Reflectance confocal microscopy as a useful diagnostic tool for monitoring of skin containing vascularized composite allograft rejection: A preliminary study on rats.

Vascularized composite allografts can undergo immune-mediated rejection, and skin biopsies are needed for monitoring of the transplant. However it is an invasive method, and requires processing time and pathological assessment. The purpose of this study is to use a new noninvasive monitoring method of the reflectance confocal microscopy (RCM) to determine severity of the allograft rejection on rats. Five groin flap allotransplantation were performed between 10 male Sprague-Dawley rats. Immunosuppressive therapy with cyclosporine A was given to the recipients during 10 days after surgery and was ended at the 10th postoperative days to allow acute transplant rejection. Following cessation of CsA, concomitant RCM evaluation and skin biopsy was performed every other day from each animal until total rejection of the allograft. Complete rejection of the allograft took nearly about 10 days and 4 or 5 RCM evaluation and skin biopsy was performed from each rat during this period. A total of 17 specimens were evaluated. A scoring system was developed based on the RCM findings. Skin biopsies were evaluated according to the Banff 2007 working classification criteria. RCM evaluation revealed epidermal irregularity and collagen destruction, however mild perivascular inflammation and degeneration of the basal epidermal layer were observed in early and late rejection period respectively with histopathologic evaluation. High correlation was found between the RCM scores and histopathologic grading. The RCM may be the useful tool to reduce the need for skin biopsy for monitoring of the skin containing vascularized composite allograft.

The Effects of Hydrogen Sulfide on Adipocyte Viability in Human Adipocyte and Adipocyte-Derived Mesenchymal Stem Cell Cultures Under Ischemic Conditions.

BACKGROUND: This study evaluated the in vitro effects of hydrogen sulfide on adipocyte survival under ischemic conditions and explored possible mechanisms of its apoptotic process. METHODS: The mesenchymal stem cell culture was prepared from a human subcutaneous adipose tissue sample. Adipose-derived mesenchymal stem cells were differentiated into the adipogenic direction, and a mature adipocyte culture was obtained. The adipose-derived mesenchymal stem cell and mature adipocyte cultures were both divided into 6 groups. Sodium hydrogen sulfide was used as a hydrogen sulfide donor. After treating the groups with sodium hydrogen sulfide (0, 0.1, 1, 10, 100, and 1000 µM), the cell cultures were incubated in 1% oxygen at 37°C for 24 hours. After the ischemia period, the cell culture groups were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test for the proliferation/cytotoxicity rates, flow cytometry for apoptosis and necrosis rates, and reverse transcriptase polymerase chain reaction for apoptotic (Bax, Caspase-3) and antiapoptotic (Bcl-2) gene expression levels. RESULTS: Statistically significant increases in proliferation rates were found in mesenchymal stem cell groups treated with low dose (0, 1, and 1 µM) sodium hydrogen sulfide (P<0.05). For each dose, a statistically significant decrease was found in late apoptosis levels on the mature adipocyte cultures (P<0.05). In both cell culture groups, Bcl-2 gene expression was increased and Caspase-3 gene expression was decreased. CONCLUSIONS: Under ischemic conditions, hydrogen sulfide has a protective effect on mesenchymal stem cells and mature adipocytes, and this effect is mediated by the elevation of antiapoptotic gene expression.

Auto-/homografting can work well even if both autograft and allograft are meshed in 4:1 ratio.

BACKGROUND: Patients suffering major burns of more than 50% total burn surface area lack an adequate skin graft donor site to resurface extensive burn wounds and usually need widely meshed autografting and allografting. Anything over the 3:1 expansion ratio is strongly associated with low graft take, poor or delayed epithelialization, and hypertrophic scarring. METHODS: In this study, both autografts and allografts were expanded at a 4:1 ratio. We aimed to use skin grafts effectively and to decrease the morbidity due to graft harvesting. Nine patients with major burns were treated with this method. Graft gain ratio and percentage of actual expansion to predicted expansion were calculated. RESULTS: Ten auto-allografting procedures were performed on a mean of day 16. Graft take was over 95% successful. Five patients survived, and four patients died. The mean total burn surface area was 58.8% in patients who recovered, and 77.5% in the patients who died. The graft gain ratio was 74.8%. The actual expansion rate was 43.7% of the predicted expansion rate. CONCLUSION: In this study, we demonstrated that the donor site morbidities were reduced and successful epithelialization was completed on the eighth day after using both autograft and allograft meshed with a 4:1 ratio.

