降钙素基因相关肽对成骨样细胞与血管内皮细胞交互作用的调控机制研究
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摘要
骨折修复重建过程中，再生神经和血管系统对骨痂发挥了严密的、实时的调控作用，相互协调，最终完成骨痂的结构和功能重建。周围神经所分泌的神经肽类物质，降钙素基因相关肽(calcitonin gene related protein，CGRP)，在骨折修复过程中发挥了促进骨痂生成的生物学作用。体外环境下CGRP能够促进成骨细胞增殖，并证明成骨细胞表面有CGRP受体的存在，大量下游细胞信号通路得到证实。但细胞实验研究中，多数实验仅围绕CGRP对成骨细胞(Osteoblasts, OB)或破骨细胞(Osteoclasts, OC)等单一成骨相关细胞展开，而对多细胞间的交互调控作用(Cross-talk)研究较少，因此未能全面完整地说明CGRP在体内多细胞环境下的促成骨机制。
成骨细胞和血管内皮细胞(Vascular endothelial cells, VECs)之间存在一种相互影响、相互调控的OB-VECs间cross-talk机制。在骨痂中VECs不仅仅构建了微血管簇为骨痂提供了营养支持，还作为一种具有成骨诱导功能细胞，参与了MSCs的募集和诱导分化。研究发现细胞间cross-talk主要发生在两个层面，一是经由细胞膜之间的蛋白完成细胞与细胞间的直接作用，二是通过自分泌和旁分泌的方式完成细胞间的间接作用。而在骨折愈合过程中，骨痂中的CGRP阳性纤维沿着血管生长，并且有大量的体外实验证实，CGRP在血管再生和形成中也发挥着重要的生物学作用，说明CGRP参与了对OB-VECs之间Cross-talk的调控。
基于骨痂中的CGRP对成骨的调控机制可能与VECs细胞相关这一推测，本实验依据课题组的前期预实验结果，建立CGRP干预的OB-VECs共培养体系体，通过对VECs细胞旁分泌成骨因子的检测，和OB细胞对应因子受体表达及细胞分化指标的检测，探讨CGRP对OB-VECs间cross-talk的调控机制。从而在体内及体外多细胞环境下，发现并证实神经通过对VECs旁分泌的调控作用发挥了促成骨效应，进一步阐述骨痂中神经对血管和成骨交互调控的作用机制。
方法：
1.细胞培养：美国ATCC公司人脐静脉血管内皮细胞HUVECs和人骨肉瘤细胞MG-63细胞株，加入含10%FCS的高糖DMEM培养基，以5×106/瓶密度接种于100ml培养瓶中，置于5%CO2孵箱中37°C孵育72或96h传代，培养传代，6-8代细胞用于共培养实验。
2、共培养体系建立：
2.1直接共培养：MG-63和HUVECs细胞经0.25％胰蛋白酶消化后，用含10％FCS的DMEM培养液配制成单个细胞悬液，按照不同混合比例的细胞悬液以1×105个/孔密度接种到12孔板，培养后24h后将细胞至于倒置显微镜下进行观察。
2.2间接共培养：将MG-63细胞以5×105浓度接种于12孔板，安装0.4μm孔径的12孔板Transtwell小室，将HUVECs细胞以5×105浓度接种于Transtwell小室内。
3.间接共培养环境下MG-63细胞的分化：
3.1MG-63细胞CollagenⅠ mRNA检测：间接培养24h，48h，72h和96h后，提取MG-63细胞mRNA用于实验检测。Real time PCR检测共培养环境对MG-63细胞CollagenⅠmRNA表达改变影响；
3.2培养液中OC检测：间接培养24h，48h，72h和96h后，提取培养液于实验检测，ELISA检测共培养环境对MG-63细胞OC表达改变影响。
4. CGRP对间接共培养环境下MG-63细胞分化的生物学作用：
4.1CGRP对间接共培养环境下MG-63细胞CollagenⅠ mRNA表达的作用：
以量效组100nM，10nM，1nM和0.1nM，和时效组0h，24h，48h，72h和96h分组，CGRP干预MG-63-HUVECs共培养体系，获取MG-63细胞mRNA，Real timePCR检测各组细胞CollagenⅠmRNA表达差异。
4.2CGRP对间接共培养环境下MG-63细胞OC表达的作用：
以量效组100nM，10nM，1nM和0.1nM，和时效组0h，24h，48h，72h和96h分组，CGRP干预MG-63-HUVECs共培养体系，获取MG-63细胞培养液，ELISA检测各组细胞OC表达差异。
5.共培养体系中CGRP所诱导的VEGF调控机制：
5.1共培养体系中CGRP所诱导的VEGF-A分泌变化：
10nM的CGRP作用于MG-63、HUVECs和MG-63-HUVECs间接共培养体系，作用0h，12h，24h，36h，和48h后收集细胞培养液或细胞，进行ELISA和Real Time-PCR测定。
5.2共培养体系中CGRP所诱导的MG-63细胞VEGF受体的表达变化：
将MG-63与HUVECs细胞Transtwell小室内间接共培养，未加Transtwell小室的MG-63培养为空白对照组(CON)，单独培养加入10nM CGRP的为CGRP作用组(CGRP)；加入Transwell及HUVECs的为共培养组(CO)，同时加入CGRP干预的为实验组(EXP)。培养48h后对MG-63细胞的VEGFR1/2进行免疫荧光检测。
结果：
1. MG-63-HUVECs细胞共培养：
1.1直接共培养： MG-63和HUVECs细胞按照1:4，1:2，1:1，2:1和4:1比例进行共培养，24h发现MG-63：HUVECs按照比例为1:1的直接共培养体系中两种细胞生长最为良好，细胞间杂生长，其中HUVECs细胞呈结节样生长，MG-63细胞生长于内皮细胞结节周围。DiI荧光标记后，MG-63-HUVECs共培养体系24h，HUVECs细胞呈结节样生长，MG-63细胞生长于内皮细胞结节周围，48h，HUVECs细胞结节中央形成细胞坏死，72h，HUVECs细胞与MG-63细胞分层，MG-63细胞向内皮细胞结节内浸润生长，96h，HUVECs细胞出现类管型样结构，MG-63细胞完全布满培养瓶底。
1.2间接共培养对MG-63细胞分化的作用：MG-63细胞在与HUVECs细胞间接共培养环境下，其CollagenⅠ mRNA表达明显增加，24h共培养组MG-63细胞所表达的CollagenⅠ mRNA为对照组的1.32，(P<0.05)；48h达到最大值，为对照组的1.57，(P<0.01)。培养液中OC表达随时间增加，且共培养组明显高于对照组，24h，共培养组培养清液中OC含量为对照组的1.54，(P<0.01)；48h为对照组的1.48，(P<0.01)；72h为对照组1.35，(P<0.05)。
2. CGRP对间接共培养体系中MG-63细胞的生物学作用：
不同浓度的CGRP作用MG-63-HUVECs细胞共培养体系48h后，MG-63细胞的CollagenⅠ mRNA和OC表达均有所增加，以10nM浓度组的MG-63细胞CollagenⅠmRNA和OC表达增高最为明显，分别为对照组1.94(P<0.01)和2.02(P<0.01)。
10nM浓度CGRP作用MG-63-HUVECs细胞共培养体系，MG-63细胞CollagenⅠmRNA表达呈现增加趋势，CollagenⅠ mRNA表达于48h达到峰值，为对照组的1.98，(P<0.01)；培养液中的OC含量自24h后呈现增加趋势(P<0.01)，培养液中的OC含量于96h达到峰值，为对照组的2.91。
3.共培养体系中CGRP所诱导的VEGF-A调控机制：
3.1共培养体系中CGRP所诱导的VEGF-A分泌变化：
在10nM CGRP的作用下，HUVECs单独作用组中培养液中VEGF-A含量相对对照组显著上升，在48h达到峰值，为对照组的2.31，VEGF-AmRNA相对对照组显著上升，在24h达到峰值，为对照组的2.71； MG-63单独作用组中培养液中的VEGF-A的含量在36h后随着时间延长出现下降，在48h达到最低值，为对照组的0.66；CGRP干预共培养组中，HUVECs细胞VEGF-A mRNA表达在24h后出现显著增高，24h达到峰值，为对照组的2.42。
3.2共培养体系中CGRP所诱导的MG-63细胞VEGF受体表达变化：
CGRP升高了共培养体系中MG-63细胞中VEGFR1的表达，10nM CGRP作用48h后，通过交叉对比，CO组VEGFR1表达显著高于CON组，0.186:0.124(p<0.01)；EXP组VEGFR1表达显著高于CON组，0.201:0.124(p<0.01)，并显著高于CGRP组，0.201:0.118(p<0.01)。
CGRP和共培养环境分别升高了MG-63细胞中VEGFR2的表达，10nM CPGR作用48h后，通过交叉对比，CGRP组和CO组均显VEGFR2表达著高于CON组，分别为0.177:0.081(p<0.01)和0.154:0.081(p<0.01)；EXP组VEGFR2表达显著高于CON组，0.194:0.081(p<0.01)，并显著高于CO组，0.194:0.154(p<0.05)。
结论
1. MG-63细胞与HUVECs细胞能够在高糖DMEM培养基中直接共培养，其中以1:1比例为优势比例；血管内皮细胞呈结节样生长，随时间延长有出现管型样结构的趋势，MG-63细胞在其周围生长。
2. MG-63-HUVECs间接共培养环境下，Collegen I mRNA表达和分泌型OC增加，促进MG-63细胞的分化成熟。
3. CGRP对共培养体系中MG-63细胞CollagenⅠ mRNA和OC表达具有显著地促进作用，以10nM为最佳浓度，CGRP和共培养两种方式均能促进MG-63细胞CollagenⅠ mRNA和OC表达，促进MG-63细胞分化成熟。
