Paul G. Scott

Mesenchymal Stem Cells Enhance Wound Healing Through Differentiation and Angiogenesis

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‐derived mesenchymal stem cells (BM‐MSCs) in wound healing. Using an excisional wound splinting model, we showed that injection around the wound and application to the wound bed of green fluorescence protein (GFP)+ allogeneic BM‐MSCs significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts or vehicle control medium. Fluorescence‐activated cell sorting analysis of cells derived from the wound for GFP‐expressing BM‐MSCs indicated engraftments of 27% at 7 days, 7.6% at 14 days, and 2.5% at 28 days of total BM‐MSCs administered. BM‐MSC‐treated wounds exhibited significantly accelerated wound closure, with increased re‐epithelialization, cellularity, and angiogenesis. Notably, BM‐MSCs, but not CD34+ bone marrow cells in the wound, expressed the keratinocyte‐specific protein keratin and formed glandular structures, suggesting a direct contribution of BM‐MSCs to cutaneous regeneration. Moreover, BM‐MSC‐conditioned medium promoted endothelial cell tube formation. Real‐time polymerase chain reaction and Western blot analysis revealed high levels of vascular endothelial growth factor and angiopoietin‐1 in BM‐MSCs and significantly greater amounts of the proteins in BM‐MSC‐treated wounds. Thus, our data suggest that BM‐MSCs promote wound healing through differentiation and release of proangiogenic factors.

HYPERTROPHIC SCARS, KELOIDS, AND CONTRACTURES: THE CELLULAR AND MOLECULAR BASIS FOR THERAPY

Keloids, hypertrophic scars, and contractures are a result of aberrations of the normal wound healing process. An understanding of the cellular and molecular events that are implicated in the development of these fibroproliferative disorders will allow for optimization of wound healing. In turn, treatment choices can be based on the most current scientific information available.

Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells.

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 ± 7.9% versus 69.9 ± 14.7%, p < 0.001). This percentage was consistently higher for patients with a ≥30% 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-β1 (TGF-β1) and the percentage of fibrocytes developing in the cultures of PBMC derived from these patients. We also demonstrated that fibrocytes were derived from CD14+ cells but not CD14− cells. Conditioned medium from CD14− cells was, however, required for fibrocyte differentiation, whereas direct contact between CD14− and CD14+ cells was not necessary. Treatment of the cell cultures with TGF-β1 enhanced the development of collagen-positive cells, whereas the inclusion of neutralizing anti-TGF-β1 antibodies in the CD14− conditioned medium suppressed fibrocyte differentiation. These data suggest that the development of fibrocytes is up-regulated systemically in burn patients. Increased TGF-β in serum stimulates the differentiation of the CD14+ cell population in PBMC into collagen-producing cells that may be important in wound healing and scarring.

Bone marrow-derived stem cells in wound healing: a review

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.

Identification of fibrocytes in postburn hypertrophic scar.

Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast‐like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two‐dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte‐specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte‐specific protein‐1 and to the N‐terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual‐labeled spindle‐shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen‐presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

transforming Growth Factor-β in Thermally Injured Patients with Hypertrophic Scars: Effects of Interferon α-2b

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

Deep dermal fibroblasts contribute to hypertrophic scarring

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-β1 and TGF-β1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more α-smooth muscle actin (α-SMA), had higher α-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS.

Fibrocytes from burn patients regulate the activities of fibroblasts

Wound healing requires an elaborate interplay between numerous cell types that orchestrate a series of regulated and overlapping events. Fibrocytes are a unique leukocyte subpopulation implicated in this process. One role proposed for these cells in wound healing is to synthesize extracellular matrix. Interestingly, using mass spectrometry to quantify hydroxyproline, we discovered that the capacity of fibrocytes from normal subjects or from burn patients to produce collagen is much less than that of dermal fibroblasts. Therefore, we investigated whether fibrocytes could play an indirect, regulatory, role in the healing of burn wounds by affecting the functions of dermal fibroblasts. Dermal fibroblasts treated with medium conditioned by burn patient fibrocytes, but not by those derived from normal subjects, showed an increase in cell proliferation and migration. Using confocal microscopy, flow cytometry, and immunoblotting, we found the level of α‐smooth muscle actin (α‐SMA) expression to be increased in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. To determine whether these effects could be attributed to transforming growth factor β (TGF‐β1) or to connective tissue growth factor (CTGF), we measured total TGF‐β1 levels in the conditioned medium by an enzyme‐linked immunosorbtion assay and assessed levels of CTGF mRNA and protein in fibroblasts and fibrocytes by reverse transcription‐polymerase chain reaction and Western blotting. The results showed significantly higher levels of TGF‐β1 and CTGF produced by burn patient fibrocytes. In addition, the application of a TGF‐β1 neutralizing antibody significantly reduced the effect of burn patient fibrocyte medium on dermal fibroblast proliferation, migration, and collagen lattice contraction. Our results suggest that in healing burn wounds, fibrocytes could regulate the activities of local fibroblasts.

The effect of mechanical stress on soft and hard tissue repair; a review

The influence of mechanical forces on intact tissue is well established. A growing body of evidence demonstrates that healing wounds also respond to the functional demands of their mechanical environment. At the present time, an understanding of the fundamental mechanism by which mechanical stress affects tissues and wounds remains elusive.

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-β1 mRNA and protein than normal skin and cells

Transforming growth factor‐β1 is a well‐known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor‐β1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription‐polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription‐polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor‐β1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen α1(I) cDNA with compatible ends was inserted into the polymerase chain reaction‐DNA fragment. The recombinant cDNA was re‐amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription‐polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor‐β1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription‐polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor‐β1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five‐fold more transforming growth factor‐β1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor‐β1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor‐β1 than normal cells (116 ± 6 vs. 97 ± 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor‐β1 mRNA in the cells and enzyme‐linked immunosorbent assay for TGF‐β1 protein in fibroblast‐conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor‐β1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription‐polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.