Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

<p>Abstract</p> <p>Background</p> <p>Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging.</p> <p>The objective of this study was 1) To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on these cells. 2) To describe a methodology to create fibroblast cell lines from patients with chronic wounds.</p> <p>Methods</p> <p>Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing.</p> <p>Results</p> <p>Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA) Cell Repository.</p> <p>Conclusion</p> <p>We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their <it>in vivo </it>phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators <it>in vitro</it>.</p

The synergism of age and db/db genotype impairs wound healing

Both diabetes and advanced age have been implicated in delaying wound repair. However, the contribution of age alone has not been shown clinically to significantly impair the ability to heal. To determine the contribution of age and db/db genotype multiple wound healing parameters were determined in young db/db mice, aged db/db mice, age-matched non-db/db control and wild-type C57BL/6 mice. Biomechanical properties (breaking load and tensile stiffness), epithelialization, and collagen deposition were determined for the four groups of mice 14 days after wounding with suture-closed incisional wounds. While neither hyperglycemia nor age alone caused impairment in biomechanical properties, the combination of age and db/db genotype resulted in a 36% reduction in stiffness and a 42% reduction in breaking load, when compared to young control mice, suggesting poor quality of healing. Statistically significant differences in the volume of granulation tissue deposited within the wound site were also observed, with the aged db/db mice displaying more than any other group, suggesting greater dermal loss from the dermal edges of incisional wounds in aged db/db mice, suggesting that the combination of age and diabetes act synergistically to impair healing in mice with type 2 diabetes. Interestingly, the impairment occurs independently of the prevailing glycemia, supporting the hypothesis that diabetes in synergy with advanced age has downstream effects, leading to further impairment, necessitating initiation of early and aggressive intervention in elderly patients with diabetic foot ulcers

Molecular Markers in Patients with Chronic Wounds to Guide Surgical Debridement

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients’ wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hyperkeratotic epidermis; dermal fibrosis; increased procollagen synthesis). Fibroblasts deriving from this location exhibit impaired migration in comparison to the cells from adjacent nonulcerated biopsies, which exhibit normalization of morphology and normal migration capacity. The nonhealing edges have a specific, identifiable, and reproducible gene expression profile. The adjacent nonulcerated biopsies have their own distinctive reproducible gene expression profile, signifying that particular wound areas can be identified by gene expression profiling. We conclude that chronic ulcers contain distinct subpopulations of cells with different capacity to heal and that gene expression profiling can be utilized to identify them. In the future, molecular markers will be developed to identify the nonimpaired tissue, thereby making surgical debridement more accurate and more efficacious

Mechanism of Sustained Release of Vascular Endothelial Growth Factor in Accelerating Experimental Diabetic Healing

In this study, we hypothesize that local sustained release of vascular endothelial growth factor (VEGF), using adenovirus vector (ADV)-mediated gene transfer, accelerates experimental wound healing. This hypothesis was tested by determining the specific effects of VEGF165 application on multiple aspects of the wound healing process, that is, time to complete wound closure and skin biomechanical properties. After showing accelerated wound healing in vivo, we studied the mechanism to explain the findings on multiple aspects of the wound healing cascade, including epithelialization, collagen deposition, and cell migration. Intradermal treatment of wounds in non-obese diabetic and db/db mice with ADV/VEGF165 improves healing by enhancing tensile stiffness and/or increasing epithelialization and collagen deposition, as well as by decreasing time to wound closure. VEGF165, in vitro, stimulates the migration of cultured human keratinocytes and fibroblasts, thus revealing a non-angiogenic effect of VEGF on wound closure. In conclusion, ADV/VEGF is effective in accelerating wound closure by stimulating angiogenesis, epithelialization, and collagen deposition. In the future, local administration and sustained, controlled release of VEGF165 may decrease amputations in patients with diabetic foot ulcers and possibly accelerate closure of venous ulcers and pressure ulcers. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclu