Dexamethasone inhibits formation of osteoclast-like cells in bone-marrow cultures

Our observations confirm the previously reported observation that multinucleated cells develop within a two-week period in cat bone-marrow cultures. Calcitonin induces cytoplasmic contraction in these multinucleated cells, which indicates that they resemble osteoclasts. After seven days of culture, cat bone-marrow cultures contain both non-attached mononuclear cells and attached mononuclear and polynuclear cells. We have cultured the attached and non-attached cell populations separately from day 7 onward and have observed the appearance of multinucleated cells in both culture systems. In both types of cultures, dexamethasone (10(-9)-10(-7) mol/L) reduced the number of multinucleated cells. This effect of dexamethasone was more rapid in the cultures derived from non-attached cells (seven days) than in cultures derived from attached cells (14 days), suggesting that two different mechanisms are involved. Dexamethasone had no effect on the survival of multinucleated cells already formed.     

Cholera toxin and dibutyryl cyclic-AMP stimulate the growth of epithelial cells derived from epithelial rests from porcine periodontal ligament

The epithelial cells (E-cells) grew best at high (greater than 5 per cent) concentrations of fetal bovine serum (FBS). Growth could be obtained at low concentrations of dialysed FBS (DFBS) if the medium (alpha-MEM) was modified so that the levels of Ca2+ and K+ were reduced to 0.1 and 1.0 mM, respectively (beta-MEM). The addition of 0.5 per cent DFBS to the beta-MEM did not initiate good growth but was sufficiently supportive to enable the effects of various growth promoters to be tested. Cholera toxin and dibutyryl cyclic-3'5'-adenosine monophosphate (Bt2cAMP), which are known to increase intracellular cAMP levels, at concentrations of 1 ng/ml and 0.5 mM, respectively increased cell number. Cholera toxin caused the E-cells to be more flattened when viewed by phase-contrast; this appeared to be due to spread of the cells. No difference in cell-size distributions obtained between the trypsinized E-cells grown in the presence or absence of cholera toxin was observed. Epithelial proliferation that occurs in dental cysts could result from a rise in intracellular cAMP levels in epithelial cell rests.     

Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

OBJECTIVES: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. RESULTS: EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.     

The induction of specific metabolic alterations in mouse calvarial organ cultures by glycosaminoglycans

Glycosaminoglycans specifically regulate the amount of calcium released from bone cultures; the mechanisms responsible for this regulation are not known. Media from glycosaminoglycan-stimulated bone organ cultures were analysed to determine (1) if specific calcium-releasing substances were selectively produced, and (2) if protein synthesis was differentially affected by glycosaminoglycans. Chondroitin sulphate B, hyaluronic acid and keratan sulphate at 100 micrograms/ml significantly increased prostaglandin release when compared with control cultures. In combination with suboptimal concentrations of PTH, chondroitin sulphate B, heparin and keratan sulphate significantly stimulated prostaglandin release. When indomethacin was included in the test assays, the stimulated prostaglandin release was abolished. Heparin-treated cultures released the greatest percentage of latent collagenase activity followed by hyaluronic acid-treated cultures. Organ cultures treated with heparin and PTH amount of active collagenase. Stimulation increased interleukin-1 above control levels but with no significant difference among the glycosaminoglycans except for keratan sulphate cultures with which had the greatest amount of interleukin-1. Collagen protein decreased between 48 and 72 h under both control and experimental conditions. Examination of the predominant [35S]-methionine labelled proteins revealed that prostaglandin E2 treatment resulted in a relative shift in labelling to higher molecular-weight proteins as time in culture increased (up to 144 h). After 48 h, when equal amounts of labelled protein were analysed, there was a predominance in labelling of a 200,000 Da protein in the prostaglandin-treated cultures. These findings demonstrate that modulation of calcium release by glycosaminoglycans results in the selective release of molecules capable of stimulating calcium release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dental follicle cell-conditioned medium enhances the formation of osteoclast-like multinucleated cells

An influx of mononuclear cells and the subsequent increase of osteoclasts around tooth germs suggests that the dental follicle (DF) regulates or influences bone resorption required for tooth eruption. In order to study the effects of DF cell products on osteoclast formation during tooth eruption, a conditioned medium (CM) was created in which DF cells were added to mouse bone marrow cultures. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated cells were formed in the presence of 10 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The CM, dose-dependently, stimulated the formation of TRAP-positive cells in the presence of 1,25(OH)2D3 for 14 days culture. The number of these cells decreased due to degradation in the control culture. A semi-solid methylcellulose assay in the presence of CM showed little expression of colony-stimulating activity. These results suggest that the DF cells of a developing tooth produce factor(s) that enhance osteoclast formation and bone resorption necessary for tooth eruption.     

