淫羊藿苷、黄芩苷和大黄素联合作用对体外骨吸收的影响

目的研究淫羊藿苷、黄芩苷和大黄素单独及联合作用对体外骨吸收的影响。方法将从新生兔长骨分离的细胞与牙本质磨片共培养,建立体外骨吸收模型,并加入白细胞介素(IL)-1β及淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml或其等比组合进行干预,动态观察并测量吸收陷窝的数量和面积。结果分离的细胞与牙本质片共同培养24 h后,牙本质片上出现少量吸收陷窝。体外共培养10 d,加入IL-1β的阴性对照组的陷窝数量和面积大于空白对照组(P<0.05)。同时加入提取物的各组陷窝数量和面积均小于阴性对照组(P<0.05)。其中,淫羊藿苷组、黄芩苷组和大黄素组3组之间无显著差异(P>0.05),而组合组陷窝数量和面积均小于上述3组(P<0.01)。结论淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml等比组合可抑制IL-1β诱导的骨吸收。     

白细胞介素-6对破骨细胞骨吸收效应及MMP-3表达影响的实验研究

目的：观察不同浓度IL－6对破骨细胞骨吸收的剂量效应及对破骨细胞基质金属蛋白酶－3表达的影响，以期进一步阐明IL－6介导基质金属蛋白酶在破骨细胞性骨吸收中的病理机制。方法：采用体外破骨细胞溶骨模型，通过原子吸收分光光度仪及免疫组化染色技术检测不同浓度IL－6对破骨细胞溶骨活性及其质金属蛋白酶－3表达的影响。结果：当IL－6＞10U／ml时，培养上清中Ca^2 浓度显著增加，牙本质片上骨吸收陷窝数目明显增多；当IL－6浓度为100U／ml、500U／ml时破骨细胞基质金属蛋白酶－3表达的阳性信号显著增强。结论：在IL－6作用下，破骨细胞表达基质金属蛋白酶－3。IL－6对破骨细胞具有激活作用，低浓度主要诱导破骨细胞形成，较高浓度刺激破骨细胞的溶骨活性。     

The effect of lipopolysaccharide from the oral bacterium Bacteroides gingivalis on osteoclastic resorption of sperm-whale dentine slices in vitro

Physiological mediators of bone resorption stimulate osteoclasts indirectly via osteoblasts. The aim was to determine whether osteoblasts are necessary for lipopolysaccharide (LPS) to stimulate osteoclastic resorption. Osteoblasts were prepared from neonatal rat calvaria by sequential digestion, and co-cultured on slices of sperm-whale dentine with osteoclasts disaggregated from neonatal rat long bones. Groups of 12 slices were cultured for 24 h in culture media alone, or media containing either 100 ng/ml parathyroid hormone (PTH) or 0.1, 1 or 10 μg/ml LPS extracted from Bacteroides gingivalis. After incubation, the slices were cleaned and viewed by scanning electron-microscopy. PTH and each concentration of LPS caused a statistically-significant increase in the mean area of resorption per dentine slice when compared with controls. When only isolated osteoclasts were cultured on dentine slices and incubated under similar conditions, none of the test media significantly stimulated bone resorption above that of controls. Thus LPS stimulates osteoclastic bone resorption indirectly via osteoblasts.     

Bone resorption stimulated by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella, and by the lipid A and the polysaccharide part of Fusobacterium lipopolyccharide

Lipopolysaccharides (LPS) isolated from oral strains of Veillonella, Fusobacterium and Bacteroides stimulated the release of ⁴⁵Ca from prelabeled fetal rat bones in culture. There was a typical dose-response relationship berween the quantities of released ⁴⁵Ca and LPS used for stimulation. Bacteroides-LPS proved to be the less active inducer of ⁴⁵Ca release. LPS had no stimulating effect on the release of ⁴⁵Ca from devitalized bone. The stimulated ⁴⁵Ca release was paralleled by an increase in the culture midium of hydroxyproline and lactate. This, together with the findings of numerous osteoclasts in stained hisological specimens of the experimental bones, indicates that LPS stimulated the osteoclasts to bone resoption. Heparin, which did not directly induce ⁴⁵Ca release, potentiated the bone resorption stimulating capability of LPS. The lipid A and the polysaccharide portion of Fusobacterium LPS also stimulated bone resorption and, remarkably, the polysaccharide portion showed the greatest activity. This may explain the mode of action of LPS lacking a typical lipid A. It is suggested that stimulation of osteoclasts by LPS may result from activation of complement by lipid A or its polysaccharide portion.     