Biomechanical analysis of the effect of mesenchymal stem cells on mandibular distraction osteogenesis.

BACKGROUND: The purpose of this study was to investigate the effects of bone marrow-derived stem cells on consolidation period by using a new biomechanical testing method on sheep mandible model. METHODS: Eight sheep underwent bilateral mandibular osteotomies. After latency period, bone distraction was activated. Mesenchymal stem cells were transplanted into the gap of the left mandibular distracted callus on the first day of consolidation period. The sheep were then randomly divided into 2 groups (group A = 4, group B = 4). Group A and group B animals were killed on the third and sixth weeks of consolidation, respectively. Fracture pattern and localization, bone regeneration ratio and density, and stress distribution of 16 distracted hemimandibles were evaluated by computed tomography and biomechanical analysis. RESULTS: Two different fracture patterns were observed in the 2 groups. The left halves of mandibles exhibited horizontal fracture out of the distraction zone, and the cross-sectional area was compact bone [H (-) C], whereas the fracture patterns of control sides were oblique, which passed through the distraction zone with a propensity of trabecular bone [O (+) T]. Stress distribution at the critical cross-section of distraction region was not different in halves of mandibles. However, bone regeneration ratios and regenerated bone densities were significantly higher in left sides (P < 0.05). CONCLUSIONS: Transplantation of mesenchymal stem cells promotes maturity of the distracted callus. The new experimental model, which allowed to test the mandible as a system by simulating in vivo loading conditions, revealed differences in the mechanical behavior of the halves of mandible.

Evaluation of breast reduction surgery effect on body posture and gait pattern using three-dimensional gait analysis.

UNLABELLED: Breast hypertrophy is a significant health burden with symptoms of back and shoulder pain, intertrigo, and shoulder grooving from the bra straps. Women often rely on surgery to relieve these symptoms, and they are mostly satisfied with the results. The satisfaction from surgery usually is evaluated by subjective measures. Objective evidence testing of the surgical outcomes is lacking. In this study, 10 women with breast hypertrophy underwent reduction mammaplasty. Their surgical outcomes were evaluated using three-dimensional gait analysis before surgery and 2 months afterward. A statistical difference was sought between the kinematic data of the spine, hip, knee, and ankle joints. The average maximum anterior pelvic tilt angles decreased 41 %, and the average maximum spine anterior flexion angles decreased 30 %. The difference between the pre- and postoperative values was statistically significant. The analysis of the kinematic data showed no significant difference in the hip, knee, or ankle joint angles postoperatively. The outcomes of breast reduction surgery have been evaluated mostly by subjective means until recently. As an objective evidence for surgical gain in the current study, reduction mammaplasty resulted in the patients' improved body posture when walking. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

One-staged silicone implant breast reconstruction following bilateral nipple-sparing prophylactic mastectomy in patients at high-risk for breast cancer.

BACKGROUND: There is little consensus regarding the indications, ideal incision, or reconstruction method for prophylactic mastectomy. In addition, the biggest limitation in studies that assess either patient satisfaction or operation results is the lack of group homogeneity. METHODS: In our center, 21 bilateral prophylactic mastectomies, due to breasts at high risk for cancer, and simultaneous breast reconstruction using submuscular medium-height high-profile silicone implants were performed between 2008 and 2012. The patients were divided into two groups: small-breasted (9 patients) and large/ptotic-breasted (12 patients). A periareolar incision was used in the 9 patients and a vertical mastopexy incision was used in the 12 patients who needed nipple-areola complex repositioning. RESULTS: The rates of general satisfaction and aesthetic outcomes, assessed using the Michigan Breast Satisfaction Questionnaire, were 100 and 90.4%, respectively. Seroma occurred in three patients and venous congestion of the nipple-areola complex developed in four patients, which resolved spontaneously. No nipple-areola necrosis, implant exposition, or capsule contractures were encountered. Only three patients underwent fat injection for contour restoration. CONCLUSION: We obtained very good aesthetic results with low complication rates using this technique in a homogeneous group of patients. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Free fibular flap vascular pedicle calcification.

