Objective To study and test novel hybrid valves in vitro and in vivo, and provide basis for clinical use in future. Methods The hybrid valves were fabricated from decellularized porcine aortic valves coated with poly (3-hydroxybutyrate-co-3hydroxyhexanoate, PHBHHx).（1）In the mechanical test in vitro, the uniaxial tensile biomechanics test of the fresh (n=12), uncoated (n=12) and hybrid valve leaflets (n=12) were investigated. (2）In study in vivo, hybrid valves(n=5) implanted in pulmonary position in sheep without cardiopulmonary bypass. Uncoated grafts (n=5) used as control. The specimens of the hybrid or uncoated valve in sheep were explanted and examined by scanning electron microscopy, histology, calcium content and immunofluorescence staining 18 weeks after surgery. Results The mechanical test in vitro revealed that coating with PHBHHx increased maximal tensile strength of hybrid valves compared with the fresh and uncoated state (P＜0.05). The results in vivo indicated the hybrid valves maintained original shape and softness. Immunofluorescence staining for CD31 confirmed that the surface of hybrid valve was covered by confluent CD31+ cells.The interstitium of hybrid valve indicated that smooth muscle actin (SMA)+ cells population were similar to native valvular tissue. The calcium content of hybrid valve was significantly lower than that of uncoated valve leaflets (P＜0.05). Conclusion Decellularized porcine aortic valves coated with PHBHHx have good biological and biomechanical characteristics. The hybrid valve may provide superior valve replacement with current techniques.
【Abstract】 Objective To evaluate the biocompatibil ity of the sheep BMSCs cultured on the surface of photografting modified copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate(PHBV). Methods BMSCs were isolated from bone marrow of the posterior il iac crest of a 6-month old sheep by whole marrow adherent culture method. The 3rd passage BMSCs were seeded onto modified PHBV and conventional PHBV films, or three-dimension scaffolds. Cell-adhesion rates were calculated by hemocytometer at 1, 2 and 6 hours after seeded. Cell morphology was examined by scanning electron microscope when the BMSCs were cultured for 3 days, 1 week and 3 weeks. Cell cycle was analyzed by flow cytometry at 5 days after seeded. The content of protein in BMSCs was determined by BCA assay and the content of DNA was quantified by Hoechst 33258 assay at 4, 8 and 12 days after seeded. Results At 1 hour after seeded, cell-adhesion rate on modified PHBV films (52.7% ± 6.0%) was significantlyhigher than that of conventional PHBV films (37.5% ± 5.3%) (P lt; 0.05); At 2 and 6 hours after seeded, cell-adhesion rate of modified PHBV films was similar to that of PHBV films (P gt; 0.05). The surface of modified PHBV film was rougher. In the early culture stage, more cells adhered to modified PHBV and the cells displayed much greater spreading morphology. Furthermore, ECM on modified PHBV were richer. There were no significant differences between the trial team and the control on the cell cycle and the content of DNA and protein of BMSCs (P gt; 0.05). Conclusion Photografting modification on PHBV can promote BMSCs’ adhesion and enhance their biocompatibil ity.
ObjectiveTo study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). MethodsPHBV and PHBHHx films were fabricated by solvent evaporation.Scanning electronic microscope (SEM) was used to study the morphology of the films.PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively.The hydrophilicity of the surface were detected by the contact angle measurements.Septal cartilage cells obtained from human septal cartilage were cultured in vitro.The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1,PHBV modified with PhaP-RGD in group A2,PHBHHx unmodified with PhaP-RGD in group B1,PHBHHx modified with PhaP-RGD in group B2,and on the cell culture plates in group C.After cultured for 3 days,the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days,the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. ResultsSEM observation showed that PHBV and PHBHHx films had porous structures.The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P<0.05).Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups.After 3 days of cultivation in vitro,the cell proliferation and viability of group B2 were the strongest among all groups (P<0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1,A2,B1,and B2 (P<0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1,A2 and C,in group B2 than group B1,and in group A1 than group A2 (P<0.05).The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1,A2,B1,and B2; group A1 and group A2 had similar staining degree,and the staining of group B2 was deeper than that of group B1.The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. ConclusionThe biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.
