ANTIMICROBIAL COMPOSITION FOR INHIBITING MICROBIAL ORGANISMS IN ALLOGRAFT AND THE METHOD THEREOF

Abstract

The present invention is a method for producing allograft tissue by applying an antimicrobial solution to allograft tissue. The antimicrobial solution exhibits antimicrobial activity to make allograft resistant to microbial organisms, such as bacterium.

Claims

1. A method for producing allograft tissue comprising the step of: applying an antimicrobial solution to allograft tissue, wherein the antimicrobial solution exhibits antimicrobial activity.

2. The method of claim 1 wherein the antimicrobial solution comprises an antimicrobial metal.

3. The method of claim 1 wherein the antimicrobial solution comprises a zinc compound.

4. The method of claim 3 wherein the zinc compound is selected from the group consisting of zinc chloride, zinc sulfate, zinc phosphate, zinc carbonate, and zinc nitrate, zinc chlorate, zinc chromate and combinations thereof.

5. The method of claim 3 wherein the zinc compound is zinc chloride.

6. The method of claim 4 wherein the zinc compound is a zinc salt selected from the group consisting of zinc acetate, zinc formate, zinc propionate, zinc gluconate, bis(maltolato)zinc, zinc acexamate, zinc aspartate, bis(maltolato)zinc(II) [Zn(ma)2], bis(2-hydroxypyridine-N-oxido)zinc(II) [Zn(hpo)2], bis(allixinato)Zn(II) [Zn(alx)2], bis(6-methylpicolinato)Zn(II) [Zn(6mpa)2], bis(aspirinato)zinc(II), bis(pyrrole-2-carboxylato) zinc [Zn(pc)2], bis(alpha-furonic acidato)zinc [Zn(fa)2], bis(thiophene-2-carboxylato) zinc [Zn(tc)2], bis(thiophene-2-acetato)zinc [Zn(ta)2], (N-acetyl-L-cysteinato) Zn(II) [Zn(nac)], zinc(II)/poly(γ-glutamic acid) [Zn(γ-pga)], bis(pyrrolidine-N-dithiocarbamate) zinc(II) [Zn(pdc).sub.2], zinc(II) L-lactate [Zn(lac).sub.2], zinc(II) D-(2)-quinic acid [Zn(qui).sub.2], bis(1,6-dimethyl-3 -hydroxy-5-methoxy-2-pentyl-1,4-dihydropyridine-4-thion-ato) zinc(II) [Zn(tanm)2], β-alanyl-L-histidinato zinc(II) (AHZ) and combinations thereof.

7. The method of claim 1 wherein the antimicrobial solution is applied by soaking the allograft tissue in the antimicrobial solution.

8. The method of claim 1 wherein the antimicrobial solution is applied by rinsing the allograft tissue with the antimicrobial solution.

9. The method of claim 1 wherein the allograft tissue is selected from the group consisting of allograft bone, autograft bone, xenograft bone, allograft cartilage, amniotic tissue, ligament tissue, tendon tissue, porous tissue and soft tissue.

10. The method of claim 1 wherein the allograft tissue is allograft bone.

11. The method of claim 1 wherein the allograft tissue is amniotic tissue.

12. The method of claim 1 wherein the allograft tissue is allograft cartilage.

13. The method of claim 1 wherein the antimicrobial activity inhibiting, controlling or combating microbial organisms.

14. The method of claim 12 wherein the microbial organisms are selected from the group consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis spores and Escherichia coli.

15. The method of claim of claim 1 wherein the antimicrobial solution is applied by soaking the allograft tissue in the antimicrobial solution for about 2 hours to about 24 hours, the antimicrobial solution comprises about 0.1% zinc chloride to about 0.5% zinc chloride.

16. The method of claim 1 wherein the antimicrobial solution is applied by soaking the allograft tissue in the antimicrobial solution for about 2 hours to about 6 hours, the antimicrobial solution comprises about 0.3% zinc chloride to about 0.5% zinc chloride.