Osseous free flaps are major reconstruction choices of the segmental mandibular defects. However, etiology of defects in mandible is variable; tumor-related surgery, trauma, radiation, or congenital anomalies are the most common causes. The advent of microvascular surgery has achieved the reconstruction of complex mandibular defects. The options for bony free-tissue transfer for mandibular reconstruction primarily depend on the fibula, iliac, scapula, and radius bone. The free fibular flap continues to be first choice in the reconstruction of mandibular defects because of its advantages. In this report, rare complication of vascular pedicle calcification after mandibular reconstruction with free fibular flap is presented.

Contour restoration of the forehead by lipofilling: our experience.

BACKGROUND: A slightly convex forehead without wrinkles adds attractiveness to a woman's face and to the general perceived image. It is also important for the balance and harmony of the face, especially in the lateral and oblique views. We used a new fat injection technique for correction of the contours of the forehead. METHODS: In this study fat injection was applied in nine female patients for restoring the contour and reducing forehead wrinkles, along with a primary rhinoplasty operation. After dissecting all forehead skin from the underlying muscle with a sharp curved-tip epidural needle, fat grafts were injected into the newly created pocket until the forehead area looked moderately ballooned and gentle digital pressure was used for feathering. The patients wore hair bandages continuously for 2 weeks after the operation. RESULTS: The minimal follow-up time was 12 months (range=12-48 months) and all the patients were pleased with the results. When we compared the photos before and after the operation, it was observed that the fat grafts were stable and retained the volume replacement. Patients declared that they had glossy and better quality skin after the operation and also had new regenerated contours. We did not observe any complications. We performed only one additional lipofilling in one patient to obtain an optimal forehead contour. CONCLUSIONS: This technique is easy to perform and effective in reducing forehead wrinkles and correcting the contour. It was also observed that better perceived nasal results could be obtained with concomitant forehead lipofilling in rhinoplasty operations.

Trifocal biplanar distraction-compression osteosynthesis: a new method for three-dimensional reconstruction of the calvarial defects.

Surgical treatment of calvarial defects and deformities has some drawbacks such as donor site morbidity or use of allogenic materials. In this experimental study, we presented a three-dimensional reconstruction of cranial defects by using trifocal biplanar distraction-compression osteosynthesis (TBDCO).Eight sheep with a mean age of 2 years were divided into 2 groups as the control (n = 3) and distraction groups (n = 5). All the animals were operated on under general anesthesia, and 80 x 50-mm full-thickness calvarial defects were created. In the control group, no treatment was performed. In the distraction group, TBDCO was performed with a custom-made distraction device with 2 transport segments. After 5 days of latency period, the anterior transport segment was distracted to a posterior and superior direction (0.7 and 0.3 mm, respectively) and the posterior transport segment was distracted obliquely to an anterior and superior direction (0.7 and 0.3 mm, respectively) with a resulting total vectorial movement of 0.75 mm/d. Active distraction was approximately 29 days, and the consolidation period was 60 days.No major complications were seen during the whole procedure. In the control group, the bone defects remained unhealed. In the distraction group, defects healed and the reconstructed calvarium was dome shaped. Preoperative and postoperative cranial volume measurements of the distraction group animals revealed an increase in cranial volume (P < 0.05). Histologic evaluation showed inductive bone regeneration and mature bone structure development within the distraction zone. Trifocal biplanar distraction-compression osteosynthesis is an effective and safe technique for three-dimensional closure of the cranial defects. In clinical practice, it could provide several advantages over currently available reconstructive procedures.

The crooked nose: the use of medial iliac crest bone graft as a supporting framework.

