Development of a Novel Pro-angiogenic SDF-1α Gene-Activated Collagen Scaffold for Diabetic Wound Healing

Abstract

Diabetes is a disease that arises due to the loss or impairment of the body’s ability to control the blood glucose level. Hyperglycemia leads to alterations in the structural functions of both macro and microvascular systems, causing vascular complications in diabetic patients such as the diabetic foot ulcer. In diabetic wounds, inadequate angiogenesis is one of the significant pathological factors hindering the progression to regular healing events. During wound healing, various cells orchestrate to promote angiogenesis. Therefore, this thesis aims to explore the impact of delivering a pro-angiogenic gene encoding for stromal-derived factor-1 alpha (SDF-1α), using a collagen-based scaffold into potential cell types involved in wound healing. Functional impact following the delivery of the gene was assessed on mesenchymal stem cells (MSCs), human endothelial cells (ECs), human Schwann cells (SCs), and ultimately, human diabetic adipose-derived stem cells (ADSCs). The first aim (Chapter 2) of this thesis was to assess the transfection efficiency of non-viral vector polyethyleneimine (PEI) based polyplex on MSCs in 2D, and develop gene-activated scaffolds by incorporating the polyplex nanoparticles to a collagen-chondroitin sulfate scaffold (3D). The results demonstrated that the use of PEI as a gene delivery vector induces transient expression of the transgene in both 2D and 3D. When the SDF-1α gene was delivered using the PEI polyplex system, the MSCs demonstrated successful translation of the SDF-1α gene into high levels of the therapeutic protein. Further, the delivery of SDF-1α gene using the gene-activated scaffold (SDF-1α GAS) induced early activation of the MSCs that exerted significantly high pro-angiogenic paracrine action on endothelial cells. The second objective (Chapter 3) of the thesis was to assess the provasculogenic impact of SDF-1α GAS on human endothelial cells and its co-culture with human ADSCs. The results demonstrated that SDF-1α GAS effectively supports endothelial angiogenesis in a well-regulated manner by desirably preserving the junctional integrity, which is essential for maturation of the endothelial network. Co-culturing with ADSCs promoted better stability and maturation. This effect was further enhanced when supported by SDF-1α GAS. Additionally, SDF-1α GAS activated co-culture group demonstrated a strong capacity to signal Schwann cells to differentiate towards a pro-neurogenic phenotype by influencing their cellular organization into bunger band-like structures. The third objective (Chapter 4) of the thesis was focused on the functional impact of SDF-1α GAS on human Schwann cells for reinnervated wound healing applications. When activated with SDF-1α GAS, SCs demonstrated a significantly enhanced production of proangiogenic VEGF that significantly induced endothelial angiogenesis. Meanwhile, Schwann cells within the SDF-1α GAS also demonstrated a strong capacity to differentiate towards a reparatory phenotype as indicated by their significantly high expression of neurotrophin receptor p75NGFR. Furthermore, this phenotypic change was associated with an extensive remodeling event that progressed through a profound increase in the deposition of the pro-neurogenic matrix laminin. The final objective (Chapter 5) of this thesis was to assess the functional impact of SDF-1α GAS on diabetic human ADSCs as an approach to develop autologous tissue-engineered graft. Overall, diabetic ADSCs on SDF-1α GAS demonstrated a strong functional resemblance to that of the healthy ADSCs on gene-free scaffold, indicating a transition towards a more healthy-like response than diabetic ADSCs without GAS. This response was noted in the secreted factors from diabetic ADSCs on SDF- 1α GAS, which became more capable of inducing strong and early proangiogenic responses on endothelial cells. Additionally, SDF-1α GAS promoted fibronectin remodeling and enhanced the deposition of collagen and elastin matrix in diabetic ADSCs. Meanwhile, healthy ADSCs on SDF-1α GAS achieved this effect with minimal activation of inflammatory and angiogenic factors, demonstrating accelerated cellular maturation for healing. When incorporated into a tri-culture angiogenesis model, diabetic ADSCs actively sustained the pro-vasculogenic response in the tri-culture construct as well as their ability to induce migration of endothelial cells. Collectively, the research presented in this thesis show that SDF-1α GAS exerts diverse functional response in potential cell types involved in wound healing. Particularly, we show that SDF-1α GAS consistently mediates the activation of strong pro-angiogenic response in these cells including diabetic stem cells, suggesting that SDF-1α GAS a highly biosinstructive platform for diabetic wound healing applications.