Stem cell-derived small extracellular vesicles embedded into methacrylated hyaluronic acid wound dressings accelerate wound repair in a pressure model of diabetic ulcer.

Over the past years, the development of innovative smart wound dressings is revolutionizing wound care management and research. Specifically, in the treatment of diabetic foot wounds, three-dimensional (3D) bioprinted patches may enable personalized medicine therapies. In the present work, a methacrylated hyaluronic acid (MeHA) bioink is employed to manufacture 3D printed patches to deliver small extracellular vesicles (sEVs) obtained from human mesenchymal stem cells (MSC-sEVs). The production of sEVs is maximized culturing MSCs in bioreactor. A series of in vitro analyses are carried out to demonstrate the influence of MSC-sEVs on functions of dermal fibroblasts and endothelial cells, which are the primary functional cells in skin repair process. Results demonstrate that both cell populations are able to internalize MSC-sEVs and that the exposure to sEVs stimulates proliferation and migration. In vivo experiments in a well-established diabetic mouse model of pressure ulcer confirm the regenerative properties of MSC-sEVs. The MeHA patch enhances the effectiveness of sEVs by enabling controlled release of MSC-sEVs over 7 days, which improve wound epithelialization, angiogenesis and innervation. The overall findings highlight that MSC-sEVs loading in 3D printed biomaterials represents a powerful technique, which can improve the translational potential of parental stem cell in terms of regulatory and economic impact.

Playing with Biophysics: How a Symphony of Different Electromagnetic Fields Acts to Reduce the Inflammation in Diabetic Derived Cells.

Several factors, such as ischemia, infection and skin injury impair the wound healing process. One common pathway in all these processes is related to the reactive oxygen species (ROS), whose production plays a vital role in wound healing. In this view, several strategies have been developed to stimulate the activation of the antioxidative system, thereby reducing the damage related to oxidative stress and improving wound healing. For this purpose, complex magnetic fields (CMFs) are used in this work on fibroblast and monocyte cultures derived from diabetic patients in order to evaluate their influence on the ROS production and related wound healing properties. Biocompatibility, cytotoxicity, mitochondrial ROS production and gene expression have been evaluated. The results confirm the complete biocompatibility of the treatment and the lack of side effects on cell physiology following the ISO standard indication. Moreover, the results confirm that the CMF treatment induced a reduction in the ROS production, an increase in the macrophage M2 anti-inflammatory phenotype through the activation of miRNA 5591, a reduction in inflammatory cytokines, such as interleukin-1 (IL-1) and IL-6, an increase in anti-inflammatory ones, such as IL-10 and IL-12 and an increase in the markers related to improved wound healing such as collagen type I and integrins. In conclusion, our findings encourage the use of CMFs for the treatment of diabetic foot.

Circulating Biomarkers of Endothelial Dysfunction and Inflammation in Predicting Clinical Outcomes in Diabetic Patients with Critical Limb Ischemia.

Critical limb ischemia (CLI) is a severe manifestation of peripheral artery disease characterized by ischemic pain, which is frequently associated with diabetes and non-healing lesions to inferior limbs. The clinical management of diabetic patients with CLI typically includes percutaneous transluminal angioplasty (PTA) to restore limb circulation and surgical treatment of diabetic foot ulcers (DFU). However, even after successful treatment, CLI patients are prone to post-procedure complications, which may lead to unplanned revascularization or foot surgery. Unfortunately, the factors predicting adverse events in treated CLI patients are only partially known. This study aimed to identify potential biomarkers that predict the disease course in diabetic patients with CLI. For this purpose, we measured the circulating levels of a panel of 23 molecules related to inflammation, endothelial dysfunction, platelet activation, and thrombophilia in 92 patients with CLI and DFU requiring PTA and foot surgery. We investigated whether these putative biomarkers were associated with the following clinical endpoints: (1) healing of the treated DFUs; (2) need for new revascularization of the limb; (3) appearance of new lesions or relapses after successful healing. We found that sICAM-1 and endothelin-1 are inversely associated with DFU healing and that PAI-1 and endothelin-1 are associated with the need for new revascularization. Moreover, we found that the levels of thrombomodulin and sCD40L are associated with new lesions or recurrence, and we show that the levels of these biomarkers could be used in a decision tree to assign patients to clusters with different risks of developing new lesions or recurrences.

