Silencing of Dicer enhances dacarbazine resistance in melanoma cells by inhibiting ADSL expression.

Dacarbazine (DTIC) is the primary first-line treatment for advanced-stage metastatic melanoma; thus, DTIC resistance is poses a major challenge. Therefore, investigating the mechanism underlying DTIC resistance must be investigated. Dicer, a type III cytoplasmic endoribonuclease, plays a pivotal role in the maturation of miRNAs. Aberrant Dicer expression may contribute to tumor progression, clinical aggressiveness, and poor prognosis in various tumors. Dicer inhibition led to a reduction in DTIC sensitivity and an augmentation in stemness in melanoma cells. Clinical analyses indicated a low Dicer expression level as a predictor of poor prognosis factor. Metabolic alterations in tumor cells may interfere with drug response. Adenylosuccinate lyase (ADSL) is a crucial enzyme in the purine metabolism pathway. An imbalance in ADSL may interfere with the therapeutic efficacy of drugs. We discovered that DTIC treatment enhanced ADSL expression and that Dicer silencing significantly reduced ADSL expression in melanoma cells. Furthermore, ADSL overexpression reversed Dicer silencing induced DTIC resistance and cancer stemness. These findings indicate that Dicer-mediated ADSL regulation influences DTIC sensitivity and stemness in melanoma cells.

Clinical Application of the Peroneal Artery Perforator Flap for Reconstruction of Lateral Malleolus Defects.

The reconstruction of defects of the lateral malleolus involving the exposed fibular bone or tendon is challenging. This study aimed to evaluate the clinical application of the peroneal artery perforator flap with or without split-thickness skin grafting for soft tissue reconstruction of the bony defect of the lateral malleolus of the ankle joints. Reconstruction using a peroneal artery perforator flap with or without split-thickness skin grafting was performed for 15 patients (10 men, 5 women) between January 2007 and December 2018. The mean age was 53.7 years, and the mean size of the flaps was 40 cm2. The flaps were elevated in the form of a perforator flap, and split-thickness skin grafting was performed over the flaps and adjoining raw areas. The flaps survived in all cases; however, partial necrosis was observed in 3 cases. In cases of small-sized defects of the lateral malleolus of the ankle joints where a flap is required for the exposed bone or tendon, reconstruction using the peroneal artery perforator flap is advantageous, since the morbidity rate of the donor site is low and soft tissue is reconstructed.

Successful Treatment of Plantar Hyperkeratosis in the Form of Recurrent Corns With Split-Thickness Sole Skin Graft.

INTRODUCTION: Plantar hyperkeratosis, such as corns and calluses, is common in older people and associated with pain, mobility impairment, and functional limitations. It usually develops on the palms, knees, or soles of feet, especially under the heels or balls. There are several treatment methods for plantar hyperkeratosis, such as salicylic acid plaster and scalpel debridement, and conservative modalities, such as using a shoe insert and properly fitting shoes. METHODS: We present an effective method of reconstructing the wound after corn excision using a split-thickness sole skin graft (STSSG). We harvested the skin graft from the arch of the sole using the dermatome with a skin thickness of 14/1000th inches. RESULTS: Because the split-thickness skin graft, harvested from the sole arch near the distal sole, is much thicker than the split-thickness skin graft from the thigh, it is more resistant to weight and friction. The healed wound with STSSG coverage over the distal sole was intact, and the donor site over the sole arch had healed without complication during the outpatient follow-up, 3 months after surgery. CONCLUSIONS: The recovery time of STSSG for corn excision is shorter than that with traditional treatment. Therefore, STSSG can be a reliable alternative treatment for recurrent palmoplantar hyperkeratosis.

Enhanced wound-healing performance of a phyto-polysaccharide-enriched dressing - a preclinical small and large animal study.

