Supercritical Carbon Dioxide-decellularized Porcine Acellular Dermal Matrix combined with Autologous Adipose-derived Stem Cells: Its Role in Accelerated Diabetic Wound Healing

Abstract

Diabetes mellitus (DM) causes impaired wound healing by affecting one or more of the biological mechanisms of hemostasis, inflammation, proliferation, and remodeling and a large number of cell types, extracellular components, growth factors, and cytokines. Interventions targeted toward these mechanisms might accelerate the wound healing process. To evaluate the wound healing efficacy of supercritical carbon dioxide (scCO2)-decellularized porcine acellular dermal matrix (ADM) combined with autologous adipose-derived stem cells (ASCs) in streptozotocin (STZ)-induced DM rats. DM was induced by injecting rats with STZ; dorsal full-thickness skin (5 × 5 cm 2) was created and treated with and without ASCs-scCO2-treated ADM to evaluate the wound healing rate through histological examination, fluorescence microscopic observation, and immunohistochemical analysis. In the present study, complete decellularization of the porcine dermal matrix was achieved through scCO2. Isolation of ASCs was conducted and evaluated using CD29 + /CD31 − /CD45 − /CD90 + markers in flow cytometry, which indicated that more than 90% of cells were ASCs. The percentage of cells labeled with CD29 + and CD90 + was found to be 97.50% and 99.69%, respectively. The wound healing rate increased in all groups relative to the group with the DM wound without treatment. DM wound treated with ADM-ASCs showed significantly higher (p < 0.01) wound healing rate than DM wound without treatment. ADM-ASC-treated rats showed significantly increased epidermal growth factor, Ki67, and prolyl 4-hydroxylase and significantly decreased CD45 compared with the group with the DM wound without treatment. The intervention comprising ADM decellularized from porcine skin by using scCO2 and ASCs was proven to improve diabetic wound healing. ADM-ASCs had a positive effect on epidermal regeneration, anti-inflammation, collagen production and processing, and cell proliferation; thus, it accelerated wound healing.

Protocols for the Preparation and Characterization of Decellularized Tissue and Organ Scaffolds for Tissue Engineering

“…The decellularized ECM tissue and organ scaffolds produced by employing scCO 2 technology are completely devoid of cellular components and noncollagenous protein substances. Decellularized tissue and organ scaffold obtained from the scCO 2 process is pure with intact native collagen scaffold, without any alteration in the content and structure of the scaffold [4,5]. We characterized the tissue and organ scaffolds by H&E and DAPI staining, depicting the removal of cells and demonstrating that the scCO 2 decellularization process was complete.…”

“…Several approaches employed in the production of natural tissue and organ scaffolds are intricate, expensive and laborious and, notably, use a variety of chemicals that are detrimental to the environment [3][4][5][6][9][10][11][12]. In the present production process, we aim to use supercritical carbon dioxide (scCO 2 ) as a platform technology to produce natural tissue and organ scaffolds, thereby eradicating the use of detrimental chemicals and without traces of chemicals in the natural tissue and organ scaffolds.…”

“…The ECM scaffolds were freeze-dried and cut to appropriate sizes to fit in the culture plates. The freeze-dried decellularized ECM scaffolds were also subjected to freeze-milling (Ultra Centrifugal Mill ZM200; Retsch, Haan, Germany) to make the powder form of the scaffold with different size sieve, packed in vials and sterilized by ␥ -irradiation (25 kGy) [3][4][5][6].…”

“…The decellularized ECM scaffold samples were fixed in 2.5% buffered glutaraldehyde at 4 • C for 1 h. Scanning electron microscopy (SEM) was done to analyze the 3D ultrastructural features of collagen scaffold. The above-mentioned experiments were done following our previously published reports regarding decellularization and characterization of biomaterials [3][4][5][6].…”

“…For example, mesenchymal stem cells or skin fibroblasts can be used in combination with the decellularized dermal matrix for the regeneration of full skin tissue. Besides, in vivo implantation of the ECM scaffold alone regenerated into the tissue [4]. The potential application of the scCO 2 decellularized natural tissue and organ scaffolds might be suitable for in vivo tissue and organ reconstruction in the near future; we summarize the potential applications for stem cell research and 3D bioprinting in Table 1.…”