Surgery for the diabetic foot: A key component of care.

Surgery for acute and chronic diabetic foot problems has long been an integral component of care. While partial foot amputations remain as important diabetic limb-salvaging operations, foot-sparing reconstructive procedures have become equally important strategies to preserve the functional anatomy of the foot while addressing infection, chronic deformities, and ulcerations. A classification of types of diabetic foot surgery is discussed in accordance with the soft tissue status and acuity of the presenting foot problem. This brief overview from the Association for Diabetic Foot Surgeons describes common conditions best treated by surgical interventions, as well as specific indications. While techniques and indications continue to evolve, effective surgical management of the diabetic foot remains an integral component of care as well as for the prevention of recurrent ulceration.

Conservative Surgical Treatment of Infected Ulceration of the First Metatarsophalangeal Joint With Osteomyelitis in Diabetic Patients.

Ulceration of the plantar aspect of the first metatarsophalangeal joint is a common localization in the diabetic foot. Conservative treatment of this lesion is a challenging problem, performed through the soft tissues and osseous debridement. The present study included a cohort of 28 patients affected by diabetes mellitus and a first ray lesion penetrating the bone. After surgical debridement with removal of the infected bone, we positioned antibiotic-loaded bone cement and stabilized the treated area with an external fixator. All patients with critical limb ischemia had their vascular disease treated before the procedure. The mean follow-up was 12.2 ± 6.9 months. Four patients developed a relapse of the ulceration after the procedure. In the postoperative period, 1 patient (3.57%) developed dehiscence of the surgical site and underwent a second procedure. In the follow-up period, 2 patients (7.14%) experienced bone cement dislocation. In 1 of these patients, a new ulceration was observed dorsally to the surgical site. The approach was surgical revision with bone cement replacement and stabilization with a new external fixator. In the other patient, given the absence of ulcerations, the cement was removed, and arthrodesis with internal stabilization using 2 cannulated screws was performed. One patient (3.57%), who had developed a relapse of ulceration after recurrent critical ischemia, underwent a percutaneous revascularization procedure and transmetatarsal amputation. During the follow-up period, no ulceration recurrences, transfer ulcerations, shoe fit problems, or gait abnormalities were detected in the other 24 patients. Our study presents the results of a technique requiring a 1-stage surgical approach to a relatively common problem, which is often difficult to solve.

Diabetic foot wounds: the value of negative pressure wound therapy with instillation.

Chronic wounds such as diabetic foot wounds are a tremendous burden to the health care system and often require a multidisciplinary approach to prevent amputations. Advanced technologies such as negative pressure wound therapy (NPWT) and bioengineered tissues have been successfully used in the treatment of these types of complex wounds. However, the introduction of NPWT with instillation (NPWTi) has provided an alternative treatment for treating complex and difficult-to-heal wounds. This article provides an overview of NPWT and the new NPWTi system and describes preliminary experience using NPWTi on patients with complicated infected diabetic foot wounds after surgical debridement and in a multidisciplinary setting.

Use of Integra Dermal Regeneration Template for Limb Salvage in Diabetic Patients With No-Option Critical Limb Ischemia.

We have analyzed in a retrospective study of consecutive diabetic patients affected by no-option critical limb ischemia (CLI) the efficacy of the dermal substitute Integra Dermal Regeneration Template for treatment of complicated foot lesions. The primary end point was limb salvage and 1-year amputation-free survival. The secondary end point was healing time of surgical site. Between October 2014 and October 2017, 1024 patients with diabetic foot ulcer (DFU) and CLI were admitted. In 84 patients (8.2%), there was a failure in distal revascularization with a persistent CLI after the procedure. Despite the persistent CLI, a group of 26 patients of this cohort obtained complete wound healing. Among them, 13 patients were treated with surgical debridement or open amputations and application of dermal substitute Integra Dermal Regeneration Template and the other 13 patients were treated without any dermal substitute. The Integra group healed within a mean time of 83.5 days, and the control group healed within a mean of 139 days (P = .028). No major amputation was carried out at 1-year follow-up in the Integra group versus 15% in the control group. A conservative foot surgery or an approach with minor amputation in diabetic patients with no-option CLI may achieve limb salvage. The use of Integra Dermal Regeneration Template in patients with DFU and no-option CLI may be a useful option in a limb salvage program.