Alginate is a natural rich anionic polysaccharide (APS), commonly available as calcium alginate (CAPS). It can maintain a physiologically moist microenvironment, which minimises bacterial infection and facilitates wound healing at a wound site. Patients with burn injuries suffer from pain and an inflammatory response. In this study, we evaluated the CAPS dressing and traditional dressing containing carboxymethyl cellulose (CMC) for wound healing and scar tissue formation in a burn model of rat and swine. In our pilot study of a burn rat model to evaluate inflammatory response and wound healing, we found that the monocyte chemoattractant protein (MCP)-1 and transforming growth factor (TGF)-ß were up-regulated in the CAPS treatment group. Next, the burn swine models tested positive for MCP-1 in a Gram-positive bacterial infection, and there was overproduction of TGF-ß during the burn wound healing process. Rats were monitored daily for 1 week for cytokine assay and sacrificed on day 28 post-burn injury. The swine were monitored over 6 weeks. We further examined the pain and related factors and inflammatory cytokine expression in a rodent burns model monitored everyday for 7 days post-burn. Our results revealed that the efficacy of the dressing containing CAPS for wound repair post-burn was better than the CMC dressing with respect to natural wound healing and scar formation. The polysaccharide-enriched dressing exerted an antimicrobial effect on burn wounds, regulated the inflammatory response and stimulated anti-inflammatory cytokine release. However, one pain assessment method showed no significant difference in the reduction in levels of adenosine triphosphate in serum of rats after wound dressing in either the CAPS or CMC group. In conclusion, a polysaccharide-enriched dressing outperformed a traditional dressing in reducing wound size, minimising hypertrophic scar formation, regulating cytokines and maximising antimicrobial effects.

Pulmonary metastatic angiosarcoma from scalp with fatal complication: A case report.

INTRODUCTION: Angiosarcoma is a rare malignant neoplasm with poor prognosis. Angiosarcoma of the scalp is frequently recurs locally, and metastasizes early despite various treatments. The common sites of metastatic are lung, liver, and lymph nodes. Pulmonary metastasis with hemoptysis and pneumothorax is rare but threatening. PRESENTATION OF CASE: A 77-year-old male had recurrent angiosarcoma of the scalp even with post operation radiotherapy. At the same time, recurrent pneumothorax was noted, thus he underwent wedge resection of the right upper lobe of the lung plus pleural biopsy. The final pathologic report of cystic lesions showed metastatic Angiosarcoma. He received intravenous paclitaxel and the lung lesions dramatically diminished subsequently. DISCUSSION: Pulmonary metastasis from soft tissue sarcoma had fatal complications and poor prognosis. Metastases of AS to the lung have a well-described morphology on CT scan, but appear to be hypometabolic on PET scan and are easily misinterpreted as benign cysts. CONCLUSION: Angiosarcoma is a rare but highly vascular invasive endothelial tumor that generally metastasizes to the lung. It could cause repeated hemoptysis pneumothorax and pleural effusion. Preoperative chest CT may be recommended routinely. Aggressive treatment resulted in not only symptoms control but also good prognosis.

A Biodegradable Hemostatic Gelatin/Polycaprolactone Composite for Surgical Hemostasis.

Massive bleeding is the leading cause of battlefield-related deaths and the second leading cause of deaths in civilian trauma centers. One of the challenges of managing severe wounds is the need to promote hemostasis as quickly as possible, which can be achieved by using hemostatic dressings. In this study, we fabricated 2 kinds of gelatin/polycaprolactone composites with 2 ratios of gelatin/polycaprolactone, 1:1 and 2:1 (GP11 and GP21, respectively). Scanning electron microscopy revealed that the GP11 composite exhibited rougher and more porous structure than the GP21 composite did. Furthermore, both composites showed similar biocompatibility as that of tissue culture polystyrene. Moreover, both GP composites tended to show a gradual decrease in contact angle to zero within 40 minutes. The in vitro blood plasma coagulation assay revealed that the prothrombin time was significantly longer for the GP composites than it was for the Quikclot composite, whereas the activated partial thromboplastin time of the GP11 composite was significantly shorter than that of the gauze. Furthermore, the GP11 had the largest platelet adsorption of all the composites. The in vivo coagulation test showed an obvious shortening of the bleeding time with the Quikclot and GP21 compared with gauze sample. In conclusion, the GP composites showed superior biocompatibility and hemostasis to the gauze and comparable effects with the Qickclot composite. Therefore, the GP composites have the potential for development as biodegradable surgical hemostatic agents.

Breast Reconstruction Using Pedicled Latissimus Dorsi Myocutaneous Flaps in Asian Patients With Small Breasts.

