成骨细胞移植在促进骨质疏松性骨折愈合过程中细胞生长因子表达特点

目的探讨碱性成纤维细胞生长因子(bFGF)在幼年成骨细胞移植、促进骨质疏松性骨折愈合过程中不同时相的动态表达及其生物学意义. 方法通过建立老年SD大鼠骨质疏松性骨折的动物模型,将体外培养幼年SD雄性大鼠颅骨成骨细胞移植到SD雌性大鼠骨质疏松性骨缺损部位,利用免疫组化及原位杂交检测骨折愈合过程中不同时相的移植标本bFGF、bFGF mRNA的表达,并用真彩色图像分析仪作图像分析. 结果实验组bFGF在7 d左右可见阳性细胞,14 d有分泌高峰,其阳性单位值达384.65±8.60,其中以软骨细胞中阳性表达最强,21 d分泌量开始下降为286.24±2.30,56 d后完全消失;对照组未见明显分泌高峰,21 d时分泌值最高,仅达到125.33±4.50. 结论 bFGF在骨折愈合过程中对促进骨折部位建立良好的血液循环具有积极意义,可促进老年性骨质疏松骨折的愈合.     

淫羊藿苷对骨质疏松性老年骨折模型愈合过程的影响

目的探讨淫羊藿苷对骨质疏松性老年骨折模型大鼠愈合过程的影响。方法取SD大鼠60只,用维A酸以80 mg/kg进行灌胃,1次/d,连续灌胃3 w,建立骨质疏松模型。造模成功后,人工造成右侧胫骨中上段骨折,建立骨质疏松性骨折模型。将模型大鼠随机分为模型组和实验组,另设假手术组,30只/组,实验组给予腹腔注射淫羊藿苷100μg/kg;假手术组和模型组给予同体积0.9%氯化钠注射液腹腔注射,1次/d,连续注射10 d。分别于术后第2、4、8、12周,收集大鼠骨标本,进行HE染色、骨保护素(OPG)免疫组织化学染色,观察切片组织学;采用X线吸收仪扫描,测量骨痂骨密度和左下肢股骨骨密度,测定血清钙磷乘积及碱性磷酸酶(ALP)。结果 HE染色显示,实验组软骨生成及软骨内成骨过程较模型组明显较快;OPG免疫组织化学染色:在各个相同时间点,实验组蛋白阳性表达的IOD值均明显高于模型组(P<0.05)。骨密度显示:各时间点,与模型组相比,实验组骨痂骨密度明显较高(P<0.05);而左侧股骨骨密度差异无统计学意义(P>0.05)。在相同的时间段,与模型组相比,实验组血清钙磷乘积略高,但差异无统计学意义(P>0.05),但实验组ALP水平明显高于模型组(P<0.05)。结论淫羊藿苷可以改善骨质疏松老年骨折大鼠愈合过程,并可能通过促进OPG的表达,加快老年大鼠骨质疏松性骨折的愈合。     

氟伐他汀对骨质疏松大鼠骨折愈合过程中骨形态发生蛋白-7的影响

目的探讨氟伐他汀对骨质疏松大鼠骨折愈合过程中骨形态发生蛋白7(BMP7)的影响。方法6个月龄雌性SD大鼠96只,随机分为去势组和非去势组,每组48只,分别行双侧卵巢切除术和假性手术;3个月后,去势组于股骨中段制造骨折模型,再随机分为给药组和对照组,每组24只,给药组手术次日给予10mg/(kg·d)氟伐他汀灌胃,疗程为2w,对照组给予安慰剂。利用免疫组化及原位杂交检测骨折愈合过程中给药组和对照组骨折后3、7、14、21、28和42d骨痂中BMP7、BMP7mRNA的表达,并用真彩色图像分析仪作图像分析。结果给药组和对照组在骨折愈合过程中BMP7及BMP7mRNA基因表达的细胞定位无差别,且表达模式基本相似。术后14d,阳性细胞的阳性程度给药组比对照组强(P<0.05)。结论氟伐他汀对骨质疏松大鼠骨折愈合过程中BMP7有一定促进作用。     