The crooked nose is one that the vertical axis of the nose inclines from the midline. As a rule, a major septal deformity almost always accompanies to impairing breathing and aesthetic appearance. To achieve total straightening of the crooked nose, the septum must become the target of the treatment. To prevent redeviation, we used rigid bone grafts instead of flexible and relatively weak cartilage to maintain the support of the corrected deformity. Between February 2005 and July 2007, 9 patients (all male) with crooked noses underwent corrective surgery. The mean age of the patients was 21 years (range, 19-23 years). The source of the bone graft was medial side of iliac crest in all patients. The mean follow-up was 18 months, ranging from 10 to 26 months. All patients had both functional and cosmetic problems. Mean operation time was 4 hours. Airway patency was improved in all cases. The grafts did not shift in any case and did not develop unsightly irregularities over time. Absorption of the grafts was not seen in the follow-up. There were no cases of extrusion or infection. No donor site complication occurred. None of the patient required secondary surgery. A residual deviation can be obvious despite all attempts, which has no deleterious effect on patient satisfaction. This article introduces a novel technique for the correction of the crooked nose.

Sutureless microvascular anastomosis with the aid of heparin loaded poloxamer 407.

BACKGROUND: Anastomosis with tissue adhesives is an alternative method for conventional anastomosis. However, this method has several technical challenges. It requires the use of suture to prevent leakage into lumen and precise application onto all surfaces of the anastomosis site. To solve these problems, poloxamer 407 (P 407) was previously used as a stent. In this study, we made heparinized P 407 (h-P 407) as a new formula. We aimed to successfully use h-P 407 as a stent in sutureless anastomosis in a rat abdominal aorta model. METHODS: Sixty Sprague-Dawley rats were used. In the first group, end-to-end anastomoses were performed with suture; in the second and third groups, sutureless anastomoses were performed with 2-octyl cyanoacrylate. As an intraluminal stent, P 407 was used in the second group, and h-P 407 was used in the third group. Anastomosis time was measured. Lumen width, intimal hyperplasia, and foreign body reaction were assessed histologically. Velocity flow rates and vessel diameters were measured radiologically. Burst strength was measured, and the results were statistically evaluated. RESULTS: Sutureless anastomosis was more rapid than conventional anastomosis. Lumen width was narrower in the suture group. Inflammation and foreign body reaction were more severe in the suture group. There was no radiologic and biomechanical difference between the groups. We found that intimal hyperplasia was less in h-P 407 than in P 407. CONCLUSION: h-P407 can be successfully used as an intraluminal stent for sutureless microvascular anastomosis with tissue adhesives.

Evaluation of the results of simultaneous open rhinoplasty and Abbe flap for the reconstruction of the secondary bilateral cleft and nasal deformity.

AIMS: We aimed to evaluate the results of simultaneous rhinoplasty and Abbe flap for the reconstruction of the secondary bilateral cleft and nasal deformity by means of a newly developed software-based method. PATIENTS AND METHODS: A total of 16 patients with the bilateral cleft lip nasal deformity received Abbe flap and simultaneous open rhinoplasty between 2004 and 2010. The mean age of the patients was 21 years, and the average follow-up time was 2.4 years. After the open rhinoplasty procedure, the upper lip was reconstructed with the Abbe flap. Preoperative and postoperative photographs that had been taken laterally were evaluated by using Adobe Photoshop(®) CS4 and Adobe InDesign(®) software. The length of the columella and the relative changes of the most anterior point of the upper lip to the vertical plane tangent to the most anterior point of the lower lip were measured in pixels on standardized preoperative and postoperative images. The differences were calculated and compared as a percentage (%). RESULTS: There was no flap loss and associated problems. The measurements of columellar length revealed an average increase of 51.8 ± 11.3%, while the relative change of the most anterior point of the upper lip revealed an average increase of 68.6 ± 11.2%. CONCLUSION: The results of the treatment modality were successfully evaluated by a newly developed software-based method.

Palmar contracture release with arterialized venous instep flap: An anatomical and clinical study.

BACKGROUND: Plantar skin has similar histologic features to the palmar area and appears to be the ideal tissue for reconstruction of the palmar region. In this study, an anatomic examination was performed to determine the superficial venous architecture of the instep area, and the use of arterialized venous instep flaps for palmar contracture release was assessed. METHODS: The anatomical study was performed on 12 fresh cadaver feet. The arterialized venous instep flap, including the skin, subcutaneous tissue and superficial venous plexus, was harvested. To determine the venous structure, dissection (n = 6) and injection-corrosion (n = 6) techniques were used. In the clinical study, nine arterialized venous instep flaps were used for palmar contracture release. All flaps were harvested above the deep fascia and included skin, subcutaneous fat, and the superficial venous plexus. At the plantar site of the flap, two or three veins, one of which was used, were dissected for a sufficient length for the arterial anastomosis. The saphenous vein was used for the venous anastomosis. RESULTS: Dissection and injection-corrosion techniques revealed that the flap had 7-12 and 4-6 veins at its plantar and superior edges, respectively, with numerous anastomoses and interconnections between the veins. The flap dimensions were between 3 × 5 cm and 4 × 6 cm. All flaps survived, with two partial flap necrosis that healed with spontaneous epithelization. No debulking procedures were undertaken and all flaps adapted well to the recipient site. CONCLUSIONS: The arterialized venous instep flap is a good alternative to reconstruct palmar contractures by adding similar tissue that is thin and pliable with minimal donor site morbidity.