Surgical OFF-LOADING of the diabetic foot.

Diabetic foot ulcer treatment is a challenge for the healthcare world. Widespread infection and the presence of critical ischemia (especially with end-stage renal disease) can lead to major amputation rather than amenable to conservative treatment. Surgical strategies of the diabetic foot have been changing over the past 10 years and are now focused on reconstructive treatment and limb salvage. These goals were achieved, thanks to an evolution of distal revascularization techniques and a distinct approach, which integrates various methods focused on limb salvage. Podoplastic techniques of the diabetic foot are focused on infection clearance, the surgical treatment of corrective deformities, soft tissue coverage and limb ischemia correction along with the management of diabetes and the comorbidities that compromise tissue repair processes. The reconstructive techniques used in diabetic foot treatment owe their effectiveness in part to the results of technological improvements such as the circular external fixator as a tool for stabilization and surgical site protection. In the last decade, many studies have shown that circular external fixation should be considered as the most useful method to protect the reconstructive surgical site in limb salvage of the diabetic foot. The objective of this review is to highlight the role of surgical offloading using circular external fixation as an adjunct to the podoplastic diabetic foot reconstruction procedures.

Challenges in the management of chronic wound infections.

OBJECTIVES: Chronic wound infections may delay the healing process and are responsible for a significant burden on healthcare systems. Since inappropriate management may commonly occur in the care of these patients, this review aims to provide a practical guide underlining actions to avoid in the management of chronic wound infections. METHODS: We performed a systematic review of the literature available in PubMed in the last 10 years, identifying studies regarding the management of patients with chronic wound infections. A panel of experts discussed the potential malpractices in this area. A list of 'Don'ts', including the main actions to be avoided, was drawn up using the 'Choosing Wisely' methodology. RESULTS: In this review, we proposed a list of actions to avoid for optimal management of patients with chronic wound infections. Adequate wound bed preparation and wound antisepsis should be combined, as the absence of one of them leads to delayed healing and a higher risk of wound complications. Moreover, avoiding inappropriate use of systemic antibiotics is an important point because of the risk of selection of multidrug-resistant organisms as well as antibiotic-related adverse events. CONCLUSION: A multidisciplinary team of experts in different fields (surgeon, infectious disease expert, microbiologist, pharmacologist, geriatrician) is required for the optimal management of chronic wound infections. Implementation of this approach may be useful to improve the management of patients with chronic wound infections.

High-Sensitivity Cardiac Troponin Predicts Major Cardiovascular Events in Diabetic Patients With Critical Limb Ischemia and Foot Lesions.

Background: Diabetic patients with critical limb ischemia (CLI) and foot lesions show a poor prognosis. Optimal risk stratification to guide tailored intervention is still uncertain. The aim of the present study was to assess the prognostic role of high-sensitivity cardiac troponin T (hs-TnT) in such a high-risk population. Methods and Results: Clinical, laboratory, and interventional data, as well as the SPINACH score, were collected. Hs-TnT was measured at hospital admission. All patients were followed up for at least 1 year. The primary endpoint was the cumulative occurrence of major cardiovascular events (MACEs, all-cause death, myocardial infarction, or stroke). The secondary endpoint was all-cause mortality. Overall, 618 patients were included and followed for a median of 981 (557-1,325) days. Diagnosis of coronary artery disease (CAD) was established in 270 (43.7%) patients. Median hs-TnT at admission was 31 (20-59) ng/L, with 525 (85%) patients over the upper reference limit. Hs-TnT values were significantly higher in patients with established CAD (39 vs. 29 ng/L, p < 0.01). Hs-TnT was an independent predictor of MACE (HR 2.440, 95% CI 1.706-3.489, p < 0.001). The best cut-offs were 40 ng/L (AUC 0.711) for patients with established CAD and 25 ng/L (AUC 0.725) for those without. Hs-TnT emerged also as an independent predictor of all-cause mortality. The addition of hs-TnT improved prognostic value of the SPINACH score. Conclusions: Hs-TnT is a powerful biomarker for prognostic stratification of diabetic CLI patients with foot lesions. This is confirmed independently to CAD diagnosis and permits the identification of higher risk patients requiring tailored intervention.