4. CGRP能够促进MG-63-HUVECs共培养体系中HUVECs细胞表达VEGF-AmRNA，上调MG-63-HUVECs共培养体系中MG-63细胞VEGFR1/2受体表达，以VEGFR2更为显著。但CGRP对MG-63-HUVECs共培养体系培养液中VEGF-A含量无显著影响。
Bone repair is a complex event that involves the interaction and coordination ofvariety many cells, and regulated by biochemical and mechanical signals. Calcitoningene-related peptide (CGRP) generated by peripheral nerve, is widely distributed in thecentral and peripheral neuronal systems and exhibits numerous biological activities inmammals, especially in osteogenese and angiogenses. In vitro experiments, CGRP has beenpromoted that can rise proliferation of osteoblasts, presence CGRP receptors on osteoblastsurface, and has a large number of downstream cell signaling pathways. But in theseexperimental studies, the majority only explored CGRP on single osteoblasts or osteoclastcells to expand the biological role of cells in fracture healing, and ignored the effectsmulti-cell interaction between cells. Therefore, these studies are unable to explainedCGRP’s osteogenesis in vivo multi-cell environment correct.
In vivo identification of regulators of bone remodeling is complex and nearlyimpossible because of many other factors in callus, such as cytokines, growth factors, andthe bone matrix itself. The co-culture system using OB and VECs provided a more simpleand useful method to explore these problems. Several studies have shown that the cross-talkbetween cells occurs at two levels, the first is completed direct role in cell-to-cell via themembrane protein, and the second is the indirect role through autocrine and paracrinemanner to complete the cell. CGRP positive fibers develop along the blood vessel growth inthe process of fracture healing callus, and play an important biological role in angiogenesisand the formation, which involved in the description that CGRP join in the regulation onCross-talk between the OB-VECs.
Therefore, we propose that CGRP exerts a biological effect on the OB-VEC co-culturesystem, and in this study, we focus on its regulation of OB differentiation. We establishedOB-VEC co-culture system to identified osteoblast differentiation via the detection of osteogenetic production, and explore the mechanism of regulation of CGRP on thecross-talk between the OB-VECs by detection the secretion of VECs cell factor andexpression these cell factor receptor on OB. At last, this study can elaborated themechanism of neural regulation on the blood vessels and osteoblasts cross-talk.
Materials and Methods
1. Cell culture: MG-63and HUVECs cells (ATCC) were cultured in Dulbecco’smodified Eagle’s Medium supplemented with10%fetal calf serum. Cells were subculturedevery72h in a humidified5%CO2incubator. Cells from passages6–8were used in theexperiments.
2. Establishment of MG-63-HUVECs co-culture.
2.1Direct contact co-culture conditions: MG-63were mixed with HUVECs inDMEC medium (10μg/mL PBS) and plated in12multiwell dishes at1×105cells/well，co-cultured24h and observed under an inverted microscope.
2.2Idirect contact co-culture conditions: A0.4μm transwell membrane insert wasused to separate HUVECs from MG-63. After MG-63cells were plated in a12multiwell at5×5×105cells/well and cultured for24h, the transwell was added, and then HUVECs wereadded to the insert chamber at5×105cells/well.
3. Detection the differentiation of MG-63in indirect co-culture sondition:
3.1Detection of CollagenⅠ mRNA in MG-63: Cells were indirect co-cultured for24h,48h,72and96h, mRNA samples in every group were extracted and via a Real timePCR to find the effect of co-culture on the expression of CollagenⅠmRNA in MG-63.
3.2Detection of Osteocalcin production in supernatants: Cells were indirectco-cultured for24h,48h,72and96h, supernatants in every group were extracted and viaELISA to find the effect of co-culture on the expression of oc in MG-63.
4. Effect of CGRP on the differentiation of MG-63in indirect co-culturesondition:
4.1Detection of CollagenⅠ mRNA in MG-63: The groups of CGRP was dividedinto dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), mRNA samples in every group were extracted and via a Real time PCR to findthe effect of CGRP on the expression of CollagenⅠmRNA in MG-63.
