Study On Preparation And Properties Of Porcine Acellular Dermal Matrix

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Scaffold material is one of the three factors of tissue engineering. Following the guidance of the performance standards of scaffold materials in tissue engineering, a novel porcine acellular dermal matrix (PADM) with native three-dimensional structure and ideal microstructure was prepared using a low-cost and simple preparation technology. The preparation technique was optimized and the properties of the scaffold material were characterized.(1) Based on pretreatments of leather technology, a PADM was prepared by the means of combined treatments with alkali, enzymes, sodium lauryl sulfate (SDS) and NaCl solution, using porcine skin as the raw material which is structurally and immunologically similar to human skin. Concentration and process time of enzymes were varied respectively, and their effects on properties of PADM were evaluated, such as porosity, mechanical properties, enzymatic degradation and collagen purity. The result indicated that with increase of alkali, enzymes concentration and process time, the porosity of PADM was enhanced and its degradation rate was quickened, but its tensile strength was weakened. NaCl treatment could enhance collagen purity of PADM obviously. Based on the results, the final technological parameters were chosen as follows,2.0 g·L-1 NaOH-0.8 g·L-1 SDS solution for 15 h,3.0 g·L-1 trypsin-3.0 g·L-1 1398 enzyme-2.0 g·L-1 SDS solution for 15 h,4.0 g·L-1 SDS for 4 h, and 58.5 g-L"1 NaCl solution for 24 h.(2) The PADM was prepared by the optimized technology, and its properties were characterized. Its appearance was observed, and its porosity, mechanical properties, enzymatic degradation and water vapor permeability were determined. The PADM was evaluated by HE staining observation. The composition of PADM was detected with an amino acid analyzer, and its microstructure was observed under SEM. To estimate its cytocompatibility, cells proliferation tests were performed by MTT assay, and cells distribution was viewed after HE stained. The result indicated that the PADM was pure white and flexible. No cells were observed in PADM, and the content of collagen was about 92.01%. The obtained PADM framework had interconnected pores at about 100μm in diameter, and the natural framework of the collagen was kept well. Its porosity was reached up to 85.54%, and the water vapor permeability was about 2923 g·m-2·24h-1. The tensile strength was about 4.63 MPa, The time of complete degradation was about 95 h. The MTT assay and HE staining observation indicated that cells cultured on PADM proliferated well and distributed evenly. The prepared PADM has good microstructure, high mechanical properties, controlled enzymatic stability and good cell compatibility, and it can achieve the performance standards of scaffold materials in skin tissue engineering.(3) Using PADM as a matrix, HAP-PADM was obtained by assembling HAP on the channel surface, and its properties were characterized. Its mechanical properties and enzymatic degradation were determined, and its microstructure was observed under SEM. To estimate its cytocompatibility, cells proliferation tests were performed by MTT assay, and cells distribution was viewed under CLSM. The result indicated that the HAP-PADM scaffold had two-level pore structure, with large channels (about 100μm in diameter) inherited from the purified PADM microstructure and small pores (<100 nm in diameter) formed by self-assembled HAP on the channel surfaces. Its compressive strength was about 7.54 MPa, and its degradation rate in collagenase solution was much slower than that of PADM. After 8 day culture of MC3T3-E1 pre-osteroblasts, MTT assay and CLSM image indicated that MC3T3-E1 cells proliferated well and distributed evenly. The prepared HAP-PADM has good microstructure, high mechanical properties, good enzymatic stability and good cell compatibility, and it can achieve the performance standards of scaffold materials in bone tissue engineering.Because of its high strength and nontoxicity, good microstructure and enzymatic stability, its simple preparation method, and designable and tailorable properties, the PADM scaffold has great potential use in the tissue engineering for further study.