Bovine Acellular Dermal Matrix
» Bovine Acellular Dermal Matrix is a remodelable collagen scaffold derived from fetal or neonatal bovine skin. It is presented to the physician as a flat white sheet that is cut to size and hydrated in room temperature sterile saline solution prior to implantation. It is utilized as a structural scaffold in orthopedic, neurosurgical, urogynecological, dermatological, plastic, and other reconstructive procedures to contribute to the repair, reinforcement, and generation of tissue. The sterile material is surgically secured, onlayed, and/or packed into deficient soft tissues such as skin, tendon, muscle, and dura mater.
The source fetal or neonatal bovine skin is mechanically and chemically processed to isolate the dermis and remove cells and cellular components. To prevent the transmission of infectious disease, the manufacturing process has been validated to inactivate viruses potentially present in the source material. To prevent the spread of transmissible spongiform encephalopathies, the source material is acquired from appropriate geographic locations in accordance with relevant guidelines subject to governmental oversight. The product is inspected and tested to assure the product meets specifications.
Packaging and storage— The package is a sealed, foil pouch that provides an effective moisture, light, gas, and sterility barrier. Store in clean, dry conditions between 15 and 30.
Labeling— Label it to indicate that it is derived from bovine origin. The product is labeled to indicate the product's intended clinical use. It is labeled with the dimensions of the product, the expiration date, the required storage conditions, lot number, part number, and the manufacturer’s name and address. The label indicates that the product is sterile and nonpyrogenic and is designed for single patient, one-time use. The labeling cautions the user to inspect the packaging for damage and to discard the product if the packaging has been compromised. The labeling also cautions the user to hydrate the product only in room temperature sterile saline solution.
USP Authentic Visual References 11— USP Bovine Acellular Dermal Matrix Reference Photomicrographs. These Photomicrographs show the histological appearance of failed, cell-containing source material (Photomicrographs 1 and 2) and of passing, processed, decellularized material (Photomicrographs 3 and 4). The samples were prepared as directed in the test for Histological evaluation.
Sterility 71: meets the requirements.
Histological evaluation—
solution preparations—
1% Acid alcohol— To 99 mL of 70% ethyl alcohol add 1 mL of hydrochloric acid (37.5%).
Potassium alum solution— Dissolve 100 g of potassium alum in 1000 mL of distilled water with the aid of heat and a magnetic stirrer.
Hematoxylin–alcohol solution— Dissolve 5 g of hematoxylin (see Reagent Specifications under Reagents, Indicators, and Solutions) in 50 mL of 100% ethyl alcohol at room temperature.
Hematoxylin solution— Slowly combine the 1000 mL of the Potassium alum solution with the 50 mL of the Hematoxylin–alcohol solution. Bring to a boil as rapidly as possible. Remove from heat and slowly add 2.5 g of mercuric oxide. Return the solution to heat until it becomes dark purple, remove from heat, and cool in a sink of cold water.
Eosin solution— Dissolve 1.0 g of eosin Y, water soluble, in 100 mL of distilled water. Dissolve 1.0 g of phloxine B in 100.0 mL of distilled water. Combine 100 mL of eosin Y solution with 10 mL of phloxine B solution, 780 mL of 95% ethyl alcohol, and 4.0 mL of glacial acetic acid. [note—Filter daily before use.]
Bluing agent— Dissolve 1.54 g of lithium carbonate in 100 mL of distilled water.
10% Neutral buffered formalin— To 6.5 g of dibasic sodium phosphate (anhydrous) and 4.0 g of monobasic sodium phosphate, add 900 mL of distilled water and 100 mL of formaldehyde (37% to 40%).