Limb salvage in Charcot foot and ankle osteomyelitis: combined use single stage/double stage of arthrodesis and external fixation.

BACKGROUND: Charcot neuroarthropathy of the foot/ankle is a devastating complication of diabetes. Along with neuroarthropathy, osteomyelitis can occur which can result in amputation. This prospective study evaluated a limb salvage procedure as an alternative to amputation through surgical treatment of osteomyelitis of the midfoot or the ankle and stabilization with external fixation. MATERIALS AND METHODS: Forty-five patients with Charcot arthropathy and osteomyelitis underwent debridement and attempted fusion with an external fixator. Chart and radiograph review was performed to assess the success of the fusion and eradication of infection. RESULTS: Out of 45 patients, 39 patients healed using emergent surgery to drain an acute manifestation of the infection while maintaining the fixation for an average of 25.7 weeks. Two patients were treated with intramedullary nail in a subsequent surgical procedure. In four patients, the infection could not be controlled, therefore a major amputation was carried out. CONCLUSION: For select patients, external fixation proved to be a reasonable alternative to below-knee amputation.

Characterization of Dermal Stem Cells of Diabetic Patients.

Diabetic foot ulcers (DFUs) are lesions that involve loss of epithelium and dermis, sometimes involving deep structures, compartments, and bones. The aim of this work is to investigate the innate regenerative properties of dermal tissue around ulcers by the identification and analysis of resident dermal stem cells (DSCs). Dermal samples were taken at the edge of DFUs, and genes related to the wound healing process were analyzed by the real-time PCR array. The DSCs were isolated and analyzed by immunofluorescence, flow cytometry, and real-time PCR array to define their stemness properties. The gene expression profile of dermal tissue showed a dysregulation in growth factors, metalloproteinases, collagens, and integrins involved in the wound healing process. In the basal condition, diabetic DSCs adhered on the culture plate with spindle-shaped fibroblast-like morphology. They were positive to the mesenchymal stem cells markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic markers CD14, CD34, CD45, and HLA-DR. In diabetic DSCs, the transcription of genes related to self-renewal and cell division were equivalent to that in normal DSCs. However, the expression of CCNA2, CCND2, CDK1, ALDH1A1, and ABCG2 was downregulated compared with that of normal DSCs. These genes are also related to cell cycle progression and stem cell maintenance. Further investigation will improve the understanding of the molecular mechanisms by which these genes together govern cell proliferation, revealing new strategies useful for future treatment of DFUs.

Limb salvage in diabetic patients with no-option critical limb ischemia: outcomes of a specialized center experience.

Objective: To describe the characteristics, the management and the outcome of a consecutive series of patients with diabetic foot lesions (DF) and no-option critical limb ischemia (CLI) treated with a multidimensional, interdisciplinary approach in a dedicated center. Research Design and Methods: The prospective database of the Diabetic Foot Unit of the Maria Cecilia Hospital (Cotignola, Italy) collects medical history, risk factors, chemistry values, angiographic data, characteristic of foot lesions, medical and surgical therapies of all patients admitted with a diagnosis of DF and CLI. All patients were followed-up for at least 1 year and/or total recovery. The primary endpoint was 1-year amputation-free survival (AFS), secondary endpoints were limb salvage and survival. Results: Between October 2014 and October 2017, 1024 patients with DF and CLI were admitted to the center. Eighty-four of them (8.2%) fulfilled the criteria for no-option CLI. At 1 year, AFS, limb salvage, and survival rates were 34%, 34%, and 83%, respectively. Lesions located proximal to the Lisfranc joint were associated with major amputation (HR 2.1 [1.2-3.6]). One-year survival of patients treated with minor procedures was significantly higher compared to patients treated with major amputation (96% vs 76%, log-rank p = 0.019). Major amputation was independently associated with mortality (HR 7.83 [1.02-59.89]). Conclusions: The application of dedicated and standardized strategies permitted limb salvage in one-third of patients with no-option CLI. Patients with stable lesions limited to the forefoot and without ischaemic pain had a greater probability to successfully receive conservative treatments. Limb salvage was associated with subsequent higher one-year survival.