17. The method of claim 1 wherein the antimicrobial solution comprises Zn.sup.2+ ions.

18. The method of claim 1 wherein the antimicrobial solution further comprises a pharmaceutically acceptable vehicle, excipient, diluent or adjuvant.

19. Allograft tissue prepared according to the method of claim 1.

20. The allograft tissue of claim 19 wherein the antimicrobial solution comprises a zinc compound.

21. The allograft tissue of claim 19 wherein the allograft tissue is selected from the group consisting of allograft bone, autograft bone, xenograft bone, allograft cartilage, amniotic tissue, ligament tissue, tendon tissue, porous tissue and soft tissue.

22. The allograft tissue of claim 19 wherein the allograft tissue is allograft bone.

23. The allograft tissue of claim 19 wherein the antimicrobial solution is applied by soaking the allograft tissue in the antimicrobial solution for about 2 hours to about 6 hours, the antimicrobial solution comprises about 0.3% zinc chloride to about 0.5% zinc chloride

24. The allograft tissue of claim 19 wherein the antimicrobial solution comprises Zn.sup.2+ ions.

25. A method of claim 1 wherein the method is used in combination with an allograft method, autograft method, xenograft method, alloplastic graft method, or orthopedic biocomposite method.

Description

DETAILED DESCRIPTION

[0009] In the first aspect the present invention relates to a method of inhibiting the growth of bacterium in allograft tissue comprising mechanically applying an antimicrobial solution to the allograft. The present invention also relates to controlling or combating microbial organisms, comprising mechanically applying an antimicrobial solution to the microbial organism, wherein the antimicrobial solution exhibits antimicrobial activity.

[0010] The term “antimicrobial activity” means in the context of the present invention that the antimicrobial of the invention is active in inhibiting, controlling or combating microbial organisms, including fungal organisms, and/or bacterial organisms, such as gram-positive and gram-negative bacteria. The antimicrobial activity can occur after the allograft is implanted into the body.

[0011] In a preferred embodiment, the antibacterial activity is the activity for bacterium selected from the group consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis spores and Escherichia coli.

[0012] The present invention also relates to an antimicrobial composition comprising, an antimicrobial agent of the invention and pharmaceutically acceptable vehicles, excipients, diluents, and adjuvants.

[0013] Zinc compounds suitable for use in the antimicrobial solution of the present invention include inorganic zinc compounds, such as mineral acid zinc salts. Examples of inorganic zinc compounds include, but are not limited to, zinc chloride, zinc sulfate, zinc phosphate, zinc carbonate, and zinc nitrate, zinc chlorate, zinc chromate or combinations thereof.

[0014] Zinc compounds which can be used in the antimicrobial solution can also be zinc salts of organic acids. Examples of organic acid zinc salts include, but are not limited to, zinc acetate, zinc formate, zinc propionate, zinc gluconate, bis(maltolato)zinc, zinc acexamate, zinc aspartate, bis(maltolato)zinc(II) [Zn(ma)2], bis(2-hydroxypyridine-N-oxido)zinc(II) [Zn(hpo)2], bis(allixinato)Zn(II) [Zn(alx)2], bis(6-methylpicolinato)Zn(II) [Zn(6mpa)2], bis(aspirinato)zinc(II), bis(pyrrole-2-carboxylato)zinc [Zn(pc)2], bis(alpha-furonic acidato)zinc [Zn(fa)2], bis(thiophene-2-carboxylato)zinc [Zn(tc)2], bis(thiophene-2-acetato)zinc [Zn(ta)2], (N-acetyl-L-cysteinato)Zn(II) [Zn(nac)], zinc(II)/poly(γ-glutamic acid) [Zn(γ-pga)], bis(pyrrolidine-N-dithiocarbamate)zinc(II) [Zn(pdc).sub.2], zinc(II) L-lactate [Zn(lac).sub.2], zinc(II) D-(2)-quinic acid [Zn(qui).sub.2], bis(1,6-dimethyl-3-hydroxy-5-methoxy-2-pentyl-1,4-dihydropyridine-4-thion-ato)zinc(II) [Zn(tanm)2], β-alanyl-L-histidinato zinc(II) (AHZ), or the like, or combinations thereof. In another embodiment, the organic acid of zinc salt is a naturally occurring fatty acid.