sample preparation and staining— Remove a sample of finished product with an 8.0-mm biopsy punch. Place the sample in a labeled tissue cassette, and fix for 24 hours in 10% Neutral buffered formalin. Dehydrate the sample in sequential soaks of the following: 70% ethyl alcohol (45 minutes), 80% ethyl alcohol (45 minutes), 95% ethyl alcohol (90 minutes), 100% ethyl alcohol (180 minutes), and xylene (90 minutes). Embed the sample in melted paraffin, cool, and cut 5-µm thick sections with a microtome. Collect sections on microscope slides. Deparaffinize the slide with xylene and hydrate with distilled water. Stain in Hematoxylin solution for 6 to 15 minutes. Wash in running tap water for 2 to 5 minutes. Dip two times in 1% Acid alcohol. Wash briefly in tap water. Place in Bluing agent until the sections are bright blue. Wash in running tap water for 10 minutes. Place in 80% ethyl alcohol for 1 to 2 minutes. Dehydrate and clear through two changes each of 95% ethyl alcohol, 100% ethyl alcohol, and xylene, 2 minutes each. Affix a coverslip over the tissue using an appropriate resinous mounting media. The nuclei stains blue, the cytoplasm stains from pink to red, and the collagen fibers stain from pink to red.
microscopic and morphological characteristics— The collagen fibers of the Bovine Acellular Dermal Matrix stain pink-red, and no evidence of cell nuclei or cytoplasm are apparent in prepared histological sections as shown in the USP Bovine Acellular Dermal Matrix Reference Photomicrographs of products with acceptable histological appearance.
Protein determination— Use the Kjeldahl nitrogen (protein) determination method to calculate the percent protein of the final product as directed under Nitrogen Determination 461 with the following specifics. Suitable equipment and procedures are readily available.1
Digestion— Prepare a rack of 15 to 20 Kjeldahl digestion tubes. In each, place 2.0 to 2.2 g of final product, 0.2 ± 0.05 g of ammonium sulfate, a metallic catalyst tablet,2 and boiling chips.3 Prepare a blank tube with catalyst tablets and boiling chips (reagent blank). To each tube add 15 mL of concentrated sulfuric acid, and then, very slowly, 3 mL of hydrogen peroxide (30% to 35%). Place the digestion tubes on a digestion block, and heat to 410. Digest for 60 ± 5 minutes. The mixture in the tubes should be a clear green.
Distillation— Add excess base (50% sodium hydroxide). Generally, for each 5 mL of concentrated sulfuric acid used in the digestion, 20 mL of 40% (w/w) sodium hydroxide is required to make the digest strongly alkaline (pH >11). Mix each tube and let cool to room temperature. Distill each tube to collect approximately 125 mL of total distillate in a flask containing 25 mL of 4% boric acid. A reagent blank is run with each set.
Titration— Titrate the collected distillate with standardized 0.2 N sulfuric acid to a neutral gray color endpoint. Record the volume of sulfuric acid used.
Calculation— Calculate the percentage of protein by the formula:
Protein % = [mL of sulfuric acid mL blank) × N of sulfuric acid × 1.4007 × 6.25] / weight of sample (g)
where 1.4007 is the milliequivalent weight N × 100(%); and 6.25 is the protein factor for meat. The percentage of protein in 2.0 to 2.2 g of Bovine Acellular Dermal Matrix sample is between 90.0% and 95.0%.
Lipid analysis— A standard Soxhlet extraction apparatus is required. Dry flasks in an oven/dessicator and weigh, recording the weight to the nearest 0.0001 g. Grind or cut into small pieces 3.0 to 4.0 g of test material and place into a thimble. Record the weight of the test material to the nearest 0.0001 g. Place the thimble of material and 80 to 90 mL of petroleum ether into an extraction flask, and place into the Soxhlet extraction tube. Reflux for 4 hours. Collect all of the ether into the flask, and evaporate. Weigh the flask, recording weight to the nearest 0.0001 g.
Calculation— For the weight of lipid, substract the weight of the clean flask from the final weight of the flask. Calculate the percent of lipid based on the weight of the starting material. The percentage of lipid in 3.0 to 4.0 g of Bovine Acellular Dermal Matrix sample is between 0% and 1.5%.
Moisture content— Proceed as directed under Loss on Drying 731 to calculate the moisture content, with the following specifics. Mince approximately 5.0 g of Bovine Acellular Dermal Matrix; place it into an aluminum dish. Dry the sample in an air oven for 16 to 18 hours at 100 to 102.
Calculation— Calculate the percentage of moisture in the sample taken by the formula:
Dry matter % = [(wt. of dried sample & pan (g) wt. of pan (g)) / g of sample] ×100
Moisture % = 100 (dry matter %).
