Sara E. Dahle

Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: study protocol for a comparative efficacy randomized controlled trial

BackgroundDiabetic foot ulcers (DFUs) represent a significant source of morbidity and an enormous financial burden. Standard care for DFUs involves systemic glucose control, ensuring adequate perfusion, debridement of nonviable tissue, off-loading, control of infection, local wound care and patient education, all administered by a multidisciplinary team. Unfortunately, even with the best standard of care (SOC) available, only 24% or 30% of DFUs will heal at weeks 12 or 20, respectively.The extracellular matrix (ECM) in DFUs is abnormal and its impairment has been proposed as a key target for new therapeutic devices. These devices intend to replace the aberrant ECM by implanting a matrix, either devoid of cells or enhanced with fibroblasts, keratinocytes or both as well as various growth factors. These new bioengineered skin substitutes are proposed to encourage angiogenesis and in-growth of new tissue, and to utilize living cells to generate cytokines needed for wound repair.To date, the efficacy of bioengineered ECM containing live cellular elements for improving healing above that of a SOC control group has not been compared with the efficacy of an ECM devoid of cells relative to the same SOC. Our hypothesis is that there is no difference in the improved healing effected by either of these two product types relative to SOC.Methods/DesignTo test this hypothesis we propose a randomized, single-blind, clinical trial with three arms: SOC, SOC plus Dermagraft® (bioengineered ECM containing living fibroblasts) and SOC plus Oasis® (ECM devoid of living cells) in patients with nonhealing DFUs. The primary outcome is the percentage of subjects that achieved complete wound closure by week 12.DiscussionIf our hypothesis is correct, then immense cost savings could be realized by using the orders-of-magnitude less expensive acellular ECM device without compromising patient health outcomes. The article describes the protocol proposed to test our hypothesis.Trial registrationClinicalTrials.gov: NCT01450943. Registered: 7 October 2011

A systematic review of low-level light therapy for treatment of diabetic foot ulcer.

Diabetes mellitus (DM) is a significant international health concern affecting more than 387 million individuals. A diabetic person has a 25% lifetime risk of developing a diabetic foot ulcer (DFU), leading to limb amputation in up to one in six DFU patients. Low‐level light therapy (LLLT) uses low‐power lasers or light‐emitting diodes to alter cellular function and molecular pathways, and may be a promising treatment for DFU. The goal of this systematic review is to examine whether the clinical use of LLLT is effective in the healing of DFU at 12 and 20 weeks in comparison with the standard of care, and to provide evidence‐based recommendation and future clinical guidelines for the treatment of DFU using LLLT. On September 30, 2015, we searched PubMed, EMBASE, CINAHL, and Web of Science databases using the following terms: “diabetic foot” AND “low level light therapy,” OR “light emitting diode,” OR “phototherapy,” OR “laser.” The relevant articles that met the following criteria were selected for inclusion: randomized control trials (RCTs) that investigated the use of LLLT for treatment of DFU. Four RCTs involving 131 participants were suitable for inclusion based upon our criteria. The clinical trials used sham irriadiation, low dose, or nontherapeutic LLLT as placebo or control in comparison to LLLT. The endpoints included ulcer size and time to complete healing with follow‐up ranging from 2 to 16 weeks. Each article was assigned a level of evidence (LOE) and graded according to the Oxford Center for Evidence‐based Medicine Levels of Evidence Grades of Recommendation criteria. Limitations of reviewed RCTs include a small sample size (N < 100), unclear allocation concealment, lack of screening phase to exclude rapid healers, unclear inclusion/exclusion criteria, short (<30 days) follow‐up period, and unclear treatment settings (wavelength and treatment time). However, all reviewed RCTs demonstrated therapeutic outcomes with no adverse events using LLLT for treatment of DFU. This systematic review reports that LLLT has significant potential to become a portable, minimally invasive, easy‐to‐use, and cost effective modality for treatment of DFU. To enthusiastically recommend LLLT for treatment of DFU, additional studies with comparable laser parameters, screening period to exclude rapid healers, larger sample sizes and longer follow‐up periods are required. We envision future stringent RCTs may validate LLLT for treatment of DFU. Systematic review registration number: PROSPERO CRD42015029825.

