The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.

Chronic wounds are characterized by failure to heal in a defined time frame. However, the pathogenic steps leading from the etiological factors to failure to heal are unknown. Recently, increasing evidence suggests that resident cells in chronic wounds display a number of critical abnormalities, including senescence and unresponsiveness to the stimulatory action of transforming growth factor-b1 (TGF-b1). In this study, we have determined some of the mechanisms that might be responsible for unresponsiveness to TGF-b1. Using Northern analysis and affinity labeling, we show that venous ulcer fibroblasts have decreased TGF-b Type II receptor expression. This finding is not the result of genetic mutation, as shown by experiments with Type II receptor satellite instability. Decreased Type II receptor expression was accompanied by failure of ulcer fibroblasts to phosphorylate Smad 2, Smad 3, and p42/44 mitogen activating protein kinase (MAPK), and was associated with a slower proliferative rate in response to TGF-b1. We conclude that venous ulcer fibroblasts show decreased Type II receptor expression and display abnormalities in the downstream signaling pathway involving MAPK and the early Smad pathway. These findings suggest ways to address and treat the abnormal cellular phenotype of cells in chronic wounds.

Recent findings point to low oxygen tension (hypoxia) as an important mechanism for the expression of several eukaryotic genes. We have previously shown that hypoxia (2% O2), when compared to standard oxygen tension (20% O2), upregulates the mRNA levels of the human alpha1(I) (COL1A1) procollagen gene and transforming growth factor-beta1 (TGF-beta1) in human dermal fibroblasts. In this report, we determined the effect of hypoxia on collagen synthesis and transcription. Exposure of human dermal fibroblasts to hypoxia for 24-72 h led to a threefold, dose-dependent increase in collagenous protein (P < 0.0001; r = 0.9794) and to enhanced type I procollagen deposition, as shown by direct immunofluorescence. Transient transfections with a series of luciferase- and CAT-promoter constructs of the human COL1A1 gene (spanning from -2.5 kb to +113 bp) showed that hypoxia increases the transcriptional activity of constructs having 5' endpoints between -804 bp and -107 bp, with loss of stimulation at -84 bp. Maximal increase in promoter activity in hypoxia was observed between -190 and -174 bp of the proximal promoter, once a cKrox repressor site (-199 to -224 bp) was deleted. Upregulation of COL1A1 mRNA levels in hypoxia was blocked by a TGF-beta1 anti-sense oligonucleotide, and failed to occur in fibroblasts from TGF-beta1 knock-out mice. Co-transfection and overexpression with a Smad7 construct abrogated the increase in COL1A1 promoter activity observed in hypoxia. Upregulated transcriptional activity of the TGF-beta1 promoter in hypoxia was found to be maximal between -453 and -175 bp from the transcriptional start site. Since hypoxia is a critical feature of the early phases of wound repair, we conclude that it may act as a potent physiologic stimulus for collagen synthesis. TGF-beta1 appears to be a critical component of this response.