Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.     

抗菌钛合金Ti6Al4V-6Cu表面构建成骨细胞膜片的实验研究

目的:探讨大鼠颌骨成骨细胞在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片的可行性。方法:体外培养大鼠颌骨成骨细胞,采用富含维生素C培养基在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片（细胞膜片组）,并以单纯培养基作对照（对照组）,检测膜片形成过程中碱性磷酸酶（ALP）和成骨相关基因ALP、I型胶原（Col-1）、骨形成蛋白2（BMP-2）的表达情况。结果:采用富含维生素C的培养基连续培养可在抗菌钛合金Ti6Al4V-6Cu表面成功构建成骨细胞膜片,该细胞膜片由多层细胞构成,富含胞外基质。相对于对照组,细胞膜片组的膜片形成过程中成骨细胞ALP活性及成骨相关基因ALP、Col-1、BMP-2的表达均显著增高。结论:在抗菌钛合金Ti6Al4V-6Cu表面可成功构建成骨细胞膜片,有望与Ti6Al4V-6Cu联合应用于引导性骨再生术。     

Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.     

雌激素对大鼠胚胎成骨细胞增殖及胶原合成的影响

目的 研究雌激素对大鼠胚胎成骨细胞增殖及I型胶原代谢的影响。方法 分离大鼠胚胎头盖骨成骨细胞，分别给予不同浓度雌激素培养；观察细胞增殖情况和ALP活性变化；苦味酸天狼星红及I型胶原免疫组织化学染色，从蛋白质水平观察成骨细胞中I型胶原的变化；地高辛标记的I型胶原基因探针进行原位杂交，从mBNA水平观察I型胶原的变化。结果 雌激素可降低大鼠胚胎成骨细胞增殖水平而使ALP活性以及I型胶原蛋白质和mRNA水平均升高。结论 雌激素可促进成骨细胞分化，而抑制增殖。     

Differentiation of osteoblasts in three-dimensional culture in processed cancellous bone matrix: quantitative analysis of gene expression based on real-time reverse transcription-polymerase chain reaction

Processed bovine cancellous bone (PBCB) is an attractive material for tissue engineering of bone. It is biocompatible, osteoconductive, nonimmunogenic, and porous and its biomechanical properties are close to those of native bone. In this study, differentiation of primary rat osteoblasts (rOBs) incubated on PBCB was investigated in vitro. rOBs were isolated and expanded in two-dimensional culture. Expanded rOBs were seeded into PBCB disks and cultured either in basal medium (BM) or differentiation medium (DM) containing ascorbic acid, beta-glycerol phosphate, and dexamethasone. Alkaline phosphatase (ALP) activity and RNA expression of ALP, bone sialoprotein (BSP), collagen type I (COL1), osteocalcin (OC), and osteopontin (OPN) were assessed by chemiluminescence assay and quantitative real-time RT-PCR over 14 days. Histologic analysis was performed on day 14. ALP increased over the observation period independent of stimulation. OPN and BSP expression was significantly higher in the DM group whereas COL1 and OC expression was significantly higher in the BM group. Matrix calcification was detectable only in the DM group by von Kossa stain. The observed expression patterns suggest a physiological response of rOBs to the differentiation stimulus. PBCB is a suitable matrix for in vitro differentiation of osteoblasts. Cell-seeded PBCB is a potential osteogenic construct for in vivo application.     

Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells

Recently, calcium sulfate dihydrate has been demonstrated as safe biodegradable osteoconductive bone void filler. However, its exact mechanism of action on bone cells is yet unknown. In this study, the influence of gypsum on gene expression and proliferation of MC3T3-E1 mouse pre-osteoblastic cells was investigated. Cells were cultured on gypsum disc, slice, polymethylmethacrylate (PMMA), or plastic culture plate for 15 days. Cell viability, alkaline phosphatase (ALP) activity and expression profile of 15 genes involved in bone metabolism were measured in cultures. Cell proliferation on gypsum was increased by almost 2-fold, while an inhibitory effect of PMMA on proliferation rate of osteoblasts was noted. Cells cultured on gypsum disc surface exhibited an increased ALP activity and markedly different gene expression profile. Quantitative real-time PCR data indicated the expression of genes that might provide a basis for an osteoinductive potential. MC3T3-E1 cells expressed genes typical of bone fracture healing like type II collagen and fibronectin 1. These effects might be related to the calcium content of gypsum and mediated likely via SMAD3. Our results suggest that gypsum can support new bone formation by its calcium content and modulatory effect on gene expression profile of bone cells.     

