Effect Of Collagen Scaffold With BCL-2 Modified Adipose-derived Stem Cells On Diabetic Wound Healing:A Study In Diabetic Mice Model

Background and purpose:Diabetes is a common clinical disease, and chronic nonhealing skin ulcers in lower extremities occur in nearly 5-10% patients with diabetes. The diabetic ulcers, mainly caused by ischemic, neuropathic or combined neuroischemic abnormalities, has become the most common reason for non-traumatic amputation. In recent years, stem cell transplantation such as bone marrow derived stem cells(BM-MSCs), adipose derived stem cells(ADSCs) and endothelial progenitor cells(EPCs) became more popular in the treatment of chronic wound. Treatment with mesenchymal stem cells(MSCs) resulted in increased wound closure, new granulation tissue formation, and increased blood vessel formation and cellularity in diabetic mice model. Compare with other MSCs, ADSCs have been widely used in wound healing, because of the characteristics such as easy collection, weak immunogenicity, and lack of morbidity at the donor site.However, using exogenous growth factors and cell transplantation for diabetic ulcer treatment has certain difficulty, mainly in two aspects:firstly, cells and growth factors were washed out by wound exudate that lead to decrease the cell survival after transplantation. Secondly, the growth factor action time is not long, and the curative effect of using single growth factor is also very limited. As the main component of the extracellular matrix, collagen can provide a suitable microenvironment for cell transplantation and tissue regenaration. Because of the weak antigenicity, excellent biocompatibility and biodegradability, collagen scaffold with bioactive molecules such as growth factors and MSCs can significantly enhance healing.Some previous studies have demonstrated that stem cells do not survive well after transplantation. The Bcl-2 antiapoptotic protein serves as critical regulator of pathways involved in apoptosis, acting to inhibit cell death. Modified MSCs by BCL-2 can not only enhance cell survival after cell transplantation,but also increase the expression of VEGF.This study aimed to construct an effective collagen-based Bcl-2-ADSCs targeting system to promote the diabetic wound healing. It was hypothesized that collagen scaffolds combine with BCL-2 modified ADSCs wound create the permissive environment for improving diabetic wound healing and enhancing neovascularization.Materials and methods:Materials:Collagen scaffolds were obtained from bovine skin(Zhenghai Biotechnology Inc, Shandong, China). They were cut into 7mm diameter rounds with a thickness of 1.0mm. The surface morphology of the scaffold was observed by scanning electron microscopy(SEM) (model S-2500, Hitachi, japan).MethodsExperiment in vitro1. Human abdominal adipose tissues were obtained from a 35-year-old healthy male patient undergoing liposuction. All experiments were approved by the Ethics Committee and informed consent was obtained from the participant. After removal of blood cells by washing with sterile phosphate-buffered saline(PBS)(Gibco), the adipose tissue was digested in 0.1% type Ⅰ collagenase(Gibco) for 45min at 37℃ with agitation. The digested tissue was centrifuged at 1500rpm for 10 min, and the suspending portion was discarded. The cells pellet was resuspended with Dulbecco’s modified Eagle medium/Ham’s F12(DMEM/F12,Gibco) supplemented with 15% fetal bovine serum(FBS)(Gibco) and 1% penicillin/streptomycin, then centrifuged at]200rpm for 5 min again. Cell pellet was resuspended in the culture medium, plated in 25-cm2 flask, and incubated at 37℃ in a humidified atmosphere with 5% CO2. After 24h, the medium was replaced and changed every 3 days thereafter. When the adherent cells reached 80% confluence, they were digestion with 0.25%trypsin/EDTA(Gibco) and passaged at a dilution of 1:3. ADSCs from passages 3 to 6 were used in the experiments.2. ADSCs were seeded in 6-well plates at 1×105 cells/well. When the confluence reached 80%, the cell were treated with an adenovirus carrying the enhanced green fluorescent protein gene(EGFP) at different multiplicities of infection (0,50,100,200 and 500). The transfection efficiency of EGFP gene was investigated by flow cytometry on day 2. The delivery of BLC-2 gene was due to the optimal MOI. ADSCs without gene transduction and those transfected with adenovirus carrying Bcl-2 (Ad/Bcl-2) or adenovirus alone were termed "ADSCs", "Bcl-2-ADSCs", and "vector-ADSCs", respectively. Levels of Bcl-2 protein expression were evaluated using Western blotting.Experiment in vivo1. Ten-week-old female db/db mice, weighing 25-30g, allow to acclimate to environment for 1 week. Mice