Abstract
BackgroundWhen 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.MethodsUnder 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 μϵ, 2000 μϵ, 3000 μϵ and 4000 μϵ 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.ResultsThe 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 μϵ and 2000 μϵ 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.ConclusionOur 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.
Highlights
Natural bone formation in vivo is a complex process in which involved contribution of multiple cell types, physical and biological environment [1]
Tetracycline hydrochloride (#0422) was purchased from Amresco (Amresco, USA), Calcein (#0875) was purchased from Sigma (Sigma, USA), Mouse Anti-Collagen I antibody [COL-1] (#ab90395) was purchased from Abcam (HK) Ltd., Rabbit Osteoprotegerin (OPG) ELISA Kit and Rabbit Bone Morphogenetic Protein-2 (Bmp-2) ELISA Kit were purchased from Cusabio Biotech Co., LTD, USA
By Micro-CT scanning, a distinct structure and rich lacuna of trabecular bone could be observed in bone explant models, it is suitable for cultivation in vitro by circulating flow (Figure 2A); And a 3D model reconstructed by Mimics and scaffold 3D model are shown in Figure 2B, C, D and E
Summary
Natural bone formation in vivo is a complex process in which involved contribution of multiple cell types, physical and biological environment [1]. 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. 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
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