[0015] In one embodiment, concentrations of zinc chloride of about 1 mM to about 10 mM can be used as the antimicrobial solution. In an alternative embodiment zinc salts can be used in the antimicrobial solution in concentrations of about 30 mM to about 100 mM.

[0016] In one embodiment, allograft bone was soaked in the antimicrobial solution. For example, the allograft bone can be soaked in the antimicrobial solutions for from about 2 hours to about 24 hours, preferably from about 2 hours to about 6 hours. In one embodiment, the antimicrobial solution is applied by soaking the allograft tissue in the antimicrobial solution for about 2 hours to about 24 hours, the antimicrobial solution comprises about 0.1% zinc chloride to about 0.5% zinc chloride, preferably about 0.3% zinc chloride to about 0.5% zinc chloride.

[0017] Alternatively, allograft bone was rinsed with the antimicrobial solution such as by The Shake Flask Method (ASTM E2149). For example, the allograft bone can be rinsed in the antimicrobial solution for from about 45 minutes to about 120 minutes.

[0018] For purposes of the following description, allograft bone is referred to as an exemplary tissue that may be processed according to the present method. However, those skilled in the art will recognize that other tissues, including but not limited to autograft bone, xenograft bone, allograft cartilage, allograft amniotic tissue, other porous tissues, synthetic porous materials, and various soft tissues, may be processed according to the principles defined herein, without departing from the spirit of the invention exemplified herein by reference to allograft bone material. A suitable allograft cartilage is manufactured by Anthrex as BioCartilage®. In one embodiment, the soft tissue is a ligament or a tendon.

[0019] In another embodiment of this aspect, the method of the present invention is used in combination with an allograft method, autograft method, xenograft method, alloplastic graft method, or orthopedic biocomposite method.

[0020] In another embodiment of this aspect, the patient is a mammalian animal. In another embodiment of this aspect, the patient is a human.

[0021] The invention can be further illustrated by the following examples thereof, although it will be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated. All percentages, ratios, and parts herein, in the Specification, Examples, and Claims, are by weight and are approximations unless otherwise stated.

EXAMPLES

Example 1

Materials and Methods

[0022] A suspension of test organism staphylococcus aureus (ATCC 6538) was obtained by adding colonies on tryptic soy agar and 5% Sheep Blood (BAP) with incubation parameters of 35° C.-37° C., aerobic in 0.85% saline to match a 3.0 McFarland standard. The suspension was exposed to a test substance of zinc chloride at specified concentrations of 0.1%, 0.3% and 0.5% from a 1% stock solution at specified exposure times of 10 minutes, 2 hours, 6 hours and 24 hours at a temperature of 37±1° C. (37.2° C.). Latheen broth and 1% sodium bicarbonate (9.9 mL) was used as a neutralizer. After exposure, an aliquot of the suspension was transferred to the neutralizer and was assayed for survivors. Tables 1-3 show results of controls used in this example.

TABLE-US-00001 TABLE 1 CONTROL RESULTS Type of Control Results Purity Staphylococcus aureus (ATCC 6538) Pure Neutralizer Sterility Control No Growth

TABLE-US-00002 TABLE 2 TEST POPULATION CONTROL RESULTS Results Average Geometric Test Organism Timepoint CFU/mL Log.sub.10 Log.sub.10 Mean Staphylococcus T.sub.0 3.5 × 10.sup.6 6.54 5.67 4.68 × 10.sup.5 aureus 24 Hour 6.2 × 10.sup.4 4.79 (ATCC 6538) CFU = Colony Forming Units

TABLE-US-00003 TABLE 3 NEUTRALIZATION CONFIRMATION CONTROL RESULTS Neutralization Confirmation Pass/ (CFU) Fail Test (Log.sub.10 Test Test Numbers Substance Differ- Substance Organism Control Results ence) Zinc Staphylococcus 34, 19 15, 13 Pass chloride aureus (0.28) Lot# (ATCC 6538) A0340879 CFU = Colony Forming Units

Results

[0023] Table 4 shows test results to evaluate antimicrobial effectiveness on staphylococcus aureus for the experiments of this example. Table 5 shows calculated data for percent and Log 10 reduction of the test results shown in Table 4.

