| Background and objective:Osteoporosis (OP) is one of metabolic diseases characterized by the decline inbone mass and bone micro-structural damage, manifested as the increase in bonebrittleness, and thus increased the risk of fracture. With the increased elderlypopulation in China and the entire world, the incidence of OP is in an uptrend, whichwas considered as one of the three diseases in the elderly by the World HealthOrganization (WHO). Pathogenesis of OP is due to an imbalance between boneresorption and bone formation. Mechanical strain and estrogen are important factorsthat affect bone remodeling. In our study we investigated the effect of a combinationof mechanical strain and α-ZAL on mechanism of bone remodeling. Tissueengineered bone in response to α-ZAL and mechanical strain was established toinvestigate biological response to OBs. The study has important theoretical andclinical values to OP and other related bone diseases, and it also provides a theoreticalbasis for war injuries.Methods:1. MC3T3-E1cells were treated with different concentrations of α-ZAL for differenttime respectively in vitro. Cell proliferation was determined by MTT method, andosteogenic differentiation was assessed by ALP activity assay. mRNA levels of ALP,OPG and RANKL were semiquantified by RT-PCR.2. MC3T3-E1cells were subjected to mechanical strain of1000or2500με at0.5Hz(generated by a four-point bending system), α-ZAL (10-6,10-8,10-10M) or acombination. Loading mode was1h/day. After72h, the capacity of cell proliferationwas assessed by flow cytometry; Alkaline phosphatase (ALP) activity was measuredby fluorometric detection kit. mRNA expressions of ALP, runt-related transcriptionalfactor2(Runx2), OPG and RANKL were determined by real time RT-PCR. Proteinexpression of Runx2, OPG and RANKL was determined by Western blotting.3. Chitosan was modified by hyaluronate acid. The structure was observed by theDifferential Scanning Calorimetry (DSC) and the Fourier transformed infraredspectroscopy (FT-IR). Three composites of HA-CS/Col/nHAP according to different ratio of chitosan and collagen solution (1:2,1:1and2:1) were prepared. Thecomposite scaffolds were co-cultured with MC3T3-E1cells, and the proliferation andcell growth curves were measured by CCK-8method.4.Dynamic strain and circulating-perfusion bioreactor was applied to3D-culturedcell-scaffold constructs, simulating mechanical microenvironment in bone tissue aswell as solving mass transfer issues. CCK-8method was used to investigate cellproliferation with three different flow rates (5,10,20ml/min) combined with3500μεof compressive strain (1Hz,2h/d). The morphology of cell-scaffold constructs wasexamined by SEM and Hematoxylin-eosin staining (HE staining). Markers ofosteogenesis were assessed by immunohistochemistry staining and Western blotting.Mineralized nodules were investigated by Von Kossa staining.5.Cell-scaffold constructs were dynamically cultured in culture medium supplementedwith different concentrations of α-ZAL(10-6,10-8,10-10M).Cell proliferation wasdetermined by CCK-8method, and osteogenic differentiation was assessed by ALPactivity assay. Protein levels of OPG and RANKL were semiquantified byWestern-blotting.Results:1. α-ZAL at the concentration of10-6to10-12M inhibited the proliferation ofMC3T3-E1cells (P<0.05), however, enhanced ALP activity (P<0.05) at differentdetected time. α-ZAL also increased ratio of OPG to RANKL (P<0.05).2. Mechanical strain promoted proliferation and osteogenic differentiation ofMC3T3-E1cells; Runx2protein expression, but not mRNA levels, was up-regulated;The OPG/RANKL ratio was also increased. Although combination of α-ZAL andstrain inhibited cell proliferation, high concentration of α-ZAL (10-6M) combinedwith high magnitude of strain (2500με) significantly enhanced ALP activity. α-ZALwith high magnitude strain increased RUNX2protein expression, but lowered theOPG/RANKL ratio. The OPG/RANKL ratio was significantly increased in responseto α-ZALwith low magnitude strain (1000με).3. Hyaluronic acid and chitosan were crosslinked with amide linkage. Pore size wason the range from50μm to250μm. Porosity was increased with the increasedcollagen content. Increased collagen content was beneficial for cell adhesion andproliferation in the primary phase of3D-culture. However, from day10, no significantdifferences were determined among the three samples.4. Perfusion at10ml/min flow rate increased the proliferation of MC3T3-E1cells;Cells adhered on the surface of scaffolds in fusiform or irregular shapes after5daysof culture, extending pseudopodia, and secreting small amounts of fuzz orextracellular matrix (ECM). On the first day of dynamic culture, both COLI and OPN have high expression while the expression of OCN is very low. Up to day7, stainingfor all three antibodies was seen in clusters around cellular nuclei or near cellularmembrane. The expression of COLI was up-regulated on day7, while the expressionof OPN was significantly down-regulated on day12. The expression of OCN did notchange during12days of in vitro culture.5. α-ZAL at high concentration inhibited proliferation but increased ALP activity on3D dynamic cultures of MC3T3-E1cells. It also increases OPG/RANKL ratio byproducing more content of OPG.Conclusions:1. The suitable concentration of α-ZAL inhibited cell proliferation, but promotedosteogenic differentiation of MC3T3-E1cells and inhibited the formation ofosteoclast activity. Therefore, it would be promising candidate for the treatment ofosteoporosis.2. Mechanical strain was effective in inducing osteogenic differentiation, andincreasing bone formation. α-ZAL combined with low magnitude strain, but not highmagnitude strain, provided benefits for regulating activity of OCs.3. HA-CS/Col/nHAP composites could improve the adhesion and proliferation of OBs.The ratio of chitosan to collagen volume at1:1was optimal.4. Dynamic culture at10ml/min flow rate combined with3500με of compressivestrain promotes proliferation of3D-cultured MC3T3-E1cells. OBs maintainosteogenesis in3D dynamically cultured cell-scaffold constructs.5. In response to α-ZAL in3D-dynamic culture the cell-scaffold constructs showenhanced or maintained osteogenesis, and increased ratio of OPG to RANKL.6. Application of perfusion and dynamic strain to cells-scaffold constructs treated withα-ZAL represents a promising approach in the studies of osteogenesis stimulation ofbone tissue engineering.7. OBs show different biological response to2D-culture and3D-culture. Engineeredmodel represents excellent physical environment similar to in vivo culture, andprovides technology platform for engineered tissue of bone.
|
PDF Full Text Request