Production of extracellular matrix powder for tissue engineering

With the aim of regenerating healthy tissues, different tissue engineering strategies pointed to extracellular matrix (ECM)-based scaffolds in tissue engineering and regenerative medicine and wound healing. It is a multidisciplinary science works to create biocompatible scaffolds with perfect physical parameters, mechanical integrity and high porosity to promote cell growth, migration and angiogenesis. With the increased incidence of obesity, subcutaneous adipose tissue is abundant and readily accessible. Liposuction surgeries yield from 100 mL to 3 L of lipoaspirate tissue. We present our prepared acellular ECM powders derived from human adipose tissue obtained from lipoaspirate, which contains large amounts of collagen suitable for induction of adipogenesis. The study had been carried out from December 2012 to March 2013 in Tissue Bank and Research Center in Imam Khomeini Hospital Tehran, Iran. Fresh human adipose tissue was obtained by liposuction of abdominal fat pad in a private Day Clinic. By using wasted material of liposuction, we obtained 100 to 200 cc fat tissue from each patient. After physical (freeze-thaw-slicing-manual massage) and chemical (enzymatic-detergent-acid digestion) treatment, an acellularized matrix was created from fat tissue. The final material lyophilized and ground to powder. We analyzed ultra structure and biochemical properties of obtained ECM powder by using electron microscopy, immunohistochemistry (IHC) examination and proteomic studies. After mechanical and chemical process of decellularization, scanning electron micrographs of the samples showed smooth and contiguous collagenous components throughout the scaffold. IHC showed strong positive labeling for collagen IV and no evidence of nuclear material in the specimen. Separation of protein complex by Blue Native Polyacrylamide gel electrophoresis (BN-PAGE) has proven type I collagen triple helices associate to form banded fibrils. RNA preparation and Gene Expression Analysis (RT-PCR) by using specific primers for laminin, fibronectin, collagen type I and IV, desmin, and actin showed strong staining of our fat tissue scaffold with collagen type I, fibronectin, collagen IV and laminin. The results show that our decellularization method produced an adipose ECM scaffold rich of collagen fibers, suitable and effective substrate for use in soft tissue engineering and regenerative medicine.