The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue

Mesenchymal stem cells (MSCs) can give rise to osteoblasts and have therefore been suggested as a cell source for bone engineering. Here we hypothesized that MSC osteoblastic differentiation and maturation can be supported by three-dimensional cultures in collagen hydrogels (hydrogel culture) to ultimately give rise to mechanically robust bone-like tissue. We first compared the osteoblastic differentiation efficiency of MSCs using osteoinductive supplements (β-glycerophosphate, vitamin C, and dexamethasone) in a hydrogel culture and in a two-dimensional culture (2D culture) by assessing surrogate parameters for osteoblastic differentiation, including osteocalcin (OC) secretion and calcium (Ca) deposition. We next constructed ring-shaped bone-like tissues using MSCs in the hydrogel cultures, and assessed their mechanical (strain-strain analysis), biochemical/molecular (OC secretion, Ca deposition, and Runx2/osterix mRNA levels), and morphological (von Kossa staining) properties. OC secretions and Ca depositions were significantly higher in the hydrogel cultures than those in the 2D cultures, suggesting better osteoblastic differentiation and maturation in the hydrogel cultures. Collagen hydrogel-based ring-shaped bone-like tissues conditioned with osteoinductive supplements developed enhanced biomechanical properties, including high tissue stiffness and ultimate burst strength, superior molecular/biochemical properties, and morphological signs typically found in mineralized bone. These results may be exploited not only to generate bioartificial bone, but also to elucidate the basic mechanisms of bone physiology.     

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.     

Effects of Remifentanil Preconditioning on Osteoblasts under Hypoxia-Reoxygenation Condition

Background: Ischemia-reperfusion of bone occurs in a variety of clinical conditions, such as orthopedic arthroplasty, plastic gnathoplasty, spinal surgery, and amputation. Usually, cellular models of hypoxia-reoxygenation reflect in vivo models of ischemia-reperfusion. With respect to hypoxia-reoxygenation conditions, the effects of remifentanil on osteogenesis have received little attention. Therefore, we investigated the effects of remifentanil on the proliferation and differentiation of osteoblasts during hypoxic-reoxygenation.Methods: After remifentanil (0.1, 1 ng/mL) preconditioning for 2 hours, human osteoblasts were cultured under 1% oxygen tension for 24 hours. Thereafter, the cells were reoxygenated for 12 hours at 37 °C. The naloxone groups were treated with naloxone for 30 minutes before remifentanil treatment. We measured cell viability via MTT assay. Osteoblast maturation was determined by assay of bone nodular mineralization. Quantitative PCR and western blot methods were used to determine BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-β1, HIF-1α, and RUNX2 expression levels.Results: Osteoblast viability and bone nodular mineralization by osteoblasts is recovered by remifentanil preconditioning from hypoxia-reoxygenation insult. During hypoxic-reoxygenation condition, remifentanil preconditioning induced the expression of BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-β1, HIF-1α, and RUNX2 in osteoblasts.Conclusions: Under hypoxia-reoxygenation conditions, remifentanil preconditioning enhanced the cell viability and maturation of osteoblasts, and stimulated the expression of proteins associated with osteoblast proliferation and differentiation of the osteoblast. Our results suggest that remifentanil may help in the treatment of bone stress injuries.     

辐射对体外培养的小鼠成骨细胞Ⅰ型胶原mRNA表达的影响

目的 肿瘤放射治疗诱发骨组织损伤,辐射后成骨细胞Ⅰ型胶原的基因表达分析揭示辐射对早期和晚期的成骨细胞功能的影响.方法 在体外诱导骨髓基质细胞生成成骨细胞,对其特性进行确定.用聚合酶链式反应(PCR)方法分析了1～4 Gy照射的早期和晚期成骨细胞的Ⅰ型胶原表达.结果与对照组相比,经1～3 Gy剂量照射后的早期成骨细胞Ⅰ型...     

Effects of coumestrol on neonatal and adult mice osteoblasts activities

Estrogen replacement therapy has been shown to reduce postmenopausal osteoporosis. In the present study, we examined the effects of the phytoestrogen coumestrol on neonatal and adult osteoblasts metabolism. Two different sources of osteoblast cells (neonatal mice calvaria and adult mice long bone) cultures were used in this study. The effects of coumestrol on the cellular activities were analyzed by the mitochondrial tetrazolium (MTT) assay, secretion of alkaline phosphatase (ALP), intracellular calcium content (Ca), and the gene expression of bone matrix protein, estrogen receptors (ER-alpha, ER-beta), and osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL). The results showed that the proliferation of neonatal mice osteoblast cells was enhanced by treatment of coumestrol. In the presence of 10(-9)M coumestrol, the osteoblast proliferation attained 139.5% of the control and that the coumestrol can increase the intracellular calcium contents. Type I collagen gene expression was upregulated 167% at the 1st day's culture; ALP gene expression was upregulated 360% at the 7th day's culture; while the osteocalcin gene expression was upregulated 222% at the 14th day's culture. When adult mice osteoblasts were cultured in the presence of 10(-9)M coumestrol, the osteoblasts population increased significantly earlier and attained its maximal effect at the 21st day's culture with 207.4% of control group. The content of ER-beta and osteoprotegerin secretion by neonatal mice control cells gradually increased during osteoblasts differentiation, whereas the ER-alpha and OPGL content were decreased in this study. The cellular responses to the estradiol and counmestrol were quite different in the osteoblasts derived from different age.     

Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.     

