| Objective:To fabricate a new graft(ASCs/CBD-bFGF/BDDM)and evaluate the characteristics of the graft and to detect its therapeutic effect in diabetic wound healing.Methods:1.Freezing microtome was used to prepare book-shaped decellularized dermal matrix(BDDM).Characteristics of BDDM were compared with NDT by H&E,Masson,IHC,DAPI and SEM.2.ASCs were acquired from 3 weeks male SD rats and identified by flow cytometry analysis CDllb,CD29,CD34,CD45,CD90 and CD105)and differentiations(osteogenic differentiation,chondrogenic differentiation and adipogenic differentiation).ASCs were implanted in Temperature controlled petri dishes to prepare ASC-sheet.The ASC-sheet was evaluated.3.The CBD-bFGF protein expression vector was transplanted into the BL21 strain of Escherichia coli to induce CBD-bFGF,which was identified by SDS and WB.IF,qRT-PCR and tube formation were applied to evaluate the endothelium inducing ability of CBD-bFGF and NAT-bFGF.Binding assay and release assay of CBD-bFGF or NAT-bFGF in BDDM were tested by Elisa.Loading CBD-bFGF or NAT-bFGF in BDDM to fabricate CBD-bFGF/BDDM or NAT-bFGF/BDDM.CCK8,LIVE/DEAD,qRT-PCR,IF,tube formation and immunogenicity test were applied to evaluate the characteristics of the compounds.4.96 healthy 8 weeks male SD rats were used to induce diabetic rat model by intraperitoneal injection with streptozotocin.Diabetic rat skin defect model was established by puncher(2cm in diameter).The skin defect models were randomly divided into 4 groups:DDM,BDDM,CBD-bFGF/BDDM,and ASCs/CBD-bFGF/BDDM.5.On postoperative days 0,7,14,and 21,the skin wounds of each rat were continuously photographed to observe the wound healing situation and the wound healing rate was calculated by statistical software.6.The wound healing was evaluated by histology(H&E,MT)on postoperative days 14 and 21.7.Angiogenesis was evaluated by immunofluorescence(CD31,α-SMA)on postoperative days 7.Results:1.The isolated ASCs showed a spindle-shaped morphology.Under osteogenic,chondrogenic or adipogenic induction,the isolated cells were positive for alizarin Red,alcian blue and oil red O staining.As determined by flow cytometry analysis,these isolated cells were positive for CD29,CD90,and CD105 but negative for CD11b,CD34,and CD45.2.H&E and DAPI stained sections of the BDDM showed that all of the cellular content had been clearly removed.MT staining and IHC staining using anti-fibronectin determined that our decellularization process did not deteriorate the structural integrity of the normal dermis.SEM images showed loose structure.3.The endothelial differentiation of ASCs was measured by evaluating CD31,vWF,and CD 144 mRNA transcript levels using qRT-PCR.Immunofluorescence staining assays confirmed the protein expression of CD31 in ASCs after NAT-bFGF or CBD-bFGF induction.More capillary-like structures formed when the ASCs were plated onto Matrigel after induction by NAT-bFGF or CBD-bFGF.4.The amount of CBD-bFGF bound on the BDDM was significantly higher than the amount of NAT-bFGF bound.CBD-bFGF can specifically tether BDDM,resulting in a more sustained release from BDDM than NAT-bFGF.5.CCK8 and Live/Dead assay demonstrated that CBD-bFGF/BDDM had no cytotoxicity.the supernatants of the TCPs and CBD-bFGF/BDDM groups showed similar levels of pro-inflammatory cytokines(TNF-α,IL-6,and IL-1β),and both of these groups showed significantly lower expression levels than those in the LPS group.6.The wound closure rates in the DDM and BDDM groups were similar without a significant difference,but both were significantly lower than those of the CBD-bFGF/BDDM and ASCs/CBD-bFGF/BDDM groups.The wound closure rate in the ASCs/CBD-bFGF/BDDM group presented a significantly higher value than that of the CBD-bFGF/BDDM group.7.H&E and MT staining showed the rate of re-epithelialization and collagen deposition in the ASCs/CBD-bFGF/BDDM group was the highest among the four groups at postoperative days 14 and 21,and the difference was statistically significant.8.At day 7 post-wounding,the total number of blood vessels and the number of mature blood vessels in the wound sites covered with DDM,BDDM,CBD-bFGF/BDDM,ASCs/CBD-bFGF/BDDM were identified by double-staining for CD31 and α-SMA,respectively.Our results showed that ASC/CBD-bFGF/BDDM-covered wounds significantly enhanced the number of total and mature blood vessels compared to other groups.Conclusion:1.The novel tissue-engineering graft(termed ASCs/CBD-bFGF/BDDM)was beneficial for enhancing diabetic wound healing via angiogenesis coordination,granulation tissue growth,collagen deposition and epithelialization.2.This study indicates that ASCs/CBD-bFGF/BDDM is a very promising graft for accelerating DFU healing in the future. |
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