BMP7腺病毒基因转染对骨髓基质干细胞生物学的影响

目的 探讨BMP7腺病毒基因转染对骨髓基质干细胞(BMSCs)的体外、体内的生物学功能影响.方法 抽取羊的骨髓,分离、培养骨髓基质干细胞.用重组骨形成蛋白7腺病毒(adenovirus bone morphogenetic protein 7;Adeno-BMP7)转染(M.O.I=100)70%～80%融合时的第二代细胞,三天后分别进行透射电镜观察、流式细胞仪检测、Western-blot、钙结节染色以及珊瑚和细胞复合物皮下回植.结果 在体外:细胞超微结构观察显示Adeno-BMP7基因转染后的BMSCs的物质合成代谢功能活跃;流式细胞仪检测表明Adeno-BMP7基因转染对BMSCs的细胞周期无明显影响;Western-blot检测证明转染Adeno-BMP7后的BMSCs有BMP7在蛋白水平上的表达;染色表明转染Adeno-BMP7后的BMSCs可形成较大的钙结节.在体内:转染Adeno-BMP7的BMSCs可明显促进新骨的形成,与没转染Adeno-BMP7的BMSCs有差异.结论 Adeno-BMP7转染可促进BMSCs的体外成骨定向分化,Adeno-BMP7转染后的BMSCs具有更强的体内成骨能力.     

Hemopoietic functions of marrow-derived osteogenic cells

Summary Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.     

Subpopulations of marrow stromal cells share a variety of osteoblastic markers

Summary A series of stromal cell lines derived from mouse bone marrow, representing subpopulations of putative stromal cell types, were examined for the expression of osteoblastic properties. The effects of dexamethasone and specific inhibitors on alkaline phosphatase activity, cAMP response to bone-seeking hormones, and the ability to mineralize extracellular matrixin vitro as well as collagen typing were used as osteoblastic markers. We found that all stromal cell types examined posses some osteoblastic features but differ in the degree of expression. The data provide support to the hypothesis of a common stem cell for marrow stromal cells.     

Growth hormone is not able to counteract osteopenia of rat cortical bone induced by Glucocorticoid with protracted effect

Osteopenia and inhibited longitudinal growth in childhood are serious side effects during glucocorticoid therapy. The effects of glucocorticoids on bone have been confirmed in animal experiments. Long-term glucocorticoid administration to rats results in reduced body weights, reduced bone growth (length and cross-sectional area), and bone strength. Glucocorticoid treatment also resulted in a reduced bending stress, indicating reduced bone quality. Growth hormone, on the other hand, increased body weights, bone dimensions, and bone strength. The aim of the present study was to evaluate if growth hormone administration would have an anabolic effect on rat bone when given to animals also receiving a high dosage of glucocorticoid. Five groups of female rats, 3.5 months old, were treated as follows: (1) saline control; (2) glucocorticoid (prednisolone: Delcortol 5 mg/kg/day); (3) growth hormone (recombinant human growth hormone 5 mg/kg/day); (4) glucocorticoid and growth hormone; and (5) food restriction, consisting of restricted access to food to reduce their weight gain to match that of the glucocorticoid injected rats. After 80 days of hormone administration the animals were sacrificed. The right femur was removed and tested biomechanically in a three-point bending procedure. The left femur was used for determination of bone dimensions. Biomechanical parameters (ultimate load and ultimate stiffness) were then normalized to diaphyseal cross-sectional diameters of the femur, giving the values of ultimate bending stress and Young's modulus. Results: administration of both hormones simultaneously could not reverse the decrease in body weights, bone length, and diameters, or the decreased bone strength induced by glucocorticoid administration. In conclusion, growth hormone cannot prevent cortical osteopenia in female rats induced by a high dose of glucocorticoid with protracted effect.     

