Improving 3D Scaffolds for Skin Tissue Engineering using Advanced Biotechnology

Abstract

Existing, dermal, regenerative scaffolds facilitate dermal repair and wound healing of severe burn injuries; however, new tissue is often functionally, mechanically and aesthetically abnormal due to irregular deposition of new extracellular matrix. In the present study two novel, elastin-containing scaffolds were developed, characterised and examined both in vitro and in vivo aiming to minimise wound contraction, improve scar appearance and increase skin elasticity post-healing. The first types of scaffolds were electrospun from a triple polymer solution of collagen, elastin and poly(ϵ-caprolactone) (CEP). Two scaffolds were chosen for characterisation: CEP 1 was fabricated using a 1.5 % (w/v) collagen, 12 % (w/v) elastin and 1.5 % (w/v) poly(ϵ-caprolactone) (PCL) solution, a flow rate of 3 mL/h, an air gap of 15 cm and an applied electric potential of 25 kV; and CEP 2 was electrospun using a 2 % (w/v) collagen, 12 % (w/v) elastin and 1 % (w/v) PCL solution at 1 mL/h, 20 cm and 20 kV. In vitro cell studies using human, dermal fibroblasts (HDFs) and immortalised, human keratinocytes (HaCaTs) revealed CEP 1 and CEP 2 supported cell-seeding and cell proliferation with significantly higher proliferation of both cell types on CEP 1. Additionally, subcutaneous implant studies in mice revealed minimal inflammation in response to both scaffolds with CEP 1 vascularised by week 2 post-surgery. However, CEP 1 was rapidly biodegraded after 2 weeks. Collagen deposition was observed in encapsulating tissue and new tissue with consistent collagen expression over 24 weeks. The second type of scaffold investigated was an elastin-modified version of the commercial, dermal substitute Integra Dermal Regeneration Template (IDRT). Elastin-IDRT (EDRT) was developed by inclusion of 10% human tropoelastin and then investigated in comparison with IDRT. Morphological analysis by scanning electron microscope and mechanical characterisation revealed EDRT had significantly enlarged pores, higher porosity and increased deformability. Higher cell seeding efficiency of HaCaTs on EDRT was observed compared to IDRT but cell proliferation rate was found to be similar over 28 days. HDFs displayed increased cell growth rate on EDRT over 28 days compared to IDRT. Enhanced and accelerated HDF infiltration of EDRT was also visualised with complete infiltration by day 14 post-seeding. An in vivo, mouse, subcutaneous implant model showed that EDRT induced minimal inflammation. Gene expression of mouse collagen was consistent over 24 weeks with non-significant increases in elastin expression from weeks 2 and 4. One-step grafting demonstrated similar contraction between EDRT-, IDRT- and autografted wounds with final contraction around 40 % compared to 100 % in open wounds. EDRT displayed significantly accelerated, early-stage angiogenesis with higher vascularisation than IDRT-grafted, autografted or open wounds 2 weeks post grafting. By week 4 EDRT- and IDRT-grafted wounds had similar levels of vascularisation which were higher than autografted and open wounds. EDRT showed improved mechanical performance, supported enhanced cell interactions in vitro and accelerated angiogenesis in vivo. In summary, investigated scaffolds demonstrated properties that could potentially improve burn wound healing. The inclusion of elastin in scaffolds produced by either electrospinning or lyophilisation improved HDF infiltration and supported formation of a confluent layer of HaCaTs which could result in increased pliability of new skin and accelerated wound healing. In EDRT elastin improved scaffold porosity, pore size and accelerated angiogenesis in vivo indicating EDRT can facilitate and improve wound remodelling. Further investigation of both scaffolds is warranted especially due to the vascular inductive effects of EDRT and the synchronous spatial and temporal biodegradation of CEP 2 observed in vivo.