骨质疏松症大鼠骨诱导能力改变的研究

目的　探讨骨质疏松性骨折愈合困难的原因 ,为临床治疗骨质疏松性骨折寻找可行性途径和方法。　方法　雌性SD大鼠 4 0只作为供体大鼠 ,随机分为卵巢切除组和对照组 ,每组 2 0只 ,卵巢切除组切除双侧卵巢以复制骨质疏松模型 ,对照组行假手术。 4个月后处死大鼠 ,取肢体长骨按Urist方法分别制备骨基质明胶。雌性SD大鼠 17只 ,双侧股部切开 ,左、右侧肌袋内分别植入卵巢切除大鼠及对照大鼠骨基质明胶。大鼠饲养 14d处死 ,分别取出肌袋植入物组织行组织学检查及骨碱性磷酸酶 (ALP)活性、Ca含量测定。　结果　卵巢切除组的和对照组的植入物组织ALP活性单位分别为 (7 2 2± 2 5 9)IU/ g和 (12 0 1± 6 18)IU/g ,两者差异有显著性 (P <0 0 1)。卵巢切除组的和对照组的植入物组织Ca含量分别为 (5 10± 0 84 ) μg/ g和 (5 73± 0 79) μg/ g ,两者差异有显著性 (P <0 0 5 )。　结论　雌激素缺失所致骨质疏松骨的骨诱导能力较正常骨明显降低 ,骨质疏松性骨折愈合困难与骨骼中骨诱导生长因子的缺乏有关。对于骨质疏松性骨折的治疗要改进骨折固定方法 ,外源补充或促进内源性骨诱导生长因子的表达 ,对于促进骨折愈合具有重要作用。     

氟伐他汀对骨质疏松大鼠骨折愈合影响的超微结构研究

目的探讨氟伐他汀促进骨质疏松大鼠骨折愈合的机制。方法卵巢切除后3个月已经出现骨质疏松的SD大鼠72只,于股骨中段制造骨折模型,随机分为给药组和对照组,每组36只,给药组手术次日给予10 mg/(kg.d)氟伐他汀灌胃,疗程为6 w,对照组给予安慰剂。采用透射电镜对给药组和对照组骨折后3、7、14、21、28和42 d骨痂进行观察。结果两组骨折愈合早期(14 d前)参与修复的细胞类型,细胞功能状态几乎相同;术后14 d给药组成骨细胞和破骨细胞功能较对照组活跃;术后28 d给药组变性坏死的软骨细胞较对照组少;术后42 d成骨细胞和破骨细胞功能均消失。结论氟伐他汀既能促进成骨细胞功能,又能促进破骨细胞功能,以前者为著。     

降钙素治疗骨折的疗效观察及机制探讨

目的观察降钙素治疗骨折的疗效,并探讨其机制。方法选用64只2个月龄SD雌性大鼠,行双侧卵巢切除（OVX）后建立骨质疏松性骨折动物模型,随机分为观察组、对照组,各32只。观察组从骨折术前1周至术后8周每天皮下注射鲑鱼降钙素2IU／kg,对照组注射等量生理盐水。采用原位杂交技术检测7、14、28、56d骨痂中Ⅰ、Ⅱ、Ⅲ型胶原mRNA的表达。结果从骨折后第7天开始至第56天,各组Ⅲ型胶原mRNA表达水平逐渐下降,Ⅱ型胶原mRNA表达水平先升高后下降,Ⅰ型胶原mRNA表达水平逐渐升高。从第28天起,Ⅰ型胶原mRNA表达水平观察组〉对照组（P均〈0．05）。术后第56天观察组骨痂已为骨性,对照组可见较多软骨性骨痂。结论降钙索能促进软骨性骨痂向骨性骨痂转换,加速骨质疏松性骨折的愈合。其机制可能与降钙素能促进Ⅰ型胶原mRNA表达,抑制Ⅱ型胶原mRNA表达有关。     