骨髓基质细胞在藻酸盐凝胶中的生物学行为

目的：探讨骨髓基质细胞（Bone Marrow Stromal Cells,MSC)在海藻酸盐（Alginate)中的生物学行为，为在组织工程研究中选用海藻酸盐做为骨髓基质细胞的载体提供理论依据。方法：将地塞米松诱导后的骨髓基质细胞种植在1%海藻酸盐中，以HE染色，BMP-2免疫组化染色，扫描电镜观察骨髓基质细胞在凝胶中的生物学行为。结果：凝胶中细胞呈“悬浮”生长状态，可见到细胞分裂和核分裂相；细胞在凝胶中增殖，4d后可以见到基质分泌；海藻酸盐中培养组与常规培养组MSC的BMP-2阳性表达率无显著差异。结论：在海藻酸盐中骨髓基质细胞的生物学行为表现良好。此特征适宜作为骨组织工程的载体。     

聚丙烯延胡索酸酯/β-磷酸三钙合成和对骨髓基质细胞生物学行为的影响

目的制备聚丙烯延胡索酸酯／β-磷酸三钙（PPF／β-TCP）复合材料。在体外检测PPF／β-TCP材料对骨髓基质细胞（BMSC）的黏附、增殖、成骨能力的影响，对PPF／β-TCP生物材料的性能进行评价。方法二步反应法合成PPF单体，加入β-TCP后进行交联。骨髓基质细胞在PPF／β-TCP材料上培养2、4、6、8、10、12h后胰蛋白酶消化，检测黏附细胞数。BMSC和PPF／β-TCP材料复合培养，用MTT法检测BMSC的增殖，绘制生长曲线，检测碱性磷酸酶的含量。结果BMSC在PPF／β-TCP材料上黏附，2～8h细胞数增加，到10h黏附数达最高，约为68％。BMSC在PPF／β-TCP材料上生长，第4、5天为对数生长期，第7、8天进入平台期。BMSC种植在PPF／β-TCP后上表达ALP并不断增加。结论PPF／β-TCP生物降解材料对BMSC的黏附、增殖、成骨功能无不良影响，是一种有前途的生物降解材料。     

The effects of tamoxifen on the osteopenia induced by sciatic neurotomy in the rat: A histomorphometric study

Summary The effects of a 3-week treatment with the nonsteroidal “antiestrogen” tamoxifen were determined on cortical and trabecular bone mass of the tibiae of growing male rats that had undergone unilateral sciatic neurotomy (USN). USN resulted in decreases in cortical area (−11.3%), cross-sectional area (−8.4%), and periosteal bone formation rate (−32.6%) in cortical bone, indicating that the disuse osteopenia results in a decrease in bone formation in cortical bone. USN significantly reduced the amount of trabecular bone in our metaphyseal sampling site (−75%), markedly increasing the amount of bone surface lined by osteoclasts (+65%) without affecting the surface lined by osteoblasts. These results suggest that trabecular bone disuse osteopenia is due, at least in part, to increased bone resorption. Tamoxifen treatment significantly reduced the loss of trabecular bone, restoring resorbing surface length to the control (sham-operated) animal levels. Tamoxifen treatment of sham-operated animals increased trabecular bone area and surface by 35.7% (±10.5) and 41.8% (±7.8), respectively, and reduced resorbing surface by 21.5% (±11.6) compared with sham-operated placebo-treated rats. Tamoxifen had no significant effect on cortical bone parameters in the sham-operated group. The results indicate that tamoxifen is able to reduce the trabecular bone loss that results from USN, but has no effect on cortical bone disuse osteopenia, or on trabecular bone formation. Moreover, tamoxifen treatment of control (intact) animals inhibited the normal levels of bone resorption occurring in these rapidly growing animals.     

转染重组人骨形态发生蛋白-7基因对骨髓基质细胞生物学行为的影响

目的 观察重组人骨形态发生蛋白-7(rhBMP-7)基因转染骨髓基质细胞(BMSCs)后的稳定表达及表达产物对BMSCs生物学行为的影响。方法利用脂质体介导法将rhBMP．7基因转染BMSCs，以G418筛选出阳性克隆并扩大培养，用免疫组织化学链霉抗生物素蛋白-生物素-过氧化物酶复合物(SABC)法检测转基因细胞的稳定表达；转染48h后，用转基因细胞的培养液上清刺激正常培养的BMSCs，利用^3H标记的胞腺嘧啶氧核苷(^3H-TdR)掺入法、Na2^35SO4掺入法以及氯胺T法检测基因表达产物对BMSCs增殖、合成蛋白多糖以及胶原的影响。结果 转基因细胞4周时仍能表达外源性基因；基因表达产物能明显促进BMSCs的增殖以及蛋白多糖和胶原的合成。结论 rhBMP-7基因转染BMSCs后可获得稳定表达，且基因表达产物能促进BMSCs增殖。诱导其向软骨细胞分化并增强其生物学活性。     

Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells

Direct grafts of human cells into immunocompromised or cortisone-treated animals, either alone or within carrier materials, have been used with some success to assess the developmental capability of the grafted cells. However, identification of the donor or host origins of the generated tissue in such direct grafts is essential. In an alternative and extensively used experimental system, cells are cultured within the isolated environments of diffusion chambers, which are surgically implanted in appropriate hosts. This system allows the direct study of the cellular potentials for differentiation as host tissues are excluded. In the present study, human osteoprogenitor cell populations derived from trabecular bone explants or marrow suspensions of 3 patients (2 females aged 14 years and 1 male aged 27 years) were cultured in the absence or the continuous presence of dexamethasone (10 nmol/L). Cells were impregnated into porous hydroxyapatite ceramics before subcutaneous implantation, or placed within diffusion chambers before intraperitoneal implantation, in athymic mice. All subcutaneous implants of cells in ceramic showed morphological evidence for the formation of bone tissue. In the diffusion chambers it was found that both marrow- and bone-derived fibroblastic cells cultured in the absence of dexamethasone generally produced fibrous tissue only. When cultured in the continuous presence of dexamethasone (10 nmol/L), these cell populations produced similar osteogenic tissues with active osteoblasts, wide osteoid seams, and mineralized tissue, with cartilage toward the interior of the chamber. These results validate the diffusion chamber as an experimental system to study human osteogenesis using appropriately primed cell populations.     

Skeletal effects of low-dose cyclosporin A therapy in children with nephrotic syndrome

Background. High doses of cyclosporin A (CsA) produce high-turnover osteopenia in rats. The aim of this study was to determine whether low-dose CsA affects the skeleton in children with nephrotic syndrome. Methods. Biochemical parameters of mineral and skeletal homeostasis, and bone mineral density (BMD) in eight boys with steroid-dependent, frequently relapsing minimal change nephrotic syndrome who had received low-dose CsA (between 1.6 and 3.1 mg/kg per day) for 2 years were compared with measurements in the same patients before CsA therapy and who had received glucocorticoids for long periods, and with measurements in age-matched controls. Results. It was possible to discontinue glucocorticoid therapy within 4 months after the start of CsA therapy. There was a significant increase in the mean serum alka-line phosphatase concentration in CsA therapy patients compared with the same patients before CsA therapy and the controls. Serum osteocalcin and tartrate-resistant acid phosphatase, and urinary deoxypyridinoline concentrations in CsA therapy patients did not differ from those in the controls. BMD in CsA therapy patients was increased significantly compared with values in the same patients before CsA therapy. BMD in CsA therapy patients was lower than that in the controls, but remained within 80% of the overall mean BMD value. Conclusions. Two years of low-dose CsA therapy without glucocorticoids does not appear to induce high-turnover osteopenia in children with steroid-dependent nephrotic syndrome. Received: February 7, 2000 / Accepted: May 10, 2000     

Electrophysiological study on differentiation of rat bone marrow stromal stem cells into neuron-like cells in vitro by edaravone

To explore the electrophysiological proper-ties of differentiation of rat bone marrow-derived stromal stem cells (rBMSCs) to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods: Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the ex-pression of Nav1.6, Kv1.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results: Cyton contraction and long processes were shown in differentiated stromal stem cells. Nav1.6, Kv1.2, Kv1.3 and Cav1.2 were expressed in both differentiated and undifferentiated cells. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion: In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiologi-cal properties of neurons.     

