Autologous Cultured Bone Marrow-Derived Mesenchymal Stem Cells in a Fibrin Spray to Treat Venous Ulcers: A Randomized Controlled Double-Blind Pilot Study.

We treated a small cohort of venous ulcers that were very unresponsive to standard and advanced therapies with autologous cultured bone marrow-derived mesenchymal stem cells (MSCs). This pilot clinical trial was randomized, controlled, and double-blinded. Subjects were treated with either normal saline (Group A), fibrin spray alone (Group B), or MSCs in fibrin (1 million cells/cm2 of wound bed surface) (Group C). The control and test materials were applied to the wound using a double-barreled syringe with thrombin and fibrinogen (with or without MSCs) in each barrel, or saline alone in both barrels. The MSCs were separated, cultured in vitro, and expanded in a dedicated Good Manufacturing Practice (GMP) facility from 30-50 ml of bone marrow aspirate obtained from the iliac crest in Group C subjects. To ensure that the study remained controlled and blinded, subjects who were randomized to one of the two control arms (saline or fibrin) underwent sham bone marrow aspiration performed by a hematologist who anesthetized the iliac crest area down to and pushing against the periosteum, but without penetrating the bone marrow. Therefore, both the clinician who evaluated wound progress and the study subjects had no knowledge of whether bone aspiration was actually performed and what treatment had been applied to the wound. The study was performed after full FDA investigational new drug (IND) approval. The primary endpoint was the rate of healing (wound closure as linear healing from the wound margins in cm/week), as measured by the Gilman equation. One-way ANOVA was used to calculate the statistical significance of differences between the mean healing rates of each of the 3 treatment groups every 4 weeks and over the 24 weeks of treatment. Overall, treatment with MSCs accelerated the healing rate by about 10-fold compared to those in the saline and fibrin control groups. Although the total number of patients in this pilot study was small (n=11), the statistical significance was surprisingly promising: p<0.01 and f-ratio of 15.9358. No serious adverse events were noted. This small but carefully performed prospective, controlled, randomized, and double-blinded pilot study in a rare population of totally unresponsive patients adds to previous reports showing the promise of MSCs in the treatment of chronic wounds and provides proof of principle for how to approach this type of very demanding clinical and translational research.

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue-engineering construct.

An FDA-approved, prototypic, living, bilayered skin construct (BSC) has been used for non-healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro 'priming' step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO2 , with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL-6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real-time PCR (qRT-PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL-6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing-related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.

Topical delivery of cultured stem cells to human non-healing wounds: GMP facility development in an academic setting and FDA requirements for an IND and human testing.

With increasing emphasis on translational research, the need for appropriate regulatory oversight and approval has become essential. The requirements of the Food and Drug Administration (FDA) for Investigational New Drug (IND) exemption in studies that are investigator-initiated have become increasingly stringent. Moreover, academic institutions have not had substantial experience in establishing Good Manufacturing Practice (GMP) facilities required for manipulating human cells in vitro and for chemical or biochemical manufacturing. GMP regulations are established by the FDA under the authority of the Federal Food, Drug and Cosmetic Act. In this report, the authors outline the general strategy and some critical steps that an investigator and the institution may find helpful in developing a GMP facility, especially in an academic center. Also, more specifically and as proof of principle, we describe our approach to culturing autologous bone marrow-derived human mesenchymal stem cells (MSCs) and delivering them to non healing wounds. The lessons learned in this often lengthy and challenging process may be helpful to other academic institutions and investigators embarking on manipulating and delivering viable cells for human experimentation.

Bone marrow cell mobilization by the systemic use of granulocyte colony-stimulating factor (GCSF) improves wound bed preparation.

Innovative approaches are needed to accelerate the healing of human chronic wounds not responding to conventional therapies. An evolving and promising treatment is the use of stem cells. Our group has previously described the use of expanded (in vitro) autologous stem cells aspirated from human bone marrow and applied topically in a fibrin spray to human acute and chronic wounds. More recently, we have sought ways to mobilize stem cells directly from the bone marrow, without in vitro expansion. In this report, we show that systemic injections of granulocyte colony-stimulating factor (GCSF) can mobilize stem cells from bone marrow into the peripheral blood and then to the wound site. Our objectives were to optimize parameters for this method by using mouse models and proof of principle in a human chronic wound situation. Mice were injected for 5 days with 2 different formulations of GCSF and compared to control saline. To monitor stem cell mobilization, flow cytometric measurements of Sca-1 and c-Kit and colony-forming cell assays were performed. Full-thickness tail wounds in mice were created and monitored for healing, and polyvinyl alcohol sponges were implanted dorsally to assess collagen accumulation. To determine bone marrow stem cell homing to the wound site, chimeric mice transplanted with Green Fluorescent Protein bone marrow cells were scanned by live imaging. Additionally, as proof of principle, we tested the systemic GCSF approach in a patient with a nonhealing venous ulcer. Our findings lay the ground work and indicate that the systemic administration of GCSF is effective in mobilizing bone marrow stem cells into the peripheral blood and to the wound site. These findings are associated with an increased accumulation of collagen and promising results in terms of wound bed preparation and healing.

Differential keratin expression during epiboly in a wound model of bioengineered skin and in human chronic wounds.