Effect of outer membrane of Treponema denticola on bone resorption

The effect of the outer membrane (outer sheath) of Treponema denticola on bone resorption was studied. Bone resorption was measured by the release of previously incorporated ⁴⁵Ca from the shafts of the radii and ulnae of 19-day fetal rats. A treated-over-control ratio (T/C ratio) significantly greater than 1 indicated the stimulation of bone resorption by the test substance. The addition of outer membrane of T. denticola increased the release of ⁴⁵Ca from the assay bones. The minimum concentrations required to yield significant ⁴⁵Ca release from the assay bones were 15, 22 and 75 μg protein/ml for serovars a, b and c, respectively. These protein values corresponded to estimated lipopolysaccharide contents of 0.6, 0.8 and 2.8 μg/ml, based on 3-deoxy-2- manno -octulosonate analysis. Heat treatment of outer membrane (60° for 30 min) did not change the effect on ⁴⁵Ca release. Parathyroid hormone or prostaglandin E₂ known to act synergistically with lipopolysaccharides in bone resorption, was also added to the assay system. Neither prostaglandin E₂ at 10^-7 M nor parathyroid hormone at 40 ng/ml, by itself, increased ⁴⁵Ca release. However, in the presence of 10 μg protein/ml of outer membrane of serovar b at 120 h, the T/C ratio was increased to 1.31±0.07 and 1.58±0.118, respectively. These results suggest that a lipopolysaccharide-like material is present in the outer membrane of T. denticola that may be responsible for bone resorption in the in vitro system.     

辛伐他汀对PTHrP诱导破骨细胞形成及小鼠颅盖骨代谢的影响

目的:探讨辛伐他汀对体外甲状旁腺素相关肽(PTHrP)诱导小鼠的破骨细胞骨吸收功能的作用及其小鼠骨代谢的影响。方法:采用PTHrP诱导小鼠骨髓细胞培养破骨细胞和小鼠颅盖骨培养体系,检测辛伐他汀作用8d后破细胞骨数目和培养上清钙的变化;检测小鼠颅盖骨培养上清碱性磷酸酶和钙含量,组织学观察小鼠颅盖骨形态学变化。结果:辛伐他汀体外可明显抑制PTHrP诱导小鼠的破骨细胞骨吸收陷窝的形成及培养上清钙的释放,辛伐他汀体外可增强小鼠颅盖骨培养上清碱性磷酸酶的活性,组织学观察到辛伐他汀使小鼠颅盖骨矿化增强。结论:辛伐他汀体外不仅可促进小鼠颅盖骨的成骨活性,并且可明显抑制PTHrP诱导小鼠的破骨细胞骨吸收功能,对骨吸收性疾病有着重要的防治作用。     

Effects of 1,25 - （OH） 2 D3 on Stimulation of Osteoclast - like Cells Formationand Expression of ODF mRNA in Murine Marrow Cells in Vitro

目的：观察不同浓度的1，25-(OH)2D3对破骨细胞形成及对骨髓细胞ODFmRNA表达的影响：进一步阐明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-(OH)2D3(0、10^-10、10^-8、10^-6mol／L)诱导大鼠骨髓细胞破骨样细胞的形成，采用体外破骨细胞溶骨模型，观察牙本质片上骨吸收陷窝数目，采用原位杂交技术检测骨髓基质细胞ODF的mRNA表达。结果：随着1，25-(OH)2D3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-(OH)2D3可以调节破骨细胞的骨吸收活性，进而调节局部骨微环境的骨吸收及骨形成平衡的变化。     

Effects of locally-delivered human macrophage products and estrogen on murine inflammatory bone resorption

