| Background and purpose:Massive full-thickness skin defect caused by extensive burns or high-energy trauma were common in clinical experience. Mesenchymal stem cells (MSCs) held a promising strategy for tissue regeneration and brought hope for the patients suffering from wounds and burns who were difficult to treat and heal. In this study, a specific MSCs affinity peptide (E7 peptide) was immobilized to collagen scaffold by collagen binding domain (CBD) to construct functional collagen scaffold. We hypothesized that the functional collagen scaffold could enrich mesenchymal stem cells (MSCs) at the wound site, and promote wound healing and skin regeneration. In porcine skin full-thickness defect model, enhanced MSCs recruitment could be observed in CBD-E7/collagen scaffold. And the functional scaffold promoted the capillaries infiltration, accelerated the healing time and improved the healing quality. In addition, cutaneous appendages were also observed in CBD-E7/collagen scaffold repairing skin at 112 days after surgery. These results demonstrated the functional collagen scaffold with collagen binding MSCs affinity peptide could be effective to promote the structural and functional recovery of skin wound.Materials and methods:Materials:Type I collagen was prepared from rat tail tendon, and then collagen scaffolds were freezing-dried and cut into 5.0 cmx5.0 cm for use. The surface morphology of the collagen scaffold was observed by scanning electron microscopy (SEM) (model S-2500, Hitachi, Japan). CBD-E7 peptides ("EPLQLKMGSAGSAAGSGGTKKTLRT") were synthesized through solidphase peptide synthesis using Fmoc Chemistry (Scilight-Peptide Inc., Beijing, China). And 250μg CBD-E7 peptides were dissolved in 100 μl PBS and added to collagen membrane. All collagen scaffolds received scanning electron microscopy test.MethodsExperiment in vitroIn a separate experiment, peripheral blood was collected using a syringe from the femoral artery of the porcine. After collagen being blocked by 5% BSA, the peripheral blood (50ml per well) was added into the plate and incubated in a shaker for 60 min. The plates were softly washed for three times and digested by collagenase type I. Then the cells were prepared for FACS analysis to analyze proportion of stem cells (CD29+, CD444) in peripheral blood mononuclear cells (CD45"cells).Experiment in vivo1. Five female domestic pigs weighing between 20 and 25 Kg were used for implantation. The pigs were intravenous anesthetized using propofol (200mg 2%) and the dorsal hair was shaved. Six full-thickness wounds (three incisions on each side) of about 5.0×5.0 cm were made on the dorsum down to the level of subcutaneous deep fascia and different collagen membranes were fixed on the defects with 4-0 silk suture. After operation, the pigs were housed in separate cages. All animals received intramuscular Penicillin for 3 days after surgery.2. For cells analysis,12 wounds of 2 animals were randomly divided into 2 groups: collagen scaffold group (Scaffold, n=3×2), and CBD-E7/collagen scaffold group (CBD-E7 peptide, n=3×2).3. At 3d after surgery, the animals were sacrificed, samples were taken out and part of the materials (1.0×1.0cm) were excised for cell infiltration analysis. The cells retained on the scaffold were stained with Hoechst 33342 (2 mg/ml, Sigma) for 15 min. After washing for three times, photos were taken.4. For skin regeneration studies, the wounds were divided into 3 groups:control group (Control, n=2x3), collagen scaffold group (Scaffold, n=2x3), and CBD-E7/collagen scaffold group (CBD-E7peptide, n=2x3).5. The wound area was examined and photographed by digital camera (Canon EOS 550D) in 7d,14d,21 d and 28d post surgery. The area of unhealed wound was measured based on the image to calculate the healing rates using Photoshop software (Adobe Photoshop 7.0). Healing rate=[(original size-non-healing area)/original area] ×100%.6. The samples (including normal skin at the wound edge and subcutaneous tissue at the bottom) were collected on day 7 postoperation under general anesthesia. Hematoxylin eosin (HE) staining was performed for analysis of inflammatory reaction and cell prolif eration. Meanwhile, immunohistochemical (IHC) staining was performed for analysis of neovascularization at different regeneration stage. Anti-von Willebrand factor antibody (vWF, 1:800, Abcam, USA) was used to evaluate the degree of vascularization in the wound granulation tissues.7. On 28d after surgery, HE staining was performed for analysis of collagen formation.8. On 112d after surgery, serial sections were stained with hematoxylin for nuclei staining, and then stained in SR. Finally, the sections were darkly observed using polarizing light microscope (Nikon E400POLS, Japan). Image-Pro Plus 6.0 software were used for picric-sirius red-positive analysis. Five non-overlapping visual fields (x200) were randomly selected from each section to analyze relative value of positive type I and III collagen fiber area, and the mean value was finally considered as the positive area.9. On 112d postoperative, HE staining was performed for analysis of observed skin adnexal (such as hair follicles, sweat glands and sebaceous glands, etc.).Results:Experiment in vitro1. The structure of the collagen scaffold was observed by SEM. The scaffold had widely interconnected pores which were well suited to cell infiltration, angiogenesis and nutrients diffusion.2. As is shown by Hoechst 33342 staining, a greater number of cells were recruited in CBD-E7/collagen scaffold group, while in collagen scaffold group, the distributions of the cells were sporadic.3. for FASC analysis, the proportion of stem cells (CD29+,CD44+) enriched in CBD-peptide group in peripheral blood mononuclear cells (CD45- cells), was significantly (98.5% ±6.5) higher than Scaffold group (76.3%±1.3) and non sorting erythrocytes-depleted peripheral blood mononuclear cells (63.5% ± 2.9).Experiment in vivo1. At 7days after surgery, the healing rate between 3 groups had no statistically significant difference (P> 0.05). At 14 days, CBD-peptide group showed the highest rate of healing (14d:67.04 ± 4.8%,21d:90.96 ± 2.7%,28d:96.90±2.3%) showed statistically significant difference(P<0.05, P<0.01)2. Histological analysis:2.1 As shown in HE staining, on 7 days postoperation, CBD-E7 group had more blood vessels in the granulation tissue and scaffold.2.2 As shown in immunohistochemisty staining, on day 7 postoperation, compared with Control group (37.30 ± 6.73) and Scaffold group (58.35 ± 5.03), higher blood vessel density in the wound granulation tissue were observed in CBD-peptide group (78.70 ± 8.77) (P<0.01).2.3 At 28d after operation, the collagen fiber arrangement of the wound sections was observed by HE and immunohistochemical staining. The collagen fibers in CBD-E7 group were arranged orderly, while in collagen scaffold and control group, the collagen fibers were randomly arranged.2.4 On the 112d after surgery, the proportion of collagen type Ⅲ in CBD-E7peptide group (34%) was much higher than Scaffold group (19%) and Control group (12%). There is statistically significant difference between the groups P< 0.01.2.5 At 112 days after the surgery, the regeneration of cutaneous appendages was also observed. Hair follicle cells and sebaceous gland cells were observed in Collagen/CBD-E7 peptide group, while only sebaceous gland cells were found in Collagen group. No skin appendages was observed in Control group.Conclusions:1. Compared with collagen scaffold group, CBD-E7/collagen scaffold could effectively enhance the recruitment of MSCs, induced a greater amount of cell migration to the wound site.2. In this work, CBD-E7/collagen scaffold group had more blood vessels in the granulation tissue and the collagen fibers were more regular than Scaffold and Control group.3. Moreover, the functional scaffold could effectively enhance the recruitment of MSCs, accelerate the healing rate and promote the regeneration of cutaneous appendages in a porcine model, which could contribute to new methods for the treatment of wound healing. |
PDF Full Text Request