辛伐他汀对合并骨量减少的老年高胆固醇血症患者骨代谢的临床研究

目的短期观察他汀类药物辛伐他汀对合并骨量减少的老年高胆固醇血症患者骨代谢指标的影响。方法入选老年高胆固醇血症合并骨量减少患者55人,平均年龄(66.8±4.7)岁,男性和女性分别为24人和31人,随机分两组分别给予辛伐他汀(20 mg.d-1)和饮食控制,所有患者每天补充钙剂1000 mg。所有受试者在治疗前后检查血脂和骨代谢指标,并进行骨密度测定。结果经过治疗6个月后,治疗组患者总胆固醇、低密度脂蛋白胆固醇、甘油三脂明显低于治疗前,骨代谢指标治疗组总碱性磷酸酶、骨钙素无明显变化,尿脱氧吡啶啉肌酐比值明显降低,相应的对照组各项骨代谢指标治疗前后均无明显变化。治疗前后腰椎和股骨骨密度虽然有所增加,但未达统计学意义。结论辛伐他汀对老年高胆固醇合并骨量减少患者骨代谢的影响可能主要通过抗骨吸收作用,对骨合成代谢无明显影响。进一步扩大研究样本数量和研究时间以了解他汀类药物是否可以真正增加骨密度从而减少骨折发生风险是必要的。     

Colony-forming efficiency response of bone marrow stromal cells to acute blood loss.

Colony-forming efficiency (CFE) was used to monitor the proliferative response of alkaline phosphatase-positive rabbit bone marrow stromal cells to acute blood loss. The CFE of animals subjected to a 1% blood loss was 0.97 compared with 0.06 (p less than 0.01) in nonbled animals. Sera obtained from animals 10 days after an initial blood loss stimulated the CFE of marrow cultures from nonbled donors to the same degree as osteogenin. Erythropoietin and control sera (from nonbled animals) had no effect. Hence, acute blood loss and sera from bled animals stimulate proliferation of alkaline phosphatase-positive marrow stromal cell colonies. The agent(s) responsible is unknown but it is present in serum in response to blood loss. Confirmation of a specific effect on osteoprogenitor cells may warrant the designation "osteopoietin."     

珍珠钙胶囊治疗男性骨质疏松和骨量减少效果观察

目的　观察“珍珠钙”治疗骨质疏松症效果。方法　选男性受试者,青年组２０～３４ 岁,中老年组４５～７４ 岁,两组共２００ 例,珍珠钙胶囊一次２ 粒,每日３ 次饭后服用,共服６ 个月。结果　治疗前后自身比较,治疗后骨密度较治疗前增高（Ｐ＜ ０．０１）,骨痛等症状明显减轻,无副作用。结论　珍珠钙是一种安全、有效的补钙剂,对防治青少年骨量减少和治疗中老年人骨质疏松症疗效显著。     

Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10⁻⁸ or 10⁻⁷ mol/L) at both P₀ and P₁ and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P₁. The highest levels of mineralized tissue formation in P₁ subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10⁻⁷ and 10⁻⁸ mol/L at P₀ and P₁, respectively, and when cultures derived from adult rats were exposed to Dex 10⁻⁸ mol/L both at P₀ and P₁. Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P₀ cultures or to P₁ cultures grown under optimal Dex conditions enhanced MBT formation in P₁ cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P₀ and P₁, whereas in cultures derived from young rats, the addition of bFGF at P₀ was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca²⁺ deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2.3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的：探讨骨髓间充质干细胞（MSCs)分化为肉芽组织中血管内皮细胞的可能性。及其参与创面修复的可能机制。方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化MSCs，体外培养扩增后，应用溴脱氧尿嘧啶（BrdU)标记技术标记细胞。于已抽取骨髓的小型香猪背部制作全层皮肤缺损创面，即刻以纤维蛋白胶为载体，将已标记的MSC回植到供体动物创面上。术后2、4、6、8、12周切取创面组织，行BrdU和因子Ⅷ（FⅧ）免疫组织化学染色，进行对比观察。结果：BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围，且有个别血管内皮细胞也呈现BrdU 阳性。部分BrdU阳性的MSCs胞浆中亦有FⅧ表达。结论：创面愈合过程中，MSCs与肉芽组织中小血管的形成密切相关。在创面微环境下，MSCs可分化为血管内皮细胞，并参与创面修复。     

Bone particles disturb new bone formation on the interface of the titanium implant after reaming of the marrow cavity