The objective of this study was to use an in vivo model of periodontitis (mouse calvaria) to quantify the effects of local release of secreted human macrophage products, 17beta-estradiol (E2), and proinflammatory lipopolysaccharide (LPS) on histologic bone resorption. Human THP-1 monocytes (106) were converted to macrophage phenotype by 500 ng/ml phorbol 12-myristate- 13-acetate (PMA) and treated as follows: no stimulation or Escherichia coli LPS (10 microg/ml) alone or in combination with a physiologic dose of E2 (100 pg/ml) for 24 h in RPMI/10% FBS, washed extensively, then incubated for 24 h in serum-free media. Supernatant products were concentrated and incorporated into a 4% (w/v) methylcellulose gel. Separate gels were incorporated with the following: LPS (500 microg/animal) alone, high dose of E2 (10 ng/animal) alone, a combination of LPS + E2, or gel only (controls). Loaded or control gels were placed into a polylactic acid occlusive dome, inserted subcutaneously over the calvaria of mature ovariectomized ICR Swiss mice (8 mice x 7 groups x 2 times [5/14 days] = 112 animals), then calvaria were evaluated histologically. Macrophage stimulation with LPS alone, but not LPS in combination with E2, produced supernatants which upregulated osteoclast numbers in the suture area compared to gel controls at 5 days (p = 0.009). The addition of LPS directly to the local delivery gels significantly upregulated osteoclasts in endosteal surfaces compared to gel controls at 5 days (p = 0.024) and at 14 days (p = 0.025). The addition of E2 to LPS down-regulated resorption to a level not different from gel controls at 14 days. This in vivo model appears effective in studying inflammatory bone resorption, which may be inhibited by E2 directly or through its influence on secreted macrophage products.     

淫羊藿对口腔各矿化组织破骨细胞性骨吸收的体外实验研究

目的 探讨中药淫羊藿抑制破骨细胞性骨吸收的作用。方法 体外分离、培养兔破骨细胞，与玻片及灭活牙片共同培养，加入不同浓度淫羊藿注射液。HE、原位末端标记染色玻片上的破骨细胞，观察其形态结构的改变，并观察破骨细胞在牙片上形成的吸收陷窝数目及面积的变化。结果 HE染色可见用药组破骨细胞胞质浓缩，核固缩深染，部分细胞出现核分裂。原位末端标记结果显示，用药组破骨细胞胞质皱缩，胞核呈棕褐色，胞质淡杂。提示淫羊藿可诱导破骨细胞凋亡，抑制骨吸收。用药组与非用药组破骨细胞凋亡率差异有显著性，吸收陷窝数目、面积差异也有显著性，随浓度增加抑制作用增强。结论 淫羊藿可诱导破骨细胞凋亡，抑制骨吸收，并随浓度增加抑制作用增强。     

Interleukin-2 stimulates osteoclastic activity; Increased acid production and radioactive calcium release

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption.     

破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究

目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。     

铜离子对牙各矿化成分破骨细胞性吸收的体外调控

目的研究铜离子对破骨细胞体外吸收人牙磨片功能的影响。方法体外分离、培养兔破骨细胞，与玻片和灭活人牙磨片共同培养，加入不同浓度的铜离子。抗酒石酸酸性磷酸酶染色鉴定玻片上的破骨细胞，显微摄影分析破骨细胞吸收造成的牙磨片上的吸收陷窝，原子吸收分光光度法测定溶出的钙，并将实验组上清液中钙离子浓度与对照组相比，定义为矿化组织吸收指数，以评价破骨细胞的功能。结果体外成功分离培养出多核的、抗酒石酸酸性磷酸酶染色（＋）的破骨细胞。破骨细胞吸收牙磨片时，首先在接近牙骨质或牙本质的部位开始形成吸收陷窝；破骨细胞在牙磨片上形成的吸收陷窝与骨片相比，数量较少，体积较小，多为正圆形；吸收深度较浅，常为大面积的浅吸收。（1×10^-14）～（1×10^-4）mol／L铜离子均能抑制矿化组织吸收，实验组矿化组织吸收指数均小于1。培养第3天，1×10^-10mol／L铜离子与对照组相比，其抑制效果差异有统计学意义（P〈0．05）。培养第7天，1×10^-4mol／L、（1×10^-10）～（1×10^-12）mol／L铜离子均能显著抑制矿化组织吸收（P〈0．05），但各浓度之间相比差异无统计学意义（P〉0．05）。结论一定浓度的铜离子可以抑制破骨细胞在牙磨片上的吸收。     

Effect of human dental bacterial plaque extract on the connective tissue of indashvitro cultured fetal rat calvaria

Plaque extract (50 βg protein/ml) stimulated the synthesis of glycosaminoglycans (GAG) in cultured calvaria expiants. The production of hyaluronic acid was increased (206–309 per cent) in the medium of bone expiants cultured without fetal calf serum. In the presence of serum the synthesis of GAG was increased in control bones to the level of plaque-treated bones. The bone nitrogen content remained relatively unchanged, but dry weights, DNA and collagen contents were all decreased in plaque-treated bones. Bone resorption measured as ⁴⁵Ca-release was enhanced in plaque-treated (173 ± 18 percent) and also parathormone-treated (155 ± 10 percent) bones. Serum in culture medium reduced the differences between treated and control bones by increasing the release of ⁴⁵Ca from control bones. Plaque-induced bone resorption may be caused by increased cell secretion of GAG and inhibition of collagen synthesis or by increased collagen breakdown. Loss of mineral accompanies this modification of the organic substrate.     

