Synergy between IL-6 and soluble IL-6 receptor enhances bone morphogenetic protein-2/absorbable collagen sponge-induced bone regeneration via regulation of BMPRIA distribution and degradation

Bone morphogenetic protein-2/absorbable collagen sponge (BMP-2/ACS) implants have been approved for clinical use to induce bone regeneration. We previously showed that exaggerated inflammation characterized by elevated level of inflammatory cytokines including TNF-α, IL-1β, and IL-6 has been shown to inhibit BMP-2/ACS-induced bone regeneration. Furthermore, unlike the negative effects of TNF-α and IL-1β, IL-6 seemed not to affect BMP-2-induced osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). We hypothesized that there may be a regulatory loop between IL-6 and BMP-2 singling to affect BMP-2/ACS-induced bone regeneration. Here, we established a BMP-2/ACS-induced ectopic bone formation model in rats and fund that IL-6 injection significantly increased BMP-2/ACS-induced bone mass. Consistent with this animal model, an in vitro study demonstrated that synergy between IL-6 and soluble IL-6 receptor (IL-6/sIL-6R) promotes BMP-2-induced osteoblastic differentiation of human BMSCs through amplification of BMP/Smad signaling. Strikingly, IL-6 injection did not activate osteoclast-mediated bone resorption in the ectopic bone formation model, and IL-6/sIL-6R treatment did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastic differentiation of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, IL-6/sIL-6R treatment did not affect expression of BMP receptors, but enhanced the cell surface translocation of BMP receptor IA (BMPRIA) and inhibited the degradation of BMPRIA. Collectively, these findings indicate that synergy between IL-6 and sIL-6R promotes the cell surface translocation of BMPRIA and maintains the stability of BMPRIA expression, leading to enhanced BMP-2/ACS-induced bone regeneration.     

骨皮质切开术辅助大鼠正畸牙移动中OPNmRNA、BSPmRNA、OCNmRNA表达的研究

目的:观察骨桥蛋白(OPN),骨涎蛋白(BSP),骨钙蛋白(OCN)在大鼠骨皮质切开术辅助正畸牙移动过程中的表达情况。方法:选取健康雄性Wistar大鼠65只,60只大鼠随机分成3组:组Ⅰ:选择性骨皮质切开术;组Ⅱ:传统的牙齿移动;组Ⅲ:混合组,剩余5只作为空白对照组(不作任何处理)。用镍钛螺旋拉簧施加约60 g力近中移动上颌实验侧第一磨牙,在第一磨牙颊腭侧行骨皮质打孔术式,分别在加载第7、14、21、28、42天颈椎脱臼处死取材,进行SYBR Green实时荧光定量PCR技术,对Ct值进行分析。结果:混合组中的成骨细胞相关细胞因子:OPN(骨桥蛋白),Bone sialoprotein(BSP骨涎蛋白),Osteocalcin(OCN骨钙蛋白)的表达早期增加暗示了同化合成反应的增加。结论:正畸牙齿移动初期骨皮质切开术加速了牙齿移动,改变了早期骨代谢状态,成骨活跃,支持区域性骨代谢加速学说。     

Silk proteins stimulate osteoblast differentiation by suppressing the Notch signaling pathway in mesenchymal stem cells

Silk fibroins are biomaterials that have been applied to surgical sutures, drug delivery systems, food supplements, and tissue engineering. Studies have shown the antiadipogenic effects of silk proteins in 3T3-L1 cells and obese mice. Furthermore, other studies have shown that silk proteins increase osteogenic marker expression in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are often regulated reciprocally, we hypothesized that silk proteins would stimulate osteoblast differentiation. The objective of this study was to evaluate the effects of silk proteins on promoting osteoblast differentiation and identify the underlying mechanism. We showed that silk proteins dose dependently stimulated alkaline phosphatase (ALP) activity, osteoblast differentiation, and induced expression of osteoblast markers in C3H10T1/2 and M2-10B4 multipotent cells. In addition, silk proteins also induced the expression of osteoblast markers in primary rat bone marrow cells isolated from tibiae. Molecular studies showed that silk proteins suppressed the expression of Notch-activated genes and blocked activation of the Notch-specific reporter. Similarly, inhibiting Notch signaling with pharmacologic inhibitors and by small interfering RNA–mediated Notch1 silencing also induced ALP activity and messenger RNA expression. Finally, induction of ALP activity and messenger RNA expression by silk proteins was blunted in Notch1 knock-downed cells, further demonstrating Notch signaling as an important mediator for the pro-osteogenic effects of silk proteins. Taken together, our data suggest that silk proteins may serve as functional foods to promote bone healing and therapeutic interventions for bone fractures and osteoporosis.     