BACKGROUND: The use of implants is still the most common procedure for breast reconstruction because they are easy, less painful than tissue transplants, and do not need a donor site. However, it is challenging to find a suitable implant for patients with small breasts, and some women fear foreign bodies and possible complications or reoperations. Autologous breast reconstruction using the pedicled latissimus dorsi (LD) myocutaneous flap without an implant provides a good option for Asian women with small breasts. MATERIALS AND METHODS: Between June 1992 and December 2015, 31 patients underwent breast reconstruction with 33 LD flaps (29 unilateral and 2 bilateral). The skin paddle of the flap was designed with an oblique or transverse pattern depending on the mastectomy defect and the elasticity of skin. The thoracodorsal nerve was divided during flap harvesting to prevent a "twitching breast" postoperatively. Patients refused to have contralateral breast augmentation except for 2 with bilateral simultaneous augmentation after mastectomy bilaterally. Outcome measures were flap survival, shape and contour, symmetry of breast, complication of flap and donor site, patient satisfaction, and any local tumor recurrence or metastasis. RESULTS: The mean patient age was 46.7 years (range, 27-72), and the mean body mass index was 22.5 kg/m (range, 18.6-30). The mean size of the harvested skin paddle was 11.9 × 5.0 cm (range, 10 × 3 cm to 15 × 9 cm). Mean operative times were 200.8 minutes (range, 112-230 minutes) and 305 minutes (range, 300-310 minutes) for unilateral and bilateral reconstructions, respectively. Pathology reports showed a negative safety margin in all cases. Most cases were of invasive duct carcinoma (58%). All LD flaps survived, and the wounds healed satisfactorily over a mean follow-up of 49.9 months (range, 3-161 months). Donor sites were closed primarily with a hidden linear scar under the dorsal bra strap. Donor site morbidities were mainly seromas (15%), which were treated conservatively in most patients. CONCLUSIONS: The LD flap produced good autologous tissue for reconstruction, and no implants were needed for Asian women with small breasts. The reconstructed breasts showed good shape, contour, and symmetry. The results of donor site were acceptable and no significant functional loss. There were no major complications, and patient satisfaction was high.

Postmastectomy Breast Reconstruction Combined With Contralateral Breast Augmentation for Taiwanese Women With Small Breasts.

BACKGROUND: Although autogenous tissue-based breast reconstruction has been widely used in the past decade, implant-based breast reconstruction is more often used in Taiwan because Asian women are generally slender with small breasts. For patients with very small breasts, it is hard to achieve the goal of reconstructing a similar breast to the contralateral one, even with the smallest size implant available commercially. Therefore, these patients need not only breast reconstruction but also contralateral breast augmentation. Here we report the surgical outcomes and cosmetic results of breast reconstruction using cohesive gel implants combined with simultaneous contralateral breast augmentation. MATERIALS AND METHODS: A retrospective chart review was conducted to identify all patients with AA-sized to B-sized breast cups undergoing expander-implant reconstruction combined with contralateral breast augmentation between 2002 and 2015. Thirty patients were included. For each patient, patient profile (age, body mass index, and initial breast size), type and stage of breast cancer, surgical information (including implant sizes and the type of reconstruction and augmentation), and postoperative subjective pain scales were recorded. Outcomes were analyzed by identifying complications, the need for surgical revision, the presence of local or distant metastases, and patient satisfaction ratings. RESULTS: At a mean 2.3-year follow-up (range, 4 months to 12 years), problems occurred in 7 of the 30 patients, with 9 complications in 8 reconstructed breasts and in 1 augmented breast. Complications were mostly capsule contracture. Aesthetic satisfaction was rated as "excellent" or "good" by most of the patients, and only 1 commented "poor" on both overall and reconstructed results because of postoperative radiotherapy-associated skin necrosis. The total mean subjective pain scale was 1.9/10; a higher mean pain scale of 3.08 was noted in those patients undergoing augmentation with no extra incision. CONCLUSIONS: This is the first report of implant-based breast reconstruction with simultaneous contralateral augmentation in Taiwan, showing its efficacy, safety, and good cosmetic outcomes with relatively low complication and revision rates.

Clinical applications of the pedicled anterolateral thigh flap in reconstruction.

