Effects of cyclosporin A on rat osteoblasts (ROS 17/2.8 cells) in vitro

Summary The effects of the immunosuppressive drug cyclosporin A (CsA) were evaluated on ROS 17/2.8 cells in vitro. ROS cells were treated with CsA (0, 0.5, 1.0, 5.0 g/ml) for 3 days with and without bovine parathyroid hormone (bPTH) (1–34) 10 nM. CsA at 0.5, 1.0, 5.0 g/ml without PTH and at 5.0 g/ml in the presence of PTH significantly inhibited proliferation, as determined by a tetrazolium colorimetric assay. In addition, ROS cell number was significantly reduced at 3 and 4 days with CsA (5.0 g/ml) without affecting cell viability. Incorporation of [³H]-thymidine into DNA was significantly reduced by 3.0 and 5.0 g/ml CsA after 12 and 24 hours exposure. Basal and 1,25-dihydroxyvitamin D₃-stimulated alkaline phosphatase levels in confluent ROS cells were reduced (P<0.05) with CsA (1.0 and 3.0 g/ml). Pretreatment of ROS 17/2.8 cells with CsA did not alter PTH-stimulated cAMP levels or [¹²⁵I]-PTHrP binding to ROS cells. CsA treatment of ROS 17/2.8 cells induced a spindle-shaped appearance with loss of attachment in confluent cultures. When ROS cells were cultured in CsA-containing media, cellular attachment at 6 and 12 hours was reduced (P<0.05) compared with untreated ROS cells. These findings indicate that CsA was capable of inhibiting proliferation, cell number, mitogenesis, alkaline phosphatase levels, and cell attachment of ROS cells without affecting PTH binding or cAMP levels. This direct effect of CsA on osteoblasts may be important in changes of bone remodeling observed in CsA-treated humans and animals.     

不同方法检测成骨细胞中碱性磷酸酶的比较

目的　探讨不同缓冲液和不同裂解方法对体外培养成骨细胞内碱性磷酸酶 (alkalinephosphatase,ALPase)活性的影响 ,建立较为理想的检测方法和条件 ,为研究骨代谢异常及骨质疏松症等骨病提供简便可靠的检测手段。方法　采取经体外培养 72h的成骨细胞 ,制备上清液、细胞裂解液和细胞反复冻融液样本 ,分别用二乙醇胺 (DEA)、碳酸盐 (CO=3)、2 氨基 2 甲基 1 内醇 (AMP)等 3种不同缓冲液种类的试剂 ,测定上述液体中的ALPase活性。结果　使用DEA缓冲液的 3种样本的测量值都为最高 ,且不精密度 (CV)较其他两种缓冲液小 ,各组比较分析 ,差异均有显著性 (P <0 .0 1 )。使用 3种缓冲液试剂 ,均以裂解液中ALP活性最高 ,而冻融液次之 ,上清液最低 ,分析结果 ,差异均有显著性 (P <0 .0 1 )。结论　用DEA缓冲液测量细胞裂解液体外培养成骨ALPase活性的效果较理想     

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (βGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of βGP supply were tested. We compared 10 mM βGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where βGP was added at day 0. Furthermore, 10 mM βGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture. Received: 15 October 1997 / Accepted: 1 July 1999     

β-半乳糖苷酶在体内外成骨细胞中的表达

目的 研究β—半乳糖苷酶(β—gal)在成骨细胞中的表达状况，为阐明MorquioB综合征的发病机制提供依据。方法 裸鼠各器官和骨组织标本行X-gal染色检测。抽取羊和人骨髓行骨髓基质细胞(BMSCs)培养，分为4组：I：Adv-hBMP-2转染组；Ⅱ：Adv—β—gal转染组；Ⅲ：未转染组；Ⅳ：地塞米松诱导组。分别行X-gal染色和RT-PCR检测β—gal的表达。结果 裸鼠骺板两侧、骨膜内面及松质骨的成骨细胞和破骨细胞可见多量β—gal的表达。未转染BMSCs组有少量β—gal的表达，其他3组细胞的β—gal表达增高。结论成骨细胞和破骨细胞可表达多量β—gal，该两种细胞的β—gal缺乏可能是MorquioB综合征骨骼异常的直接原因。     

