| ObjectiveThe purpose of the study is preliminary assessing of PCDM as a dermal substitute. We provide the feasibility of PCDM as dermal substitute in experiment and clinical application by observing histological structure and evaluating cytotoxicity about PCDM. After isolated and cultured, the fibroblasts were seeded onto the papillary surface and reticular surface of PCDM. Through observing the morphology, structure, growth character of fibroblasts adhering to two sides of PCDM by HE staining, SEM and TEM, we supply strategy for modifying structure and investigate the treatment of improving cell compatibility of porcine dermal scaffold.Methods1) Observing histological structure of PCDM by HE staining, immunohistochemistry staining, SEM and TEM. The histological structure of PCDM was compared to that of PADM and HADM.2) The cell growth curves of L-929 with different concentration were measured by MTT assay in order to decide an appropriated cell seeding concentration. The leaching liquor of PCDM was made and used as test group, blank control group, positive control group and negative control group were set. The effects of different levels of leaching liquor on L-929 were detected by MTT. The cytotoxicity of PCDM was evaluated in vitro according to the national standard of biomaterial.3) Human fibroblasts were isolated and cultivated by tissue mass cell culture.4) Culture plate was used as a control group, the adhersion of FB adhered to papillary side of PCDM was measured by MTT in order to decide the seeding density on PCDM.5) The papillary dermis was set as test group A, the reticular dermis was set as test group B. The fibroblasts were seeded on two sides of PCDM ,after seeded for 1 7 14 days, the FB- PCDM composites were observed by HE staining, SEM and TEM.Results1) PCDM was pure white and smooth with good tenacity and soft feeling. In the light microscope , the side of papillary dermis was smooth and intact, immunohistochemical staining showed filiform brown region on papillary dermis, it suggest intact basal membrane was remained ,but the cell, blood vessel, appendages of the skin were removed completely, The three dimensional structure of collagenic matrix was intact. The side of reticular dermis was rough, ruptured collagen fiber were seen. In transmission electron microscope, collagenic structure remained as normal porcine dermis was observed. A comparison of PCDM, HADM and PADM by SEM reveals that they all have a tightly packed network of thick collagen bundles, however, the PCDM and HADM appears to have a more loosely packed surface than PADM.2) The number of cells in 5×10~3/well to 1×10~4/well were appropriate for MTT, The cell growth curves of L-929 showed straight line correlation of direct proportion according to the curve of cell number vs OD in a week, the cell proliferation was active and the measured data was stable according to the standard detection method of cytotoxicity, cell proliferation degree of test group were higher than positive control group significantly, the value among test group , blank control group and negative control group was no significant difference. The toxicity grade of PCDM was grade 0-1, there was no significant toxicity to body.3) The cell adhersion of test group (PCDM) was less significantly than control group (culture plate), The numbers of test group was only 50% of control group.4) HE staining showed: on day 1, there were more samples adhered single cell in group A than group B. the difference was significant(P<0.05); on day 7, there were more samples with continual monolayer of cell in group A than group B, the difference was significant(P<0.05), stratified cells could be seen between group A and group B, but no cell infiltrated into dermal matrix in group A. there were 6/25 samples with cell infiltration in group B. on day 14,there were 18/27 samples without cell adhered in group A and only 9/30 samples like that in group B, more samples with cell infiltration were seen in group B. scanning electron microscope showed: stellar FB attached to PCDM tightly after seeded for 3 days, some cells connected to each other by the long processes. Fusiform FB covered the papillary dermis in longitudinal direction densely and regularly after seeded for 7 days, reticular collagen fibrils were seen around active FB. Transmission electron microscope showed: FB attached to two sides of PCDM functioned actively, there were abundant free ribosomes and Golgi's complex in intracytoplasm, the rough endoplasm icreticulum became dilat at day 3 after seeding. Conclusion1) PCDM prepared by physical-chemical process has no cytotoxicity. It is a safe xenogenic dermal substitute.2) FB proliferated well on PCDM, small amounts human fibroblasts infiltration was seen in few samples in this study. This find shows PCDM has good cell compatibility in vitro and it also suggests that there is no xenogenic barrier to human fibroblast migration on porcine collagen but possibly a collagenic structural obstacle to widespread infiltration of FB.3) Human fibroblasts adhere better to papillary dermis than the reticular dermis in the primary stage of cell seeding, however, in advanced stage of cell seeding, the papillary side become barrier to fibroblasts proliferation and infiltration on porcine collagen. So discarding papillary dermis is favorable to improve the cell compatibility of xenogenic dermal replacement, but the surface of reticular dermis should be modified to promote fibroblasts adherence.
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