Themis R. Kyriakides

Chronic wounds, a major healthcare burden, are characterized by impaired fibroblast function and ECM remodeling. Thrombospondin‐2 (TSP2), a matricellular glycoprotein, has been shown to negatively regulate wound healing. Here, we investigated the cellular and transcriptomic consequences of TSP2 deficiency in dermal fibroblasts, key cells in tissue repair and extracellular matrix (ECM) remodeling. Using bulk RNA sequencing of wild‐type (WT) and TSP2 knockout (TSP2 KO) murine primary fibroblasts, we identified upregulation of pro‐regenerative molecules and signaling pathways, specifically TGF‐β3 and Wnt4/β‐catenin, in the latter. To overcome the inherent variability of primary cells and establish a robust model, we generated a stable CRISPR/Cas9‐engineered TSP2 knockout in NIH3T3 fibroblasts. This system confirmed that TSP2 depletion enhances fibroblast proliferation and migration, associated with increased activity of TGF‐β3 and Wnt/β‐catenin signaling pathways. These findings not only provide novel mechanistic insights into the role of TSP2 in regulating fibroblast function and ECM interactions during tissue repair, but also highlight TSP2 as a potential therapeutic target for promoting regeneration in healing‐impaired or chronic wounds.

Fibroblasts display complex functions linked to distinct gene expression profiles. These profiles influence matrix production, cell communication, and tissue development and repair. Thrombospondin-2 (TSP2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell RNA sequencing (scRNA-seq) on WT and TSP2 KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10 + multipotent progenitor cells, identified as Schwann precursor cells, in TSP2 KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of mouse back skin wounds and cells validated the increase of this Sox10 + population. Furthermore, in silico analysis suggested enhanced PDGF-β-mediated pro-survival and inhibited BMP4-mediated differentiation signaling pathways. These molecular and functional alterations likely contribute to improved healing and increased neurogenesis in TSP2 KO wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts (DFs) and identify pro-regenerative features of TSP2 KO fibroblasts.

Encapsulating fibroblasts in alginate hydrogels is a promising strategy to promote wound healing. However, improving the cell function within the alginate matrix remains a challenge. In this study, we engineer an injectable hydrogel through mixing alginate function with collagen and fibronectin, creating a better microenvironment for enhancing fibroblast function and cytokine secretion. We systematically analyze microstructure, mechanical properties, and fibroblast behavior of the developed hydrogel and compare it to alginate control. Our results demonstrate that inclusion collagen and fibronectin lead to the formation of fibrils on macroporous structures with pore sizes ranging from 100 to 500 μm. Compared to collagen hydrogel, the composite hydrogel shows approximately 12-fold increase in storage modulus. After encapsulating fibroblasts into the modified hydrogels, we observed increased fibroblast spreading, proliferation, and cytokine secretion when compared to neat alginate hydrogel. In addition, VEGF secretion of encapsulated fibroblasts is upregulated, indicating its pro-angiogenic potential. These findings suggest that the alginate/collagen/fibronectin hydrogel-encapsulated fibroblasts might serve as a promising therapeutic approach for wound healing.

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-β, and PDGF-β, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-β3 and Wnt4, and enrichment of nuclear β-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

Impaired wound healing in diabetic patients is the leading cause of diabetes‐associated hospitalizations and approximately 50% of lower limb amputations. This is due to multiple factors, including elevated glucose, sustained hypoxia, and cell dysfunction. Previously, diabetic wounds were found to contain excessive levels of the matricellular protein thrombospondin‐2 (TSP2) and genetic ablation of TSP2 in diabetic mice or treatment of wounds with a hydrogel derived from TSP2‐null mouse skin improved healing. Previously, TSP2 has been shown to be repressed by hypoxia, but in the present study we observed sustained hypoxia and overlapping TSP2 deposition in diabetic wounds. We determined this observation was due to the insufficient HIF‐1α activation verified by western blot and immunofluorescent analysis of wound tissues and in vitro hypoxia experiments. Application of Dimethyloxalylglycine (DMOG), which can stabilize HIF‐1α, inhibited TSP2 expression in diabetic fibroblasts in hypoxic conditions. Therefore, we prepared DMOG‐containing TSP2KO hydrogel and applied it to the wounds of diabetic mice. In comparison to empty TSP2KO hydrogel or DMOG treatment, we observed improved wound healing associated with a reduction of TSP2, reduced hypoxia, and increased neovascularization. Overall, our findings shed light on the intricate interplay between hyperglycemia, hypoxia, and TSP2 in the complex environment of diabetic wounds.

Tissue Engineering Part C: Methods

Tissue Engineering Part B: Reviews