Homocysteine– A Stealth Mediator of Impaired Wound Healing: A Preliminary Study

4/1/2006
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Author(s):

Joseph V. Boykin, Jr., MD,1,2 and Chris Baylis, PhD3

Methods and Materials

Dermal substitute therapy. During the authors’ observations, 12 consecutive patients who were scheduled for topical human fibroblast-derived dermal substitute therapy for lower-extremity ulcers (LEUs) at the Retreat Hospital Wound Healing Center were selected for study. The product is a tissue-engineered human (neonatal) fibroblast-derived dermal substitute designed as an interactive therapy for the treatment of full-thickness diabetic foot ulcers greater than 6 weeks duration. When applied to the wound bed, the dermal substitute delivers a diverse elaboration of growth factors, cytokines, matrix proteins, and glycosaminoglycans. These components are found in healthy human dermis and have been demonstrated to encourage dermal matrix development, angiogenesis, epithelial cell migration, and wound closure. For ulcer treatment, patients received 8 weekly applications of dermal substitute to the debrided wound bed. Patients with signs of soft tissue infection or osteomyelitis were excluded from treatment. Patients whose ulcers displayed enhancements of wound healing parameters and a progressive demonstration of wound granulation tissue deposition, increased wound vascularity, and decreasing wound area and volumes following the initiation of dermal substitute therapy were considered “responders” to dermal substitute therapy. Patients whose wounds displayed minimal or no improvements with wound healing parameters were considered “non-responders” to dermal substitute therapy. Investigational review board approval and informed patient consent were obtained prior to the performance of these observations. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
Wound fluid NOx determinations. During this study, baseline (pre-treatment) wound fluid NOx determinations were obtained from patients receiving dermal substitute therapy. Additionally, 4 study patients were selected for serial evaluation of wound fluid NOx and wound area determinations following dermal substitute therapy. Two of these patients (1 responder and 1 non-responder) are presented with illustrations of the serial profile of wound fluid NOx values and wound area measurements following treatment (Table 1, WFN patients). For this demonstration, wound fluid NOx determinations were made prior to dermal substitute treatment (baseline value) and at weeks 2, 4, 6, and 8 of dermal substitute treatment. Wound fluid from the study ulcer was collected with nitrate-free filter paper. After placing the filter paper on the wound for 24 hours with the routine dressing (in a silver- [Ag+] free environment), the wound fluid was eluted from the filter paper and the wound fluid NOx was determined by using the Griess assay.¹⁸ Wound fluid NOx samples were stored at -70˚C until the assay was performed. Laboratory personnel performing the assays were blinded to the identification of study subjects.
Wound area measurements. Measurements were performed directly on the wound (multiple measurements with averaged data). Estimations were then made of the wound area (cm²) for comparative analysis. Wound area measurements were performed immediately prior to initial dermal substitute application (baseline measurement) and at weeks 2, 4, 6, and 8 of therapy.

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