RESUMO
BACKGROUND: When studying and designing an artificial bone in vitro with similar features and functionality of natural bone by tissue engineering technology, the culturing environment, especially the mechanical environment is supposed to be an important factor, because a suitable mechanical environment in vitro may improve the adaptability of the planted-in tissue engineering bone in the body. Unfortunately, up to now, the relationship between mechanical stimuli and natural bone growth has not yet been precisely determined, and it is so imperative for a prior study on effect of mechanical loading on growth of the natural bone cultured in vitro. METHODS: Under sterile conditions, explant models of rabbit cancellous bone with 3 mm in thickness and 8 mm in diameter were prepared and cultured in a dynamic loading and circulating perfusion bioreactor system. By Micro-CT scanning, a 3D model for finite element (FEM) analysis was achieved. According to the results of FEM analysis and physiological load bearing capacity of the natural bone, these models were firstly subjected to mechanical load with 1Hz frequency causing average apparent strain of 1000 µÎµ, 2000 µÎµ, 3000 µÎµ and 4000 µÎµ respectively for 30 min every day, activities of alkaline phosphatase (AKP) were detected on the 5th and the 14th loading day and on the 14th and the 21st day, mechanical properties, tissue mineral density (TMD) of the bone explant models were investigated and Von-kossa staining and fluorescence double labeling assays were conducted to evaluate whether there were fresh osteoid in the bone explant models. In addition, Western blot, Elisa and Real-time PCR were employed to analyze expression of Collagen-I (COL-1), bone morphogenetic protein-2 (BMP-2) and osteoprotegerin (OPG) protein and RNA. RESULTS: The explant models of rabbit cancellous bone prepared under sterile conditions grew well in the bioreactor system. With the increasing culturing time and load levels, bone explant models in groups with 1000 µÎµ and 2000 µÎµ average apparent strain experienced improving mechanical properties and TMD (P<0.05), and results of Von-kossa staining and fluorescence double labeling also showed apparent fresh osteoid formation. Under the same loading conditions, a up-regulations in protein and RNA of COL-1, BMP-2 and OPG were detected, especially, relative genes notably expressed after 21 days. CONCLUSION: Our study demonstrated that mechanical load could improve function and activity of osteoblasts in explant models of cancellous bone. Through regulations of COL-1, OPG and BMP-2 secreted by osteoblasts, the mechanical load could improve the tissue structural density and stiffness due to formation of fresh osteoid.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/fisiologia , Osteogênese/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Colágeno/genética , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática , Análise de Elementos Finitos , Regulação da Expressão Gênica , Modelos Biológicos , Osteoblastos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Engenharia Tecidual/métodos , Suporte de CargaRESUMO
<p><b>OBJECTIVES</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>