4.2Detection of Osteocalcin production in supernatants: The groups of CGRP wasdivided into dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), supernatants in every group were extracted and via ELISA to find theeffect of CGRP on the expression of oc in MG-63.
5. Regulation of CGRP on VEGF-A in MG-63-HUVECs Cross-talk:
5.1Detection of VEGF-A production in supernatants:MG-63, HUVECs, andMG-63-HUVECs indirect co-culture cells were induced by CGRP for0h,12h,24h,36h and48h, mRNA samples and supernatants in every group were extracted, and via Real timePCR or ELISA to find the effect of CGRP on the expression of VEGF-A and VEGF-AmRNA in cells.
5.2Expression of VEGF1/2on MG-63s: The CGRP groups (CGRP) were induced by10nM CGRP for48hours, and the control group (CON) was treated with DMEC medium.Co-culture groups (CO) were indirect co-cultured with HUVECs and without CGRP for48hours, and the experimental groups (EX) were indirect co-cultured with HUVECs and with10nM CGRP. Expression of VEGFR1/2in MG-63cells were detected byimmunofluorescence after induced for48h.
Results:
1. MG-63-HUVECs co-culture system.
1.1Direct co-culture: MG-63and HUVECs cells were co-cultured in accordancewith1:4,1:2,1:1,2:1and4:1for24h. MG-63:HUVECs got the best growth condition as in1:1proportion, which MG-63and HUVECs cells intermingled with growth, and HUVECscell grew as nodular and MG-63cells grew around these endothelial cell nodules. AsMG-63-HUVECs co-culture system were labeled by the DiI fluorescent,24h, the HUVECsgrew as nodular, the MG-63cells grew around endothelial cell nodules;48h, cell necrosiswere found in the center of HUVECs nodules;72h, HUVECs were separated from MG-63cells layer, MG-63cells invades into endothelial nodules;96h, HUVECs formed tube-likestructures, the MG-63cells completely distributed the bottom of the bottle.
1.2Indirect co-culture: MG-63cells were indirect co-cultured with HUVECs cells,its Collagen Ⅰ mRNA expression was significantly increased, expression of Collagen ⅠmRNA in co-cultured groups was1.32of the control group,(P <0.05);48h achieve maximum,1.57of the control group (P <0.01). OC production in supernatants groupsincreased with time, and the co-culture groups was higher than that in the control groups,24h, OC in co-culture groups was1.54of the control group,(P <0.01);48h,1.48of thecontrol group (P <0.01);72h,1.35of the control group (P <0.05).
2. Effect of CGRP on MG-63in indirect co-culture system.
Different concentrations of CGRP effect on MG-63-HUVECs co-culture for48h,Collagen Ⅰ mRNA in and OC expression in MG-63cells both increased. In10nM CGRPgroup, expression of Collagen Ⅰ mRNA and OC in MG-63cells increased the mostobvious,1.94(P <0.01) and2.02(P <0.01).
MG-63-HUVECs cells were induced by10nM CGRP, Collagen Ⅰ mRNA expressionin MG-63cells presented an increasing trend, reached peak for the control group1.98at48h,(P <0.01); OC production in supernatants contented of an increasing trend since24h(P <0.01), peaked2.91for the control group at96h.
3. Regulation of CGRP on the VEGF-A in MG-63-HUVECs co-culture:
3.1Effect of CGRP on the production of VEGF-A:
Induced by10nM CGRP, in HUVECs groups, VEGF-A production were raised, andreached peak for the control group2.31at48h, VEGF-A mRNA were raised, peaked2.71for the control group at24h; in MG-63groups, VEGF-A production were decreased from36h, and decline to he lowest level,0.66of control groups at48h; CGRP induced co-culturegroup, VEGF-A mRNA expression in HUVECs increased significantly after24h, andpeak for the2.42control group at24h.
3.2Effect of CGRP on the expression of VEGFR1/2in MG-63:
CGRP and co-culture environment both increased VEGFR1expression in MG-63cells.Induced by the10nM CPGR for48hours, through cross-comparison, VEGFR1expressionin CO group was higher than the CON group,0.186:0.124(p <0.01); VEGFR1expressionin EXP group was significantly higher than the CON group,0.201:0.124(p <0.01), and washigher than CGRP group,0.201:0.118(p <0.01).
The CGRP and co-culture environment both increased the expression of VEGFR2inMG-63cells. Induced by the10nM CPGR for48hours, through cross-comparison,VEGFR2in CGRP and CO groups were significantly increase compared to the CON group,0.177:0.081(p <0.01) and0.154:0.081(p <0.01); VEGFR2in EXP groups noticeable higher than the CON group,0.194:0.081,(p <0.01), and significantly higher than the COgroup,0.194:0.154(p <0.05).
Conclusion:
1. MG-63cells and HUVECs cells can be co-cultured in high glucose DMEM mediumdirectly, and the1:1grow ratio is the dominant proportion. Vascular endothelial cells growinto cells noduls, and become tube-like structures with time. MG-63cells grow aroundthese cells noduls.
2. In indirect MG-63-HUVECs co-culture environment, Collegen I mRNA expressionand secreted OC increased, and promoted the differentiation and maturation of the MG-63cells.
3. In co-culture system, CGRP can increase Collagen Ⅰ mRNA and OC expression inMG-63cells, whose optimal concentration is10nM. Under induction of CGRP orco-culture with HUVECs, Collagen Ⅰ mRNA and OC expression in MG-63cells can beincreased, and promoted the differentiation and maturation of the MG-63cells.
4. CGRP can promote the VEGF-A mRNA expression in HUVECs cell, and raised theVEGFR1/2in MG-63in MG-63-HUVECs co-culture system, VEGFR2more significant.But CGRP can not effect on the VEGF-A production in supernatants of MG-63-HUVECsco-culture.