Differentiation of osteoblasts and formation of mineralized bone in vitro

Summary Periostea consisting of the osteogenic layer and the fibrous layer of the periosteum were dissected from 17-day-old embryonic chick calvariae, leaving the osteoblasts behind on bone. The dissected periostea were folded with the osteogenic cells in apposition. The explants were cultured on plasma clots for up to 6 days, during which time osteodifferentiation was observed followed by osteoid formation in between the two layers. These cultures consistently mineralized in the presence of 5 or 10 mMβ-glycerophosphate. The mineralization and osteoid formation displayed many characteristics identical with those seen in vivo. Specifically, the osteoid formed was birefringent under polarized light, the central zone of osteoid became mineralized within 24 h of formation in vitro, and a clear border between mineralized and non-mineralized osteoid suggestive of a mineralization front was present. The unmineralized osteoid at the periphery was surrounded by osteoblasts. These data suggest that physiologic mineralization of osteoid produced in vitro did occur in this system by the addition of the alkaline phosphatase substrateβ-glycerophosphate.     

A Bone Replaceable Artificial Bone Substitute: Osteoinduction by Combining with Bone Inducing Agent

Bone inducing agent (BIA) isolated from Saos-2 human osteosarcoma cells was added to an artificial bone substitute composed of 980 degrees C-heated carbonate apatite (CAp) and Type I atelocollagen (AtCol) extracted from bovine tail skins (88/12 in wt/wt %), and a CAp-AtCol-BIA substitute was prepared as an osteoinductive bone substitute. Rat calvaria osteoblasts treated by the isolated BIA demonstrated significantly increased alkaline phosphatase (ALP) activity after 3 days (p < 0.05). In vitro cell attachment and proliferation and ALP activity were investigated for the bone substitute combined with BIA. Osteoblasts cultured onto the surface of the CAp-AtCol-BIA substitute demonstrated remarkable morphological changes such as radial spreading, flattening, and projecting filopodia after 5 days. In comparison with the substitute without BIA, osteoblasts grown in the BIA-combined CAp-AtCol substitute expressed significantly increased proliferation and ALP activity, respectively (p < 0.05). Both the substitutes combined with and without BIA were implanted into artificial defects created in rabbit radii. After 4 weeks, the CAp-AtCol-BIA substitute implanted lesion was completely replaced by regenerated host bone in radiological observation whereas the substitute without BIA was partially resorbed. No histologic abnormalities appeared in the substitute either with or without BIA.     

绿色荧光蛋白体外转染与体内示踪成骨细胞的研究

目的观察经腺病毒介导的绿色荧光蛋白(greenfluorescentprotein,GFP)转染的成骨细胞的体外表达及体内示踪情况,探讨GFP作为组织工程种子细胞示踪剂的可行性。方法以腺病毒为载体,293A细胞为包装细胞,介导GFP转染成人骨髓源成骨细胞,与未转染的同期细胞作对照,在相差显微镜和荧光显微镜下观察,流式细胞术检测GFP表达效率;分别检测转染后两组细胞的碱性磷酸酶(ALP)活性与骨钙素(OCN)的含量;并将GFP转染8d后的成骨细胞植入裸鼠股部肌袋内,术后4、8周取材进行荧光显微镜、HE染色组织学和免疫组织化学染色观察。结果GFP转染的骨髓源成骨细胞表达绿色荧光的阳性率达75%以上,转染8d后的成骨细胞表面抗原标志CD29、CD44高效表达,而CD34不表达;转染后4、8d细胞内ALP活性与OCN含量与未转染组比较差异无显著性意义(P>005)。GFP转染8d后的成骨细胞植入裸鼠体内4、8周均可表达明显的绿色荧光,并具有成骨细胞的形态特征,ALP免疫组化染色呈黄褐色。结论GFP能在体外转染、裸鼠体内示踪成骨细胞,是一种可用于组织工程研究的理想的活细胞示踪剂。     