颗粒酶B基因检测在心脏移植急性排异诊断中的作用

目的探讨心肌颗粒酶B基因mRNA表达水平在大鼠心脏移植急性排异中的变化及意义。方法应用大鼠腹腔异位心脏移植模型。实验组供体为Wistar大鼠,受体为SD大鼠,对照组供体与受体均为SD大鼠。半定量RT-PCR法检测术后第1、3、5、7、9、11天心肌颗粒酶B基因mRNA表达。病理学检查,心肌间质浸润细胞计数。结果实验组各时间点均有颗粒酶B基因mRNA表达,术后第1天表达水平较低,第3天明显升高,第11天达高峰,与移植心肌病理组织学损害程度正相关;实验组与对照组各时间点颗粒酶B基因表达均存在显著差异。结论检测心肌颗粒酶B基因表达水平可以用来诊断心脏移植急性排异。     

miR-126及VEGF在大鼠心肌梗死交界区表达的动态变化

目的 观察大鼠心肌梗死(MI)后MI交界区微小RNA-126(miR-126)及血管内皮生长因子（VEGF）表达的动态表达变化,初步探讨miR-126及VEGF对缺血局部血管新生的影响。方法 雄性SD大鼠结扎冠状动脉左前降支建立急性心肌梗死模型组(AMI组),另设假手术对照组(Sham组),每组30只。于术后7 d、14 d和28 d分别处死10只大鼠,进行MI面积百分比测定,实时荧光定量PCR法检测心梗交界区miR-126、VEGF mRNA表达水平,免疫组织化学方法检测各组大鼠MI交界区VEGF蛋白的表达。结果 Sham组术后7 d、14 d和28 d心肌组织 miR-126 mRNA和VEGF mRNA的表达均无明显区别,AMI组术后7 d、14 d和28 d,MI交界区mir-126 mRNA表达与相应的Sham组相比,均有不同程度的下降(P<0.01),而VEGF mRNA的表达则均有不同程度的增高(P<0.01);在AMI组,随着MI时间的延长,miR-126和VEGF mRNA的表达均逐渐升高,且在AMI 14 d达到高峰（P<0.05）。VEGF 的蛋白表达情况与其基因表达基本一致。结论 大鼠MI后VEGF mRNA及其蛋白的表达明显升高的同时伴随miR-126基因表达的降低,随着缺血时间延长,miR-126 mRNA表达有所恢复。     

老年骨质疏松患者股骨Runt相关转录因子2水平与骨密度的关系

目的探讨老年骨质疏松Runt相关转录因子(Runx)2水平与骨密度的关系。方法对19例老年骨质疏松(PO组)患者股骨干骨组织Runx2表达水平进行检测,并与11例无骨质疏松的创伤性骨折者(非PO组)比较。结果两组Runx2均于骨折后5~7 d于手术中获得。PO组Runx2表达值低于非PO组(P0.05),并且PO组Runx2水平与骨密度具有相关性。结论骨质疏松患者前成骨细胞分化为成骨细胞的能力下降。     

Involvement of activin in the regulation of bone metabolism

Osteogenic activities of activin, a member of TGF-ß superfamily, have been shown in both in vivo and in vitro studies. Local injection of activin promoted fracture healing in rat fibula fracture models. Since both activin and its receptor are expressed during fracture healing, activin would be involved in the healing process via autocrine and/or paracrine mode of action. Activin was abundantly stored also in normal bone matrix, presumably produced by osteoblasts in the process of normal bone formation. It was observed that activin was released in the culture of neonatal mouse calvaria, and the release was strongly coupled with bone resorption. Thus, activin could be involved in the regulation of bone remodeling as one of coupling factors, as was suggested for TGF-ß. Systemic administration of activin in aged ovariectomized rats, in which bone mass decreases due to uncoupling between bone resorption and formation, increased both bone mass and mechanical strength of vertebral bodies. These findings suggest physiological roles of activin in the regulation of bone formation, and further, its possible usefulness for the therapy of fracture and osteoporosis.     

