In vitro release and cellular bioactivity of a composite adipose-derived hydrogel scaffold

Abstract

Event Abstract Back to Event In vitro release and cellular bioactivity of a composite adipose-derived hydrogel scaffold Christopher Mahoney1, Malik Snowden1, Kacey Marra1, 2, 3, 4 and J Peter Rubin1, 2, 3, 4 1 University of Pittsburgh, Bioengineering, United States 2 University of Pittsburgh, Plastic Surgery, United States 3 University of Pittsburgh, McGowan Institute of Regenerative Medicine, United States 4 University of Pittsburgh, School of Medicine, United States Introduction: Soft tissue reconstruction for the repair of congenital deformities or defects from tumor resections/trauma often require adequate replacement of adipose tissue[1],[2]. Standards of care include vascularized flaps or prosthetic implants consisting of silicone or saline. Although tissue flaps can have favorable results, complications may lead to flap failure, infections, pulmonary embolisms, and morbidity of the donor site. Autologous fat grafting using lipoaspirate is minimally invasive in reconstructive surgery but results are unpredictable due to resorption up to 10% volume retention[3]. These limitations serve as motivation for developing therapies to regenerate adipose tissue within the tissue engineering field[2],[4]. Materials and Methods: Abdomen whole fat was donated from a non-diabetic female (age: 41, BMI: 26.3) undergoing elective cosmetic surgery at the University of Pittsburgh Medical Center. The decellularization process includes four main phases consisting of alcohol rinses, delipidization, and disinfection of the adipose matrix. After processing, the matrix was snap frozen using liquid nitrogen and then lyophilized. A Mini Wiley Mill (Thomas Scientific, Swedesboro, NJ) breaks down the lyophilized matrix into a powder for pepsin digest and hydrogel formation. PLGA (50:50) (Sigma Aldrich, St. Louis, MO) was used as the base polymer to encapsulate fluorescent dexamethasone in microscopheres using a single emulsion mixing technique. Adipose-derived stems cells (ASCs) were acquired using an isolation protocol on abdominal fat donated from a non-diabetic female (age: 38, BMI: 24.8) undergoing elective cosmetic surgery. Adipocyte quantification of ASCs study was conducted in a 12-well tissue culture plate along with Transwell tissue culture inserts to suspend the composite hydrogel above the cells in culture medium. The following culture conditions were used for comparison: cells with adipogenic medium (StemPro Adipogenesis Differentiation Kit, Invitrogen, Inc.), cells + composite hydrogel with adipogenic medium, cells + composite hydrogel without adipogenic medium (maintenance medium), and cells in maintenance medium (n=3). Cells were kept incubated at 37 °C and 5% CO2. At day 7 and 14, mature adipocytes were stained using the AdipoRed™ Reagent Assay (Lonza Brand distributed by Fisher Scientific). Results and Discussion: SEM images of the lyophilized hydrogel indicates porosity throughout the structure (Fig. 1). As expected, higher concentrations of MS in hydrogel displayed a lower presence of porosity which may generate challenges for progenitor adipocytes migration into the scaffold. The 14-day differentiation study demonstrated higher amounts of adipogenesis in groups containing hydrogel and hydrogel with microspheres when compared to the positive control of ASCs alone (Fig. 2 & 3). It is also important to note that adding the hydrogel to ASCs in maintenance medium resulted increased differentiation compared to the ASC in maintenance medium. Conclusion: The objective of this research project is to develop a composite hydrogel scaffold from discarded human adipose tissue for an enhanced adipogenic effect in the form of microspheres containing dexamethasone for the application of soft tissue engineering. The decellularization process has been found to be a reproducible process with consistent material production. These bioactivity studies allowed for further characterization of the composite hydrogel. A 14-day differentiation study confirmed the scaffold’s potential to increase proliferation and differentiation of adipose stem cells into adipocytes. Overall, the results warrant further investigation of the composite and its possible use in adipose regeneration/retention in autologous fat grafts. CATER Training Program; Badylak Lab for Wiley Mill Access; Center of Biological Imaging for Microscopy Images