The moisture loss is not less than 10.0% and not more than 12.0% of the original sample weight.
Ash determination— Place a sample of the final product, about 5.0 g, in a kiln-dried, porcelain crucible. Record the weight to the nearest 0.0001 g. Place the crucible containing the sample into an oven at 125 for 2 to 4 hours. Then place the crucible containing the sample into a cool muffle furnace. Heat the furnace to 350, and maintain the temperature until smoking ceases (generally about 20 minutes). Heat the furnace to 550. Maintain the temperature for 2 hours. Cool the crucible in a desiccator. Weigh the crucible, and record the weight to the nearest 0.0001 g.
Calculation— Calculate the percentage of ash by the following formula:
Ash % = [(wt. of crucible & residue (g) wt. of crucible (g)) / g of sample] × 100
The percentage of ash is between 0% and 0.3%.
Carbohydrates—
Calculation— The percentage of carbohydrates is determined by the following formula:
Carbohydrate % = 100% (lipid % + protein % + moisture % + ash %)
The percentage of carbohydrates is equal to or less than 0.0%. Because this is a calculated value, influenced by the error inherent in the test methods above (Lipid analysis, Moisture content, and Ash determination), a calculated value less than 0.0% is acceptable.
Gel electrophoresis— Use the electrophoresis determination method as directed under Biotechnology Derived Articles—Polyacrylamide Gel Electrophoresis 1056 with the following specifics.
solution preparations—
Collagen extraction solution— Prepare a 0.5 M acetic acid solution containing 2 mM ethylenediaminetetraacetic acid (EDTA).
2X Tris-glycine sample buffer— Prepare a 2X solution containing 63 mM Tris-HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 0.05% 2-mercaptoethanol, and 0.25% bromophenol blue.4
1X Sample buffer— Prepare a solution containing a mixture of 2X Tris-glycine sample buffer and water (1:1).
SDS-PAGE running buffer— Prepare a solution containing 25 mM Tris pH 8.3, 192 mM glycine, and 0.1% SDS.5
Polyacrylamide gel— Prepare a Tris-HCl polyacrylamide gel with a 4% to 20% gradient.6
Molecular weight marker— Use a suitable molecular weight marker containing protein bands between 10 and 250 kilodaltons (kDa).
Staining solution— Prepare a solution containing 0.25% (w/v) Coomassie brilliant blue R-250 (see Reagent Specifications under Reagents, Indicators, and Solutions) in 10% acetic acid and 10% n-propanol.
Destain solution— Prepare a mixture of water, acetic acid, and n-propanol (8:1:1).
collagen preparations— Mince 0.5 g of Bovine Acellular Dermal Matrix final product. Weigh a sample of minced Bovine Acellular Dermal Matrix, and add to a volume of Collagen extraction solution to obtain a concentration of 5 mg per mL (dry weight of Bovine Acellular Dermal Matrix). Extract on a rocking platform at room temperature for 72 hours.
Procedure— Dilute acid-extracted collagen samples in 2X Tris-glycine sample buffer to a concentration of 0.5 mg per mL, and incubate for 5 minutes at 100. Load the Polyacrylamide gel in the electrophoresis apparatus, and add SDS-PAGE running buffer to the top and bottom reservoirs. Load 10 µL of Molecular weight markers in the first well of the Polyacrylamide gel and 10 µL of 1X Sample buffer in the second well. Load 10 µL (5 µg) of each collagen sample into subsequent gel wells. Attach the cathode and anode to the appropriate terminals, and apply 110 V to the gel. Run the gel until the bromophenol blue reaches the bottom of the gel. Remove the gel from the electrophoresis apparatus, and place it in a tray containing enough Staining solution to cover the gel. Incubate the gel for 3 hours on a rocker at room temperature. Completely remove the Staining solution from the tray, cover the gel with Destain solution, and slowly rock the gel for 20 minutes. Remove the Destain solution, and repeat the destaining procedure three times. Inspect the gel for bands that have migrated from the origin.
System suitability— All bands between 20 and 200 kDa are present. The lane containing 1X Sample buffer does not contain any bands.