were induced type I diabetes by once intraperitoneal injection of streptozocin(STZ:165mg/kg, Sigma), dissolved in citrate buffer(0.1 M at pH4.5). The blood glucose levels were measured once daily during the experiment with a blood glucose meter. After injecting STZ, there was a permanent hyperglycemic condition(blood glucose>21mmol/L) and the mice were randomly divided into four groups:A(control groups, n=12),B(scaffold groups, n=12),C(ADSCs/scaffold groups, n=12), D(BCL-2/ADSCs/scaffold groups, n=12). Full-thickness circular wound(0.7cm) were made on dorsal skin. PBS was sprayed on the wounds in Group A. Scaffolds with PBS were applied to the wound in Group B. Scaffolds with 1×105 ADSCs suspended were covered on the wounds in Group C Scaffolds with 1×105 BCL-2-ADSCs suspended were covered on the wounds in Group D. All wounds were dressed with vaseline gauze and sterile gauze. All the animal protocols were performed in accordance with the guidelines of Chines Ministry of Public Health(CMH) and Use of Laboratory Animals.2. The wound area was photographed by digital camera on day 3,7,10 and 14 postoperation. The wound area was analyzed by Photoshop software(Adobe Photoshop 7.0) to determine the healing rate. Wound healing rate= [(original areas-non-healing areas)/original area] ×100%.3. On day 7 postoperation, the skin samples, including normal skim at the wound edge, were taken as 7 X 7mm biopsied under general anesthesia.The skin samples were fixed in 4% paraformaldehyde over night, at 4℃, then embedded in paraffin for hematoxylineosin (HE) staining to detect capillaries. The slices were observed in light microscopy at 100 X magnification. Dil-positive cells were identified with fluorescent microscope.4. A portion of the wound samples(7 X 7mm) was excised at day 7 after operation. The neovascularization level of wound granulation tissues was detect by immunolabelling with rabbit anti human factor VIII related antigen polyclonal antibody (vWF,1:800, Abeam, USA). The countess of capillaries were made for six randomly chosen fields by two independent researches under a magnification of 200 X.Results:Experiment in vitro1. The collagen scaffolds was full of pores under scanning electron microscopy. The sizes of the internal pores ranged 150-200 u m, that permitted cell infiltration and guaranteed the diffusion of nutrient in the interior of the scaffold.2. Human ADSCs were isolated from human lipoaspriye tissue. Flow cytomerty analysis for the surface phenotypes of human ADSCs indicated that ADSCs expressed stem cell specific markers CD29, CD44 and CD90 but did not express CD34, HLA-DR and the hematopoietic stem cell marker CD133.3. Transduction efficiency of Ad/EGFP, conformed by flow cytometry, was 0,54%,79% and 81.5% at MOIs of 0,100 and 200. The ADSCs express green fluorescence after transduced with Ad/EGFP. The optimal multiplicity of infection was conformed to MOI= 200. Compared to ADSCs, there was no pathological changes in Bcl-2-ADSCs and vector/ADSCs after transduction. Bcl-2 expression was enhanced in Bcl-2-ADSCs as early as 24h and the level of expression remained high on day 7. Weak protein expression of Bcl-2 wast detected in vector-ADSCs and ADSc at 24h after transduction. This result indicated a low level of endogenous Bcl-2 protein in ADSCs.Experiment in vivo1. In our study, the wound healing rate did not differ significantly between Group A and B on day 7 postoperation(P>0.05). The wound healing rate in Group C was significantly greater than Group A and B(P<0.05, P< 0.01).Group D showed the highest wound healing rate. The results demonstrated that BCL-2-ADSCs groups can effectively improve wound healing compared with other groups.2. On the seventh day, the viability of engrafted cells was observed with a fluorescence microscope. In granulation tissue of the diabetic wound of Group C and D, the Dil-labeling ADSCs released specific red fluorescence.3. Similar to the results of wound healing rate, the blood vessel density in Group C was significantly higher than Group A and B. Group D showed the highest of wound vascular density on day 7 postoperation. No significant difference in vascular density between Group A and B.Conclusions:1. The exogenous BCL-2 gene resulted in a high of Bcl-2 expression at the protein level. In addition, Bcl-2 upregulation through gene transfection significantly reduced ADSC apoptosis after transplantation.2. The BCL-2 modified ADSCs could migrate to the scaffold, enhance cell survival, improve wound repair and shorten the healing time after cell transplantation.3. The BCL-2-ADSCs-scaffold effectively accelerated the healing rate and promoted neovascularization after implantation at the wound.