Acute Wounding Alters the Beta2-Adrenergic Signaling and Catecholamine Synthetic Pathways in Keratinocytes

Keratinocyte migration is critical for wound re-epithelialization. Previous studies showed that epinephrine activates the beta2-adrenergic receptor (B2AR), impairing keratinocyte migration. Here, we investigated the keratinocyte catecholamine synthetic pathway in response to acute trauma. Cultured keratinocytes were scratch wounded and expression levels of the B2AR and catecholamine synthetic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were assayed. The binding affinity of the B2AR was measured. Wounding downregulated B2AR, tyrosine hydroxylase, and phenylethanolamine-N-methyltransferase expression, but pre-exposure to timolol, a beta-adrenergic receptor antagonist, delayed this effect. In wounded keratinocytes, B2AR-binding affinity remained depressed even after its expression returned to prewounding levels. Keratinocyte-derived norepinephrine increased after wounding. Norepinephrine impaired keratinocyte migration; this effect was abrogated with B2AR-selective antagonist ICI-118,551 but not with B1AR-selective antagonist bisoprolol. Finally, for clinical relevance, we determined that norepinephrine was present in freshly wounded skin, thus providing a potential mechanism for impaired healing by local B2AR activation in wound-edge keratinocytes. Taken together, the data show that keratinocytes modulate catecholamine synthetic enzymes and release norepinephrine after scratch wounding. Norepinephrine appears to be a stress-related mediator that impairs keratinocyte migration through activation of the B2AR. Future therapeutic strategies evaluating modulation of norepinephrine-related effects in the wound are warranted.

Reclaiming Autologous Amputated Tissue for Limb Salvage of a Diabetic Foot Burn with Underlying Critical Limb Ischemia

BACKGROUND: Diabetes mellitus is a worldwide pandemic that impacts more than 387 million people, with 29 million individuals affected in the United States alone. Diabetic patients have a 25% lifetime risk of developing a diabetic foot ulcer (DFU). Having a DFU is associated with a risk of recurrence approaching 70%. In addition, 1 in 6 patients with DFU will have a lower-limb amputation, with an associated increase in mortality ranging from 47% to 70%. Therefore, limb salvage is critical in patients with DFU. CASE STUDY: This article describes the case of a 70-year-old man with diabetes mellitus, end-stage renal disease, and peripheral arterial occlusive disease who presented with a 1.5% total-body-surface-area, third-degree burn to the left hallux with dry gangrene extending to the midfoot. Ankle brachial indexes were 0.66 on the left and 0.64 on the right. Toe pressures on the left were absent because of extensive dry gangrene. His right foot had a prior transmetatarsal amputation. Using a retrograde pedal approach, a chronic total occlusion of the left posterior tibial artery was recanalized with balloon angioplasty. He then underwent a transmetatarsal amputation with closure, except that the plantar medial side could not be closed without tension. Therefore, an autologous full-thickness skin graft, from the amputation specimen, was used to bridge the defect. DISCUSSION: At 32-week follow-up, the wound was healed, the graft had fully incorporated, and the patient was ambulating well using custom orthotic footwear. The creative use of amputated tissue to assist with wound coverage has not been well described in the literature.