Data analysis— Where a protein band appears in the gel, the molecular weight of this protein is determined by comparing the position of the band to that of the known Molecular weight marker.
Specificity— The lanes of the Polyacrylamide gel that correspond to Bovine Acellular Dermal Matrix show four major protein bands. Two bands, when compared to the Molecular weight marker, appear at 96 and 94 kDa. These two bands correspond to the monomeric alpha 1 and alpha 2 chains of collagen Type I, respectively. Another two bands appear close together at 200 kDa, which correspond to alpha 1 and alpha 1/alpha 2 collagen dimers.
Tensile strength— Cut test specimens 5-mm wide × 50-mm long from representative pieces from final product lots. Measure the thickness of the specimen. Test the specimens with a commercially available material test system.7 Mount and align the specimen, gripping 1 cm of the test specimen on both ends to ensure a test specimen gauge length of 3 cm. Pull the grips apart at 30 mm per minute while concurrently measuring the force exerted on the specimen. Record the maximum force (N) measured during the test.
Calculation— Calculate the tensile strength by the formula:
Tensile strength (N/mm2) = maximum force (N)/5 (mm) × thickness (mm)
The measured tensile strength for each lot is not less than 5 N per mm2.
Suture retention force— Cut representative 1 × 1 cm test specimens from final product lots. Using an appropriate suture material (e.g., 40 polypropylene suture), thread the suture 3 mm from the edge of the sample in the center and pull through. Clamp approximately 5 mm of the opposite, unsutured end of the test specimen in the upper pneumatic grip of a commercially available material test system.8 The suture tails are hanging freely. Clamp the suture tails to the lower grip. Pull the grips apart at 20 mm per minute while concurrently measuring the force exerted. Record the maximum force (N) measured. The suture retention force measured for each lot is not less than 5 N for a 1 × 1 cm test sample of the Bovine Acellular Dermal Matrix.
Thermal analysis— A final product sample of approximately 10 to 20 mg is heated at 2 per minute from 30 to 90, hydrated with water, and the thermal characteristics of each processed skin is measured with a differential scanning calorimeter as directed under Thermal Analysis 891. Bovine Acellular Dermal Matrix displays a single thermal transition peak between 58 and 67.
Visual inspection— Each piece of final product is visually inspected under a white light at a distance of 30 to 45 cm for color, the presence of particulates, and holes. Bovine Acellular Dermal Matrix is white, and neither particulates nor holes are visible.
Hydration rate— Cut a sample of finished product lot approximately 1 × 1 cm. The sample fully hydrates, as indicated by a change in color from white to gray, in less than 3 minutes when placed in room temperature saline solution.
1  A suitable device and associated procedures can be obtained from Labconoco, 8811 Prospect Ave., Kansas City, MO.
2  A suitable catalyst is Pro-Pac CT-37, Alfie Packers, 8901 J St., Omaha, NE.
3  Commonly referred to as Henger granules.
4  A suitable sample buffer can be obtained from Invitrogen Corporation, 1600 Faraday Ave., P.O. Box 6482, Carlsbad, CA 92008.
5  A suitable gel running buffer can be obtained from Bio-Rad Laboratories, 1000 Alfred Nobel Dr., Hercules, CA 94547.
6  A suitable precast acrylamide gel can be obtained from Bio-Rad Laboratories, 1000 Alfred Nobel Dr., Hercules, CA 94547.
7  A suitable material test system is available from Instron Corporation, 825 University Ave., Norwood, MA.
8  A suitable material test system is available from Instron Corporation, 825 University Ave., Norwood, MA.
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Topic/Question Contact Expert Committee
Monograph Fouad Atouf, Ph.D.
Senior Scientific Associate
1-301-816-8365		 (BBCGT05) Biologics and Biotechnology - Cell Gene and Tissue Therapies
Reference Standards Lili Wang, Technical Services Scientist
1-301-816-8129
RSTech@usp.org
85 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
1-301-816-8339		 (MSA05) Microbiology and Sterility Assurance
71 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
1-301-816-8339		 (MSA05) Microbiology and Sterility Assurance
USP32–NF27 Page 1701
Pharmacopeial Forum: Volume No. 33(5) Page 898