Abstract
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 in vitro and in vivo 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.
Highlights
Porous collagen-glycosaminoglycan scaffolds were amongst the first scaffolds developed for tissue engineering applications (Yannas et al, 1982)
The PRP-derived fibrin was found filling the pores throughout the collagen-GAG structure (Figure 1F), with thin fibers more homogenously distributed within the pores of the composite scaffold than in the PRP gel
scanning electron microscopy (SEM) analysis shows the collagenGAG strut (Figure 1J) and the fibrin network in PRP gel (Figure 1K), which is homogenously incorporated within the pores of a well-preserved collagen-GAG strut in our composite scaffolds (Figure 1L)
Summary
Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds were amongst the first scaffolds developed for tissue engineering applications (Yannas et al, 1982). These scaffolds are produced by combining collagen type I with glycosaminoglycan followed by a controlled freeze drying procedure, resulting in highly porous and biomimetic scaffolds (O’brien, 2011) These scaffolds readily allow for cell infiltration and their degradation is balanced by the formation of new tissue by the infiltrating cells. Their use has seen widespread positive clinical results in treating burns (Heimbach et al, 1988), scar contractures (Stiefel et al, 2010), and diabetic foot ulcers (DFU) (Driver et al, 2015), and it is currently FDA-approved for these applications. Reducing the duration of healing and/or eliminating the requirement for a second procedure would be a clear advantage clinically, reducing infection risk, costs, and enhancing patient welfare
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