成骨细胞和血管内皮细胞(Vascular endothelial cells, VECs)之间存在一种相互影响、相互调控的OB-VECs间cross-talk机制。在骨痂中VECs不仅仅构建了微血管簇为骨痂提供了营养支持，还作为一种具有成骨诱导功能细胞，参与了MSCs的募集和诱导分化。研究发现细胞间cross-talk主要发生在两个层面，一是经由细胞膜之间的蛋白完成细胞与细胞间的直接作用，二是通过自分泌和旁分泌的方式完成细胞间的间接作用。而在骨折愈合过程中，骨痂中的CGRP阳性纤维沿着血管生长，并且有大量的体外实验证实，CGRP在血管再生和形成中也发挥着重要的生物学作用，说明CGRP参与了对OB-VECs之间Cross-talk的调控。
基于骨痂中的CGRP对成骨的调控机制可能与VECs细胞相关这一推测，本实验依据课题组的前期预实验结果，建立CGRP干预的OB-VECs共培养体系体，通过对VECs细胞旁分泌成骨因子的检测，和OB细胞对应因子受体表达及细胞分化指标的检测，探讨CGRP对OB-VECs间cross-talk的调控机制。从而在体内及体外多细胞环境下，发现并证实神经通过对VECs旁分泌的调控作用发挥了促成骨效应，进一步阐述骨痂中神经对血管和成骨交互调控的作用机制。
方法：
1.细胞培养：美国ATCC公司人脐静脉血管内皮细胞HUVECs和人骨肉瘤细胞MG-63细胞株，加入含10%FCS的高糖DMEM培养基，以5×106/瓶密度接种于100ml培养瓶中，置于5%CO2孵箱中37°C孵育72或96h传代，培养传代，6-8代细胞用于共培养实验。
2、共培养体系建立：
2.1直接共培养：MG-63和HUVECs细胞经0.25％胰蛋白酶消化后，用含10％FCS的DMEM培养液配制成单个细胞悬液，按照不同混合比例的细胞悬液以1×105个/孔密度接种到12孔板，培养后24h后将细胞至于倒置显微镜下进行观察。
2.2间接共培养：将MG-63细胞以5×105浓度接种于12孔板，安装0.4μm孔径的12孔板Transtwell小室，将HUVECs细胞以5×105浓度接种于Transtwell小室内。
3.间接共培养环境下MG-63细胞的分化：
3.1MG-63细胞CollagenⅠ mRNA检测：间接培养24h，48h，72h和96h后，提取MG-63细胞mRNA用于实验检测。Real time PCR检测共培养环境对MG-63细胞CollagenⅠmRNA表达改变影响；
3.2培养液中OC检测：间接培养24h，48h，72h和96h后，提取培养液于实验检测，ELISA检测共培养环境对MG-63细胞OC表达改变影响。
4. CGRP对间接共培养环境下MG-63细胞分化的生物学作用：
4.1CGRP对间接共培养环境下MG-63细胞CollagenⅠ mRNA表达的作用：
以量效组100nM，10nM，1nM和0.1nM，和时效组0h，24h，48h，72h和96h分组，CGRP干预MG-63-HUVECs共培养体系，获取MG-63细胞mRNA，Real timePCR检测各组细胞CollagenⅠmRNA表达差异。
4.2CGRP对间接共培养环境下MG-63细胞OC表达的作用：
以量效组100nM，10nM，1nM和0.1nM，和时效组0h，24h，48h，72h和96h分组，CGRP干预MG-63-HUVECs共培养体系，获取MG-63细胞培养液，ELISA检测各组细胞OC表达差异。
5.共培养体系中CGRP所诱导的VEGF调控机制：
5.1共培养体系中CGRP所诱导的VEGF-A分泌变化：
10nM的CGRP作用于MG-63、HUVECs和MG-63-HUVECs间接共培养体系，作用0h，12h，24h，36h，和48h后收集细胞培养液或细胞，进行ELISA和Real Time-PCR测定。
5.2共培养体系中CGRP所诱导的MG-63细胞VEGF受体的表达变化：
将MG-63与HUVECs细胞Transtwell小室内间接共培养，未加Transtwell小室的MG-63培养为空白对照组(CON)，单独培养加入10nM CGRP的为CGRP作用组(CGRP)；加入Transwell及HUVECs的为共培养组(CO)，同时加入CGRP干预的为实验组(EXP)。培养48h后对MG-63细胞的VEGFR1/2进行免疫荧光检测。
结果：
1. MG-63-HUVECs细胞共培养：
1.1直接共培养： MG-63和HUVECs细胞按照1:4，1:2，1:1，2:1和4:1比例进行共培养，24h发现MG-63：HUVECs按照比例为1:1的直接共培养体系中两种细胞生长最为良好，细胞间杂生长，其中HUVECs细胞呈结节样生长，MG-63细胞生长于内皮细胞结节周围。DiI荧光标记后，MG-63-HUVECs共培养体系24h，HUVECs细胞呈结节样生长，MG-63细胞生长于内皮细胞结节周围，48h，HUVECs细胞结节中央形成细胞坏死，72h，HUVECs细胞与MG-63细胞分层，MG-63细胞向内皮细胞结节内浸润生长，96h，HUVECs细胞出现类管型样结构，MG-63细胞完全布满培养瓶底。
1.2间接共培养对MG-63细胞分化的作用：MG-63细胞在与HUVECs细胞间接共培养环境下，其CollagenⅠ mRNA表达明显增加，24h共培养组MG-63细胞所表达的CollagenⅠ mRNA为对照组的1.32，(P<0.05)；48h达到最大值，为对照组的1.57，(P<0.01)。培养液中OC表达随时间增加，且共培养组明显高于对照组，24h，共培养组培养清液中OC含量为对照组的1.54，(P<0.01)；48h为对照组的1.48，(P<0.01)；72h为对照组1.35，(P<0.05)。
2. CGRP对间接共培养体系中MG-63细胞的生物学作用：
不同浓度的CGRP作用MG-63-HUVECs细胞共培养体系48h后，MG-63细胞的CollagenⅠ mRNA和OC表达均有所增加，以10nM浓度组的MG-63细胞CollagenⅠmRNA和OC表达增高最为明显，分别为对照组1.94(P<0.01)和2.02(P<0.01)。
10nM浓度CGRP作用MG-63-HUVECs细胞共培养体系，MG-63细胞CollagenⅠmRNA表达呈现增加趋势，CollagenⅠ mRNA表达于48h达到峰值，为对照组的1.98，(P<0.01)；培养液中的OC含量自24h后呈现增加趋势(P<0.01)，培养液中的OC含量于96h达到峰值，为对照组的2.91。
3.共培养体系中CGRP所诱导的VEGF-A调控机制：
3.1共培养体系中CGRP所诱导的VEGF-A分泌变化：
在10nM CGRP的作用下，HUVECs单独作用组中培养液中VEGF-A含量相对对照组显著上升，在48h达到峰值，为对照组的2.31，VEGF-AmRNA相对对照组显著上升，在24h达到峰值，为对照组的2.71； MG-63单独作用组中培养液中的VEGF-A的含量在36h后随着时间延长出现下降，在48h达到最低值，为对照组的0.66；CGRP干预共培养组中，HUVECs细胞VEGF-A mRNA表达在24h后出现显著增高，24h达到峰值，为对照组的2.42。
3.2共培养体系中CGRP所诱导的MG-63细胞VEGF受体表达变化：
CGRP升高了共培养体系中MG-63细胞中VEGFR1的表达，10nM CGRP作用48h后，通过交叉对比，CO组VEGFR1表达显著高于CON组，0.186:0.124(p<0.01)；EXP组VEGFR1表达显著高于CON组，0.201:0.124(p<0.01)，并显著高于CGRP组，0.201:0.118(p<0.01)。
CGRP和共培养环境分别升高了MG-63细胞中VEGFR2的表达，10nM CPGR作用48h后，通过交叉对比，CGRP组和CO组均显VEGFR2表达著高于CON组，分别为0.177:0.081(p<0.01)和0.154:0.081(p<0.01)；EXP组VEGFR2表达显著高于CON组，0.194:0.081(p<0.01)，并显著高于CO组，0.194:0.154(p<0.05)。
结论
1. MG-63细胞与HUVECs细胞能够在高糖DMEM培养基中直接共培养，其中以1:1比例为优势比例；血管内皮细胞呈结节样生长，随时间延长有出现管型样结构的趋势，MG-63细胞在其周围生长。
2. MG-63-HUVECs间接共培养环境下，Collegen I mRNA表达和分泌型OC增加，促进MG-63细胞的分化成熟。
3. CGRP对共培养体系中MG-63细胞CollagenⅠ mRNA和OC表达具有显著地促进作用，以10nM为最佳浓度，CGRP和共培养两种方式均能促进MG-63细胞CollagenⅠ mRNA和OC表达，促进MG-63细胞分化成熟。
4. CGRP能够促进MG-63-HUVECs共培养体系中HUVECs细胞表达VEGF-AmRNA，上调MG-63-HUVECs共培养体系中MG-63细胞VEGFR1/2受体表达，以VEGFR2更为显著。但CGRP对MG-63-HUVECs共培养体系培养液中VEGF-A含量无显著影响。
Bone repair is a complex event that involves the interaction and coordination ofvariety many cells, and regulated by biochemical and mechanical signals. Calcitoningene-related peptide (CGRP) generated by peripheral nerve, is widely distributed in thecentral and peripheral neuronal systems and exhibits numerous biological activities inmammals, especially in osteogenese and angiogenses. In vitro experiments, CGRP has beenpromoted that can rise proliferation of osteoblasts, presence CGRP receptors on osteoblastsurface, and has a large number of downstream cell signaling pathways. But in theseexperimental studies, the majority only explored CGRP on single osteoblasts or osteoclastcells to expand the biological role of cells in fracture healing, and ignored the effectsmulti-cell interaction between cells. Therefore, these studies are unable to explainedCGRP’s osteogenesis in vivo multi-cell environment correct.
