do Amaral Ronaldo J F C, Zayed Noora M A, Pascu Elena I, Cavanagh Brenton, Hobbs Chris, Santarella Francesco, Simpson Christopher R, Murphy Ciara M, Sridharan Rukmani, González-Vázquez Arlyng, O'Sullivan Barry, O'Brien Fergal J, Kearney Cathal J
Kearney Lab, Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
Front Bioeng Biotechnol. 2019 Dec 3;7:371. doi: 10.3389/fbioe.2019.00371. eCollection 2019.
Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds have shown promising clinical results for wound healing; however, these scaffolds do not replace the dermal and epidermal layer simultaneously and rely on local endogenous signaling to direct healing. Functionalizing collagen-GAG scaffolds with signaling factors, and/or additional matrix molecules, could help overcome these challenges. An ideal candidate for this is platelet-rich plasma (PRP) as it is a natural reservoir of growth factors, can be activated to form a fibrin gel, and is available intraoperatively. We tested the factors released from PRP (PRPr) and found that at specific concentrations, PRPr enhanced cell proliferation and migration and induced angiogenesis to a greater extent than fetal bovine serum (FBS) controls. This motivated us to develop a strategy to successfully incorporate PRP homogeneously within the pores of the collagen-GAG scaffolds. The composite scaffold released key growth factors for wound healing (FGF, TGFβ) and vascularization (VEGF, PDGF) for up to 14 days. In addition, the composite scaffold had enhanced mechanical properties (when compared to PRP gel alone), while providing a continuous upper surface of extracellular matrix (ECM) for keratinocyte seeding. The levels of the factors released from the composite scaffold were sufficient to sustain proliferation of key cells involved in wound healing, including human endothelial cells, mesenchymal stromal cells, fibroblasts, and keratinocytes; even in the absence of FBS supplementation. In functional and vascularization assays, our composite scaffold demonstrated increased angiogenic and vascularization potential, which is known to lead to enhanced wound healing. Upon pro-inflammatory induction, macrophages released lower levels of the pro-inflammatory marker MIP-1α when treated with PRPr; and released higher levels of the anti-inflammatory marker IL1-ra upon both pro- and anti-inflammatory induction when treated with the composite scaffold. Finally, our composite scaffold supported a co-culture system of human fibroblasts and keratinocytes that resulted in an epidermal-like layer, with keratinocytes constrained to the surface of the scaffold; by contrast, keratinocytes were observed infiltrating the PRP-free scaffold. This novel composite scaffold has the potential for rapid translation to the clinic by isolating PRP from a patient intraoperatively and combining it with regulatory approved scaffolds to enhance wound repair.
多孔胶原-糖胺聚糖（胶原-GAG）支架在伤口愈合方面已显示出良好的临床效果；然而，这些支架不能同时替代真皮层和表皮层，而是依赖局部内源性信号来引导愈合。用信号因子和/或其他基质分子对胶原-GAG支架进行功能化，可能有助于克服这些挑战。富血小板血浆（PRP）是实现这一目标的理想候选物，因为它是生长因子的天然储存库，可被激活形成纤维蛋白凝胶，且可在术中获取。我们测试了PRP释放的因子（PRPr），发现特定浓度下，PRPr比胎牛血清（FBS）对照组更能增强细胞增殖、迁移并诱导血管生成。这促使我们开发一种策略，将PRP成功均匀地整合到胶原-GAG支架的孔隙中。复合支架可在长达14天的时间内释放伤口愈合的关键生长因子（FGF、TGFβ）和血管生成因子（VEGF、PDGF）。此外，复合支架的机械性能得到增强（与单独的PRP凝胶相比），同时为角质形成细胞接种提供了连续的细胞外基质（ECM）上表面。复合支架释放的因子水平足以维持参与伤口愈合的关键细胞的增殖，包括人内皮细胞、间充质基质细胞、成纤维细胞和角质形成细胞；即使在不补充FBS的情况下也是如此。在功能和血管生成试验中，我们的复合支架显示出增强的血管生成和血管化潜力，已知这会促进伤口愈合。在促炎诱导下，用PRPr处理的巨噬细胞释放的促炎标志物MIP-1α水平较低；而用复合支架处理时，在促炎和抗炎诱导下，巨噬细胞释放的抗炎标志物IL1-ra水平较高。最后，我们的复合支架支持人成纤维细胞和角质形成细胞的共培养系统，形成了类似表皮的层，角质形成细胞局限于支架表面；相比之下，观察到角质形成细胞浸润无PRP的支架。这种新型复合支架有可能通过术中从患者体内分离PRP并将其与监管部门批准的支架结合以增强伤口修复，从而迅速转化应用于临床。
