ABSTRACT
Aims: To analyze the effect of rhBMP-2 and Chitosan in differentiation of Periodontal Ligament Stem Cells (PDLC) into an osteoblastic lineage. Study Design: This study was designed as in vitro study and osteogenic biomarkers were determined in the culture supernatant. Place and Duration of Study: Laboratory of Oral Biology Faculty of Dentistry Universitas Indonesia. Jakarta 10430 Indonesia, January – September 2016. Methodology: Human periodontal ligament stem cells (PDLC) were isolated from the root of vital teeth, followed by identification of stem cells by antibody anti STRO-1. Chitosan was used at the concentration of 0.15%. The culture cells were divided into four groups as follow, the control group (PDLC) and treatment groups with recombinant human Bone Morphogenic protein 2 (rhBMP-2), the combination chitosan-rhBMP-2 and chitosan only. The levels of alkaline phosphatase (ALP) was determined by colorimetry and osteocalcin and collagen type I were measured using ELISA. Results: The results showed that levels of ALP tended to increase is in all groups. At day 14, the highest levels of ALP was in chitosan treated group. The concentration of collagen type 1 managed to raise is in all groups on days 14, and the highest levels Collagen type 1 occurred in RH BMP-2 and chitosan treated cells, after that decrease in all groups until day 21(p < 0.05). Osteocalcin concentration tended to increase is in all groups, and at days 21, the highest levels in with rhBMP-2 + chitosan. Conclusion: The rhBMP-2, chitosan, and its combination induce differentiation of periodontal ligament stem cells into the osteoblastic lineage.
ABSTRACT
Background: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine. Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp). E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene. Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene. Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine.
Subject(s)
Contraception , Family Planning Services , MenABSTRACT
Background: This research was done to study the influence of Acalypha indica Linn root extract towards relative cell viability and proliferation as parameters of neurogenesis in post-hypoxic hippocampal tissue culture. Methods: Experimental in vitro study using 24 primary neuronal cell cultures obtained from adult Sprague Dawley rat exposed to hypoxia with 5% O2/5% CO2/N2 balance gas for 24 hours. Post-hypoxia, Acalypha indica Linn root extract was added at doses of 10, 15, and 20 mg/mL to 3 treatment groups. No treatment was given to the control group. Each group consists of 6 samples. After 90 hours of incubation, relative cell viability was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) examination, and cell proliferation was measured by using 5-bromo2’-deoxy-uridine (BrdU) for cell proliferation. Data was analyzed using one way ANOVA parametric tests, then further analyzed with post-hoc analysis. Results: The relative cell viability of rat hippocampal tissue culture treated with Acalypha indica Linn root extract with dose of 10, 15, and 20 mg/mL was significantly higher than control (176.95%, 220.62%, and 386.02% vs. 100%). Cell proliferation of rat hippocampal tissue culture treated with Acalypha indica Linn root extract with dose of 10, 15, and 20 mg/mL was significantly higher than control (0.132, 0.117, 0.114 vs 0.096). Conclusion: Acalypha indica Linn root extract with doses of 10, 15, and 20 mg/mL can increase relative cell viability and proliferation in post-hypoxic hippocampal tissue culture.