| Background and purpose:Diabetic wound is a common chronic and refractory wound in clinic. It often happens in the patient’s lower extremity, especially in the weight-bearing parts. Nowadays, the diabetic wound has become the most common reason for non-traumatic amputation. Peripheral vasculopathy and neuropathy, combined with a lower level of endogenous growth factors and persistent opportunistic infections result in the critical complication of diabetic patients.In recent years, cell therapy has been considered as an effective method of tissue regeneration. There is considerable interest in the treatment of diabetic ulcers with stem cell transplantation such as bone marrow derived stem cells (BM-MSCs), adipose-derived stem cells (ADSCs) and endothelial progenitor cells (EPCs). Previous publications have showed that ADSCs improve the blood flow perfusion of ischemic flaps and promote wound healing effectively. However, cell expansion in virro is needed for two to three weeks before transplantation, which is not only time-consuming, but also increases the risk of contamination and differentiation of stem cells. Moreover, Food and Drug Administration (FDA) approved facilities and techniques are required, which may be an obstacle for clinicians who culture cells in a laboratory dedicated to research. Fortunately, stromal vascular fraction (SVF) derived from adipose tissue has a group of heterogeneous cells such as ADSCs, EPCs, hematopoietic progenitors, anti-inflammatory cells and T cells. It is easily harvested with a less invasion and is consider as a better reserve of ADSCs. Abundant cell component in adipose tissue and avoidance of in vitro culture make SVF become a good cell source to promote the diabetic wound healing.However, as we all know that the survival percentage of the engrafted cells is not high. Cell migration and tissue regeneration require a suitable microenvironment. Collagen is the main component of the extracellular matrix, which represents30%of the total body protein in mammals. The collagen has been widely used as a cell carrier or as a composite of various bioactive substances for restoring and reconstruction of tissue because of its weak antigenicity, excellent biocompatibility and biodegradability.This study aimed to construct an effective collagen-based SVF targeting system to accelerate the diabetic wound healing. It was hypothesized that the improvement of local wound microenvironment with an active provisional matrix would create the permissive environment for enhancing neovascularization and improving diabetic wound healing.Materials and methods:Materials:Collagen scaffolds were freezing-dried from the purified collagen extracted from bovine skin (Zhenghai Biotechnology Inc. Shandong, China). They were cut into1.5cm×1.5cm for use. The surface feature of the scaffold was observed by scanning electron microscope.Methods:I. Six female domestic pigs, allowed to be acclimatized for1week before the initiation of the experiment. After the diabetic porcine models were successfully established, the adipose tissues were cut from the neck region to obtain the SVF. Before transplantation. SVF were labeled with Dil for tracking. The diabetic wound models were made by removal of full thickness skin (1.5cm×1.5cm) on the pigs’ back under general anesthesia. The wounds were randomly divided into four groups. The body mass have been measured before operation and the serum glucose concentrations were measured once daily during the experiment.2. All collagen scaffolds received scanning electron microscopy test.3. The percentage of SVF attaching to collagen scaffold was tested by seeding the SVF cells on the collagen scaffold and incubated for5-120minutes.4. Group A (control groups, n=6×3), Group B (SVF groups, n=6×3), Group C (scaffold groups, n=6×3). Group D (SVF-scaffold groups, n=6×3)5. PBS was applied to the wounds in Group A. l×105SVF suspended in PBS were sprayed on the wounds in Group B. Scaffolds only with culture medium were covered on the wounds in Group C. Scaffolds with equal volume of cell suspension containing1×105SVF (incubated under the standard culture condition for1h) were covered on the wounds in Group D. In Group C and D, the scaffolds were fixed with4-0suture respectively. Five minutes later all wounds were dressed with two layers of vaseline gauze and sterile gauze.6. The samples (including normal skin at the wound edge and subcutaneous tissue at the bottom) were collected on day7postoperation under general anesthesia.7. Photographs were taken on day7postoperation by digital camera, and then were analyzed by computer image analysis system to calculate the healing rates.8. On day7postoperation. the tissue specimens were biopsied by embedding in optimal cutting temperature compound and cutting into10μm slices for hematoxylin-eosin staining. The frozen slices were observed by a fluorescence microscope to observe Dil-positive cells.9. The wound granulation tissues were harvested on day7postoperation and take histological analysis.10. On day7after surgery, the specimen tissues were collected for the assay of VEGF and bFGF using ELISA kit according to the manufacturer’s recommendations.Results:1. Scanning electron microscopy shows that the collagen scaffold has widely interconnected porous stereoscopic structure and the pore is20to200μn in size. The fibrous of collagen scaffold structure permits cell ingrowth and facilitates the diffusion of nutrients into the matrix.2. The percentage of SVF cell attachment increased to41.11±2.3%(mean±SEM) at60minutes. There were significantly more cells attaching to the collagen scaffolds after60minutes. This result was used to guide the study in vivo.3. On day7postoperation, compared with Group A, Group B and Group C respectively, group D showed the highest rate of healing (34.67±4.9%)showed statistically significant difference(P<0.05, P<0.01).4. On the seventh day, the viability of engrafted cells was observed with a fluorescence microscope. In Group B and D, the Dil-labeling SVF released specific red fluorescence in granulation tissue of the diabetic wound.5. Histological analysis:5.1As show in HE staining, on day7postoperation, Group D had more blood vessels in the granulation tissue.5.2As show in immunohistochemisty staining, on day7postoperation, compared with Group A (10.11±3.98). Group B (11.50±4.36)and Group C(14.81±4.87) respectively. Group D (20.69±5.50) had higher blood vessel density in the wound granulation tissue (P<0.01).6. Alter7days, the protein expression of VEGF in Group D (285.44+56.52pg/ml) was significantly higher than that in the other three groups (Group A:158.75±39.25pg/ml, Group B:172.09±52.34pg/ml and Group C:215.50±43.01pg/ml)(P<0.05. P<0.01). Similarly, Group D showed the higher level of bFGF (16.07±3.36pg/ml) in the diabetic wound granulation zone than Group A, B and C respectively (Group A:10.12±2.46pg/ml. Group B:10.23±3.13pg/ml and Group C:12.25±2.02pg/ml)(P<0.05, P<0.01).Conclusions:1. In our study, a collagen-targeting autologous adipose SVF system induced a greater amount of blood capillaries formation and proangiogenesis factors expression, which accelerated tissue regeneration in diabetic wound repair in pig.2. Compared with SVF, SVF-collagen scaffold was better able to remain in the wound site, and thus improved its therapeutic efficiency.3. Moreover, with lower invasiveness to obtain adipose tissues from patients as well as the avoidance of in vitro culture, the SVF-collagen scaffold may offer a simple and effective treatment for targeting tissue regeneration and diabetic wound repairing. |
PDF Full Text Request