ObjectiveTo assess the suitability of P (3HB-co-4HB) combined with embryonic stem cells (ESCs) for myocardial patch formation and whether adding vitamin C would improve inductivity or not. Method We extracted mouse embryonic fibrous cell from three clean female white Kunming mouses at a mean body weight of 37.5 grams. We recovered and cultured mouse ESCs. Those mouse embryonic stem cells were obtained from Shanghai Institutes of Biological Sciences. We took pendant-drop method to form embryonic bodies (EBs) and co-cultured them with myocardial patch. The experimental group were cultured in the substate with vitamin C while the control group were cultured in the substate without vitamin C. We immunostained the myocardial patch and observed them by scanning electron microscope. We calculated the differentiation efficiency and mapped the distribution curve of induction time. ResultsThe scattergram showed that the differentiation efficiency increased gradually. The differentiation efficiency of the group with vitamin C was 71.1% and the group without vitamin C was 17.8%. There was a statistical difference between the two groups (P < 0.05). ConclusionOn the biological patch of P (3HB-co-4HB), ESCs could grow, proliferate, and differentiate into myocardial cell and adding vitamin C into it could improve the differentiation efficiency.
ObjectiveTo study the external biocompatibility bewteen the mouse induced pluripotent stem cells (miPSCs) and poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx). MethodsAfter we recovered and subcultured miPSCs, we divided them into two groups. There was one group cultured with material of PHBHHx films outside the body. We observed the adhesive pattern of miPSCs on film by fluorescence of 4, 6-diamidino-2-phenylindole (DAPI) staining. The cell vitality was detected by cell counting kit-8 (CCK-8). The morphology of miPSCs attached on the film was visualized under scanning electron microscope (SEM). We used the traditional petri dish to culture miPSCs and detect the cell activity by CCK-8. ResultsMiPSCs can adhere and proliferate on PHBHHx films. The result of cell vitality which detected by CCK-8 showed that there was a statistical difference in OD value between culturing on PHBHHx films and traditional cultivation (0.617±0.019 vs. 0.312±0.004, P < 0.05). ConclusionThere are adhesion and proliferation on the surface of cells patch made by miPSCs co-culturing with PHBHHx film. Compared with traditional culturing in the cell culture dish, culturing in PHBHHx films have great advantages in the process of adhesion and proliferation. PHBHHx can be used as one of the scaffold for stem cells treating various disease.
ObjectiveTo investigate the effects of hypoxic three-dimensional culture microenvironment on the proliferation of bone marrow mesenchymal stem cells and its mechanism. MethodsP5 generation mouse bone marrow mesenchymal stem cells and P (3HB-co-4HB) were co-cultured under normoxic three-dimensional (20%) and hypoxic three-dimensional microenvironment (4%) respectively. After 24 hours, the proliferation of the two groups was determined by CCK-8 method. The expression of HIF-1α gene was detected by real-time quantitative PCR after 12 hours. Western blotting was used to detect the expression of HIF-1α protein after 24 hours. ResultsAfter 24 hours, the CCK-8 method showed that the OD value of the hypoxia group was significantly higher than that of the normoxia group (0.455±0.027 vs. 0.352±0.090, n=12, P<0.05). After 12 hours of hypoxic culture, the expression level of HIF-1α mRNA in the hypoxia group was significantly higher than that in the normoxia group (P<0.05). Compared with the normoxia group (0.47± 0.05), the relative expression level of HIF-1α protein in the hypoxia group (0.63±0.06) significantly increased in the Western blotting after 24 hours (n=3, P<0.05). ConclusionThe hypoxic three-dimensional microenvironment can promote the proliferation of bone marrow mesenchymal stem cells, which may be related to the activation of HIF-1α signaling pathway.
ObjectiveTo review the research progress of natural biomaterial polyhydroxyalkanoate (PHA) in orthopedics. Methods The literature concerning PHA devices for bone defects, bone repair, and bone neoplasms, respectively, in recent years was extensively consulted. The three aspects of the advantages of PHA in bone repair, the preparation of PHA medical devices for bone repair and their application in orthopedics were discussed. ResultsDue to excellent biodegradability, biocompatibility, and potential osteoinduction, PHA is a kind of good bone repair material. In addition to the traditional PHA medical implants, the use of electrostatic spinning and three-dimensional printing can be designed to various functional PHA medical devices, in order to meet the orthopedic clinical demands, including the bone regeneration, minimally invasive bone tissue repair by injection, antibacterial bone repair, auxiliary establishment of three-dimensional bone tumor model, directed osteogenic differentiation of stem cells, etc. ConclusionAt present, PHA is a hotspot of biomaterials for translational medicine in orthopedics. Although they have not completely applied in the clinic, the advantages of repair in bone defects have been gradually reflected in tissue engineering, showing an application prospect in orthopedics.