TABLE-US-00004 TABLE 4 TEST RESULTS FOR Staphylococcus aureus DILUTION Exposure Time (VOLUME 10 minutes 2 hours 6 hours 24 hours PLATED) Number of Survivors Test Substance: 0.1% Zinc Chloride 10.sup.0 (1.00 mL) T, T 56, 42 22, 23 4, 3 10.sup.0 (0.100 mL) T, T 3, 3 3, 4 0, 0 10.sup.1 (0.100 mL) T, T 1, 0 2, 0 0, 0 10.sup.2 (0.100 mL) 66, 69 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL)  9, 19 0, 0 0, 1 0, 0 Test Substance: 0.3% Zinc Chloride 10.sup.0 (1.00 mL) T, T 0, 2 0, 0 0, 0 10.sup.0 (0.100 mL) T, T 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 60, 57 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 5, 6 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 1, 3 0, 0 0, 0 0, 0 Test Substance: 0.5% Zinc Chloride 10.sup.0 (1.00 mL) T, T 2, 0 0, 0 0, 0 10.sup.0 (0.100 mL) T, T 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 32, 26 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 5, 5 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 0, 1 0, 0 0, 0 0, 0 T = Too Numerous To Count (>300 colonies) A value of <1 was used in place of zero for calculation purposes.

TABLE-US-00005 TABLE 5 CALCULATED DATA FOR Staphylococcus aureus CFU/mL in Test Population Exposure Control CFU/mL of Log.sub.10 Percent Log.sub.10 Test Substance Time (Log.sub.10) Survivors Survivors Reduction Reduction 0.1% Zinc Chloride 10 minutes 4.68 × 10.sup.5 6.8 × 10.sup.5 5.83 None None  2 hours (5.67) 4.9 × 10.sup.2 2.69  >99.8% 2.98  6 hours 2.3 × 10.sup.2 2.36  >99.9% 3.31 24 hours   4 × 10.sup.1 1.60   99.99% 4.07 0.3% Zinc Chloride 10 minutes 5.9 × 10.sup.4 4.77    87.4% 0.90  2 hours   1 × 10.sup.1 1.00 >99.99% 4.67  6 hours <5 <0.70 >99.99% >4.97 24 hours <5 <0.70 >99.99% >4.97 0.5% Zinc Chloride 10 minutes 2.9 × 10.sup.4 4.46    93.8% 1.21  2 hours   1 × 10.sup.1 1.00 >99.99% 4.67  6 hours <5 <0.70 >99.99% >4.97 24 hours <5 <0.70 >99.99% >4.97 CFU = Colony Forming Units The geometric mean and average log.sub.10 values were used for the population control.

Example 2

Materials and Methods

[0024] A suspension of test organism staphylococcus aureus (ATCC 6538) was obtained by adding colonies on tryptic soy agar and 5% Sheep Blood (BAP) with incubation parameters of 35° C.-37° C., aerobic in 0.85% saline to match a 3.0 McFarland standard. The suspension was exposed to a test substance of zinc chloride (4.75 mL) with allograft bone 0.25 g at specified concentrations of 0.3% and 0.5% from a 1% stock solution at specified exposure times of 2 hours, 6 hours and 24 hours at a temperature of 37±1° C. Latheen broth and 1% sodium bicarbonate (9.9 mL) was used as a neutralizer. After exposure, an aliquot of the suspension was transferred to the neutralizer and was assayed for survivors. Tables 6-8 show results of controls used in this example.