BACKGROUND: Reconstruction of extensive defects of the lower abdomen, penoscrotum, trochanter, groin and knee without using complex microsurgery is a reconstructive challenge. Pedicled anterolateral thigh (ALT) flaps offer many advantages over other regional flaps for this purpose, such as the large skin area and soft-tissue availability, a remarkable pedicle length, and possessing multiple components and reliability. We present our experience of using pedicled ALT flaps for repairing various defects. METHODS: From September 2006 to December 2013, 42 pedicled ALT flaps were used in 41 patients for defects of the lower abdomen (three patients), trochanter (26 patients), penoscrotum (10 patients), groin (one patient) and knee (one patient). Twenty-eight were men and 13 were women, and their mean age was 70.5 years (range, 22-103 years). The characteristics of the patients' age, sex, cause, flap size, flap component, follow-up and donor sites were recorded. RESULTS: The flap size ranged from 8 × 5 cm (40 cm2 ) to 11 × 18 cm (198 cm2 ). The length of the pedicle ranged from 9 to 16 cm, which was enough to reach the defect without tension. No surgery-related mortality occurred. In 34 flaps, donor sites were closed primarily and eight underwent split-skin grafting. Satisfactory coverage was achieved in all patients. CONCLUSION: Our experience has shown the wide arc of rotation, large skin replacement potential, multiple components and reliability of pedicled ALT flaps. They are technically simple to apply as myocutaneous/fasciocutaneous flaps with minimal donor site morbidity.

Chylous ascites after pedicled transverse rectus abdominis myocutaneous flap harvest.

Chylous ascites is a rare clinical condition that occurs as a consequence of disruption of the abdominal lymphatics. Here, we present the case of a 58-year-old woman with sustained chylous ascites after pedicled transverse rectus abdominis myocutaneous (TRAM) flap harvest. The chylous ascites did not decrease despite conservative therapy. For the diagnosis and localization of the chyle leakage, lymphangiography with lipiodol delivery was performed. Chylous drainage continued in a decreasing manner for the next 4 d because lipiodol accumulated to the point of leakage outside the lymphatic vessel causing a regional inflammatory reaction and obstructing the lymphatic vessels. To our knowledge, there is no case of chylous ascites related to pedicled TRAM flap harvest that has been reported in the English literature, in which the chyle leakage spontaneously resolved after lymphangiography.

Cranioplasty Using a Novel Osteoconductive Scaffold and Platelet Gel.

BACKGROUND: Commonly used materials for cranioplasty include autogenous bone grafts, methyl methacrylate, and titanium mesh. We evaluated a novel osteoconductive scaffold [N-isopropylacrylamide cross-linked with acrylic acid using γ-rays (ANa powder)] mixed with platelet gel for cranioplasty. METHODS: ANa powder mixed with platelet gel was implanted into a 15 × 15-mm, full-thickness calvarial bone defect in 5 New Zealand white rabbits. ANa powder mixed with phosphate-buffered saline was implanted in 5 rabbits. The calvarial bone defect was left unreconstructed in another 5 rabbits. Twelve weeks after surgery, computed tomography examination was used to evaluate the radiographic evidence of bone healing in vivo. Bone specimens were then retrieved for histologic study. RESULTS: The ANa scaffold mixed with platelet gel is biocompatible, biodegradable, and both osteoconductive and osteoinductive, leading to progressive growth of new bone into the calvarial bone defect. CONCLUSION: The use of this novel osteoconductive scaffold combined with osteoinductive platelet gel offers a valuable alternative for the reconstruction of calvarial bone defects.

Partial Calcanectomy Facilitates Wound Closure of Heel Defects in High-Risk Patients.

BACKGROUND: Heel ulcers in patients with severe peripheral artery occlusive disease represent a challenge to the treating physician. They become more difficult to treat with underlying medical comorbidities. The purpose of this report is to document evidence that partial calcanectomy is simple to perform and clears infected bone, tissue, and ulceration. MATERIALS: Between July 2011 and August 2013, 30 consecutive patients presented to our department with heel wounds caused by diabetes mellitus and pressure. After evaluation by a vascular surgeon, 12 patients diagnosed with near total occlusive peripheral vascular disease were included in this report. Of the 12 patients, 7 were women. Their ages ranged from 65 to 79 years (mean, 73.3 years). After admission, surgical debridement was performed emergently with subsequent partial calcanectomy and wound closure. RESULTS: Eight heel wounds (75%) healed completely with no further surgery to achieve defect coverage. Wound dehiscence developed in 4 patients (25%). The mean number of debridements was 1.75 (range, 1-3) with a total operation time of 71.5 minutes (range, 45-114 min). One patient died of acute myocardial infarction 2 weeks after discharge. The mean length of hospital stay was 8.3 days (range, 5-16 days). CONCLUSION: In this study, we demonstrate that partial calcanectomy is practical for the treatment of plantar heel ulcers in patients with severe comorbidities. With proper surgical planning and postoperative care, partial calcanectomy is a viable alternative to below-the-knee amputation and may better serve the patient who would otherwise be restricted to a sedentary lifestyle.

