2.1The CBD-E7 functional collagen scaffolds
The main material used in the study, collagen scaffold, which is also the focus of this research, was provided by Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Beijing, China) [10, 12] by synthesizing CBD-E7 peptides (“EPLQLKMGSAGSAAGSGGTKKTLRT”) through solidphase peptide synthesis using Fmoc Chemistry (Scilight-Peptide Inc., Beijing, China), after which 250 μg CBD-E7 peptides were dissolved in 100 µl PBS and added to collagen scaffold.
The scaffold was cut into squares of 5.0×5.0 cm, and the surface morphology both pre-and post-treatment was observed by scanning electron microscopy (SEM) (model S-2500, Hitachi, Japan) (Fig. 1).
2.2 Animal models and surgical procedures
In order to ensure the results of the experiment as accurate as possible without being disturbed by other factors, researchers selected 12 female pigs weighing between 20 and 25 kg for the experiment. 3 full-thickness excisions (6 wounds totally on each individual animal) of about 5.0×5.0 cm were performed with sharp dissection on each side of dorsum, each wound was observed to have reached the level of subcutaneous deep fascia. Then different collagen scaffolds were applied and pressurized on the skin defects. The pigs were placed in separate cages after operation，and intramuscular Penicillin was applied on each subject for 3 days. All animals and experimental procedures used in this research were approved by the local Institutional Animal Care.
2.3 Evaluation of healing rate in porcine models
72 wounds of 12 animals were divided into 3 groups: control group (Control, n=2×12), collagen group (Collagen, n=2×12), and Collagen/CBD-E7 peptide group (Collagen/CBD-E7 peptide, n=2×12). The wound area was photographed weekly by digital camera (Canon EOS 550D) at the first 4 weeks post surgery. The area of unhealed wound was measured based on the image using Photoshop software (Adobe Photoshop 7.0). Healing rate = [(original size - non-healing area)/original area] ×100% .
2.4 Analysis of cell infiltration
At the first week after surgery, 3 subjects were sacrificed. A portion of wounds (1.0×1.0 cm) with normal tissues and collagen scaffolds at the margin were excised for cell infiltration observation. We evaluated the cellularization by counting cells infiltrated on the samples (hematoxylin eosin staining) in six random selected areas at 200× magnification level in a blinded fashion. The cells on the remained materials were obtained for cell enrichment analysis. After washing softly for three times, the remaining collagen was digested by collagenase type I. Then the cells were prepared for FACS (CD45‑ /CD29+, CD44+).
2.5 Histological observation
At the 1w, 4w, 16w, 64w after operation, the wounds (1.0×1.0 cm) were excised at the margin contained the scaffolds and normal tissues. After fixed in 10% formalin, 5 mm paraffin sections were stained by hematoxylin eosin (HE, 1 w, 4 w, 16 w, 64 w), immunohistochemical (IHC, 1 w), masson trichrome (MT, 4 w, 64 w) and sirius red (SR, 16 w, 64 w) respectively for analysis of tissue vascularization, collagen formation and scar hyperplasia.
The capillaries were detected by immunostaining of anti-von Willebrand factor antibody (anti-vWF, 1:800, Abcam, USA) at 1 w. Two independent observers quantified the number of blood vessels for each section which was counted in six random selected areas at 200× magnification level in a blinded fashion.
MT and SR staining were used to evaluate the formation of collagen fibers. The presence and absence of collagen fibrillar structures within the wound bed was detected by MT staining. SR staining, considered to be one of the best understood techniques of collagen histochemistry, was use to assess the proportion between collagen type I and type III . After dewaxed, serial sections were stained with hematoxylin for nuclei staining, and then stained by SR. Finally, the sections were observed under no-light environment polarizing light microscope (Nikon E400POLS, Japan)．Image-Pro Plus 6.0 software was used for sirius red-positive analysis.
2.6 Statistical analysis
Statistics were calculated with SPSS computer software for Windows (version 13.0, SPSS Inc, Chicago, IL). The data were expressed as means ± standard deviation (SD). Statistical differences between the groups were discerned by one-way analysis of variance (ANOVA). A probability value (P) of less than 0.05 was considered to be statistically significant. Statistically significant values were defined as *P < 0.05 and **P < 0.01.