丹参对骨髓培养中破骨细胞生成的影响

目的：研究丹参对体外骨髓细胞培养中破骨细胞生成的作用。方法：采用小鼠长骨骨髓体外培养方法,通过检测抗酒石酸磷酸酶（ＴＲＡＣＰ）阳性细胞生成数,反映破骨细胞的生成情况。结果：１．０ｇ／Ｌ丹参可明显抑制ＴＲＡＣＰ阳性细胞的生成,其生成数仅为对照组的２９．８％;ＰＧＥ２（１０－７ｍｏｌ／Ｌ）及１．２５（ＯＨ）２Ｄ３（１０－８ｍｏｌ／Ｌ）产生明显的促进作用,与对照组比较,ＴＲＡＣＰ阳性细胞生成数分别增加了６．０４和６．６８倍;丹参还可明显抑制ＰＧＥ２和１．２５（ＯＨ）２Ｄ３的作用,混合培养组ＴＲＡＣＰ阳性细胞生成数仅仅是单纯加ＰＧＥ２和１．２５（ＯＨ）２Ｄ３组的３５．２％和３９．３％,特别是多核破骨细胞数大量减少。结论：丹参可抑制骨髓细胞体外培养中破骨样细胞的生成,主要抑制破骨母细胞向成熟破骨细胞的转化,而成骨细胞可能在其中发挥了作用     

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces

ObjectivesOsteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs).MethodsVarious cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro.ResultsOsteoclast formation was promoted on pillars (diameter, 1 μm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm).ConclusionsWe demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 μm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.     

Changes in protein kinase C activity in rat calvarial bone cells cultured in a low-calcium environment.

This enzyme activity was examined in bone cells cultured for 8-10 days; the calcium concentration was 1.87 +/- 0.05 (n = 10) mM in the control medium and 0.34 +/- 0.02 (n = 10) mM in the low-calcium medium. The activity was significantly lower in the low-calcium group than in the control (p less than 0.01). The cytosolic fraction decreased more than the membranous fraction. After restoration to a regular calcium environment, the protein kinase C activity recovered rapidly to near the control value. The extent of recovery was greater in the membranous than in the cytosolic fraction. These results suggest that the enzyme was inhibited in bone cells placed in a low-calcium environment, while the sensitivity in the membrane was enhanced.     

培养状态下成人骨髓基质细胞的生长特性

目的研究培养状态下成人骨髓基质细胞的生长特性,为自体骨组织工程的功能细胞培养提供实验基础研究。方法培养健康成人骨髓基质细胞连续8代,测定第3、6代细胞的生长曲线、分裂指数、贴壁率及倍增时间。结果所测第3、6代细胞生长曲线基本相同,12h内细胞贴壁率达88.54%,分裂指数最高达15‰。结论在本实验条件下,细胞生长较稳定,接种成活率较高,增殖速度较快,可用于自体骨组织工程。     

应用珊瑚／骨髓基质细胞／富血小板血浆组织工程骨修复兔颅骨缺损

目的：评价珊瑚／骨髓基质细胞（MSCs）／富血小板血浆（PRP）组织工程骨修复骨缺损的效果。方法：将兔MSCs悬液与同一供体来源的PRP混合后滴加到珊瑚团片上,形成珊瑚／MSCs／PRP复合物,用其修复MSCs和PRP来源兔颅骨极限缺损。自体颅骨或单纯珊瑚植入作为对照。术后6周和12周,通过组织学观察和组织形态测量分析．评价其骨修复效果。实验数据采用SPSS11．0软件包处理,两组之间差异采用t检验。结果：组织学观察显示,珊瑚／MSCs／PRP组,术后第6周,在整个缺损区珊瑚表面及其孔洞内有大量新骨形成;第12周,缺损区珊瑚完全降解吸收,被成熟的板层骨取代,缺损表现为完全的骨修复.图像分析显示：术后第6周和第12周,珊瑚／MSCs／PRP组的成骨量显著多于珊瑚组（P〈0．01）;术后第12周,珊瑚／MSCs／PRP组成骨量与自体骨组接近（P〉0．05）。结论：应用珊瑚作为支架材料、MSCs作为种子细胞、PRP作为内源性生长因子构建的组织工程骨修复骨缺损效果良好,与自体骨移植相近．是较理想的骨移植替代材料．     

陶瓷化骨/水凝胶与骨髓基质细胞修复自体颅骨缺损实验

目的:观察以陶瓷化骨(ceramic bovine bone,CBB)/水凝胶为支架复合骨髓基质细胞修复颅骨极量缺损效果。方法:将滴有rhBMP-2(10μg)/TGF-β(50ng)、经矿化诱导的SD大鼠自体骨髓基质细胞分别复合在陶瓷化骨/水凝胶和陶瓷化骨上,修复大鼠的颅骨缺损(8mm直径),以CBB/MSCs植入另8只SD大鼠颅骨缺损处为对照组,未修复组为空白对照。分别在术后4、8周取材,应用常规HE染色、改良Mallory三色法染色观察骨缺损修复情况,图像分析测量单位面积骨形成量。结果:4周时2组均有骨样组织形成,陶瓷化骨/水凝胶组形成的骨基质(6813.09±96.32)μm2明显多于陶瓷化骨组(3839.25±104.52)μm2。8周时2组骨缺损均为骨性修复,组间差别与4周时一致,C组骨缺损为纤维覆盖。结论:陶瓷化骨/水凝胶、陶瓷化骨复合骨髓基质细胞能够修复颅骨缺损。     