TABLE-US-00006 TABLE 6 CONTROL RESULTS Type of Control Results Purity Staphylococcus aureus (ATCC 6538) Pure Neutralizer Sterility Control No Growth

TABLE-US-00007 TABLE 7 TEST POPULATION CONTROL RESULTS Results Average Geometric Test Organism Timepoint CFU/mL Log.sub.10 Log.sub.10 Mean Staphylococcus T.sub.0 1.15 × 10.sup.6  6.06 5.83 6.76 × 10.sup.5 aureus 24 Hour 3.9 × 10.sup.5 5.59 (ATCC 6538) CFU = Colony Forming Units

TABLE-US-00008 TABLE 8 NEUTRALIZATION CONFIRMATION CONTROL RESULTS Neutralization Confirmation Pass/ (CFU) Fail Test (Log.sub.10 Test Test Numbers Substance Differ- Substance Organism Control Results ence) Zinc Staphylococcus 33, 34 30, 21 Pass chloride aureus (0.12) (ATCC 6538) CFU = Colony Forming Units

Results

[0025] Table 9 shows test results for staphylococcus aureus for the experiments of this example. Table 10 shows calculated data of percent and Log 10 reduction for the test results shown in Table 9.

TABLE-US-00009 TABLE 9 TEST RESULTS FOR Staphylococcus aureus Number of Survivors DILUTION Exposure Time (VOLUME 2 hours 6 hours 24 hours PLATED) Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Test Substance: 0.3% Zinc Chloride with allograft bone 10.sup.0 (1.00 mL) T, T T, T T, T T, T T, T T, T 12, 2  23, 25 8, 7 10.sup.0 (0.100 mL) T, T T, T T, T T, T T, T T, T 0, 4 1, 8 0, 0 10.sup.1 (0.100 mL) T, T T, T T, T 117, 151 210, 224 254, 206 0, 0 1, 1 0, 0 10.sup.2 (0.100 mL) 69, 63 54, 71  85, 101 14, 3 33, 30 25, 32 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 8, 9 7, 9 13, 12 1, 1 3, 3 3, 5 0, 0 0, 0 0, 0 Test Substance: 0.5% Zinc Chloride with allograft bone 10.sup.0 (1.00 mL) T, T T, T T, T T, T T, T T, T  9, 15 5, 5 5, 3 10.sup.0 (0.100 mL) T, T T, T T, T T, T T, T T, T 3, 2 0, 1 1, 0 10.sup.1 (0.100 mL) T, T T, T T, T 78, 73 67, 66 68, 59 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 74, 70 130, 117 86, 74 6, 4 4, 8 2, 2 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 15, 9  11, 17 13, 9  1, 0 0, 0 1, 0 0, 0 0, 0 0, 0 Rep = Replicate T = Too Numerous To Count (>300 colonies)

TABLE-US-00010 TABLE 10 CALCULATED DATA FOR Staphylococcus aureus CFU/mL in Test Geometric Percent Population Mean Reduction Exposure Control CFU/mL of Log.sub.10 (Average (Log.sub.10 Test Substance Time (Log.sub.10) Survivors Survivors Log.sub.10) Reduction) 0.3% Zinc Chloride 2 hours 6.76 × 10.sup.5 6.6 × 10.sup.5 5.82 7.24 × 10.sup.5 No with allograft bone (5.83) 6.3 × 10.sup.5 5.80 (5.86) Reduction 9.3 × 10.sup.6 5.97 6 hours 1.34 × 10.sup.5  5.13 1.91 × 10.sup.5 71.7% 2.17 × 10.sup.5 5.34 (5.28) (0.55) 2.30 × 10.sup.5 5.36 24 hours  1.2 × 10.sup.2 2.08 1.32 × 10.sup.2 >99.9%   2.4 × 10.sup.2 2.38 (2.12) (3.71)   8 × 10.sup.1 1.90 0.5% Zinc Chloride 2 hours 7.2 × 10.sup.5 5.86 8.91 × 10.sup.5 No with allograft bone 1.24 × 10.sup.6  6.09 (5.95) Reduction 8.0 × 10.sup.5 5.90 6 hours 7.6 × 10.sup.5 4.88 6.92 × 10.sup.4 89.8% 6.7 × 10.sup.4 4.83 (4.84) (0.99) 6.4 × 10.sup.4 4.81 24 hours  1.2 × 10.sup.2 2.08 6.17 × 10.sup.1 99.99%    5 × 10.sup.1 1.70 (1.79) (4.04)   4 × 10.sup.1 1.60 CFU = Colony Forming Units The geometric mean and average log.sub.10 values were used for the test replicates and population control.