纳米淡水珍珠粉对成骨相关基因表达的影响

背景:珍珠中高含量的钙离子可以促进钙盐沉积,抑制破骨细胞的骨吸收活性,促进骨再生,且其含有的水溶性蛋白具有骨诱导作用,可促进成骨细胞的分化.目的:观察纳米淡水珍珠粉对成骨细胞成骨相关基因表达的影响.方法:取第3代小鼠成骨细胞MC3T3-E1细胞,分别与纳米淡水珍珠粉(实验组)、纳米羟基磷灰石(对照组)共培养,以单独培养...     

In vitro analysis of anionic collagen scaffolds for bone repair

Collagen has been extensively described as a beneficial material in bone tissue engineering due to its biocompatibility, biodegradability, low antigenicity, and high tensile strength. However, collagen scaffolds in their pure form have some drawbacks and improvements in the physical, chemical, and biologic properties of collagen are necessary to overcome those inadequacies. Recently, the selective hydrolysis of carboxyamides of asparagine and glutamine residues of collagen has been employed to increase the number of negative sites and enhance the piezoelectric properties of collagen. Anionic collagen scaffolds were prepared by use of a hydrolysis treatment for either 24 h [bovine pericardium (BP 24)] or 48 h (BP 48). Bovine osteoblasts were cultured on them and on native matrices to understand the cellular interactions responsible for the good osteoconductivity and biocompatibility reported with in vivo tests. Based on the data obtained on cell adhesion, alkaline phosphatase (ALP) and extracellular matrix macromolecule production, and cellular proliferation through histological analysis, we may conclude that the materials tested reveal sufficient biocompatibility level for bone repair. Further, the evidence of some connection between ALP activity and the mineralization process should be emphasized. BP 48 presented the most promising results stimulating in vitro mineralization, ALP production, and possible osteoblast differentiation.     

密骨打老儿丸含药血清对共育体系中成骨细胞和破骨细胞功能的影响

 目的： 观察密骨打老儿丸（Migu-Dalaoer pill,MDP）含药血清在成骨-破骨细胞共同培养体系中对成骨细胞（osteoblasts,OB）增殖和破骨细胞（osteoclasts,OC）骨吸收功能的影响。方法： 利用分段酶消化法从胎鼠颅骨中分离出OB,取1日龄SD大鼠四肢股骨和胫骨分离培养OC,建立培养上清相通但2种细胞间互不接触的成骨-破骨细胞共育模型。实验分为不同浓度（低、中、高）的MDP含药血清组和对照组进行比较,以细胞增殖（MTT 法）和碱性磷酸酶（alkaline phosphatase,ALP）活性代表OB的成骨活性,以抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase,TRAP）活性和骨吸收陷窝数目代表OC的破骨能力进行测定。结果： 与对照组相比,中、高浓度MDP含药血清在成骨-破骨细胞共同培养体系中6和7 d 能显著提高OB数目和 ALP 活性（P<0.01）。与对照组相比,中、高浓度MDP含药血清在成骨-破骨细胞共同培养体系中6和7 d 能显著降低OC骨吸收陷窝的数目和分泌 TRAP 的活性（P<0.01）。结论： 密骨打老儿丸含药血清在共育体系中能够促进OB增殖和骨形成,同时抑制OC骨吸收功能。     

Osteointegration of titanium implant is sensitive to specific nanostructure morphology

An important aspect of orthopedic implant integration is the enhancement of functional activity of osteoblasts at the tissue-implant interface without any fibrous tissue intervention. Nanostructured implant surfaces are known to enhance osteoblast activity. Previously, we have reported a simple hydrothermal method for the fabrication of non-periodic nanostructures (nanoscaffold, nanoleaves and nanoneedles) on titanium implants showing good biocompatibility and a distinct osteoblast response in vitro in terms of osteoblast adhesion to the surface. In the present work, these nanostructures have been evaluated for their detailed in vitro cellular response as well as in vivo osteointegration. Our studies showed that a specific surface nanomorphology, viz. nanoleaves, which is a network of vertically aligned, non-periodic, leaf-like structures with thickness in the nanoscale, provided a distinct increase in osteoblast cell proliferation, alkaline phosphatase (ALP) activity and collagen synthesis compared to several other types of nanomorphology, such as nanotubes, nanoscaffold and nanoneedles (rods). Gene expression analysis of ALP, osteocalcin, collagen, decorin and Runx2 showed ~20- to 40-fold up-regulation on the leaf-like topography. Cytoskeletal arrangement studies on this substrate again revealed a unique response with favorable intracellular protein expressions of vinculin, FAK and src. In vivo osteointegration study over 12 weeks on rat model (Sprague-Dawley) showed early-stage bone formation (60% bone contact by week 2 and ~85% by week 8, p<0.01) in the leaf-like nanopattern, without any inflammatory cytokine production.     

Sesamin Stimulates Osteoblast Differentiation Through p38 and ERK1/2 MAPK Signaling Pathways

ABSTRACT: BACKGROUND: Osteoporosisis a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function. METHOD: Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 hour of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 hour of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stain with Alizarin Red S staining. MAPK signaling pathway activation was observed by using western blotting. RESULTS: Sesamin promoted the expression COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19. CONCLUSIONS: The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the MAPK signaling pathway (p38 and ERK) and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for osteoporotic therapy.