Use of a retrograde nail for ankle arthrodesis in Charcot neuroarthropathy: a limb salvage procedure.

BACKGROUND: Charcot neuroarthropathy is a serious complication associated with diabetic neuropathy. This complication probably is most serious when the ankle is involved because of the instability and progressive deformity, which often leads to ulceration, osteomyelitis, and amputation. Arthrodesis before the ulcerated lesion appears is considered a limb salvage treatment. One of the most effective techniques for an unstable ankle in Charcot neuroarthropathy is retrograde transcalcaneal nailing. METHODS: Eighteen diabetic patients, without a history of ulceration, were treated from July, 2003, to November, 2005, with panarthrodesis of the ankle using intramedullary retrograde transcalcaneal nailing. The average follow up was 14 +/- 10.1 months. All patients completed the unloaded postoperative period with a fiberglass cast (3 months nonweightbearing and 3 months partial weightbearing) and commenced walking in shoes with a stiff rocker sole and a molded insole. RESULTS: During the followup period there were no major complications. In three patients, removal of one of the proximal screws used for anchoring the nail to the tibia was done because of protrusion causing skin breakdown. Fourteen patients had a stable fusion and four patients had fibrous union. The percentage of limb salvage was 100% in the followup period. CONCLUSIONS: Our study confirms that this operative technique is effective and safe.

Extension and grading of osteomyelitis are not related to limb salvage in Charcot neuropathic osteoarthropathy: A cohort prospective study.

AIMS: Charcot neuropathic osteoarthropathy (CN) represents a complication for diabetic patients which involves a progressive alteration of the osteoarticular apparatus with high risk of amputation. The aim of the study was to assess whether the localization of CN and the extent or grading of the osteomyelitis have an influence on the rate of limb salvage and the time to recovery. METHODS: We treated a diabetic population affected by CN complicated by ulceration and widespread osteomyelitic involvement. All patients were treated surgically to eliminate infected tissues, stabilize and correct the bone deformities. Histopathological and microbiological analyses were carried out on the bone specimens. RESULTS: Thirty-three patients affected by CN complicated by large osteomyelitic involvement of midfoot and/or ankle were treated between January 2010 and May 2014. The mean follow-up was 409,35 ± 154,06 days. Thirty patients had complete recovery (90.91%) at the end of follow-up. No difference in limb salvage rate and time to recovery was observed when stratifying the population according to CN localization, extent and grading of osteomyelitis. CONCLUSIONS: In this cohort prospective study we observed a high percentage of limb salvage in patients affected by CN complicated by diffuse midfoot/hindfoot osteomyelitis. The localization of Charcot deformity and the extent/stage of osteomyelitis did not change the rate of limb salvage.

Combination of Open Subtotal Calcanectomy and Stabilization With External Fixation as Limb Salvage Procedure in Hindfoot-Infected Diabetic Foot Ulcers.

Diabetic hindfoot ulcers, complicated by osteomyelitis, are associated with a high risk of major amputation. Partial calcanectomy, preceded by an effective management of the infection and of the eventual peripheral artery disease, can be considered as valid therapeutic option. We have evaluated a therapeutic protocol for diabetic hindfoot ulcers complicated by osteomyelitis, which, besides an adequate surgical debridement, considers a reconstructive pathway assisted by the positioning of a circular external fixator. We made a prospective study of a cohort of diabetic patients affected by heel ulcer complicated by osteomyelitis. All patients underwent open partial calcanectomy associated with the positioning of a circular external frame specifically designed for hindfoot stabilization and offloading. A reconstructive procedure was implemented starting with the application of negative pressure wound therapy and coverage with dermal substitute and split thickness skin grafting. From November 2014 to November 2015, 18 consecutive patients were enrolled. Mean follow-up period was 212.3 ± 64.0 days. Healing was achieved in 18 (100%) patients. The mean healing time was 69.0 ± 64.0 days. No major amputation had to be performed during the follow-up. Open partial calcanectomy associated with external fixation and skin reconstruction was as efficient as limb salvage in patients with infected lesions of the hindfoot complicated by calcaneal osteomyelitis.

