Although chronic wounds are common, treatment for these disabling conditions remains limited and largely ineffective. In this study, we examined the benefit of bone marrow-

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-α, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-postive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor) cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.

The mouse excisional wound healing model has been used extensively to study wound healing and cutaneous regeneration. However, as mouse skin is mobile, contraction accounts for a large part of wound closure. In the mouse excisional wound splinting model, a splinting ring tightly adheres to the skin around the wound, preventing local skin contraction. The wound therefore heals through granulation and re-epithelialization, a process similar to that occurring in humans. The model, which takes 2-4 weeks to carry out, can be used to study the effects of stem cells on cutaneous repair or regeneration. In this protocol, we also describe how to implant stem cells onto the wound bed in Matrigel and/or into the surrounding tissue through injection. Serial wound tissue samples at different time points can be harvested to monitor the engraftment and the effects of stem cells in angiogenesis and wound healing.

Skin stem cells can regenerate epidermal appendages; however, hair follicles (HF) lost as a result of injury are barely regenerated. Here we show that macrophages in wounds activate HF stem cells, leading to telogen–anagen transition (TAT) around the wound and de novo HF regeneration, mostly through TNF signalling. Both TNF knockout and overexpression attenuate HF neogenesis in wounds, suggesting dose-dependent induction of HF neogenesis by TNF, which is consistent with TNF-induced AKT signalling in epidermal stem cells in vitro. TNF-induced β-catenin accumulation is dependent on AKT but not Wnt signalling. Inhibition of PI3K/AKT blocks depilation-induced HF TAT. Notably, Pten loss in Lgr5+ HF stem cells results in HF TAT independent of injury and promotes HF neogenesis after wounding. Thus, our results suggest that macrophage-TNF-induced AKT/β-catenin signalling in Lgr5+ HF stem cells has a crucial role in promoting HF cycling and neogenesis after wounding.

SUMMARY:Peripheral blood fibrocytes are a newly identified leukocyte subpopulation that displays fibroblast-like properties.These blood-borne cells can rapidly enter the site of injury at the same time as circulating inflammatory cells. We hypothesize that circulating fibrocytes represent an important source of fibroblasts for healing of extensive burn wounds where it may be difficult for fibroblasts to migrate from the edges of uninjured tissue. In this study we identified and quantified fibrocytes among the adherent cells cultured from human peripheral blood mononuclear cells (PBMC) obtained from 18 burn patients and 12 normal individuals, based on their ability to express type I collagen. Our results showed that adherent cells cultured from PBMC of burn patients differentiated to fibrocytes more efficiently than did those from normal individuals. The percentage of type I collagen-positive fibrocytes was significantly higher for patients than for controls (89.7 Ϯ 7.9% versus 69.9 Ϯ 14.7%, p Ͻ 0.001). This percentage was consistently higher for patients with a Ն30% total body surface area burn until 1 year, with the highest percentage appearing within 3 weeks of injury. A positive correlation was found between the levels of serum transforming growth factor-␤1 (TGF-␤1) and the percentage of fibrocytes developing in the cultures of PBMC derived from these patients. We also demonstrated that fibrocytes were derived from CD14 ϩ cells but not CD14 Ϫ cells. Conditioned medium from CD14 Ϫ cells was, however, required for fibrocyte differentiation, whereas direct contact between CD14 Ϫ and CD14 ϩ cells was not necessary. Treatment of the cell cultures with TGF-␤1 enhanced the development of collagen-positive cells, whereas the inclusion of neutralizing anti-TGF-␤1 antibodies in the CD14 Ϫ conditioned medium suppressed fibrocyte differentiation. These data suggest that the development of fibrocytes is up-regulated systemically in burn patients. Increased TGF-␤ in serum stimulates the differentiation of the CD14 ϩ cell population in PBMC into collagen-producing cells that may be important in wound healing and scarring. (Lab Invest 2002, 82:1183-1192.

Our understanding of the role of bone marrow (BM)-derived cells in cutaneous homeostasis and wound healing had long been limited to the contribution of inflammatory cells. Recent studies, however, suggest that the BM contributes a significant proportion of noninflammatory cells to the skin, which are present primarily in the dermis in fibroblast-like morphology and in the epidermis in a keratinocyte phenotype; and the number of these BM-derived cells increases markedly after wounding. More recently, several studies indicate that mesenchymal stem cells derived from the BM could significantly impact wound healing in diabetic and nondiabetic animals, through cell differentiation and the release of paracrine factors, implying a profound therapeutic potential. This review discusses the most recent understanding of the contribution of BM-derived noninflammatory cells to cutaneous homeostasis and wound healing.

Optimum healing of a cutaneous wound requires a well-orchestrated integration of the complex biological and molecular events of cell migration and proliferation, and of extracellular matrix deposition and remodeling. Several studies in recent years suggest that bone marrow derived stem cells such as mesenchymal stem cells, progenitor cells such as endothelial progenitor cells and fibrocytes may be involved in these processes, contributing to skin cells or releasing regulatory cytokines. Stem/progenitor cells may be mobilized to leave the bone marrow, home to injured tissues and participate in the repair and regeneration. Direct injection of bone marrow derived mesenchymal stem cells or endothelial progenitor cells into injured tissues shows improved repair through mechanisms of differentiation and/or release of paracrine factors. Enhanced understanding of these cells may help develop novel therapies for difficult cutaneous conditions such as non-healing chronic wounds and hypertrophic scarring as well as engineering cutaneous substitutes.

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