In vivo identification of regulators of bone remodeling is complex and nearlyimpossible because of many other factors in callus, such as cytokines, growth factors, andthe bone matrix itself. The co-culture system using OB and VECs provided a more simpleand useful method to explore these problems. Several studies have shown that the cross-talkbetween cells occurs at two levels, the first is completed direct role in cell-to-cell via themembrane protein, and the second is the indirect role through autocrine and paracrinemanner to complete the cell. CGRP positive fibers develop along the blood vessel growth inthe process of fracture healing callus, and play an important biological role in angiogenesisand the formation, which involved in the description that CGRP join in the regulation onCross-talk between the OB-VECs.
Therefore, we propose that CGRP exerts a biological effect on the OB-VEC co-culturesystem, and in this study, we focus on its regulation of OB differentiation. We establishedOB-VEC co-culture system to identified osteoblast differentiation via the detection of osteogenetic production, and explore the mechanism of regulation of CGRP on thecross-talk between the OB-VECs by detection the secretion of VECs cell factor andexpression these cell factor receptor on OB. At last, this study can elaborated themechanism of neural regulation on the blood vessels and osteoblasts cross-talk.
Materials and Methods
1. Cell culture: MG-63and HUVECs cells (ATCC) were cultured in Dulbecco’smodified Eagle’s Medium supplemented with10%fetal calf serum. Cells were subculturedevery72h in a humidified5%CO2incubator. Cells from passages6–8were used in theexperiments.
2. Establishment of MG-63-HUVECs co-culture.
2.1Direct contact co-culture conditions: MG-63were mixed with HUVECs inDMEC medium (10μg/mL PBS) and plated in12multiwell dishes at1×105cells/well，co-cultured24h and observed under an inverted microscope.
2.2Idirect contact co-culture conditions: A0.4μm transwell membrane insert wasused to separate HUVECs from MG-63. After MG-63cells were plated in a12multiwell at5×5×105cells/well and cultured for24h, the transwell was added, and then HUVECs wereadded to the insert chamber at5×105cells/well.
3. Detection the differentiation of MG-63in indirect co-culture sondition:
3.1Detection of CollagenⅠ mRNA in MG-63: Cells were indirect co-cultured for24h,48h,72and96h, mRNA samples in every group were extracted and via a Real timePCR to find the effect of co-culture on the expression of CollagenⅠmRNA in MG-63.
3.2Detection of Osteocalcin production in supernatants: Cells were indirectco-cultured for24h,48h,72and96h, supernatants in every group were extracted and viaELISA to find the effect of co-culture on the expression of oc in MG-63.
4. Effect of CGRP on the differentiation of MG-63in indirect co-culturesondition:
4.1Detection of CollagenⅠ mRNA in MG-63: The groups of CGRP was dividedinto dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), mRNA samples in every group were extracted and via a Real time PCR to findthe effect of CGRP on the expression of CollagenⅠmRNA in MG-63.
4.2Detection of Osteocalcin production in supernatants: The groups of CGRP wasdivided into dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), supernatants in every group were extracted and via ELISA to find theeffect of CGRP on the expression of oc in MG-63.
5. Regulation of CGRP on VEGF-A in MG-63-HUVECs Cross-talk:
5.1Detection of VEGF-A production in supernatants:MG-63, HUVECs, andMG-63-HUVECs indirect co-culture cells were induced by CGRP for0h,12h,24h,36h and48h, mRNA samples and supernatants in every group were extracted, and via Real timePCR or ELISA to find the effect of CGRP on the expression of VEGF-A and VEGF-AmRNA in cells.
5.2Expression of VEGF1/2on MG-63s: The CGRP groups (CGRP) were induced by10nM CGRP for48hours, and the control group (CON) was treated with DMEC medium.Co-culture groups (CO) were indirect co-cultured with HUVECs and without CGRP for48hours, and the experimental groups (EX) were indirect co-cultured with HUVECs and with10nM CGRP. Expression of VEGFR1/2in MG-63cells were detected byimmunofluorescence after induced for48h.
Results:
1. MG-63-HUVECs co-culture system.
1.1Direct co-culture: MG-63and HUVECs cells were co-cultured in accordancewith1:4,1:2,1:1,2:1and4:1for24h. MG-63:HUVECs got the best growth condition as in1:1proportion, which MG-63and HUVECs cells intermingled with growth, and HUVECscell grew as nodular and MG-63cells grew around these endothelial cell nodules. AsMG-63-HUVECs co-culture system were labeled by the DiI fluorescent,24h, the HUVECsgrew as nodular, the MG-63cells grew around endothelial cell nodules;48h, cell necrosiswere found in the center of HUVECs nodules;72h, HUVECs were separated from MG-63cells layer, MG-63cells invades into endothelial nodules;96h, HUVECs formed tube-likestructures, the MG-63cells completely distributed the bottom of the bottle.
