LLP2A functionalized polyethylene glycol hydrogels induce differentiation and angiogenic factor release in mesenchymal stem cells

Chronic wounds are a growing problem caused by increases in the incidence of diabetes, obesity, and the aging population, and they represent a disproportionately large burden to the healthcare system. The risk of complications including infections that result in hospitalization and amputation make chronic wounds particularly pernicious. Many attempts have been made to identify both the molecular basis behind chronic wounds and an effective treatment to improve prognosis. As yet, the exact cause of chronic wounds is idiopathic and may be varied, but research indicates many if not most result from a failure to advance from the inflammatory phase to the proliferative phase of wound healing. Current standard care and even the most advanced treatments currently on the market provide little value to chronic wounds, leaving a tremendous need for both a better understanding of the physiology of chronic wounds and relief for patients suffering with chronic wounds and their complications. One avenue being pursued for chronic wound healing is the application of mesenchymal stem cells (MSCs) due to their ability to release growth factors and anti-inflammatory cytokines. MSCs normally home to wound sites, but they require a structural support that may be damaged in chronic wounds. Therefore, research is pursuing scaffolds, such as hydrogels, to provide an appropriate niche comparable to a normal wound to hold MSCs at the wound site and stimulate them to release appropriate factors for that point in the healing process. MSCs like other cells anchor to their environment through membrane bound proteins including integrins, which provide signals about the nature of the extracellular matrix (ECM) within which the cell resides. Hydrogels are polymers of either synthetic or natural organic compounds that can serve as anchors for MSCs and delivery vehicles for factors that influence healing. Polyethylene glycol (PEG) is a synthetic hydrogel that can be tuned to varying degrees of stiffness and used to deliver MSCs. However, PEG requires the conjugation of ligands that integrins can bind. These ligands can be designed to be highly specific to only one or a few relevant integrins. Research in the Maverakis lab has used the one-bead-one-compound method to screen potential ligands for integrin-binding specificity and assessed their capacity to induce differentiation in vitro. However, the capacity of these ligand-conjugated hydrogels to modulate MSC function in vitro or facilitate wound healing has not yet been determined. The overall goal of the Maverakis lab is to develop hydrogels that can be administered in a clinical setting to facilitate healing of chronic wounds. Previous work in the has suggested MSCs differentiate toward chondrogenic and osteogenic lineages using tentagel beads conjugated with integrin binding ligands. Here the effect of 30 cell culture in PEG-hydrogels conjugated to integrin binding ligand specific to a4~ 1 integrin is explored through PCR and RNA-seq. To characterize MSCs received from collaborating labs, flow cytometry was used for cell surface expression of MSC markers: C044, CD73, C090, and C0105. The presence of MSC markers and the absence of markers for ematopoietic cells suggests experiments were conducted on true MSCs. To characterize the effect of specific integrin stimulation in 30 cell culture on hydrogel encapsulated MSCs PCR and RNA-seq was used. The results from RNA-seq analysis support differentiation of MSCs into osteogenic and chondrogenic lineages when treated with LLP2A in support of previous finding in the Maverakis lab from 20 tengel bead cultures. The finding that a4~ 1 integrin stimulation results in osteogenic and chondrogenic differentiation suggests that LLP2A may not be effective for dermal wound healing. In contrast, RGD conjugated hydrogel, a control for LLP2A, showed differentiation toward smooth muscle and adipose with enhanced angiogenic signaling, suggesting a potentially effective treatment of improving dermal wound healing. While these findings were not born out in the in vivo studies using hydrogels alone without MSCs, there remains the possibility that hydrogel encapsulated MSCs will be able to enhance wound healing as proposed. Moving forward, additional putative integrin binding ligands will continue to be screened. Specific integrin binding ligands that have been screened will be tested for their effect on MSCs in vitro, and hydrogel encapsulated MSCs will be used in in vivo studies on mouse splint wounds. LLP2A conjugated hydrogel may be studied for in vivo healing in bone and cartilage dysfunction.