高纯度破骨样细胞体外培养及功能表达的研究

目的建立大量高纯度破骨样细胞体外培养的方法,并运用分子生物学技术观察体外诱导培养的破骨样细胞标志酶的基因表达。方法按照巨噬细胞集落刺激因子（M-CSF） 30 ng/mL、核因子κB受体活化因子配基（RANKL） 50 ng/mL的质量浓度对骨髓单个核细胞诱导培养6 d,利用形态学观察、抗酒石酸酸性磷酸酶（TRAP）染色、Giemsa染色、骨吸收陷窝检测以及破骨细胞标志酶基因表达的检测,对生成的破骨样细胞进行鉴定。结果实验获得的破骨样细胞中,TRAP阳性的单核破骨样细胞多见、TRAP阳性的多核破骨样细胞数量相对较少,胞核从2个到十几个不等;光镜下牙本质片上可见各种形态的骨吸收陷窝;应用RT-PCR方法证实破骨样细胞表达膜型基质金属酶（MT1-MMP）、基质金属蛋白酶-9（MMP-9）、TRAP和组织蛋白酶K（CK）4种标志酶。结论以M-CSF和RANKL作为诱导因子,对大鼠骨髓单个核细胞进行诱导培养可以获得具有典型的破骨细胞形态特征,可以表达特征性标志酶基因,且数量大、纯度高。     

Inhibitory effect of titanium particles on osteoclast formation generated by treatment of mouse bone marrow cells with PGE2

OBJECTIVE: The present study was designed to evaluate the effect of titanium (Ti) particles with no endotoxin on osteoclast differentiation and osteoclast activity in in vitro experiments. METHODS: Osteoclast formation as well as osteoclastic bone resorbing activity were examined using the mouse bone marrow culture system and purified rabbit osteoclasts treated with Ti particles (2.5-20 microgram cm-2). RESULTS: Ti particles, with no adherent endotoxin, inhibited osteoclastogenesis and receptor activator of NF-kappaB ligand (RANKL) expression in bone marrow cells treated with prostaglandin E2 (PGE2) (100 nM). The inhibitory effect of Ti particles was concentration-dependent (5-20 microgram cm-2), and was observed only on the generation of osteoclasts by PGE2, but not by 1,25-dihydroxyvitamin D3 or soluble RANKL. This suggests that Ti particles did not act uniformly on a common process in the generation of osteoclasts, but specifically on signal transduction for PGE2 in generating osteoclasts. In highly purified osteoclasts, Ti particles showed no effect on survival and bone resorbing activity. CONCLUSION: Ti particles inhibited osteoclast differentiation and RANKL expression in mouse bone marrow cells treated with PGE2, without affecting mature osteoclast survival or activity. Thus, Ti particles may alter the osteoclastogenetic action of PGE2, which is one of the regulatory factors of bone remodeling.     

不同浓度1,25-二羟基维生素D3对体外培养破骨样细胞骨吸收作用的影响

目的:观察不同浓度的1,25-二羟基维生素D3对体外培养破骨样细胞骨吸收作用的影响.进一步阐述骨吸收刺激因子在正畸牙周组织改建中的作用.方法:应用不同浓度的1,25-二羟基维生素D3(0、10-10、10-8、10-6mol/L)诱导大鼠骨髓破骨样细胞的形成,观察细胞在牙本质片上形成骨吸收陷窝的数目以及陷窝面积的大小;并采用原位杂交技术检测大鼠骨髓细胞的ODF mRNA表达.结果:随着1,25-二羟基维生素D3浓度的增加,骨陷窝数明显增多,陷窝面积增大;ODF mRNA表达的阳性信号显著增强.结论:在体外,1,25-二羟基维生素D3可以调节大鼠骨髓破骨样细胞的形成及骨吸收活性.     

Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration

The role of cultured bone marrow stromal cells (BMSCs) in peripheral nerve regeneration was examined using an established rabbit peroneal nerve regeneration model. A 15-mm peroneal nerve defect was bridged with a vein filled with BMSCs (1 x 10(6)), which had been embedded in collagen gel. On the contralateral side, the defect was bridged with a vein filled with collagen gel alone. When the regenerated tissue was examined 4, 8 and 12 weeks after grafting, the number and diameter of the myelinated fibers in the side with the BMSCs were significantly higher than in the control side without the BMSCs. This demonstrates the potential of using cultured BMSCs in peripheral nerve regeneration.