Example 3

Materials and Methods

[0026] A suspension of test organism staphylococcus aureus (ATCC 6538) was obtained by adding colonies on tryptic soy agar and 5% Sheep Blood (BAP) with incubation parameters of 35° C.-37° C., aerobic in 0.85% saline to match a 3.0 McFarland standard. The suspension was exposed to a test BioCartilage® from Anthrex substance of 4.75 mL of zinc chloride at specified concentrations of 0.3% and 0.5% containing 0.25 g BioCartilage ® at specified exposure times of 6 hours and 24 hours at a temperature of 37±1° C. Latheen broth and 1% sodium bicarbonate (9.9 mL) was used as a neutralizer. After exposure, an aliquot of the suspension was transferred to the neutralizer and was assayed for survivors. The suspension was also exposed to a test Amnion Matrix from Anthrex substance of 9.5 mL of zinc chloride at specified concentrations of 0.3% and 0.5% containing Amnion Matrix (1 count) at specified exposure times of 6 hours and 24 hours at a temperature of 37+−1 C. Latheen broth and 1% sodium bicarbonate (9.9 mL) was used as a neutralizer. After exposure, an aliquot of the suspension was transferred to the neutralizer and was assayed for survivors. A

[0027] Tables 11-13 show controls used in this example.

TABLE-US-00011 TABLE 11 CONTROL RESULTS Type of Control Results Purity Staphylococcus aureus (ATCC 6538) Pure Neutralizer Sterility Control No Growth

TABLE-US-00012 TABLE 12 TEST POPULATION CONTROL RESULTS Results Test Organism Time Point CFU/mL Log.sub.10 Staphylococcus Time Zero 1.54 × 10.sup.6  6.19 aureus 24 Hours 9.8 × 10.sup.4 4.99 (ATCC 6538) Average Log.sub.10: 5.59 Geometric Mean: 3.89 × 10.sup.5 CFU = Colony Forming Units

TABLE-US-00013 TABLE 13 NEUTRALIZATION CONFIRMATION CONTROL RESULTS Neutralization Confirmation Pass/ (CFU) Fail Test (Log.sub.10 Test Test Numbers Substance Differ- Substance Organism Control Results ence) Zinc Staphylococcus 36, 29 38, 36 Pass chloride (with aureus (−0.05) BioCartilage ®) (ATCC 6538) Zinc 36, 29 59, 46 Pass chloride (with (−0.20) Amnion Matrix) CFU = Colony Forming Units

[0028] Table 14 shows test results for staphylococcus aureus for the BioCartilage® experiments of this example. Table 15 shows test results for staphylococcus aureus for the Amnion Matrix experiments of this example. Table 16 shows calculated data of percent and Log 10 reduction for the test results shown in Table 14. Table 17 shows calculated data of percent and Log 10 reduction for the test results shown in Table 15.