Recruitment of host progenitor cells in rat liver transplants

Despite major histocompatibility complex incompatibility, liver transplants from Lewis rats to dark agouti (DA) rats survive indefinitely without immunosuppression, and the studies we report sought the mechanism(s) responsible for this. At 1 year, most of the liver reacted positively to host anti-DA antibody. When small (50%) grafts were transplanted, recruitment was more rapid because most of the organ assumed the host phenotype at 3 months. After transplantation, the Y chromosome was detected in the hepatocytes of XX to XY grafts by both in situ hybridization and polymerase chain reaction. Further, livers from transgenic Lewis rats carrying strong green fluorescent protein (GFP) markers lost the marker with time after transplantation to DA, GFP-negative hosts. Few liver cells contained the Y chromosome in syngeneic XX to XY liver grafts or when the hosts of Lewis XX to DA XY allografts were treated with cyclosporine A at 10 mg/kg/day. This dosage also impeded enlargement of the liver at 10 days. Using GFP-positive XX Lewis donors transplanted to GFP-negative XY DA hosts, we found little Y DNA in GFP-positive cells at 10 days. Host-derived OV-6-positive and c-kit-positive, albumin-positive cells were present at 3-10 days, but cells with the CD34 marker were less common and some clearly still had the donor phenotype at 10 days. Cells positive for chemokine cysteine-X-cysteine receptor-4 increased with time and were abundant 1 month after transplantation. We conclude: (1) extrahepatic cells can differentiate into liver tissues; (2) regenerative stimuli accelerate stem cell recruitment; (3) both regeneration and recruitment are impeded by cyclosporine A immunosuppression, and (4) donor GFP-positive cells contained little host Y chromosome after transplantation, suggesting that cell fusion was uncommon and, therefore, unlikely to be the mechanism leading to the changes in genotype and phenotype we observed.     

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.     

Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration

The hypoxia-inducible factor-1alpha (HIF-1alpha) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1alpha pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1alpha activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1alpha in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro. One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1alpha pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.     

Molecules which are involved in osteoclastic bone resorption: from the aspect of targets of treatment for osteoporosis

Bone in vertebrates is continuously destroyed and reformed to maintain bone volume throughout their life. In normal bone remodeling, osteoblastic bone formation follows osteoclastic bone resorption in a precise and quantitative manner. In osteoporosis, the bone resorption surpasses the bone formation. Recent studies have shown that osteoclastic bone resorption is tightly regulated by bone-forming osteoblasts. Osteoblasts express RANKL (receptor activator of nuclear factor-KappaB ligand), which plays central roles in osteoclast differentiation and function. In this review article, we explain molecules which are involved in the RANKL-RANK interaction in the regulation of osteoclastic bone resorption.     

Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass

Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use "Rs1," an engineered GPCR with constitutive G(s) signaling, to evaluate the temporal and skeletal effects of G(s) signaling in murine osteoblasts. In vivo, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.     

Effect of Topical PTH 1-34 Functionalized to Biogran® in the Process of Alveolar Repair in Rats Submitted to Orchiectomy

(1) Background: There are many therapies for osteoporosis control and bone maintenance; anabolic drugs such as teriparatide and bone grafts help in the repair process and stimulate bone formation. Thus, the aim of the present study was to evaluate the behavior of repaired bone in the presence of PTH (teriparatide) associated with Biogran^® (biomaterial) through a sonochemical procedure after extraction in rats. (2) Methods: The insertion of Biogran^® with PTH in the alveolus was performed 30 days after incisor extraction. Euthanasia occurred after 60 days. (3) Results: The use of local treatment of PTH loaded with Biogran® in healthy rats promoted good results for micro-CT, with an increase in percentage and bone volume, number and trabecular separation and less total porosity. Greater immunostaining for Wnt, β-Catenin and osteocalcin proteins and lower expression for Thrombospondin-Related Adhesive Protein (TRAP), which shows an increase in the number of osteoblasts and inhibition of osteoclast action. However, the treated orchiectomized groups did not obtain such expressive results. (4) Conclusion: The use of Biogran^® with PTH improved alveolar repair in rats. However, new researches with more efficient doses must be studied to collaborate effectively with the formation of a quality bone after the orchiectomy.     

Experimental studies on the long term fate of the vein and artery grafts and its choice in aortocoronary bypass surgery.