1.2Indirect co-culture: MG-63cells were indirect co-cultured with HUVECs cells,its Collagen Ⅰ mRNA expression was significantly increased, expression of Collagen ⅠmRNA in co-cultured groups was1.32of the control group,(P <0.05);48h achieve maximum,1.57of the control group (P <0.01). OC production in supernatants groupsincreased with time, and the co-culture groups was higher than that in the control groups,24h, OC in co-culture groups was1.54of the control group,(P <0.01);48h,1.48of thecontrol group (P <0.01);72h,1.35of the control group (P <0.05).
2. Effect of CGRP on MG-63in indirect co-culture system.
Different concentrations of CGRP effect on MG-63-HUVECs co-culture for48h,Collagen Ⅰ mRNA in and OC expression in MG-63cells both increased. In10nM CGRPgroup, expression of Collagen Ⅰ mRNA and OC in MG-63cells increased the mostobvious,1.94(P <0.01) and2.02(P <0.01).
MG-63-HUVECs cells were induced by10nM CGRP, Collagen Ⅰ mRNA expressionin MG-63cells presented an increasing trend, reached peak for the control group1.98at48h,(P <0.01); OC production in supernatants contented of an increasing trend since24h(P <0.01), peaked2.91for the control group at96h.
3. Regulation of CGRP on the VEGF-A in MG-63-HUVECs co-culture:
3.1Effect of CGRP on the production of VEGF-A:
Induced by10nM CGRP, in HUVECs groups, VEGF-A production were raised, andreached peak for the control group2.31at48h, VEGF-A mRNA were raised, peaked2.71for the control group at24h; in MG-63groups, VEGF-A production were decreased from36h, and decline to he lowest level,0.66of control groups at48h; CGRP induced co-culturegroup, VEGF-A mRNA expression in HUVECs increased significantly after24h, andpeak for the2.42control group at24h.
3.2Effect of CGRP on the expression of VEGFR1/2in MG-63:
CGRP and co-culture environment both increased VEGFR1expression in MG-63cells.Induced by the10nM CPGR for48hours, through cross-comparison, VEGFR1expressionin CO group was higher than the CON group,0.186:0.124(p <0.01); VEGFR1expressionin EXP group was significantly higher than the CON group,0.201:0.124(p <0.01), and washigher than CGRP group,0.201:0.118(p <0.01).
The CGRP and co-culture environment both increased the expression of VEGFR2inMG-63cells. Induced by the10nM CPGR for48hours, through cross-comparison,VEGFR2in CGRP and CO groups were significantly increase compared to the CON group,0.177:0.081(p <0.01) and0.154:0.081(p <0.01); VEGFR2in EXP groups noticeable higher than the CON group,0.194:0.081,(p <0.01), and significantly higher than the COgroup,0.194:0.154(p <0.05).
Conclusion:
1. MG-63cells and HUVECs cells can be co-cultured in high glucose DMEM mediumdirectly, and the1:1grow ratio is the dominant proportion. Vascular endothelial cells growinto cells noduls, and become tube-like structures with time. MG-63cells grow aroundthese cells noduls.
2. In indirect MG-63-HUVECs co-culture environment, Collegen I mRNA expressionand secreted OC increased, and promoted the differentiation and maturation of the MG-63cells.
3. In co-culture system, CGRP can increase Collagen Ⅰ mRNA and OC expression inMG-63cells, whose optimal concentration is10nM. Under induction of CGRP orco-culture with HUVECs, Collagen Ⅰ mRNA and OC expression in MG-63cells can beincreased, and promoted the differentiation and maturation of the MG-63cells.
4. CGRP can promote the VEGF-A mRNA expression in HUVECs cell, and raised theVEGFR1/2in MG-63in MG-63-HUVECs co-culture system, VEGFR2more significant.But CGRP can not effect on the VEGF-A production in supernatants of MG-63-HUVECsco-culture.
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43.王福科, et al.,体外血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞成骨分化的影响.中国修复重建外科杂志,2010.24(4): p.399-405.
44.郭英, et al.,血管化人工骨的体外构建和体内异位成骨.中国组织工程研究与临床康复,2011.15(20).
45. Schipani, E., et al., Regulation of Osteogenesis ngiogenesis coupling by HIFs andVEGF. Journal of bone and mineral research,2009.24(8): p.1347-1353.
46. C Keramaris, N., et al., Endothelial Progenitor Cells (EPCs) and Mesenchymal StemCells (MSCs) in Bone Healing. Current Stem Cell Research& Therapy,2012.7(4): p.293-301.
47. Mayr-Wohlfart, U., et al., Vascular endothelial growth factor stimulates chemotacticmigration of primary human osteoblasts. Bone,2002.30(3): p.472-477.
48. Kleinheinz, J., et al., VEGF-activated angiogenesis during bone regeneration. Journalof oral and maxillofacial surgery,2005.63(9): p.1310-1316.
49. Hiltunen, M.O., et al., Adenovirus-mediated VEGF-A gene transfer induces boneformation in vivo. The FASEB journal,2003.17(9): p.1147-1149.
50. Tombran-Tink, J. and C.J. Barnstable, Osteoblasts and osteoclasts express PEDF,VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis andmatrix remodeling in the bone. Biochemical and biophysical research communications,2004.316(2): p.573-579.
51. Kanczler, J.M., et al., The effect of mesenchymal populations and vascular endothelialgrowth factor delivered from biodegradable polymer scaffolds on bone formation.Biomaterials,2008.29(12): p.1892-1900.
52. Mac Gabhann, F. and A.S. Popel, Model of competitive binding of vascular endothelialgrowth factor and placental growth factor to VEGF receptors on endothelial cells.American Journal of Physiology-Heart and Circulatory Physiology,2004.286(1): p.H153-H164.
53. Plouet, J. and H. Moukadiri, Characterization of the receptor to vasculotropin onbovine adrenal cortex-derived capillary endothelial cells. Journal of biologicalchemistry,1990.265(36): p.22071.
54. Park, J.E., et al., Placenta growth factor. Potentiation of vascular endothelial growthfactor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1but not toFlk-1/KDR. Journal of Biological Chemistry,1994.269(41): p.25646-25654.
1. Langer, R., and Vacanti, J. P. Tissue engineering. Science.1993,260,920–926.
2. Sittinger, M., Bujia, J., Rotter, N., Reitzel, D., Minuth, W. W.,and Burmester, G. R.Tissue engineering and autologous transplant formation: Practical approaches withresorbable biomaterialsand new cell culture techniques. Biomaterials.1996,17,237–242.
3. Reeve, J., and Metz, D. AgeNet workshop on ageing fragility and the biomechanics ofbone. AgeNet Report.1998,1,1–3.
4. Jackson, D. W., and Simon, T. M. Tissue engineering principles orthopaedic surgery.Clin. Orthop.1999,367(Suppl.),S31–S45.