TABLE-US-00014 TABLE 14 SHOWS TEST RESULTS FOR ZINC CHLORIDE (with BioCartilage ®) Test Organism: Staphylococcus aureus (ATCC 6538) Number of Survivors DILUTION Exposure Time (VOLUME 6 hours 24 hours PLATED) Replicate 1 Replicate 2 Replicate 1 Replicate 2 Test Substance: 0.3% Zinc chloride with BioCartilage ® 10.sup.0 (1.00 mL) 35, 42 51, 28 0, 0 0, 0 10.sup.0 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 Test Substance: 0.5% Zinc chloride with BioCartilage ® 10.sup.0 (1.00 mL) 29, 21 35, 41 0, 0 0, 0 10.sup.0 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 0, 0 0, 0 0, 0 0, 0

TABLE-US-00015 TABLE 15 SHOWS TEST RESULTS FOR ZINC CHLORIDE (with Amnion Matrix) Test Organism: Staphylococcus aureus (ATCC 6538) Number of Survivors DILUTION Exposure Time (VOLUME 6 hours 24 hours PLATED) Replicate 1 Replicate 2 Replicate 1 Replicate 2 Test Substance: 0.3% Zinc chloride with Amnion Matrix 10.sup.0 (1.00 mL) 8, 7 19, 18 0, 0 0, 0 10.sup.0 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 Test Substance: 0.5% Zinc chloride with Amnion Matrix 10.sup.0 (1.00 mL) 84, 74 4, 2 0, 0 0, 0 10.sup.0 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.1 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.2 (0.100 mL) 0, 0 0, 0 0, 0 0, 0 10.sup.3 (0.100 mL) 0, 0 0, 0 0, 0 0, 0

TABLE-US-00016 TABLE 16 CALCULATED DATA FOR ZINC CHLORIDE (with BioCartilage ®) CFU/mL in Test Geometric Percent Population Mean Reduction Exposure Control CFU/mL of Log.sub.10 (Average (Log.sub.10 Test Substance Time (Log.sub.10) Survivors Survivors Log.sub.10) Reduction) 0.3% Zinc chloride  6 hours 3.89 × 10.sup.5 3.9 × 10.sup.2 2.59 3.98 × 10.sup.2  >99.8% with BioCartilage ® (5.59) 4.0 × 10.sup.2 2.60   (2.60)   (2.99) 24 hours <5 <0.70 <5.01 >99.99% <5 <0.70 (<0.70) (>4.89) 0.5% Zinc chloride  6 hours 2.5 × 10.sup.2 2.40 3.09 × 10.sup.2    99.9% with BioCartilage ® 3.8 × 10.sup.2 2.58   (2.49)   (3.10) 24 hours <5 <0.70 <5.01 >99.99% <5 <0.70 (<0.70) (>4.89) CFU = Colony Forming Units A value of <1 was used in place of zero for calculation purposes. The geometric mean and average log.sub.10 values were used for the population control. The geometric mean and average log.sub.10 values were used for the test replicates to determine reductions.

TABLE-US-00017 TABLE 17 CALCULATED DATA FOR ZINC CHLORIDE (with Amnion Matrix) CFU/mL in Test Geometric Percent Population Mean Reduction Exposure Control CFU/mL of Log.sub.10 (Average (Log.sub.10 Test Substance Time (Log.sub.10) Survivors Survivors Log.sub.10) Reduction) 0.3% Zinc chloride  6 hours 3.89 × 10.sup.5   8 × 10.sup.1 1.90 1.23 × 10.sup.2  >99.9% with Amnion Matrix (5.59) 1.9 × 10.sup.2 2.28   (2.09)   (3.50) 24 hours <5 <0.70 <5.01 >99.99% <5 <0.70 (<0.70) (>4.89) 0.5% Zinc chloride  6 hours 7.9 × 10.sup.2 2.90 1.55 × 10.sup.2  >99.9% with Amnion Matrix   3 × 10.sup.1 1.48   (2.19)   (3.40) 24 hours <5 <0.70 <5.01 >99.99% <5 <0.70 (<0.70) (>4.89) CFU = Colony Forming Units A value of <1 was used in place of zero for calculation purposes. The geometric mean and average log.sub.10 values were used for the population control. The geometric mean and average log.sub.10 values were used for the test replicates to determine reductions.

[0029] It is to be understood that the above-described embodiments are illustrative of only a few of the many possible specific embodiments, which can represent applications of the principles of the invention. Numerous and varied other arrangements can be readily devised in accordance with these principles by those skilled in the art without departing from the spirit and scope of the invention.