Successful Pedicled Anterolateral Thigh Flap Reconstruction for a Recurrent Ischial Pressure Ulcer: A Case With Multiple Recurrences Over a 7-year Follow-up.

Ischial pressure ulcers are difficult ulcers to treat and have a low treatment success rate compared to sacral and trochanteric ulcers; regional flap failure further complicates the treatment. Reported here is a case of a 65-year-old man who experienced a spinal injury with paraplegia due to trauma 20 years ago. The patient experienced a recurrent ischial ulcer since 2007, and underwent several types of flap reconstruction with poor outcomes over a 7-year period. Therefore, the chosen intervention was a pedicled anterolateral thigh (pALT) fasciocutaneous flap reconstruction for the ischial ulcer via a subcutaneous route. Over the 10-month follow-up, the recurrent ischial ulcer healed without wound dehiscence. Island pALT reconstruction appears to be an alternative technique for treating recurrent ischial pressure ulcers. Though reconstruction of ischial ulcers via the pALT technique has been described previously, this may be the first case report to describe pALT flap in a patient with recurrent ischial ulcers after failed reconstructions using a gluteus maximus flap, V-Y advancement flap, and hatchet flap.Ischial pressure ulcers are difficult to treat and have a low treatment success rate1 compared to sacral and trochanteric ulcers. In addition, there are many different techniques that can be used to treat ischial pressure ulcers, including primary wound closure, gluteus maximus flaps, V-Y advancement flaps, or inferior gluteal artery perforator flaps. However, several experts have recently described using the pedicled anterolateral thigh (pALT) flap for reconstruction of recurrent ischial pressure ulcers.1,2 In the presented case, the authors followed a single patient with paraplegia with a recurrent ischial ulcer who had undergone several types of wound treatment over a 7-year period. The indurated ulcer was ultimately resolved by pALT reconstruction.

Oncological safety and outcomes of nipple-sparing mastectomy with breast reconstruction: a single-centered experience in Taiwan.

INTRODUCTION: Nipple-sparing mastectomy has become a contemporary surgical treatment that achieves improved cosmetic outcomes for patients with breast cancer in Western countries. We examined oncological and cosmetic outcomes in Asian women who underwent nipple-sparing mastectomy in Taiwan. METHODS: Between 2006 and 2011, 42 patients with breast cancer who underwent 44 nipple-sparing mastectomy operations with immediate reconstruction at the Tri-Service General Hospital were reviewed. The cancer type, tumor stage, reconstruction method, presence of local recurrence, presence of distant metastasis, mortality, and complications were assessed and documented. Questionnaires were used to assess and rate patients' satisfaction with regard to appearance, sensation, symmetry, color, arousal, and texture. RESULTS: The mean follow-up period was 40.9 months (median, 45.5 months; range, 13-72 months). Among the 42 cases, only one case (2.4%) of local recurrence was observed and treated by nipple-areola complex resection. The overall complication rate was 25%, with nipple necrosis comprising 13.6%. CONCLUSION: Nipple-sparing mastectomy is a safe procedure in properly selected patients with breast cancer. This procedure yields similar oncological safety and cosmetic outcomes among Asians and women from Western countries.

Bipedicled flap for the reconstruction of soft tissue defects of the Achilles tendon.

Soft tissue defects exposing the Achilles tendon are common in patients who have undergone trauma or in those with pressure ulcers associated with vascular diseases. The purpose of this article was to present our experience of 11 patients who underwent reconstruction of soft tissue defects of the Achilles tendon using bipedicled fasciocutaneous flaps. Between August 2008 and August 2012, 11 patients were admitted to our hospital, presenting with soft tissue defects overlying the Achilles tendon. After adequate debridement, the 11 patients underwent bipedicled fasciocutaneous flap placement to resurface the complex soft tissue defects and provide a gliding surface for the exposed Achilles tendon. The patients' age, comorbidity, etiology, defect size and location, wound culture, skin graft size, complications, surgery duration, and follow-up period were reviewed. The 11 fasciocutaneous bipedicled flaps survived completely, and the wounds healed satisfactorily at a mean follow-up period of 20.9 months (range, 6-48 months). Only 1 flap was complicated with wound dehiscence and superficial necrosis of its lateral edge, which healed conservatively. The donor sites were covered with split-thickness skin grafts and healed well without complications. The bipedicled fasciocutaneous flap is a reliable flap for coverage of defects overlying the Achilles tendon, especially in patients with vascular problems and/or elderly patients. The ease of handling, short operative time, and early recovery of mobilization function are of great benefit to patients. Thus, the bipedicled fasciocutaneous flap can be a valuable alternative for defect reconstructions overlying the Achilles tendon, with satisfactory results both functionally and cosmetically.