Treatment modalities for post-burn axillary contractures and the versatility of the scapular flap.

Inappropriate treatment of axillary burns frequently results in adduction contractures. In this clinical study we have reviewed 32 patients with different types of axillary post-burn adduction contractures. We have used a variety of surgical treatments for reconstruction of axillary contracture releasing defects such as simple grafting, Z-plasties and locally pedicled flaps. Among these alternatives, we preferred to use scapular island flap most frequently. In addition to conventional harvest of this flap, extension of its pedicle up to the subscapular ramification by passing it through the triangular space allowed its transfer even to the anterior axillary line defects in a vertical orientation without pedicle kinking. In conclusion, the island scapular flap is a good choice for reconstruction of all types of axillary contracture, releasing defects with satisfactory results in terms of function and cosmesis.

Prolongation of skin xenograft survival with modified cultured fibroblasts.

Cyclosporine A, one of the most potent immunosuppressive drugs, mediates some of its immunosuppressive and nephrotoxic effects by enhancing transforming growth factor-beta secretion and receptor expression. In this experimental study, the effect of cyclosporine pretreatment of cultured dermal fibroblasts on xenogeneic tissue rejection after microimplantation beneath skin grafts was investigated. The effects of the site-specific immunosuppressive strategy on skin xenograft survival were tested. Because the skin is an immunological indicator of the rejection of composite tissue allografts, it was considered that this strategy could be used as a supportive therapy for composite tissue allotransplantation in the future. In the first stage of the study, fibroblast cultures obtained from skin biopsy samples from five rats were treated with different single doses of cyclosporine (100 to 3000 ng/ml), and transforming growth factor-beta levels were measured in culture supernatants after 72 hours. In the second stage, 60 Sprague-Dawley rats were divided into six groups, as follows. For group I (sham), only the standard grafting procedure was performed. For group II, after the standard grafting procedure, rats were treated with intraperitoneal injections of 30 mg/kg (n = 5) or 10 mg/kg (n = 5) cyclosporine for 10 days. For group III, cultured fibroblasts obtained from skin biopsy samples from rats were treated with 100 or 500 ng/ml cyclosporine, and the cells were collected by light trypsinization and centrifugation after 72 hours. After the standard skin grafting procedure, modified fibroblasts were implanted between the graft and the recipient bed with a Pasteur pipet. For group IV, the same procedures as for group III were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. For group V, in addition to standard grafting, unmodified fibroblasts (not treated with cyclosporine) were implanted between the graft and the recipient bed. For group VI, the same procedures as for group V were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. The rejection process was observed macroscopically, and statistical significance was determined with the Mann-Whitney test (p < 0.01). Graft survival times were significantly prolonged in groups III and IV, compared with groups I, II, V, and VI (p < 0.001). No difference between groups III and IV was observed. The novel finding of this investigation is that xenogeneic skin graft survival times could be prolonged with microimplantation of cyclosporine-treated cultured fibro-blasts.

Versatility of the medial plantar flap: our clinical experience.

The medial plantar flap presents an ideal tissue reserve, particularly for the reconstruction of the plantar and palmar areas, which require a sensate and unique form of skin. In the past 5 years, the authors performed 16 free flaps, 10 locally pedicled flaps, and five cross-leg flaps on 31 patients for the reconstruction of palmar and plantar defects. All flaps transferred to the palmar area survived, providing good color match and sufficient bulkiness. The overall results were satisfactory in terms of function and sensation, and no complications related to flap survival in the plantar area were observed. All flaps used to cover defects in the heel and ankle region adapted well to their recipient areas, and all lower extremities remained functional. Because the medial plantar flap presents glabrous, sensate skin with proper bulkiness and permits the movement of underlying structures, the authors advocate its use and view this procedure as an excellent alternative in the reconstruction of palmar and plantar weight-bearing areas.

