Ravi K. Garg

Stem cell recruitment after injury: lessons for regenerative medicine.

Tissue repair and regeneration are thought to involve resident cell proliferation as well as the selective recruitment of circulating stem and progenitor cell populations through complex signaling cascades. Many of these recruited cells originate from the bone marrow, and specific subpopulations of bone marrow cells have been isolated and used to augment adult tissue regeneration in preclinical models. Clinical studies of cell-based therapies have reported mixed results, however, and a variety of approaches to enhance the regenerative capacity of stem cell therapies are being developed based on emerging insights into the mechanisms of progenitor cell biology and recruitment following injury. This article discusses the function and mechanisms of recruitment of important bone marrow-derived stem and progenitor cell populations following injury, as well as the emerging therapeutic applications targeting these cells.

Capillary Force Seeding of Hydrogels for Adipose-Derived Stem Cell Delivery in Wounds

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan‐collagen hydrogel scaffold for improving bone marrow‐derived mesenchymal stem cell survival within ischemic skin wounds by creating a “stem cell niche” that enhances regenerative cytokine secretion. Adipose‐derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel‐based therapies. To optimize hydrogel cell seeding, a rapid, capillary force‐based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis‐related genes, including Oct4, Vegf, Mcp‐1, and Sdf‐1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC‐seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan‐collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC‐seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

Objective:To investigate how epithelial mechanotransduction pathways impact wound repair. Background:Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. Methods:A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Results:Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Conclusions:Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

Cellular response to a novel fetal acellular collagen matrix: implications for tissue regeneration.

Introduction. PriMatrix (TEI Biosciences Inc., Boston, MA, USA) is a novel acellular collagen matrix derived from fetal bovine dermis that is designed for use in partial- and full-thickness wounds. This study analyzes the cellular response to PriMatrix in vivo, as well as the ability of this matrix to facilitate normal tissue regeneration. Methods. Five by five mm squares of rehydrated PriMatrix were implanted in a subcutaneous fashion on the dorsum of wild-type mice. Implant site tissue was harvested for histology, immunohistochemistry (IHC), and flow cytometric analyses at multiple time points until day 28. Results. PriMatrix implants were found to go through a biological progression initiated by a transient infiltrate of inflammatory cells, followed by mesenchymal cell recruitment and vascular development. IHC analysis revealed that the majority of the implanted fetal dermal collagen fibers persisted through day 28 but underwent remodeling and cellular repopulation to form tissue with a density and morphology consistent with healthy dermis. Conclusions. PriMatrix implants undergo progressive in vivo remodeling, facilitating the regeneration of histologically normal tissue through a mild inflammatory and progenitor cell response. Regeneration of normal tissue is especially important in a wound environment, and these findings warrant further investigation of PriMatrix in this setting.

Abstract 77: A NOVEL FETAL ECM PROVIDES A NICHE FOR STEM CELL RECRUITMENT AND TISSUE REGENERATION

Methods and Results: To elucidate the molecular mechanisms of cleft soft palate, we have used Tgfbr2 / ;K14Cre mice as an established animal model for cleft soft palate. Based on imaging analyses using both three-dimensional microCT and histological reconstruction images, we have found that Tgfbr2 / ;K14-Cre mice exhibit decreased muscle mass, following defects in cell proliferation, differentiation, and maturation of myotubes into myo bers. Dickkopf (Dkk)1 and Dkk4, negative regulators of WNT/Œ≤-catenin signaling, are up-regulated in Tgfbr2 mutant cells and WNT/ Œ≤catenin signaling is disrupted in palatal mesenchymal cells of Tgfbr2 / ;K14-Cre mice. Moreover, neutralizing antibodies for DKK1 and DKK4 can rescue the muscle defect in the soft palate of Tgfbr2 / ;K14-Cre mice.