Development of a mesenchymal stromal cell / collagen scaffold product for treatment of diabetic wounds

Du, Shanshan
Foot ulceration is a major complication of diabetes mellitus, which results in significant human suffering and a major burden on healthcare systems. Mesenchymal stromal cells (MSCs) emerged as a promising therapeutic agent for treatment of diabetic wound healing because of their paracrine properties, including the secretion of angiogenic, immunomodulatory and anti-inflammatory factors. However, topical administration of MSCs via direct injection result in low cell viability and poor cell localization at the wound bed, which hinder the efficacy and clinical translation of a MSC therapy product. Here in, we used macromolecular crowding (MMC) concept to develop a MSC-scaffold bio-complex containing the native extracellular matrix (ECM) and investigated its therapeutic effect in a diabetic wound model. Firstly, we investigated the optimal MMC agent in human umbilical cord derived MSC (hUC-MSC) cultures. Seven types of carrageenan at five different concentrations were supplemented to MSC medium. 50 μg/ml l-MV carrageenan was identified to be the optimal cell culture condition, as it did not adversely affect (p > 0.05) cell viability, metabolic activity, proliferation, and the expression of MSC surface markers and immune phenotype markers, whereas it significantly increased (p < 0.05) the deposition of collagen types I, III and IV, fibronectin and laminin. The shortest cell culture time was 4 days. These data highlight the potential of l-MV carrageenan as crowding agents for developing ECM-rich cell therapy product and describes the optimal conditions for use. Secondly, as hUC-MSCs grown under the optimum MMC conditions, can only develop a thin cell layer, which was not suitable for transfer from the tissue culture dish directly to the wound. A collagen porous scaffold was utilised to deliver this ECM-rich MSC layer. Scaffolds were fabricated using either neutralised or non-neutralised collagen. Compared to neutralized scaffold, the non-neutralized scaffold is pliable, thicker and has significantly (p < 0.05) smaller pore size, which is suitable for pre-clinical wound healing studies. hUC-MSCs grown on the non-neutralized scaffold under MMC conditions maintained the cell viability and proliferation at three cell doses (150k, 300k, 600k) groups. hUC-MSCs deposited significantly higher (p < 0.05) fibronectin in the presence of 50 μg/ml l-MV carrageenan at 300k and 600k cell dose groups and no significant difference was observed between the two groups. These data suggest this MSC-scaffold bio-complex at 300k cell dose may have augmented therapeutic potential for the treatment of diabetic wounds. Thirdly, to evaluate the therapeutic effect of this MSC-scaffold bio-complex, we developed a diabetic model using immunodeficient mice, and reproduced the excisional splinting wound model to avoid the confounding effect of wound contraction. Diabetes was induced in animals using low-dose streptozotocin. Wounds were treated as per groups: Sham (no treatment control), SC (scaffold only), SC+MSC (MSC grown on scaffold), and SC+MSC+MMC (MSC grown on scaffold under MMC conditions). 60% of mice successfully developed diabetes and exhibited mild diabetic signs (polydipsia and polyuria) with no loss of body weight and fatality. Gross view of wounds on day 7 showed no significant difference in wound closure rate. Histology and immunohistochemistry analysis revealed no significant difference in re-epithelialization, granulation tissue area, blood vessel density and ECM deposition between the four groups. Donor comparison analysis revealed no significant difference in therapeutic effects between MSCs from 3 different donors. Correlation analysis revealed no correlation between wound gap and blood glucose level, and each group had similar blood glucose levels. Although current pre-clinical data did not support this MSC-scaffold bio-complex to improve diabetic wound healing, these results direct investigation of alternative scaffolds and cell doses as a next step. Collectively, this study provides a new method for the development of a novel MSC therapy product with augmented therapeutic potential. Further improvement of the wound model and optimization of this MSC therapy product are needed for future study.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland