微量恒流直流电刺激仪的研制及应用

微量直流电刺激治疗骨不连、促进骨折愈合的方法，已在临床应用中得到显著疗效，但对刺激电流量的选择尚缺乏定量研究，电流刺激促进骨折愈合的机制仍不清楚。本实验中研制的微量恒流直流电刺激仪（ＣＤＭＣ－１型），具有自行反馈调节稳定电流的功能，刺激电流强度选择范围广（０～２０００Ａ），精确度高（±１Ａ），便于操作使用等优点。直流电刺激具有促进体外培养成骨细胞生长和代谢作用，影响细胞排列方向。该仪器为定量研究刺激电流强度及作用机制，提供了条件和方法。     

Endosteal surfaces in hyperparathyroidism: An enzyme cytochemical study on low-temperature-processed,glycol-methacrylate-embedded bone biopsies

Summary Alkaline phosphatase (AIP) and tartrate-resistant acid phosphatase (TRAP) activities have been studied comparatively in needle biopsies of the iliac crest of four cases of secondary hyperparathyroidism (renal osteodystrophy). AIP activity was associated with the plasma membrane of osteoblasts and their processes, of reticular cells of bone marrow and of young osteocytes of osteoid borders and woven bone. Moreover, it was detected in the fibroblast-like cells of the endosteal fibrosis. These cells were orderly in arrangement and were parallel to the endosteal surfaces near zones of bone formation. They were disorderly near zones of bone resorption. A strong TRAP-positive reaction was present in osteoclasts and mononuclear cells of endosteal fibrosis and in osteocytes located near active osteoclasts and in woven bone. These results suggest that the socalled fibrosis of hyperparathyroidism, rather than representing reparative, inert tissue, consists of osteoblastlike cells, probably precursors of osteoblasts derived by parathormone-stimulated proliferation of AIP-positive stromal cells of bone marrow, and of TRAP-positive, mononuclear cells, probably preosteoclasts. Moreover, they show that TRAP activity can be present in osteocytes, probably under stimulation by the same factors which stimulate osteoclast activity. The histochemical demonstration of AIP and TRAP facilitates the morphological diagnosis of metabolic bone disease and may improve knowledge of bone physiopathology.     

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture

The effects of ipriflavone (IP) (10^–5 M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of -glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.     

益骨胶囊对成骨细胞增殖影响的时效及量效关系研究

目的 :运用中药血清药理学方法 ,观察益骨胶囊含药血清对新生大鼠颅骨成骨细胞 (OB)体外增殖的影响 ,并探讨用于OB药效观察的益骨胶囊含药血清制备条件。方法 :雌性 12月龄SD大鼠 80只 ,随机分为生理盐水组、益骨胶囊低剂量 (11 6g/kg)、中剂量 (34 8g/kg)、高剂量 (10 4 4g/kg)组 ,每日灌胃一次 ,连续 12天 ,分别在灌胃后的 0 5h、1h、1 5h、2h采血 ,分离血清 ,用含不同浓度益骨胶囊的血清培养SD新生大鼠颅骨OB ,MTT法检测细胞增殖情况。结果 :末次灌胃后 1 5h采血组的吸收度 (A)值明显高于各组其它时间 (P <0 0 5 ) ;不同剂量各浓度的益骨胶囊含药血清对体外培养的OB增殖均有显著的促进作用 ,其中以高剂量组的稀释比为 2 5 %的血清浓度最佳。结论 :益骨胶囊含药血清对OB增殖具有明显的促进作用 ;益骨胶囊用于OB药效观察的大鼠含药血清制备条件以人等效剂量 9倍连续 12天灌胃、末次灌胃后 1 5h采血 ,血清添加浓度以 2 5 %为宜。     

Modulation of Isoflavones on Bone—nodule Formation in Rat Calvaria Osteoblasts in vitro