异黄酮类药Genistein对原代培养大鼠成骨细胞增殖、分化、钙含量及矿化功能的影响

目的：了解异黄酮类药Genistein对体外培养成骨细胞的作用。方法：应用MTT法、对硝基苯磷酸盐法，原子吸收分光光度法及茜素红染色方法观察Genistein对体外培养成骨细胞的增殖、ALP表达，基质钙含量及矿化结节形成的影响。结果：Genistein具有刺激成骨细胞增殖，提高ALP活性，细胞基质钙含量及矿化结节形成的数量的作用。结论：Genistein具有刺激体外培养成骨细胞增殖，分化成熟及促进矿化的作用。     

胶原酶阶梯消化法体外培养成骨细胞的研究

目的 采用胶原酶阶梯消化法进行鼠颅盖骨体外培养。方法 将新生的健康Wistar大鼠处死,无菌条件下取出颅骨,剔净,粉碎漂洗。依次用1．0％、0．5％及0．1％的Ⅰ型胶原酶分3次对颅骨进行消化。制成细胞悬液,接种培养,然后进行细胞内碱性磷酸酶（ALP）染色,最后将成骨细胞纯化、鉴定。结果 纯化处理的培养皿中,ALP染色阳性区域不少于95％,而对照皿中,不超过62％。结论 胶原酶阶梯消化法培养 的成骨细胞具有典型的成骨细胞特征,且成份单一。     

The effect of ascorbic acid on the metabolism of rat calvarial bone cells in vitro

Addition of 50 micrograms/ml sodium ascorbate to confluent cultures of isolated rat calvarium bone cells resulted in a 21% increase in DNA production, a 50-60% increase in incorporation of [14C]proline into collagenous and noncollagenous proteins, and a 200% increase in alkaline phosphatase activity; under identical conditions, [35S]sulfate incorporation into proteoglycans (glycosaminoglycans) was not affected. These results suggest that ascorbate may be important in maintaining or stimulating the osteogenic phenotype of normal bone cells.     

Low-dose parathyroid hormone and estrogen reverse alkaline phosphatase activity suppressed by dexamethasone in mouse osteoblastic cells

Glucocorticoid (GC)-induced osteoporosis (GIO) is frequently seen in patients with excessive GC. Numerous questions remain to be clarified about the pathogenesis and treatment of GIO, and the mechanism of GC-inhibited bone formation is not well known. Several studies suggest that parathyroid hormone (PTH) and hormone replacement therapy are effective for GIO. We therefore investigated whether PTH and estrogen would affect cell proliferation and alkaline phosphatase (ALP) activity inhibited by dexamethasone (Dex) in mouse osteoblastic cell-line MC3T3-E1 cells. Low-dose (10⁻¹¹ M) PTH as well as 10⁻⁸ M 17-β-estradiol (17β-E₂) significantly attenuated Dex-inhibited ALP activity, although 10⁻⁸ M PTH did not affect it. ICI 182780 (10⁻⁸ M) antagonized the effects of 17β-E₂ on Dex-suppressed ALP activity. Neutralizing anti-IGF-I antibody (3 μg/ml) blocked the reverse effects of 17β-E₂ on ALP activity suppressed by Dex. PTH (10⁻¹¹ M), but not 17β-E₂, significantly attenuated [³H]thymidine incorporation inhibited by Dex. On the other hand, PTH and estrogen did not affect the level of 11-β-hydrosteroid dehydrogenase type I mRNA increased by Dex. In conclusion, the present study demonstrated that low-dose PTH and estrogen reversed Dex-inhibited ALP activity in the mouse osteoblastic cell-line.     