5. Einhorn, T. A. Clinically applied models of bone regenerationin tissue engineeringresearch. Clin. Orthop.1999,367(Suppl.),S59–S67.
6. Fialkov, K. A., Holy, C. E., and Antonyshyn, O. Strategiesfor bone substitutes incraniofacial surgery. In Bone Engineering,2000,2(1),548–557.
7. Perry, C. R. Bone repair techniques, bone graft, and bonegraft substitutes. Clin. Orthop.1999,10(8),71–86.
8. Vacanti, C. A., Bonassar, L. J., and Vacanti, J. P.(2000) Structural tissue engineering. InPrinciples of Tissue Engineering1999,10(8),71–86.
9. Burg, K. J., Porter, S., and Kellam, J. F. Biomaterial developments for bone tissueengineering. Biomaterials21.2000,3(14),102–106.
10. Newspaper report10th April (2001) Stem cells in fat transformedinto muscle and bone.The Times,2001,17(22)15.
11. Chapekar, M. S. Tissue engineering: Challenges and opportunities.J. Biomed. Mater.Res.2000,53,617–620.
12. Cohen, S., Bano, M. C., Cima, L. G., Allcock, H. R., Vacanti, J. P.,Vacanti, C. A., andLanger, R. Design of synthetic polymericstructures for cell transplantation and tissueengineering.Clin. Mater.1993,13,3–10.
13. Chrischilles, E., Shireman, T., and Wallace, R. Costs and health effects of osteoporoticfractures. Bone,1994,15(2),377–386.
14. Jinling M, Jeroen J, Beucken, et al. Coculture of Osteoblasts and Endothelial Cells:Optimization of Culture Medium and Cell Ratio. TISSUE ENGINEERING,2010,17(3),1-9.
15. Dbouk, H.A. et al. Connexins: a myriad of functions extendingbeyond assembly of gapjunction channels. Cell Commun Signal,2009,7,4.
16. Stains, J.P. and Civitelli, R. Gap junctions in skeletaldevelopment and function.Biochim Biophys Acta,2005,17(19),69–81
17. Yeh, H.I. et al. Reduced expression of endothelial connexins43and37in hypertensiverats is rectified after7-day carvedilol,2006,3(10),122–129.
18. Stains, J.P. and Civitelli, R. Gap junctions regulate extracellular signal-regulated kinasesignaling to affect gene transcription. Mol Biol Cell,2005,16,64–72.
19. Guillotin, B. et al. Interaction between human umbilical vein endothelial cells andhuman osteoprogenitors triggers pleiotropic effect that may support osteoblasticfunction. Bone.2008,42,1080–1091.
20. Kaigler, D. et al. VEGF scaffolds enhance angiogenesis and boneregeneration inirradiated osseous defects. J Bone Miner Res.2006,21,735–744.
21. Clarkin, C.E. et al. Evaluation of VEGF-mediated signaling inprimary human cellsreveals a paracrine action for VEGF in osteoblastmediated crosstalk to endothelialcells. J Cell Physiol,2008,214,537–544.
22. Clarkin, C.E. et al. Heterotypic contact reveals a COX-2-mediated suppression ofosteoblast differentiation by endothelial cells: A negative modulatory role forprostanoids in VEGF-mediated cell: cell communication? Exp Cell Res,2008,314,3152–3161.
23. Bouletreau, P.J. et al. Hypoxia and VEGF up-regulate BMP-2mRNA and proteinexpression in microvascular endothelial cells:implications for fracture healing. PlastReconstr Surg,2002,109,2384–2397.
24. Veillette, C.J. and von Schroeder, H.P. Endothelin-1downregulatesthe expression ofvascular endothelial growth factor-A associated with osteoprogenitor proliferation anddifferentiation.Bone,2004,34,288–296.
25. Fiedler, J. et al. IGF-I and IGF-II stimulate directed cellmigration ofbone-marrow-derived human mesenchymal progenitor cells. Biochem Biophys ResCommun,2006,345,1177–1183
26. Soker S. Neuropilin in the midst of cell migration and retraction.Int J Biochem Cel lBiol,2001,33(4):433-437.
27. Boul et reau PJ, Warren SM, Spect or JA, et al. Hypoxi a and VEGF up-regul at eBMP-2mRNA and prot ei n expressi on i n mi crovascul ar en-dot hel i al cel l s: Impl icat i ons for fract ure heal i ng. Plast i c&Re-const ruct i ve Surg,2002,109(7):2384-2397.
28. Peng H, Wri ght V, Usas A, et al. Synergi st i c enhancement of boneformat i on andheal i ng by st em cel l-expressed VEGF and bone mor-phogenet i c prot ei n-4. J Cl i nInvest,2002,110(6):751-759.
29. Grellier. M, Bordenave. L, Ame′de′e. J. Cell-to-cell communication betweenosteogenic and endothelial lineages: implications for tissue engineering. Trends inBiotechnology,2009,27(10),562-71.
30. Kanaan, R.A. et al. The role of connective tissue growth factor in skeletal growth anddevelopment.2006, Med Sci Monit12, RA277–281.
31. Lebrin, F. et al. TGF-beta receptor function in the endothelium.Cardiovasc Res,2005,65,599–608
32. Qu, G. and von Schroeder, H.P. Role of osterix in endothelin-1-induced downregulationof vascular endothelial growth factor in osteoblastic cells. Bone,2006,38,21–29
33. Niger, C. et al. Endothelin-1inhibits human osteoblastic celldifferentiation: influenceof connexin-43expression level. J CellBiochem,2008,103,110–122
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42. Ying, G., et al., Construction of vascularization artificial bone in vitro and ectopicosteogenesis in vivo. Journal of Clinical Rehabilitative Tissue Engineering Research,2010.15(20): p.23-26.
43.王福科, et al.,体外血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞成骨分化的影响.中国修复重建外科杂志,2010.24(4): p.399-405.
44.郭英, et al.,血管化人工骨的体外构建和体内异位成骨.中国组织工程研究与临床康复,2011.15(20).
45. Schipani, E., et al., Regulation of Osteogenesis ngiogenesis coupling by HIFs andVEGF. Journal of bone and mineral research,2009.24(8): p.1347-1353.
46. C Keramaris, N., et al., Endothelial Progenitor Cells (EPCs) and Mesenchymal StemCells (MSCs) in Bone Healing. Current Stem Cell Research& Therapy,2012.7(4): p.293-301.
47. Mayr-Wohlfart, U., et al., Vascular endothelial growth factor stimulates chemotacticmigration of primary human osteoblasts. Bone,2002.30(3): p.472-477.
48. Kleinheinz, J., et al., VEGF-activated angiogenesis during bone regeneration. Journalof oral and maxillofacial surgery,2005.63(9): p.1310-1316.
49. Hiltunen, M.O., et al., Adenovirus-mediated VEGF-A gene transfer induces boneformation in vivo. The FASEB journal,2003.17(9): p.1147-1149.