Objective:to observe the effects of two main isoflavones,daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro.Methods:Osteoblasts obtained from newborn Sprague-dawley rat calvarias were cultured for several generations.The second generation cells were cultured in Minimum Essential Medium supplemenmted with ascorbic acid and Na-beta-glycerophosphate for several days,in the presence of daidzein and genistein,with or without the estrogen receptor antagonist ICI 182780.Number of nodules was counted at the end of the incubation period(day 20) by staining with Alizarin Red S calcium stain.The release of osteocalcin,as a marker of osteoblast activity,was also determined on day 7 and 12 during the incubation period.Results:compared with the control,the numbers of nodules were both increased by incubation with daidzin and genistein,17β-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation than daidzein and genisterin.The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein,as well as 17β-estradiol on day 7 and day 12(day 12 were higher).The estrogen receptor antagonist ICI 182780 completely blocked the genistein-and 17β0estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts.Howerver,the effects induced by daidzein could not be inhibited by ICI 182780.Conclusion:These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts.The effects,like those induced by 17β-estradiol,are mediated by the estrogen receptor dependent pathway,Daidzin also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts,but it is not,at least not merely,mediated by the estrogen receptor dependent pathway.     

Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.     

Osteoblast Gene Expression in Rat Long Bones: Effects of Ovariectomy and Dihydrotestosterone on mRNA Levels

The steroid sex hormones exert major effects on bone formation although the molecular events associated with their activity remain unclear. We have investigated the effects of ovariectomy and dihydrotestosterone (DHT) administration to both sham-operated and ovariectomized (ovx) rats on the bone mRNA levels of osteoblast genes. Rats were randomly allocated to either sham or ovariectomy operations and were administered either vehicle or 40 mg/kg body weight DHT by silastic tube implants at the time of operation for 8 weeks, at which time they were killed and total RNA was extracted from the long bones. Northern blot analysis indicated that the mRNA levels of the bone cell genes α1(I) collagen, alkaline phosphatase, osteocalcin, and osteopontin were markedly increased in ovx rats between 6- and 30-fold. DHT administration to ovary-intact, estrogen-sufficient rats increased the mRNA levels of α1(I) collagen, alkaline phosphatase, osteopontin, and osteocalcin between 3- and 9-fold. In contrast, DHT did not alter levels of these mRNA species in ovx rats. The data demonstrate that estrogen deficiency increased mRNA levels of genes expressed during osteoblast development and suggest an interplay between estrogen and androgen action in regulating the expression of a number of bone cell genes. Received: 20 May 1999 / Accepted: 21 January 2000     

Mechanical Loading Stimulates Differentiation of Periodontal Osteoblasts in a Mouse Osteoinduction Model: Effect on Type I Collagen and Alkaline Phosphatase Genes

The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer. Received: 9 August 1999 / Accepted: 4 February 2000     

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     

Einfluß verschiedener Knochendesinfektions- und Sterilisationsverfahren auf die Osteoblastenfunktion Eine vergleichende In-vitro-Studie

All sterilization and disinfection procedures for bone grafts are different in regard to influence of bone graft features, which may influence the function of different cell types. We used an in-vitro approach to assess the influence of bone matrix, which was sterilized or disinfected, on osteoblastic activities in-vitro by simulating a cell-transplant-interface. Primary bovine osteoblast cell cultures were established from periosteum. Bone graft specimens made of bovine cortical bone (O 15 mm, 300 microns thickness) were treated in 5 different ways: autoclaved, ethylene-oxide-sterilized, demineralized and low-temperature-plasma-sterilized (DEM-LTP), chemically sterilized (modified Tutoplast method), and 80 degrees C-temperature disinfected. The following cell function parameters were assayed: plating efficiency proliferation by measuring the DNA-content, and MTT-activity, soluble protein and extracellular matrix synthesis, alkaline phosphatase, and osteocalcin expression. All disinfected bone grafts were biocompatible with primary periosteal osteoblasts. Measured cell activities upon bone specimens showed better results than cells of the plastic surface control. The DEM-LTP-bone showed better results in comparison to other groups, and stimulated the proliferation and differentiation.