Histomorphometric studies were conducted in rats to determine whether bone particles would disturb new bone formation on the interface of titanium implants inserted after reaming of the marrow cavity. In eighty 10-week-old female Wistar rats, smooth-surfaced titanium alloy implants were inserted bilaterally into the marrow cavity after reaming in the distal femur. There were three experimental groups: in the irrigated femora, sterile saline was flushed through the medullary canal; in the particle femora, autologous bone particles were inserted into the intramedullary cavity; and in the reamed femora, the implant was inserted without procedures after reaming. The rats were sacrificed at one, two, four or eight weeks postoperatively, and Villanueva bone staining was applied for histomorphometric studies. The bone volume of new bone on the interface of the implant in the irrigated femora was greater than that in the particle or the reamed femora throughout the study period. The results suggest that clearance of bone particles by irrigation after reaming of the marrow cavity significantly facilitates new bone formation on the interface of implants by one week. The findings also suggest the potential clinical application of total canal irrigation prior to insertion of cementless femoral components as well as cemented prosthesis.     

rhBMP-2对不同培养时间骨髓基质干细胞成骨能力的影响

目的：探讨不同培养阶段骨髓基质干细胞成骨能力的变化及BMP-2对其成骨能力的影响。方法：培养兔骨髓基质干细胞，测定第3代和第18代细胞碱性磷酸酶及骨钙素活性；测定BMP-2对不同培养时间骨髓基质干细胞成骨能力的影响；测定rhBMP-2对细胞增殖的影响。结果：细胞传至第18代后，分泌的骨钙素（OC）水平及ALP活力明显降低，与第3代细胞相比，差异显著（P〈0．05）。第3代细胞在rhBMP-2的诱导下，分泌的OC水平及ALP活力在原基础上进一步升高，与对照组相比，差异显著（P〈0.05）。结第18代细胞在rhBMP-2的诱导下，分泌的OC水平及ALP活力在原基础上升高，与对照组相比，差异不显著（P〉0．05）；随培养时间的延长，各组细胞数量均有所增加，rhBMP-2诱导组与对照组细胞无显著性差异（P〉0．05）。细胞的传代次数对细胞增殖无明显影响（P〉0．05）。结论：rhBMP-2对细胞增殖无影响。随着传代次数的增加，骨髓基质干细胞的成骨能力下降，rhBMP-2促进其成骨的能力亦下降。     

补肾方药对绝经后妇女卵巢功能和骨密度的影响

本根据ＷＨＯ的骨质疏松诊断标准,利用双能Ｘ射线骨密度仪,对２５０例绝经后妇女做骨密度检查,筛选出临床诊断为原发性骨质疏松症和骨量减少的妇女１３１例,在筛选过程中,确定肝肾功能正常,排除风湿病、肾病、甲旁亢、甲亢、胃癌及胃炎、卵巢除等继发性骨质疏松的妇女,采用随机双育对照实验,分成３组：补肾中药组、依普拉封组、对照组,目的是观察补肾中药对原发性骨质疏松症和骨量减少的效果和作用机制,并与治疗骨质疏     

Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells

PURPOSE: This study aimed to investigate the capability of combining marrow stromal cells (MSC) and partially demineralized bone matrix (PDBM) to fill bone defect and enhance bone ingrowth using a canine non-weight-bearing gap model. METHODS: Custom-made implants with 3mm gap between the porous surface and the host bone were used. The implants were inserted into the distal femurs of 25 mongrel dogs and the gaps were randomly assigned to be filled with culture-expanded autologous MSC-loaded PDBM, autograft, fresh-frozen allograft, PDBM alone, or nothing as controls. Histomorphometry using backscattered scanning electron microscopic examination, and mechanical push-out test were performed at 6 months after surgery. RESULTS: Histomorphometry showed that amounts of bone regeneration in the gap and bone ingrowth into the porous-coated surface in the MSC-loaded PDBM-treated group were comparable to those of autograft-treated group and were significantly greater than those of allograft-treated, PDBM-treated, or non-grafted groups. Mechanical test showed the same differences. CONCLUSION: The results of this study showed that combining PDBM and autologous culture-expanded MSC restored bone stock and enhanced bone ingrowth into the porous-coated area in a canine non-weight-bearing gap model. This combination may provide an option for reconstructing bone defect when we perform a cementless revision arthroplasty.