| Background: One of the main causes of calcification and collagen fibrous degeneration is that the surface of the bioprostheses(homografts and heterografts) is lack of endothelial cells (ECs) and the matrix is lack of interstitial cells. So, since 1990s, to seed autologous cells onto the valvular scaffold to fabricate a new bioprosthcsis with excellent biocompatibility, anti-calcification and self-repairing functions has been a focus of heart valve surgery. But the different scaffolds which are investigated still have some disadvantages. The study on acellular and fibrous scaffold which is tanned and recellularized hasn't been reported. The aim of this experiment is to study the biocompatibility and the characters of seeded cells on the tanned, biological and fibrous scaffold.Method:(1) Fresh bovine pericardia were divided into two groups: Group I (n=24,1×1cm per patch): fresh bovine pericardium was acellularized with Courtman's method and fixed with GA; GroupII(n=24, 1×1cm2 per patch): first treated as Group I , then was modified with 2,3-butanediol.(2) Cell isolating, culture and seeding: Myofibroblast cells (MFC) and ECs were isolated from porcine carotid artery separately, then cultured in vitro; cell seeding: on the first, second and third day, MFC were seeded onto each patch at a cell density of 1.0×105/cm2, then the patch with MFC were cultured until 17 days; On the 17th day, ECs were seeded onto each patch at a cell density of 1.5× 105/cm2, then the patch with MFC and ECs were cultured until 27 days.(3) Analysis of specimens: Viable cells were counted by Trypan blue staining; Hematoxylin-eosin (HE) staining was performed for histological analysis; VIII-factor and a -actin staining was performed to evaluate ECs and MFC separately; Scanning electron microscope (SEM) and transmission electron microscope (TEM) were also performed for ultrastructural analysis. Results: (1) Effects of acellularization: Courtman's method could get rid of most of the pericardial cells, the matrix structure became a biological fibrous scaffold which was lightly loosed with many fissures. (2) The amount of viable cells: On the 4th day, the amount of cells in group I (1.5± 0.03×105/cm2) was significantly less than group II (2.9 ± 0.04×105/cm2) (p<0.05); In Goup I no MFC was detected on the 7th day and no viable ECs was detected on the 20th day. The cells of group II proliferated after seeding: The amount of cells (3.4± 0.04×105/cm2) on the 17th day was significantly more than the amount (2.9 ± 0.04×105/cm2) on the 4th day (p<0.05), the amount on the 27th day (5.4 ± 0.01×105/cm2) was significantly more than on the 18th day (4.5 ± 0.02× 105/cm2) (p<0.05). (3) On the 27th day, HE staining and immunohistochemistry examination showed that no cells grew in group I, and 2-3 layers of cells grew on the pericardial fibrous scaffold in group II, cells on the surface were ECs, in the matrix were MFC, MFC had migrated into the pericardial matrix. (4) SEM and TEM: On the 17th day, SEM showed that no cells grew in group I and no cells were found on the surface of the specimen in Group II, but some interfiber fissures disappeared in Group II; On the 27th day, SEM showed that no cells grew in Group I , however in Group II there was an intact confluent monolayer of ECs, some of the ECs were spindle-shaped; TEM showed that on the 17th day, no cells grew in Group I , but some MFC grew into the matrix in Group II.Conclusion:(l)Biological fibrous scaffold which is from acellular, fixed and modified bovine pericardium has excellent biocompatibility.(2)Separate isolating, separate culture and separate seeding of MFC and ECs is a practical tissue engineering technique for the re-cellularization on biological fibrous scaffold, and this scaffold can be completely endothelialized and partly interstitial cellularized in vitro.
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