Reconstruction of the one-digit foot with an osteocutaneous fibula flap: a case report.

The decision to amputate or reconstruct after high-energy foot injuries is controversial. A 25-year-old male patient was admitted to our clinic with a complex injury to his left foot sustained during a mine explosion, and the second to fifth digits and metatarsals of the left foot had been traumatically amputated before admission to our facility. The complex left foot defect was reconstructed with an osteocutaneous fibula flap during a single session. An osteotomy was performed on the bone segment of the flap, and both lateral longitudinal and transverse arches were repaired. Both aesthetic and functional outcomes were very satisfactory, including independent ambulation, light jogging, and full performance of activities of daily living without limitation. Many factors, including comorbidities, should be considered during the decision-making process of amputating or reconstructing complex foot injuries.

A new approach to objective evaluation of the success of nasal septum perforation.

BACKGROUND: Perforations in the nasal septum (NSP) give rise not only to disintegration of the septum anatomy but also impairment in normal nasal physiology. The successes of these surgical techniques are usually equated to anatomical closure of the perforation. The goal of this study is to evaluate the subjective and objective results of our surgical technique for septal perforation surgery. METHODS: All NSPs in the six patients were closed by inferior turbinate flap. The Nasal Obstruction Symptom Evaluation (NOSE) instrument was used to evaluate the preoperative and postoperative subjective sensation of nasal obstruction. Measurement of preoperative and postoperative nasal airway resistance was performed using active anterior rhinomanometry which is an objective test. Wilcoxson signed rank test and Spearman correlation test were used to analyze correlation between NOSE scores and rhinomanometric measurements. RESULTS: The full closure of the septal perforations was noted in 100% of patients. The total NOSE score was 14 preoperatively and one postoperatively. The improvement in NOSE scores was statistically significant (P≤0.002). The mean preoperative total resistance (ResT150) value was 0.13 Pa/cm(3)s(-1), which is below the normal range (0.16-0.31 Pa/cm(3)s(-1)), while the mean postoperative ResT150 value was 0.27 Pa/cm(3)s(-1). The correlation between the improvement in NOSE scores and improvements in ResT150 values was statistically significant. CONCLUSIONS: Surgical approaches should aim to solve both the anatomical and physiological problems of NSP. The application of subjective and objective tests in the postoperative period will help surgeons assess the applied techniques.

Experimental assessment of the neo-vascularisation of acellular dermal matrix in the wound bed pretreated with mesenchymal stem cell under subatmospheric pressure.

Neo-vascularisation of the acellular dermal matrix (ADM) is an essential procedure if a full-thickness wound is closed with ADM and skin is grafted over the ADM. In this study, we aimed to improve the neo-vascularisation of ADM by combining the effects of negative pressure wound therapy (NPWT) and mesenchymal stem cells (MSCs) on angiogenesis. In this study, 28 female Sprague-Dawley rats were used and divided into four groups. Full-thickness dorsal skin defects were created in 2 × 2 cm dimensions. The wounds were treated with only the ADM in group 1, the ADM and NPWT in group 2, the ADM and MSCs in group 3 and the ADM, NPWT and MSCs in group 4. By the ninth day of surgery, the excisional biopsy samples were histologically examined to identify the rates of ADM adherence to the recipient bed; the newly formed blood vessels which penetrate the ADM vertically and vascularisation were evaluated by immunohistochemical staining. The graft adherence rates were higher in group 4 than in the other groups statistically, p = 0.003. The numbers of cluster of differentiation 31 (CD31)-stained newly formed microvessels were higher in group 4 than in the other groups statistically, p < 0.05. All subjects in group 4 had the vertical vessels in normal calibration with open lumen vessels which penetrate the ADM. These findings suggest that MSC transplantation induces angiogenesis more efficiently than NPWT. The combination of the NPWT with MSC in this study has shown a synergistic effect on angiogenesis and has affected the neo-vascularisation of the ADM significantly.