Acellular dermal matrix for skin and surgical reconstruction and methods of making same
An acellular dermal matrix (ADM) is produced using a series of steps that maintains a high percentage of collagen, hydrates quickly and maintains high mechanical properties. The ADM is aseptically processed and requires no terminal sterilization. The basement membrane may be preserved in the ADM. The ADM may hydrate very quickly, wherein the ADM doubles in weight within 10 seconds or less of soaking in water at standard temperature and pressure and may triple in weight within 30 minutes. Also, the ADM may have limited thickness swelling. The ADM may preserve mechanical properties and may have mechanical properties comparable to human skin. The ADM may be decellularized and have very minimal DNA content and be non-immunogenic. The ADM may be manufactured in various sizes and thickness. The ADM may be manufactured without lyophilization or freeze-drying.
1 . A method of making the manufactured acellular dermal matrix, said method comprising:
a) providing a mammalian skin tissue comprising:
i) a collagen;
ii) proteins including glycoproteins, and lipids;
iii) genetic material including DNA;
iv) cells with a cell membrane; and
v) extracellular matrix;
b) disrupting the cell membranes of the mammalian skin tissue by washing the mammalian skin tissue with an ionic solution at a concentration of 1M or more at a temperature of 40° C. or higher for at least 3 hours to disrupt the cell membranes and produce an epidermis free tissue;
c) washing the epidermis free tissue in a recombinant serine protease enzyme to destroy the cells while preserving the extracellular matrix to produce a processed dermal tissue;
d) removing residual lipids from the processed dermal tissue using an organic solvent or surfactant-based solution containing one or more of chloroform, methanol, hexane, or surfactants to produce a lipid extracted tissue;
e) washing the lipid extracted tissue with an acid solution containing 0.01% to 2% peracetic acid for at least 45 minutes to achieve microbial inactivation to produce a microbe-free acellular dermal matrix; and
f) dehydrating the microbe-free acellular dermal matrix by washing the microbe-free acellular dermal matrix in a dehydration solution containing one or more of ethanol, methanol and acetone, and subsequently washing in xylene to selectively remove cellular debris and membrane lipids without disrupting collagen fiber organization to produce the manufactured acellular dermal matrix comprising:
i) at least 70% of the collagen;
ii) no more than 20 ng DNA/mg of the manufactured acellular dermal matrix;
iii) a solvent concentration of no more than 10 mg/g of manufactured acellular dermal matrix;
iv) a hydration rate, a time to double in weight when the manufactured acellular dermal matrix is soaked in water, of no more than 60 second;
v) an elongation at break when said manufactured acellular dermal matrix is a hydrated acellular dermal matrix of 50% or more as determined by a tensile test with a 25.4 mm (1 in) jaw gap and strain rate of 10 mm/min with a 25.4 mm (1 in)×101.6 mm (4 in) sample; and
wherein said hydrated manufactured acellular dermal matrix is soaked in water for 5 minutes; and
vi) a dry Youngs Modulus of at least 40 MPa for a dry manufactured acellular dermal matrix having no more than 20% moisture and a wet Youngs Modulus of at least 15 MPa for the hydrated manufactured acellular dermal matrix, wherein both the wet Youngs modulus and dry Youngs modulus are measured by said tensile test,
wherein the manufactured acellular dermal matrix has less than 20% moisture as determined by a Thermogravimetric Analyzer; and
wherein the manufactured acellular dermal matrix is not freeze dried or lyophilized and maintains a dry condition having no more than 20% water when maintained at standard temperature and pressure at less than 50% Rh, for a shelf life of at least 6 months.
2 . The method of claim 1 , wherein the enzymatic processing step preserves at least 70% of the native collagen structure.
3 . The method of claim 1 , wherein dehydrating the microbe-free acellular dermal matrix in a dehydration solution containing one or more of ethanol, methanol and acetone includes a series of washing in said dehydration solution with an increasing concentration of one or more of ethanol, methanol and acetone.
4 . The method of claim 3 , wherein the concentration of the dehydration solution ranges from 70% to 100%.
5 . The method of claim 1 , wherein the final processed acellular dermal matrix is characterized by a swelling ratio of no more than 50% in thickness after 30 minutes of hydration at room temperature, ensuring minimal expansion upon implantation.
6 . The method of claim 1 , wherein the sterilization process is performed using pera acetic acid and without gamma irradiation or ethylene oxide exposure, preserving native matrix bioactivity and mechanical integrity.
7 . The method of claim 1 , wherein the ionic solution comprises a sodium chloride solution.
8 . The method of claim 1 , further comprising meshing the acellular dermal matrix to produce a plurality of apertures through the acellular dermal matrix.
9 . A method of making the manufactured acellular dermal matrix, the method comprising:
a) providing a mammalian skin tissue comprising:
i) a collagen;
ii) proteins including glycoproteins, and lipids;
iii) genetic material including DNA;
iv) cells with a cell membrane; and
b) washing the mammalian skin tissue with an ionic solution at a concentration of 1M or more at a temperature of 40° C. or higher for at least 3 hours to disrupt the cell membranes and produce an epidermis free tissue;
c) washing the epidermis free tissue to a recombinant serine protease enzyme to destroy the cells while preserving the extracellular matrix to produce a processed dermal tissue;
d) removing residual lipids from the processed dermal tissue using an organic solvent or surfactant-based solution containing one or more of chloroform, methanol, hexane, or surfactants to produce a lipid extracted tissue;
e) washing the lipid extracted tissue with an acid solution containing 0.01% to 2% peracetic acid for at least 45 minutes to achieve microbial inactivation to produce a microbe-free acellular dermal matrix; and
f) finishing the microbe-free acellular dermal matrix consisting of dehydrating the microbe-free acellular dermal matrix by washing the microbe-free acellular dermal matrix in a dehydration solution containing one or more of ethanol, methanol and acetone, and subsequently washing in xylene to selectively remove cellular debris and membrane lipids without disrupting collagen fiber organization to produce the manufactured acellular dermal matrix comprising:
i) at least 70% of the collagen;
ii) no more than 20 ng DNA/mg of the manufactured acellular dermal matrix;
iii) a solvent concentration of no more than 10 mg/g of manufactured acellular dermal matrix;
iv) a hydration rate, a time to double in weight when the manufactured acellular dermal matrix is soaked in water, of no more than 60 second;
v) an elongation at break when said manufactured acellular dermal matrix is a hydrated acellular dermal matrix of 50% or more as determined by a tensile test with a 25.4 mm (1 in) jaw gap and strain rate of 10 mm/min with a 25.4 mm (1 in)×101.6 mm (4 in) sample; and
wherein said hydrated manufactured acellular dermal matrix is soaked in water for 5 minutes; and
vi) a dry Youngs Modulus of at least 40 MPa for a dry manufactured acellular dermal matrix having no more than 20% moisture and a wet Youngs Modulus of at least 15 MPa for the hydrated manufactured acellular dermal matrix, wherein both the wet Youngs modulus and dry Youngs modulus are measured by said tensile test, and
wherein the manufactured acellular dermal matrix has less than 20% moisture as determined by a Thermogravimetric Analyzer; and
wherein the manufactured acellular dermal matrix is not freeze dried or lyophilized and maintains a dry condition having no more than 20% water when maintained at standard temperature and pressure at less than 50% Rh, for a shelf life of at least 6 months.