IL-6 levels in gingival crevicular fluid (GCF) from patients with non-insulin dependent diabetes mellitus (NIDDM), adult periodontitis and healthy subjects

Cytokines play an important role in the pathology associated with chronic inflammatory diseases. One of these cytokines, interleukin 6 (IL-6) is a major mediator of the host response to tissue injury, infection and bone resorption. In the present study, gingival crevicular fluid (GCF) level of IL-6 was determined in patients with non-insulin dependent diabetes mellitus (NIDDM) with periodontitis, adult periodontitis, and healthy controls by use of an enzyme linked immunosorbent assay (ELISA). Twenty-four NIDDM patients with periodontitis, twenty-four adult periodontitis and twenty-four healthy controls were selected for the study. GCF sampling was performed on the vestibular aspects of maxillary incisors and canine teeth. Plaque index (PI), gingival index (GI), gingival bleeding time index (GBTI), probing depth (PD) and probing attachment levels (PAL) were recorded from each sampling area and also the entire dentition. NIDDM and adult periodontitis patients had numerous sites with radiographic evidence of alveolar bone resorption, loss of attachment and pocket depth greater than 3 mm. The mean GCF IL-6 level was 2.43 +/- 0.97 ng/ml in NIDDM patients, 1.31 +/- 0.92 ng/ml in adult periodontitis and 0.62 +/- 0.58 ng/ml in healthy subjects, respectively (p < 0.05). GCF IL-6 levels were markedly higher in NIDDM and adult periodontitis groups compared to the healthy controls. No correlation was found between GCF IL-6 levels and all clinical parameters. These findings suggested that GCF IL-6 levels were significantly higher in the area of inflammation and periodontal destruction locally. The high IL-6 levels in NIDDM patients might be due to different microbial flora in periodontal pockets and altered immune system. Future studies are needed to evaluate the complex interaction among IL-6 GCF levels, host response and local microbial environment in the NIDDM patients.     

BioAggregate和MTA对体内破骨细胞分化和功能的影响

目的研究口腔生物陶瓷材料Bio Aggregate及MTA对体内LPS诱导的炎性骨吸收的影响。方法 6周龄C57/BL6雄性小鼠随机分为四组:PBS组、LPS组、Bio Aggregate+LPS组和MTA+LPS组,每组6只。LPS干预小鼠7 d后,取出颅盖骨进行显微-CT扫描、HE染色、酶组织化学染色和组织免疫荧光染色,观察骨破坏面积、破骨细胞的形成以及组织蛋白酶K(cathepsin K)的表达。结果 Bio Aggregate和MTA浸提液明显减少LPS诱导的破骨细胞形成和小鼠颅盖骨炎性骨破坏。与破骨细胞功能密切相关的组织蛋白酶K的表达在Bio Aggregate和MTA浸提液的作用下被显著下调。结论新型口腔生物陶瓷材料Bio Aggregate和传统材料MTA能够抑制体内炎性骨吸收。     

前列腺素E2对鼠头盖骨钙代谢的作用

目的：观察前列腺素E2（PGE2）对骨细胞钙代谢的作用。方法：原子吸收分光光度计检测骨器官培养上清中Ca^2 浓度。结果：0．01－0．1ng/ml浓度PGE2促进Ca^2 吸收，10－100ng/ml浓度PGE2促进Ca^2 释放。结论：PGE2在骨钙代谢中的作用与其局部浓度有关。     

Characterization of bone resorbing activity in gingival crevicular fluid from patients with periodontitis

