Objectives To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vitro. Methods Cancellous bone were extracted from rabbit femoral head and cut into 1-mm-thick and 8-mm-diameter slices under sterile conditions. HE staining and scanning electron microscopy were employed to identify the histomorphology of the model after being cultured with a new dynamic load and circulating perfusion bioreactor system for 0, 3, 5, and 7 days, respectively. We built a three-dimensional model using microCT and analyzed the loading effects using finite element analysis. The model was subjected to mechanical load of 1000, 2000, 3000, and 4000 με respectively for 30 minutes per day. After 5 days of continuous stimuli, the activities of alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRAP) were detected. Apoptosis was analyzed by DNA ladder detection and caspase-3/8/9 activity detection. Results After being cultured for 3, 5, and 7 days, the bone explant model grew well. HE staining showed the apparent nucleus in cells at the each indicated time, and electron microscope revealed the living cells in the bone tissue. The activities of AKP and TRAP in the bone explant model under mechanical load of 3000 and 4000 με were significantly lower than those in the unstressed bone tissues (all P<0.05). DNA ladders were seen in the bone tissue under 3000 and 4000 με mechanical load. Moreover, there was significant enhancement in the activities of caspase-3/8/9 in the mechanical stress group of 3000 and 4000 με (all P<0.05). Conclusions The cancellous bone explant model extracted from the rabbit femoral head could be alive at least for 7 days in the dynamic load and circulating perfusion bioreactor system, however, pathological mechanical load could affect the bone tissue growth by apoptosis in vitro. The differentiation of osteoblasts and osteoclasts might be inhibited after the model is stimulated by mechanical load of 3000 and 4000 με.

Key words: bone tissue engineering, mechanical load, bone explant culture, apoptosis, osteoblast, osteoclast

Funding:

△Supported by grants from the National Natural Science Foundation Key Project of China (10832012) and the National Natural Science Foundation of China (31370942 and 11072266).