Experimental studies of aorto-coronary bypass were performed using the technique of distal coronary artery perfusion in 47 dogs to ellucidate the flow characteristics at various grafts and long term fate of grafts up to 440 days after operation. On short term results, early graft occlusion seems to occur within 2 weeks with 6.5 days of mean in Group I and II (free grafts) when inflammatory reaction is most prominent. After 2 weeks patency rate was 65% in Group I and II, more than 80% of patency rate were obtained in Group III. In Group IV (prosthetic grafts), all but few (3 cases) could survive for more than 24 hours. In flow characteristic, artery and vein graft of which diameter is larger than that of the coronary artery can maintain enough the basal flow after ligation of the proximal coronary artery. In Group III (IMA-Circumflex anastomosis group), the internal mammary artery must be large enough to carry the blood to coronary artery lest the graft flow becomes below the basal flow. In Group IV, grafts can carry full flow but long term studies were not done because of early graft occlusion. There are much differences between free artery and vein grafts. In artery graft except IMA proper arterial structure is being kept until 440 days but has arteriosclerotic wall changes similar to human being. On the contrary, almost all free vein grafts have subintimal hyperplasia with collagen deposit and fibrous tissue and still progress after one year. These findings demonstrate that ischemia and hydrauiic effect in the graft must be important factor for the irreversible graft wall changes.     

Possible benefits of strontium ranelate in complicated long bone fractures

Osteoporosis drugs are prescribed to prevent fragility fractures, which is the principal aim of the management of osteoporosis. However, if fracture does occur, then it is also important to promote a fast and uneventful healing process. Despite this, little is known about the effect of osteoporosis drugs on bone healing in humans. Strontium ranelate is an osteoporosis agent that increases bone formation and reduces bone resorption and may therefore be beneficial in fracture healing. We report four cases of fracture non-union for up to 20 months. Treatment with strontium ranelate (2 g/day) for between 6 weeks and 6 months appeared to contribute to bone consolidation in the four cases. Animal studies support beneficial effects of strontium ranelate on bone healing via improvement of bone material properties and microarchitecture in the vicinity of the fracture. The clinical cases described herein provide new information on these effects, in the absence of randomized controlled studies on the clinical efficacy of pharmacological treatments in osteoporosis in fracture repair. Further studies are necessary. Fracture healing is an important topic in orthopedic research and is also a concern for patients with postmenopausal osteoporosis. Evidence from case reports and animal studies suggests that strontium ranelate improves bone microarchitecture and accelerates fracture healing. A positive effect of osteoporosis treatments on bone healing is an interesting possibility and merits further clinical research.     

Alcohol-induced bone loss and deficient bone repair

BACKGROUND: Chronic consumption of excessive alcohol eventually results in an osteopenic skeleton and increased risk for osteoporosis. Alcoholics experience not only increased incidence of fractures from falls, but also delays in fracture healing compared with non-alcoholics. In this review the term "alcohol-induced bone disease" is used to refer to these skeletal abnormalities. Alcohol-induced osteopenia is distinct from osteoporoses such as postmenopausal osteoporosis and disuse osteoporosis. Gonadal insufficiency increases the rate of bone remodeling, whereas alcohol decreases this rate. Thus, histomorphometric studies show different characteristics for the bone loss that occurs in these two disease states. In particular, alcohol-induced osteopenia results mainly from decreased bone formation rather than increased bone resorption. Human, animal and cell culture studies of the effects of alcohol on bone strongly suggest alcohol has a dose-dependent toxic effect on osteoblast activity. The capacity of bone marrow stromal cells to differentiate into osteoblasts has a critical role in the cellular processes involved in the maintenance of the adult human skeleton by bone remodeling. Chronic alcohol consumption suppresses osteoblastic differentiation of bone marrow cells and promotes adipogenesis. In fracture healing, the effect of alcohol is to suppress synthesis of an ossifiable matrix, possibly due to inhibition of cell proliferation and maldifferentiation of mesenchymal cells in the repair tissue. This results in the deficient bone repair observed in animal studies, characterized by repair tissue of lower stiffness, strength and mineral content. Current knowledge of cellular effects and molecular mechanisms involved in alcohol-induced bone disease is insufficient to develop interventional strategies for its prevention and treatment. OBJECTIVES: The objectives of this review are 1) to identify the characteristics of alcohol-induced bone loss and deficient bone repair as revealed in human and animal studies, 2) to determine the current understanding of the cellular effects underlying both skeletal abnormalities, and 3) to suggest directions for future studies to resolve current ambiguities regarding the cellular basis of alcohol-induced bone disease.