Histochemical and functional improvement of adipose-derived stem cell-based tissue-engineered cartilage by hyperbaric oxygen/air treatment in a rabbit articular defect model.

Cartilage is exposed to compression forces during joint loading. Therefore, exogenous stimuli are frequently used in cartilage tissue engineering strategies to enhance chondrocyte differentiation and extracellular matrix (ECM) secretion. In this study, human adipose-derived stem cells were seeded on a gelatin/polycaprolactone scaffold to evaluate the histochemical and functional improvement of tissue-engineered cartilage after hyperbaric oxygen/air treatment in a rabbit articular defect model. Behavior tests showed beneficial effects on weight-bearing and rear leg-supporting capacities after treatment of tissue-engineered cartilage with 2.5 ATA oxygen or air. Moreover, positron emission tomography images and immunohistochemistry staining demonstrated hydroxyapatite formation and increased ECM synthesis, respectively, at the tissue-engineered cartilage graft site after high pressure oxygen/air treatment. Based on these results, we concluded that hyperbaric oxygen and air treatment can improve the quality of tissue-engineered cartilage in vivo by increasing the synthesis of ECM.

A simple protocol for the management of deep sternal surgical site infection: a retrospective study of twenty-five cases.

BACKGROUND: Deep sternal incisional surgical site infection is a serious and potentially life-threatening complication after open heart surgery. Although a rare post-operative complication, the rates of post-operative morbidity and mortality are greater in patients who develop a deep sternal incisional surgical site infection than in those who do not. METHODS: We evaluated retrospectively the results of patients who developed a deep sternal incisional surgical site infection who were treated with either a pectoralis major flap or delayed primary closure after previous negative-pressure wound therapy (NWPT). From July 2007 to July 2012, 25 patients had a deep sternal incisional surgical site infection after open heart surgery in the Departments of Plastic Surgery and Cardiac Surgery of the Tri-Service General Hospital Medical Center. Sternal refixation was not performed in our patients. RESULTS: In 15 patients, a unilateral or bilateral pectoralis major advancement flap with a myocutaneous or muscle flap was used. In seven patients, delayed primary closure was performed after NPWT. One patient received a rectus abdominis myocutaneous flap and another received a free anterior lateral thigh flap. One patient died after developing nosocomial pneumonia with severe sepsis after debridement. CONCLUSIONS: In our series, no patient required sternal re-fixation. Our findings suggest that delayed primary closure and use of a unilateral or bilateral pectoralis major flap following NPWT for a deep sternal incisional surgical site infection are simple and quick methods for managing such difficult surgical incisions even if the deep sternal surgical site infection is located in the lower one-third of the sternum.

Tailored design of electrospun composite nanofibers with staged release of multiple angiogenic growth factors for chronic wound healing.

The objective of this research study is to develop a collagen (Col) and hyaluronic acid (HA) inter-stacking nanofibrous skin equivalent substitute with the programmable release of multiple angiogenic growth factors (vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and endothelial growth factor (EGF)) either directly embedded in the nanofibers or encapsulated in the gelatin nanoparticles (GNs) by electrospinning technology. The delivery of EGF and bFGF in the early stage is expected to accelerate epithelialization and vasculature sprouting, while the release of PDGF and VEGF in the late stage is with the aim of inducing blood vessels maturation. The physiochemical characterizations indicate that the Col-HA-GN nanofibrous membrane possesses mechanical properties similar to human native skin. The design of a particle-in-fiber structure allows growth factors for slow controlled release up to 1month. Cultured on biodegradable Col-HA membrane with four kinds of growth factors (Col-HA w/4GF), endothelial cells not only increase in growth rate but also form a better network with a thread-like tubular structure. The therapeutic effect of Col-HA w/4GF membrane on streptozotocin (STZ)-induced diabetic rats reveals an accelerated wound closure rate, together with elevated collagen deposition and enhanced maturation of vessels, as revealed by Masson's trichrome stain and immunohistochemical analysis, respectively. From the above, the electrospun Col-HA-GN composite nanofibrous skin substitute with a stage-wise release pattern of multiple angiogenic factors could be a promising bioengineered construct for chronic wound healing in skin tissue regeneration.