Deferoxamine: potential novel topical therapeutic for chronic wounds

Although chronic wounds have multifactorial aetiologies, they share a common characteristic of compromise of the local vasculature leading to diminished oxygen tension. In pressure ulcers (PU), the obstruction of capillary blood flow and lymphatics from the local shear pressure results in ischaemia. The subsequent reperfusion causes hyperaemia and oedema, further reducing the regional oxygen level. Similarly, in venous leg ulcers (VLU), vascular permeability and increase in venous hypertension generate local oedema. In diabetic foot ulcers (DFU), hypoxia results from local pressure and high metabolic stress on the wound bed. During the initial phase of healing for all three wound types, the local and acute hypoxic climate results in the release of numerous growth factors by endothelial cells, fibroblasts and macrophages. One of those essential factors is the hypoxia inducible factor-1 (HIF-1). The regulation of HIF-1 activity resides in its oxygen-dependent degradation, by prolyl 4-hydroxylase (PHD) enzymes that require iron (Fe) as a cofactor. Under hypoxic conditions, HIF-1a is stabilized against PHD enzymatic degradation and moves to the nucleus, where it dimerizes with HIF-1b and binds to a hypoxia response element. For its transcriptional activity, HIF-1a also needs to bind to the coactivators p300/cyclic adenosine monophosphate response element binding protein (CREB) binding protein (CBP). This results in the upregulation and the activation of more than 60 HIF-1a target genes for tissue repair, cell growth and proliferation, and important angiogenic factors such as vascular endothelial growth factor (VEGF; Fig. 1). Although VEGF induces angiogenesis, studies have shown that prolonged hypoxia results in increased acidosis, decrease adenosine triphosphate production and inhibition of neovascularization that cannot be reversed by VEGF. This explains the need for a therapeutic agent that would maintain the multitarget activity of HIF-1a within the chronic wound environment (Fig. 1). Another characteristic of chronic wounds is the local deposition of free iron. In VLU, leakage of erythrocytes into the interstitium and their degradation by macrophages leads to the release of the iron bound to ferritin. In patients with diabetes, insulin resistance results in the increase in ferritin synthesis and iron stores, which concurrently reduce the hepatic extraction and metabolism of insulin, leading to hyperinsulinaemia. We speculate that the local tissue ischaemia with vessel occlusion encountered in PU also leads to increased iron deposition (Fig. 1). Iron is a transition metal that has the ability to donate or accept single electrons. This allows iron to neutralize free radicals and thus to serve as a critical antioxidant defence. In order to maintain the physiological redox status of the cells, iron is sequestered bound with either proteins or cofactors. If released from its bound form, free iron serves as a chemoattractant for neutrophils and macrophages. The combination of iron and hydrogen peroxide (from activated neutrophils) generates ferric ion (Fe) and reactive oxygen species (ROS), the prolonged production of which results in a chronically inflamed environment. A vicious cycle is created by the reduction of ferric to ferrous ion (Fe), generating more ROS, and the subsequent ROS enhancement of release of iron from carriers (Fig. 1). Iron also induces an unrestrained proinflammatory M1 macrophage phenotype, which contributes to perpetual tissue damage and compromised tissue regeneration as the cells fail to switch to the proreparative M2 phenotype. All of the above sustain the chronic inflammatory state encountered in chronic wounds (Fig. 1). We postulate that the topical application of deferoxamine (DFO), which simultaneously acts as an iron chelator and effective HIF-1a inducer and stabilizer, is a potential therapeutic approach to improve healing of chronic wounds. DFO could be topically administered to the impaired wound bed at concentrations 100-fold lower than the therapeutic parenteral infusion warranted in the treatment of thalassaemia or iron poisoning, and with a better safety profile. Preclinical studies have demonstrated that DFO topically applied or injected improved wound healing in db/db mice, with increased granulation tissue and neovascularization. DFO modulated the expression of several cytokines and growth factors (including HIF-1a and VEGF). As a result, DFO led to decreased inflammation, enhanced angiogenesis and wound maturation. The authors proposed that DFO ointment could potentially heal cutaneous wounds in patients with diabetes. The addition of DFO to fibroblasts derived from diabetic db/db mice cultured in hyperglycaemic and hypoxic conditions resulted in increased expression of HIF-1a target genes responsible for motility and angiogenesis. DFO enhanced wound healing rates in the db/db mouse, even in the presence of continuous hyperglycaemia. Human aortic endothelial cells cultured in high glucose and hypoxia with DFO, resulted in a twofold elevation in HIF-1a binding to p300 coactivator, approaching levels found in cells cultivated in low glucose. DFO prevented the methylglyoxal modification of p300, which has been shown to impact negatively on the heterodimerization of the two HIF-1 subunits. Additionally, local injection of DFO into ischaemic flaps of diabetic mice resulted in wound healing enhancement

Radioprotection with Amifostine Enhances Bone Strength and Regeneration and Bony Union in a Rat Model of Mandibular Distraction Osteogenesis

Background Using distraction osteogenesis (DO) to regenerate robust endogenous bone could greatly enhance postoncologic reconstruction of head and neck cancer. However, radiation (XRT) corrosive effects still preclude DOs immense potential. We posit that adjunctive pretreatment with the radioprotectant amifostine (AMF) can optimize wound healing and allow for successful DO with quantifiable enhancements in bony union and strength despite previous surgical bed irradiation. Methods Two groups of murine left hemimandibles were exposed to a human equivalent radiation dosage fractionated over 5 daily doses of 7 Gy. AMF-XRT-DO (n = 30) received AMF before radiation, whereas XRT-DO (n = 22) was untreated. All animals underwent left hemimandibular osteotomy and external fixator placement, followed by distraction to a 5.1-mm gap. Left hemimandibles were harvested and mechanically tested for parameters of strength, yield, and breaking load. Results Radiation-related complications such as severe alopecia were significantly increased in XRT-DO compared with the AMF-treated group (P = 0.001), whereas infection and death were comparable (P = 0.318). Upon dissection, bony defects were grossly visible in XRT-DO distraction gap compared with AMF-XRT-DO, which exhibited significantly more complete unions (P = 0.004). Those results were significantly increased in the specimens prophylactically treated with AMF (yield: 39.41 N vs 21.78 N, P = 0.023; breaking load: 61.74 N vs 34.77 N, P = 0.044; respectively). Conclusions Our study revealed that AMF enhances biomechanical strength, regeneration, and bony union after radiation in a murine model of DO. The use of prophylactic AMF in combination with DO offers the promise of an alternative reconstructive option for patients afflicted with head and neck cancer.