubation the solution that contained the bone sample (saturated method) was frozen and lyophilized afterwards. After freeze drying the bone was taken out from the well together with the chitosan-antibiotic mixture that was in part attached to the surface of the bone. Thus a short-term drug release coating was prepared.

Example 8: Pectin-Alginate Two Component Film

(33) 0.8 ml 4% Na-Alg and 0.2 ml pectin was mixed together until a homogenous gel was formed. The mixture was poured onto the surface of a previously prepared freeze dried chitosan matrix and dried in an oven at 40° C. to form a composite film. The film was soaked in 5% calcium ion containing solution for 2 minutes and dried again afterwards. Thus a 2 component partly water insoluble film was produced.

Example 9: Pectin-Alginate Two Component Microsphere

(34) 0.9 ml 4% Na-Alg and 0.1 ml pectin with 50 mg vancomycin was mixed together until a homogenous gel/suspension was formed. The mixture was poured into a syringe. The content of the syringe was added at a constant dropwise rate into a 15% calcium ion containing solution for 30 seconds and filtered off immediately and dried afterwards in an oven at 40° C. Thus a 2 component partly water insoluble microsphere was produced.