Elevation of alkaline phosphatase activity induced by parathyroid hormone in osteoblast-like cells from the spinal hyperostotic mouse TWY (twy/twy)

We have examined the alkaline phosphatase (AP) activity of primary calvaria-derived osteoblast-like cells from the twy (tip-toe walking Yoshimura) and normal ICR control mouse. The twy mouse displays elevated osseous formation particularly in the spine, and the pathophysiological features resemble that of human ankylosing spinal hyperostosis. In the proliferative stage of cultured bone cells, parathyroid hormone (PTH) stimulation induced the elevation of AP activity of both twy and ICR mouse-derived cells. When they reached confluence, the AP activity of ICR mouse-derived cells ceased to increase with PTH stimulation. The twy mouse-derived cells, however, continued to respond to PTH, with the enzyme activity increasing even in the confluent, stationary stage. PTH stimulation also increased the intracellular cAMP content of twy mouse-derived cells but it did not influence that of ICR mouse-derived cells in the stationary stage. Moreover, stimulation with dibutyryl cAMP, but not with phorbol myristate acetate, increased the AP activity of both twy and ICR-derived bone cells irrespective of culture conditions, either in the proliferative or in the confluent stage. These data suggest that the protein kinase A-mediated pathway plays a pivotal role in bone cells with PTH stimulation, and that the uninhibited AP activity observed in twy mouse-derived bone cells might be due to some deviating process between the PTH ligand/receptor interaction and cAMP generation.     

负载BMP-2掺锶磷酸钙复合材料对成骨细胞增殖及功能的影响

目的探索负载BMP-2掺锶磷酸钙复合材料对成骨细胞增殖及功能的影响。方法获取SD大鼠成骨细胞,随机分为空白对照组(Con组)、磷酸钙组(CPC组)和复合材料组(BSCPC组);培养基中分别添加安慰剂、磷酸钙和负载BMP-2掺锶磷酸钙共培养一段时间,通过CCK-8法检测成骨细胞的增殖情况,碱性磷酸酶(alkalinephosphatase,ALP)及茜素红染色观察细胞的功能状态,蛋白电泳观察骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子(receptor activator of nuclear factor-KB,RANK)及核因子κB受体活化因子配体(ligand of receptor activator of nuclear factor-κB,RANKL)蛋白的表达情况。结果共培养1、3、5和7 d,和Con组比较,CPC组和BSCPC组的成骨细胞数目明显增加(P0.05),且以BSCPC组成骨细胞数目最多(P0.05);共培养14 d及21 d,和Con组比较,CPC组和BSCPC组的成骨细胞的矿化能力及ALP活性明显增加(P0.05),且以BSCPC组细胞钙化能力最强及ALP活性最高(P0.05);共培养7 d,和Con组比较,CPC组和BSCPC组的成骨细胞的OPG表达明显增加,而RANK及RANKL蛋白表达明显降低(P0.05),且以BSCPC组的成骨细胞蛋白OPG、RANK及RANKL蛋白表达量改善最为显著(P0.05)。结论负载BMP-2掺锶磷酸钙复合材料促进成骨细胞增殖分化和改善细胞活性和功能。     

Effects of parathyroid hormone on alkaline phosphatase activity and mineralization of cultured chick embryo tibiae

