Unique cytological behavior of MC3T3-E1 osteoblasts on H_2O_2-modified C/C composites in vitro

MC3T3-E1 osteoblasts were cultured on H2O2- modified and unmodified carbon/carbon （H-C/C and C/C） composites for one week in order to evaluate differences in cell adhesion, spreading, and proliferation. The results indicated a certain degree of enhancement in the cell adhesion capability of osteoblasts cultured on H-C/C samples. Cellu- lar morphologies after cell adhesion were observed via scan- ning electron microscopy （SEM）, which showed that the cells adhered more closely and spread more widely on the H-C/C sample surface. However, no cell appeared in several mul- tiple and continuous types of minor pores on both the C/C and H-C/C surfaces. In addition, two unique situations were observed on the H-C/C samples： an outline change of the osteoblasts was observed when the cells spread across some minor pores, and the cells entered and adhered well in some larger pores.

Effects of Quenching Temperature and Time on Pore Diameter of Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) Porous Scaffolds and MC3T3-E1 Osteoblast Response to the Scaffolds

Poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （PHBHHx） scaffolds were prepared by thermally inducing phase separation （TIPS） for bone reconstruction. Scanning electron microscopy and porosity measurements were used to analyze the structure and properties of the scaffolds. The pore diameter of the scaffolds could be easily controlled by changing the quenching temperature and time. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 osteoprogenitor cells seeded on the scaffolds. Cultures grown in the presence of a source of phosphate ions showed the formation of a mineralized extracellular matrix. The results indicate that PHBHHx scaffolds prepared using TIPS are a promising candidate for bone reconstruction.

Osteogenic effects of antihypertensive drug benidipine on mouse MC3T3-E1 cells in vitro

Hypertension is a prevalent systemic disease in the elderly,who can suffer from several pathological skeletal conditions simultaneously,including osteoporosis.Benidipine(BD),which is widely used to treat hypertension,has been proved to have a beneficial effect on bone metabolism.In order to confirm the osteogenic effects of BD,we investigated its osteogenic function using mouse MC3T3-E1 preosteoblast cells in vitro.The proliferative ability of MC3T3-E1 cells was significantly associated with the concentration of BD,as measured by methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay and cell cycle assay.With BD treatment,the osteogenic differentiation and maturation of MC3T3-E1 cells were increased,as established by the alkaline phosphatase(ALP)activity test,matrix mineralized nodules formation,osteogenic genetic test,and protein expression analyses.Moreover,our data showed that the BMP2/Smad pathway could be the partial mechanism for the promotion of osteogenesis by BD,while BD might suppress the possible function of osteoclasts through the OPG/RANKL/RANK(receptor activator of nuclear factor-κB(NF-κB))pathway.The hypothesis that BD bears a considerable potential in further research on its dual therapeutic effect on hypertensive patients with poor skeletal conditions was proved within the limitations of the present study.

Comparative evaluation of the physicochemical properties of nano-hydroxyapatite/collagen and natural bone ceramic/collagen scaffolds and their osteogenesis-promoting effect on MC3T3-E1 cells

The use of various types of calcium phosphate has been reported in the preparation of repairing materials for bone defects.However,the physicochemical and biological properties among them might be vastly different.In this study,we prepared two types of calcium phosphates,nano-hydroxyapatite(nHA)and natural bone ceramic(NBC),into 3D scaffolds by mixing with type I collagen(CoL),resulting in the nHA/CoL and NBC/CoL scaffolds.We then evaluated and compared the physicochemical and biological properties of these two calcium phosphates and their composite scaffold with CoL.Scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS),Fourier-transform infrared spectroscopy(FTIR),X-ray diffraction spectroscopy(XRD)and compressive tests were used to,respectively,characterize the morphology,composition,distribution and the effect of nHA and NBC to collagen.Next,we examined the biological properties of the scaffolds using cytotoxicity testing,flow cytometry,immunofluorescence staining,biocompatibility testing,CCK-8 assays and RT-PCR.The results reflected that the Ca2t released from nHA and NBC could bind chemically with collagen and affect its physicochemical properties,including the infrared absorption spectrum and compression modulus,among others.Furthermore,the two kinds of scaffolds could promote the expression of osteo-relative genes,but showed different gene induction properties.In short,NBC/CoL could promote the expression of early osteogenic genes,while nHA/CoL could upregulate late osteogenic genes.Conclusively,these two composite scaffolds could provide MC3T3-E1 cells with a biomimetic surface for adhesion,proliferation and the formation of mineralized extracellular matrices.Moreover,nHA/CoL and NBC/CoL had different effects on the period and extent ofMC3T3-E1 cell mineralization.

Aqueous extract of freeze-dried Protaetia brevitarsis larvae promotes osteogenesis by activating β-catenin signaling

Objective:To investigate the effect of an aqueous extract of Protaetia brevitarsis(AEPB)on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae.Methods:Flow cytometric analysis was used to measure the cytotoxicy.Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate.Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells.In addition,vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression.Results:AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2,osterix,and alkaline phosphatase,along with elevated levels of mineralization in MC3T3-E1 cells.Moreover,AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes.FH535,an inhibitor of Wnt/β-catenin,suppressed AEPB-induced osteogenic gene expression and vertebral formation,indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway.Conclusions:AEPB stimulates osteoblast differentiation and bone formation by activatingβ-catenin.Therefore,AEPB is a promising material that induces osteogenesis,and is useful for the treatment of bone resorption diseases.