Establishment Of Antituberculosis Bone Tissue-engineering Complex In Vitro

Objective:To establish antituberculosis bone tissue-engineering complex in vitro based on rabbit adipose-derived stem cells(rADSCs), rifapentine polylactic-acid microspheres and partial decalcified allograft bone scaffold.Methods:The adipose tissue was obtained from inguinal fat pads of the rabbit, and digested with type I collagenase. The characteristics of rADSCs were certified by the results of cell cycle, anti-CD44antibody and multipotential differentiation. The rADSCs were randomly divided into four group according to drug concentration of rifapentine, including group0μg/ml, group20μg/ml, group40μg/ml and group60μg/ml. The cell growth curves, cell apoptosis and alkaline phosphatase(ALP)acti vity were used for determining the impact of rifapentine on rADSCs. Rifapentine p olylactic acid microspheres were prepared using a double-emulsion solvent-extractio n technique. The appearance of the microspheres was observed by scanning electro n microscope(SEM)and the average diameter was measured. The drug-carried rate a nd envelopment rate were determined by UV spectrophotometry. The two-dimension al(2D)complex of rADSCs-rifapentine microspheres was established and the osteoge nic differentiation potential was also observed. The three-dimensional(3D)complex of rADSCs-rifapentine microspheres-bone scaffold was established, the adhesion rate was calculated. The appearance of3D complex was observed by SEM and hemato xylin-eosin(HE)staining. The release characteristics were studied in vitro. The releas e quantity was determined every three days, and calculated the mean day release r ate, the total release rate.Results:The rADSCs grew spindle-shaped in vitro, G0/G1cycle was around(80.3±3.2)%, expression of CD44was positive. ALP and Alizarin red staining demonstrated positive expression of ALP and formation of mineralized nods, the results of Oil red O staining and Alcian blue staining were also positive. The results of cell growth curves showed the proliferation in group60μg/ml were lower than that in other groups from day4to12. The cell apoptosis rate was higher in group60μg/ml than that in other groups(P<0.01). The expression of ALP was fewer on day7and14in group60μg/ml than that in other groups(P<0.05). The critical concentration was around40μg/ml. The average diameter of microspheres was(26.4±3.6)μm, drug-carried rate was(40.89±0.63)%, envelopment rate was(75.24±0.18)%. Alizarin red staining was positive in2D complex after osteogenic induction. The3D complex had good compatibility according to the appearance of SEM and HE staining, and the adhesion rate was(96.94±1.24)%. The total release rate on day3,6and39was8.54%、20.12%and93.66%respectively. The drug concentration at each time was lower than critical concentration, but much higher than minimum inhibition concentration.Conclusion:The antituberculosis bone tissue-engineering complex has the characteristic of slowly rifapentine releasing, and the released concentration has no impact on cell proliferation or osteogenic activity.