Summary The objectives of this study were (a) to determine if decreased bone alkaline phosphatase (AlPase) activity, resulting either from exposure to parathyroid hormone (PTH) or from direct inhibition of AlPase with levamisole, was concomitant with net changes in bone Ca and P content; and (b) to determine the duration of the effect of PTH on bone AlPase activity after the hormone was removed. Tibiae from 7-day chick embryos were incubated in chemically defined medium for 3–4 days. The Ca and P content of bones incubated for 72 h in this system increased from 2-to 9-fold. Addition of PTH (1 U/ml) to the medium resulted in a 60% decrease in bone AlPase activity, and this decrease was accompanied by a marked inhibition of Ca and P accumulation in bone. When levamisole, a specific inhibitor of AlPase activity, was added to the medium, a similar inhibition of bone mineral accumulation resulted. A 24-h incubation in medium containing PTH (1 U/ml) resulted in a 30% decrease in bone AlPase activity. Following the 24-h exposure to PTH, incubation was continued in medium containing no hormone. After 72 h in hormone-free medium, bones that had been exposed to PTH earlier contained more AlPase activity than paired control bones never exposed to PTH. The PTH-treated bones also recovered their ability to accumulate Ca and P. The results of this study show that: (a) decreased bone AlPase activity resulting from either exposure to PTH or direct inhibition by levamisole is associated with a marked decrease in mineral accumulation in bone; (b) exposure to PTH followed by removal of the hormone leads to recovery and even an increase in bone AlPase activity; and (c) following removal of PTH from the culture system, tibiae recover their ability to accumulate Ca and P. These results suggest that one of the ways PTH may alter mineral accretion is through its effects on bone AlPase activity.     

双膦酸盐的长期治疗增加了骨单位的矿化程度

目的评估双膦酸盐的长期应用对骨单位的次级矿化程度的影响。方法30只1岁龄猎犬按体重随机分成三组(每组雌雄各5只):对照组犬每天口服乳糖,低剂量组和高剂量组犬分别每天口服因卡膦酸钠incadronate0.3mgkg和0.6mgkg。所有犬持续给药3a。处死前进行四环素双标,处死后取左侧第9肋骨进行组织形态计测和次级矿化程度评估。结果组织形态计测表明两个双膦酸盐治疗组的骨激活频率(Ac.f)都明显低于对照组,分别降低了40%和82%。矿化程度的测定表明,低剂量和高剂量双膦酸盐组骨单位内的平均矿化程度(MDMB)都明显高于对照组,分别增加了22%和30%。双膦酸盐组MDMB分布曲线与对照组保持了相同的峰值,并且随治疗剂量的增大逐渐向高矿化端漂移。结论双膦酸盐长期应用明显抑制了骨转换,增加了骨单位的矿化程度。     

Efficacy of wheat germ lectin-precipitated alkaline phosphatase in serum as an estimator of bone mineralization rate: Comparison to serum total alkaline phosphatase and serum bone Gla-protein

Summary Serum levels of total alkaline phosphatase activity (S-T-AP), wheat germ lectin-precipitated alkaline phosphatase activity (S-L-AP), and bone Gla-protein immunoreactivity (S-BGP) were measured in 26 patients (23 females and 3 males) aged 35–73 years (mean 59 years) with primary hyperparathyroidism (n=7), hyperthyroidism (n=9), and hypothyroidism (n=10) in whom the bone mineralization rate (m) was determined by⁴⁷Ca-kinetics (continuously expanding calcium pool model). A weak positive correlation (r=0.42,P<0.05) was found between S-T-AP and m, which in the range from 0–18 mmol Ca/day could be estimated with a standard error of 4.6 mmol/day. A closer correlation (r=0.65,P<0.001) was found between S-L-AP and m which was estimated with an error of 3.9 mmol Ca/day. The AP activity in the supernatant showed no significant correlation to m (r=0.11,P>0.50). The highest correlation coefficient (r=0.81,P<0.001) was found between S-BGP and m which could be predicted with an error of 3.4 mmol Ca/day. S-BGP showed a closer correlation to S-L-AP (r=0.71,P<0.001) than to S-T-AP (r=0.58,P<0.01). We concluded that S-L-AP predicts bone mineralization at organ level better than S-T-AP in selected metabolic bone disorders and that the supernatant activity shows no relation to bone turnover. We find the assay easy to handle and suitable for large-scale use in the diagnosis and monitoring of metabolic bone disease.     