BACKGROUND: In attempts to elucidate factors stimulating bone resorption in patients with different inflammatory diseases in the vicinity of the skeleton, e.g., peridontal disease and rheumatoid arthritis, we are investigating the presence of bone-resorbing activity in a variety of inflammatory exudates. The aim of the present study was to characterize the bone-resorbing activity present in patients with periodontitis. METHODS: Bone-resorbing activity was assessed in gingival crevicular fluids (GCFs) collected from patients with periodontitis and from patients with no signs of gingivitis. Bone-resorbing activity was evaluated by analyzing the capacity of GCFs to stimulate the release of minerals and the breakdown of bone matrix proteins in cultured neonatal mouse calvariae. The concentrations of IL-1alpha, IL-1beta and PGE2 were determined with ELISA and RIA techniques, respectively. RESULTS: GCF eluates from 24 different healthy sites caused a 1.23+/-0.05 fold stimulation of 45Ca release, whereas GCF eluates from 45 different diseased (periodontitis) sites caused a 2.46+/-0.10 fold stimulation. The effect on 45Ca release was time- and concentration-dependent, inhibited by 3 different osteoclast inhibitors and associated with enhanced release of 3H from [3H]-proline-labelled bones. The activity in GCF causing enhanced 45Ca release was unaffected, or in some samples partially reduced, by ultrafiltration using a filter with a molecular weight cut-off of 3000 Daltons. The bone-resorbing activity was temperature sensitive (+90degrees C, 10 min). The concentrations of prostaglandin E2 (PGE2) in the diluted GCF eluates, used in the bone resorption bioassay, were too low to be responsible for the release of 45Ca. Antisera specifically neutralizing human IL-1a inhibited the stimulatory effect of GCF pooled from several diseased sites. The specific, recombinant human IL-1 receptor antagonist completely inhibited the effect of pooled GCFs. GCF eluates from diseased sites contained human IL-1alpha and IL-1beta at concentrations of 1838+/-294 pg/ml and 512+/-91 pg/ml, respectively. CONCLUSIONS: These data show that GCF contains activity(ies) stimulating osteoclastic bone resorption in vitro. The factor primarily responsible for this activity seems to be IL-1alpha, but IL-1alpha is not the sole activator of bone resorption in GCF.     

Calcium deficiency, pregnancy and lactation in rats

Adult female rats were subjected to severe calcium deprivation by feeding them a calcium-deficient diet containing oxalate (Test group a), and in addition by subjecting some of them to pregnancy and lactation (Test group b). The mandibles were collected for determination of ash and cortical bone area.
The ash content of the mandible was reduced in both test groups, but the additional effect of pregnancy and lactation was less than that previously found in long bones. Planimetric analysis revealed that bone removal had not occurred from the periosteal surfaces of the mandible. Most of the bone loss had taken place by resorption from its endosteal surfaces.     

Secretion of a bone resorbing factor by epithelial cells cultured from porcine rests of Malassez

In the present study we have investigated the effect of factors released by cells derived from porcine epithelial rests of Malassez (ERM) on bone resorption in vitro . The osteolytic effect of these cells was tested by co-culturing them with fetal rat calvariae and then measuring the cumulative change in calcium concentration of the culture medium. When ERM cells were cultured with calvariae for 4 days, there was a significant increase in calcium release from the bones compared with bones cultured without ERM. ERM cells alone did not change the calcium concentration of the culture medium. When calvariae there cultured in medium that had been collected from ERM cultures, significant bone resorption could also be obtained. Indomethacin (200 ng/ml), an inhibitor of prostaglandin synthesis, inhibited calcium release from control calvariae and from calvariae cultured with ERM. However, calvariae cultured in the presence of ERM and indomethacin released significantly more calcium than those cultured in the presence of indomethacin alone. Thus, factors other than PG account for the osteolytic effect of ERM. Other cells cultured from oral tissues, such as gingival fibroblasts and fibroblasts from periodontal ligament as well as rat osteosarcoma cells also stimulated calcium release from calvariae. These results show that many cell types have the capacity to release bone resorbing factors in vitro and support the belief that under suitable conditions in vivo breakdown of alveolar bone may be stimulated by tissues proximal to it.     

Stimulation of bone resorption in cultured mouse calvaria by met-lys-bradykinin

Met-lys-bradykinin, which may be released from kininogen due to cleavage by neutrophil leukocyte acid proteases, stimulated ⁴⁵Ca release from prelabelled mouse calvarial bones. The stimulatory effect of met-Iys-bradykinin on ⁴⁵Ca release was inhibited by indomethacin, meclofenamic acid, flurbiprofert, and calcitonin. Met-lys-bradykinin caused a dose-dependent stimulation of prostaglandin E₂ production in osteoblast-enriched mouse calvarial bone cells. These findings indicate that increased biosynthesis of prostaglandins, capable of stimulating osteoclast-mediated bone resorption, may be implicated in the mechanism by which metlys-bradykinin stimulates bone resorption. Our observations suggest that, in addition to bradykinin and lys-bradykinin, met-lys-bradykinin may be a potential mediator of bone resorption in inflammatory lesions.