Epiboly represents the process by which keratinocytes migrate to envelop a surface. The authors have been investigating a living bilayered skin construct (BSC) that is used in the treatment of lower extremity wounds due to venous insufficiency and diabetes. The construct demonstrates epiboly after injury and incubation in vitro, and this model may be useful for studying epidermal migration and the process of skin maturation. Punch biopsies of the construct in vitro were cultured and immunostained for specific keratins at baseline and at 24 to 72 hours. For comparison, skin biopsy specimens from human chronic venous ulcers and acute healing wounds were similarly processed. The authors found that K1 and K10 were fully expressed in the epidermis of the fully epibolized surface on BSC. K1 was also present in the migrating edge of specimens, whereas K10 was not detectable. K16 and K6 were evident in normal skin and the epibolized area of the construct; K6 expression was very prominent in the migrating edge. Importantly, K17 was distinctly limited to the epibolized surface and the migrating edge, and its expression was very similar to that observed in healing human wounds. In conclusion, differential expression of keratins in this epiboly model closely reflects in vivo studies and supports keratin specificity in the processes of migration and differentiation of new epidermis. Therefore, these findings provide further and important validity for the study of epithelialization and the hope of developing prognostic markers for venous ulcer healing.

Fibroblasts from non-healing human chronic wounds show decreased expression of beta ig-h3, a TGF-beta inducible protein.

BACKGROUND: Increasing evidence shows persistent phenotypic alterations in fibroblasts from non-healing human chronic wounds, which may result in faulty extracellular matrix deposition and keratinocyte migration. We have previously shown that these cells are characterized by morphological changes, low proliferative potential and unresponsiveness to TGF-beta1, and down regulated phosphorylation of Smad 2/3 and p42/44 MAPK from decreased expression of the TGF-beta type II receptor. OBJECTIVE: To identify genes and proteins that may be differentially expressed in chronic wounds and their cultured fibroblasts. METHODS: Differential display analysis with 120 random primer sets was used in fibroblasts from human venous ulcers and acute wounds created on the ipsilateral thighs of the same patients. Positive differential results were confirmed by RT-PCR. Immunohistochemistry of cultured fibroblasts and tissues was used to determine the expression of differentially expressed proteins. RESULTS: A total of 16 differentially expressed genes were identified and cloned. The only candidate gene that was differentially expressed in all patients and confirmed by repeated differential display testing and RT-PCR was beta ig-h3, a TGF-beta-induced gene involved in cell adhesion, migration, and proliferation. Decreased expression of beta ig-h3 in chronic wounds and their fibroblasts was further confirmed by Western blot and immunostaining. CONCLUSION: These findings point to beta ig-h3 as an important gene characterizing the abnormal phenotype of chronic wound fibroblasts. Corrective measures to increase the expression of this protein might have therapeutic potential.

Migration of the epidermal over the dermal component (epiboly) in a bilayered bioengineered skin construct.

A bilayered bioengineered living skin construct (LSC) consisting of viable human neonatal keratinocytes over a collagenous dermis seeded with dermal fibroblasts has been used extensively in difficult-to heal human wounds. Its biological properties include production of several mediators, cytokines, and growth factors and the ability to heal itself upon injury. In this study, we investigated the process of keratinocyte migration in LSC. At baseline, 6-mm punch biopsies of the construct were placed in serum-free medium (AIM-V) or Dulbecco's modified Eagle medium. At varying time points, the LSC samples were processed and analyzed using histology and immunohistochemistry. By 72 h, in a time-dependent manner, the overlying epidermis had migrated over and enveloped the entire underlying dermis, a process known as epiboly. Increasing concentrations of neutralizing antibodies to epidermal growth factor or interleukin-1 alpha down-regulated the extent of epiboly, as measured using computerized planimetry, but antibodies to transforming growth factor-beta 1 did not affect it. The consistent expression of laminin V, alpha3beta1 integrin, and vitronectin (epibolin) and its integrin receptor (alphavbeta5) characterized the tongue of migrating epidermis. Increasing concentrations of antibodies to vitronectin blocked the process of epiboly, as did antibodies to the alphavbeta5 integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. This process of epiboly provides novel mechanisms of action for bioengineered skin constructs.

Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds.

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.

Human beta-defensin-2 expression is increased in chronic wounds.

First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human beta-defensin-2 (hbetaD-2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hbetaD-2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hbetaD-2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hbetaD-2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hbetaD-2 expression but that marked up-regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate-to-strong immunostaining for hbetaD-2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hbetaD-2 immunostaining after meshing. We conclude that the expression of hbetaD-2 is up-regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hbetaD-2, possibly due to ongoing tissue injury and bacterial colonization.

Fibroblasts from chronic wounds show altered TGF-beta-signaling and decreased TGF-beta Type II receptor expression.

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-beta1 (TGF-beta1). In this study, we have determined some of the mechanisms that might be responsible for unresponsiveness to TGF-beta1. Using Northern analysis and affinity labeling, we show that venous ulcer fibroblasts have decreased TGF-beta 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-beta1. 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.

Transcriptional activation of alpha 1(III) procollagen gene in Tsk2/+ dermal fibroblasts.

Transient transfection experiments into Tsk2/+ and normal dermal fibroblasts were performed using four successively shorter Col3a1 promoter deletion constructs: #103, #110, #114, and #120 fused to the chloramphenicol-acetyl-transferase (CAT) reporter gene. The transcriptional activity in Tsk2/+ and normal dermal fibroblasts driven by the three longer constructs was equal. With the shortest construct, #120 (-96 to +16bp) the transcriptional activity in Tsk2/+ fibroblasts was 25 times higher than in normal fibroblasts. Electrophoretic mobility shift assays with a labeled #120 probe revealed that increased DNA-protein binding occurred with nuclear extracts prepared from Tsk2/+ fibroblasts and that this binding was displaced by consensus Sp1 and NF-1 oligonucleotide sequences. These data indicate that sequences from -96 to +16bp of the Col3a1 promoter play an important role in the upregulated expression of this gene in Tsk2/+ fibroblasts and that the promoter contains sequences which bind the trans-acting nuclear factors, Sp1(like) and NF-1(like).

Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-beta1.

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.