Alkaline phosphatase and ATPase activities of rat bone: Separation and characterization

Summary Extraction with Triton X-100 has proved effective in solubilizing alkaline phosphatase from rat bone particles, whereas ATPase with optimum activity at pH 8 remains attached to the bone particles. The kinetic characteristics of the ATPase activity of the Triton extracts are different from those of the same enzyme attached to bone particles, but the kinetic characteristics of the particle-bound and solubilized alkaline phosphatases are similar. The results suggest that the Triton extracts do not have true ATPase activity and provide a means of separating the ATPase and alkaline phosphatase activities.     

Hydrolysis of pyrophosphate and ester phosphates by bone extracts

A study was conducted of the enzymatic hydrolysis of pyrophosphate and ester phosphates by extracts of bones of young rabbits. The proximal ends of humeri and distal ends of femurs were homogenized in barbital buffer (7.5 mM) at pH 7.4. The extracted pyrophosphatase activity had acid and alkaline pH optima. The acid pyrophosphatase activity at pH 5.0 was independent of magnesium up to a concentration of 3 mM and was inhibited by an excess of this ion. Pyrophosphatase activities at both pH 5.0 and 7.4 were inhibited by fluoride. The Michaelis constant of pyrophosphatase with pH 5.0 optimum activity was 1.4×10^–3 M. The bone extract, when loaded on a column of Sephadex G-200 and eluted with barbital buffer (7.5 mM) at pH 7.4 containing 1% sodium chloride, allowed a separation of the two pyrophosphatase activities; that at pH 5.0 optimum activity was associated with acid phosphatase activity. Results of experiments of the two pyrophosphatase activities and alkaline phosphatase in bones of rabbits as they aged from 5 to 60 days are presented.This investigation was supported by Grant DE-1850 from the National Institute of Dental Research, National Institute of Health, Bethesda, Maryland.The authors wish to acknowledge the help of Miss Doris Bauder in the preparation of the Sephadex columns.     

Formation of bone by isolated,cultured osteoblasts in millipore diffusion chambers

Summary Osteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6–7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an “inducer.” After 20 days, 11 of 18 chambers containing the osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.     

Influence of flexible nailing in the later phase of fracture healing: strength and mineralization in rat femora

In this experimental study, the influence of flex-ible nailing in the later phase of femoral fracture healing was investigated. Sixty rats were randomly assigned to three groups. In 20 rats no intervention was performed, and they served as a control group. Fracture and reamed nailing with a rigid steel nail was performed in the left femur in the other 40 rats. These rats were reoperated after 30 days, and the medullary nail was removed. In one group (20 rats) a flexible polyethylene nail was installed (flexibly nailed group), while the rats in the other group received a steel nail identical to the one that was removed (rigidly nailed group). At 60 and 90 days, the left femurs of 10 animals in each group were studied clinically, radiologically, and biomechanically, and bone mineralization was measured by dual-energy X-ray absorptiometry. Radiographs in two planes revealed a clearly visible fracture line in both intervention groups at 60 days. At 90 days, the fracture line was clearly visible in the flexibly nailed group, while bridging callus was apparent after the rigid nailing. At 60 and 90 days, the callus area in the flexibly nailed group was significantly larger than that in the rigidly nailed bones. Biomechanically, flexible nailing reduced maximum bending load and fracture energy at 60 and 90 days compared with findings in rigidly nailed bones, while bending rigidity was similar in the two groups. All values for biomechanical characteristics were reduced at 60 and 90 days in flexibly nailed bones compared with intact femurs, while in the rigid nailing group, bending load and fracture energy were similar to those in intact bones at 90 days. Bone mineral content in the callus segment and diaphysis was greater in the rigidly nailed bones than in the flexible nailing group at 60 days, while at 90 days, no differences were detected. In conclusion, this animal study indicates that: (1) flexible nailing in the later phase of fracture healing increases callus formation, while (2) the quality of bone healing is reduced. Received: March 1, 2001 / Accepted: July 26, 2001