Ulcer recurrence following first ray amputation in diabetic patients: a cohort prospective study.

OBJECTIVE: To evaluate the reulceration and reamputation rates in a cohort of diabetic patients following first ray amputation. RESEARCH DESIGN AND METHODS: We evaluated a cohort of 89 diabetic patients, 63 men and 26 women, who underwent first ray amputation in the period from January 2000 to December 2001. The first ray lesions were Wagner grade 2 in 3 patients, Wagner grade 3 in 47 patients, and Wagner grade 4 in 39 patients. Following surgical wound healing, all patients wore special footwear with rocker bottom soles and custom molded insoles and were put on an intensive secondary prevention program. RESULTS: The mean follow-up duration was 16.35 +/- 6.76 months (range 7-28). Fifteen patients developed new ulcerations, with 11 lesions occurring ipsilaterally and 4 contralaterally to the first ray amputation. In seven patients, the new lesion was treated and healed with dressing. Eight patients underwent a new surgical procedure: panmetatarsal head resection in four patients, toe amputation in two patients, a transmetatarsal amputation in one patient, and Lisfranc's amputation in one patient. CONCLUSIONS: In the population studied, the first ray amputation presented a lower reulceration and reamputation rate with respect to that reported in the literature. This finding should therefore be attributed to the follow-up program, which uses shoes with a rocker bottom sole and custom molded insoles and intensive ambulatory check-ups.

HYAFF 11-based autologous dermal and epidermal grafts in the treatment of noninfected diabetic plantar and dorsal foot ulcers: a prospective, multicenter, controlled, randomized clinical trial.

OBJECTIVE: To evaluate the clinical efficacy and safety of HYAFF 11-based autologous dermal and epidermal grafts in the management of diabetic foot ulcers. RESEARCH DESIGN AND METHODS: A total of 79 patients with diabetic dorsal (n = 37) or plantar (n = 42) ulcers were randomized to either the control group with nonadherent paraffin gauze (n = 36) or the treatment group with autologous tissue-engineered grafts (n = 43). Weekly assessment, aggressive debridement, wound infection control, and adequate pressure relief (fiberglass off-loading cast for plantar ulcers) were provided in both groups. Complete wound healing was assessed within 11 weeks. Safety was monitored by adverse events. RESULTS: Complete ulcer healing was achieved in 65.3% of the treatment group and 49.6% of the control group (P = 0.191). The Kaplan-Meier mean time to closure was 57 and 77 days, respectively, for the treatment versus control groups. Plantar foot ulcer healing was 55% and 50% in the treatment and control groups, respectively. Dorsal foot ulcer healing was significantly different, with 67% in the treatment group and 31% in the control group (P = 0.049). The mean healing time in the dorsal treatment group was 63 days, and the odds ratio for dorsal ulcer healing compared with the control group was 4.44 (P = 0.037). Adverse events were equally distributed between the two groups, and none were related to the treatments. CONCLUSIONS: The autologous tissue-engineered treatment exhibited improved healing in dorsal ulcers when compared with the current standard dressing. For plantar ulcers, the off-loading cast was presumably paramount and masked or nullified the effects of the autologous wound treatment. This treatment, however, may be useful in patients for whom the total off-loading cast is not recommended and only a less effective off-loading device can be applied.

Using hyaluronic acid derivatives and cultured autologous fibroblasts and keratinocytes in a lower limb wound in a patient with diabetes: a case report.

Patients with diabetes have an impaired wound healing process that contributes to the pathophysiology that may lead to amputation. In this case study, an extensive (103.49 cm2) full-thickness cutaneous wound with exposure of a necrotic Achilles' tendon in a patient with diabetes, neuropathy, and infrapopliteal vascular disease of the lower limbs was healed using a two-stage autologous skin substitute technique. The scaffolds on which the autologous fibroblasts and keratinocytes were grown comprised an ester derivative of hyaluronic acid. Two applications of the cultured autologous fibroblasts and one of the cultured autologous keratinocytes were placed on the wound at 7-day intervals. The ulcer healed completely 60 days following the first fibroblast graft application. After 16 months of follow-up, no recurrence was noted and the patient can walk without ancillary support. This novel tissue engineering technique is a promising treatment for wound healing.