中药骨康含药血清培养脂肪干细胞移植促进骨质疏松性骨折愈合过程中cbfa1和OCN的表达变化

目的 探讨中药骨康含药血清培养脂肪干细胞移植促进骨折愈合过程中骨折端cbfa1和OCN的动态变化。方法 制备中药骨康含药血清并进行脂肪干细胞培养,移植脂肪干细胞到骨折部位,用免疫组化观察骨折部cbfa1和OCN的动态变化。结果 第3 代脂肪干细胞流式鉴定CD90阳性,CD34、CD45低表达。免疫组化观察骨折部cbfa1和OCN的动态变化：第一周干预组Cbfal和OCN组均可见血肿、肉芽组织及纤维性骨痂组织中出现阳性细胞和基质阳性信号,阳性较强,对照组可见阳性细胞和基质阳性信号,阳性较弱,空白组基本上无阳性表达;第二周Cbfal和OCN组均出现软骨岛和新生骨小梁,胶原纤维排列整齐,大量阳性细胞和基质阳性信号,强阳性表达, 对照组骨小梁和软骨岛均出现,但比干预组少,亦出现阳性细胞和基质阳性信号,信号比干预组弱,空白组少见软骨岛和骨小梁,胶原纤维排列混乱,阳性细胞和基质阳性信号亦可见,但比前两组要弱;第四周干预组Cbfal组阳性表达减弱,OCN组继续强阳性表达,对照组两指标均阳性表达,空白组阳性细胞和基质阳性信号存在,阳性程度比以往稍强。结论 通过免疫组化观察骨折部cbfa1和OCN的动态变化,表明脂肪干细胞移植可促进骨质疏松性骨折愈合,而中药骨康含药血清培养脂肪干细胞移植效果更理想。     

Drynaria fortunei J. Sm. promotes osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative stress-induced apoptotic insults

Aim of the study

Drynaria fortunei J. Sm. is one variety of the traditional Chinese medical herb Gusuibu. This study was aimed to evaluate the effects of water extracts of Kunze on regulation of osteoblast maturation and its possible mechanisms.

Materials and methods

Primary osteoblasts prepared from neonatal rat calvarias were exposed to the water extracts of Kunze (WEK), and the cytotoxicity was assayed. Osteoblast maturation was evaluated by analyzing cell mineralization. RT-PCR was executed to determine the effects of WEK on regulation of osteoblast differentiation-related gene expression. Nitrosative stress and apoptotic cells were quantified using flow cytometry.

Results

Exposure of rat calvarial osteoblasts to WEK did not affect cell viability, but significantly promoted osteoblast mineralization. WEK induced osteoprogenitor proliferation-related insulin-like growth factor-1 mRNA, but did not affect collagen type 1 mRNA expression. Treatment with WEK likewise induced the expression of matrix maturation-related bone morphogenetic protein (BMP)-2 and BMP-6 mRNA. Consequently, WEK enhanced the levels of mineralization-related alkaline phosphatase, ostepontin, and osteocalcin mRNA in osteoblasts. In addition, exposure of osteoblasts to WEK alleviated nitrosative stress-caused apoptotic insults.

Conclusions

This study shows that WEK can promote osteoblast maturation by regulating bone differentiation-related gene expression and defending against nitrosative stress-induced apoptotic insults.     

A traditional Chinese herbal preparation, Er-Zhi-Wan, prevent ovariectomy-induced osteoporosis in rats

Ethnopharmacological relevance

Er-Zhi-Wan (EZW), a classic Chinese formulation, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various kidney diseases for its actions of nourishing the kidney and strengthening tendon and bone. Although recent reports indicate that EZW restrains osteoclastic bone resorption, its effects on the protection against define OVX-induced bone loss in mature rats have not been systematically investigated.

Materials and methods

Sixty 3-month-old female Sprague-Dawley rats were randomly assigned into sham-operated group (Sham) and five OVX subgroups, OVX with vehicle (OVX); OVX with Estradiol Valerate (EV, 0.4 mg/kg body weight/day); OVX with EZW of graded doses (9.0, 4.5, or 2.25 g/kg/day). Daily oral administration of EV and EZW on 5th week for 26 weeks. Bone turnover markers (Serum alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OCN), deoxypyridinoline (DPD)), other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), phosphorus (U-P), and bone mineral density (BMD) of the femur, 4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture parameters were measured.

Results

Administration of EZW could significantly prevent ovariectomy-induced bone loss, biomechanical reduction, deterioration of trabecular microarchitecture and the body weight gain without affecting the weight of the uterus, and increased S-Ca, S-P levels, decreased level of bone turnover markers and U-Ca, U-P levels in ovariectomized rats.

Conclusion

The present study indicated that EZW had a definite antiosteoporotic effect without hyperplastic effect on uterus, and it might be a potential alternative medicine for treatment of postmenopausal osteoporosis.     

骨钙素(OCN)与非酒精性脂肪肝(NAFLD)关系的研究进展

 骨钙素(osteocalcin,OCN)是由成骨细胞生成并特异性分泌的非胶原蛋白,参与调节胰岛素分泌、葡萄糖代谢和能量消耗,成为骨和调节能量代谢器官的复杂信号通路之间的重要话串的关键分子。肝脏作为代谢中枢器官参与多种物质代谢。动物及细胞分子学实验显示,OCN具有改善肝脏脂肪过量沉积的作用,其机制可能与OCN降低血清三酰甘油水平、促进胰岛素分泌、改善胰岛素抵抗、减轻氧化应激及炎性反应等相关。多项临床研究表明,OCN与糖代谢、脂代谢、肥胖等代谢异常密切相关,但OCN与非酒精性脂肪肝病(non alcoholic fatty liver disease,NAFLD)的关系研究目前仍存在争议。