[EXPERIMENTAL STUDY ON THREE DIMENSINONAL CULTURE OF RABBIT ANNULUS FIBROSUS CELLS ON KLD-12 POLYPEPTIDE NANOFIBER GEL IN VlTRO].

OBJECTIVE: To investigate the feasibility to culture rabbit annulus fibrosus cells on the KLD-12 polypeptide nanofiber gel so as to search for the seed cells and the scaffolds for tissue engineering. METHODS: The rabbit annulus fibrosus cells were isolated with pancreatin and cultured; the cells at passage 3 were seeded on the KLD-12 polypeptide nanofiber gel to prepare the KLD-12 polypeptide/annulus fibrosus cells gel. The cell morphology change was observed by inverted microscope. The cell counting kit 8 (CCK-8) was used to detect the cell proliferation, and Calcein-AM/propidium iodide (PI) fluorescent staining to observe the cell vitality. The alcian blue method was used to measure the glycosaminoglycan (GAG) content, immunofluorescence technique to observe the collagen type II level, and real-time fluorescence quantitative PCR (RT-qPCR) to measure the mRNA expressions of Aggrecan and collagen type II. RESULTS: The cells on the scaffolds grew well, showing round shape on the scaffolds and spindle or fusiform shape at the edge of the scaffold. The cell proliferation exhibited increasing trend with time, and it was significantly higher at 14 days than the other time points (P < 0.05), and on KLD-12 polypeptide nanofiber gel than on blank gel (P < 0.05). The ratios of living cells were 89.32% ± 8.58% at 5 days and 97.81% ± 1.09% at 14 days, showing no significant difference (t = - 1.962, P = 0.097). The GAG content gradually increased with culture time, reached the peak at 8 days, and then gradually decreased; the GAG content at 5, 8, and 11 days was significantly higher than that at 2 and 14 days (P < 0.05). The level of collagen type II was normal. The mRNA expressions of collagen type II and Aggrecan could be measured at 5 and 14 days; the relative expression levels of collagen type II and Aggrecan mRNA were significantly higher at 14 days than 5 days (P < 0.05). CONCLUSION: The rabbit annulus fibrosus cells on KLD-12 polypeptide nanofiber gel are able to grow well and to produce extracellular matrix, so KLD-12 polypeptide nanofiber gel has the potential to serve as a scaffold for the treatment of intervertebral disc degeneration.

[EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL].

OBJECTIVE: To investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs). METHODS: Bone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. RESULTS: Under the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P>0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P<0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000). CONCLUSIONS: BMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.