50. Tombran-Tink, J. and C.J. Barnstable, Osteoblasts and osteoclasts express PEDF,VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis andmatrix remodeling in the bone. Biochemical and biophysical research communications,2004.316(2): p.573-579.
51. Kanczler, J.M., et al., The effect of mesenchymal populations and vascular endothelialgrowth factor delivered from biodegradable polymer scaffolds on bone formation.Biomaterials,2008.29(12): p.1892-1900.
52. Mac Gabhann, F. and A.S. Popel, Model of competitive binding of vascular endothelialgrowth factor and placental growth factor to VEGF receptors on endothelial cells.American Journal of Physiology-Heart and Circulatory Physiology,2004.286(1): p.H153-H164.
53. Plouet, J. and H. Moukadiri, Characterization of the receptor to vasculotropin onbovine adrenal cortex-derived capillary endothelial cells. Journal of biologicalchemistry,1990.265(36): p.22071.
54. Park, J.E., et al., Placenta growth factor. Potentiation of vascular endothelial growthfactor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1but not toFlk-1/KDR. Journal of Biological Chemistry,1994.269(41): p.25646-25654.
1. Langer, R., and Vacanti, J. P. Tissue engineering. Science.1993,260,920–926.
2. Sittinger, M., Bujia, J., Rotter, N., Reitzel, D., Minuth, W. W.,and Burmester, G. R.Tissue engineering and autologous transplant formation: Practical approaches withresorbable biomaterialsand new cell culture techniques. Biomaterials.1996,17,237–242.
3. Reeve, J., and Metz, D. AgeNet workshop on ageing fragility and the biomechanics ofbone. AgeNet Report.1998,1,1–3.
4. Jackson, D. W., and Simon, T. M. Tissue engineering principles orthopaedic surgery.Clin. Orthop.1999,367(Suppl.),S31–S45.
5. Einhorn, T. A. Clinically applied models of bone regenerationin tissue engineeringresearch. Clin. Orthop.1999,367(Suppl.),S59–S67.
6. Fialkov, K. A., Holy, C. E., and Antonyshyn, O. Strategiesfor bone substitutes incraniofacial surgery. In Bone Engineering,2000,2(1),548–557.
7. Perry, C. R. Bone repair techniques, bone graft, and bonegraft substitutes. Clin. Orthop.1999,10(8),71–86.
8. Vacanti, C. A., Bonassar, L. J., and Vacanti, J. P.(2000) Structural tissue engineering. InPrinciples of Tissue Engineering1999,10(8),71–86.
9. Burg, K. J., Porter, S., and Kellam, J. F. Biomaterial developments for bone tissueengineering. Biomaterials21.2000,3(14),102–106.
10. Newspaper report10th April (2001) Stem cells in fat transformedinto muscle and bone.The Times,2001,17(22)15.
11. Chapekar, M. S. Tissue engineering: Challenges and opportunities.J. Biomed. Mater.Res.2000,53,617–620.
12. Cohen, S., Bano, M. C., Cima, L. G., Allcock, H. R., Vacanti, J. P.,Vacanti, C. A., andLanger, R. Design of synthetic polymericstructures for cell transplantation and tissueengineering.Clin. Mater.1993,13,3–10.
13. Chrischilles, E., Shireman, T., and Wallace, R. Costs and health effects of osteoporoticfractures. Bone,1994,15(2),377–386.
14. Jinling M, Jeroen J, Beucken, et al. Coculture of Osteoblasts and Endothelial Cells:Optimization of Culture Medium and Cell Ratio. TISSUE ENGINEERING,2010,17(3),1-9.
15. Dbouk, H.A. et al. Connexins: a myriad of functions extendingbeyond assembly of gapjunction channels. Cell Commun Signal,2009,7,4.
16. Stains, J.P. and Civitelli, R. Gap junctions in skeletaldevelopment and function.Biochim Biophys Acta,2005,17(19),69–81
17. Yeh, H.I. et al. Reduced expression of endothelial connexins43and37in hypertensiverats is rectified after7-day carvedilol,2006,3(10),122–129.
18. Stains, J.P. and Civitelli, R. Gap junctions regulate extracellular signal-regulated kinasesignaling to affect gene transcription. Mol Biol Cell,2005,16,64–72.
19. Guillotin, B. et al. Interaction between human umbilical vein endothelial cells andhuman osteoprogenitors triggers pleiotropic effect that may support osteoblasticfunction. Bone.2008,42,1080–1091.
20. Kaigler, D. et al. VEGF scaffolds enhance angiogenesis and boneregeneration inirradiated osseous defects. J Bone Miner Res.2006,21,735–744.
21. Clarkin, C.E. et al. Evaluation of VEGF-mediated signaling inprimary human cellsreveals a paracrine action for VEGF in osteoblastmediated crosstalk to endothelialcells. J Cell Physiol,2008,214,537–544.
22. Clarkin, C.E. et al. Heterotypic contact reveals a COX-2-mediated suppression ofosteoblast differentiation by endothelial cells: A negative modulatory role forprostanoids in VEGF-mediated cell: cell communication? Exp Cell Res,2008,314,3152–3161.
23. Bouletreau, P.J. et al. Hypoxia and VEGF up-regulate BMP-2mRNA and proteinexpression in microvascular endothelial cells:implications for fracture healing. PlastReconstr Surg,2002,109,2384–2397.
24. Veillette, C.J. and von Schroeder, H.P. Endothelin-1downregulatesthe expression ofvascular endothelial growth factor-A associated with osteoprogenitor proliferation anddifferentiation.Bone,2004,34,288–296.
25. Fiedler, J. et al. IGF-I and IGF-II stimulate directed cellmigration ofbone-marrow-derived human mesenchymal progenitor cells. Biochem Biophys ResCommun,2006,345,1177–1183
26. Soker S. Neuropilin in the midst of cell migration and retraction.Int J Biochem Cel lBiol,2001,33(4):433-437.
27. Boul et reau PJ, Warren SM, Spect or JA, et al. Hypoxi a and VEGF up-regul at eBMP-2mRNA and prot ei n expressi on i n mi crovascul ar en-dot hel i al cel l s: Impl icat i ons for fract ure heal i ng. Plast i c&Re-const ruct i ve Surg,2002,109(7):2384-2397.
28. Peng H, Wri ght V, Usas A, et al. Synergi st i c enhancement of boneformat i on andheal i ng by st em cel l-expressed VEGF and bone mor-phogenet i c prot ei n-4. J Cl i nInvest,2002,110(6):751-759.
29. Grellier. M, Bordenave. L, Ame′de′e. J. Cell-to-cell communication betweenosteogenic and endothelial lineages: implications for tissue engineering. Trends inBiotechnology,2009,27(10),562-71.
30. Kanaan, R.A. et al. The role of connective tissue growth factor in skeletal growth anddevelopment.2006, Med Sci Monit12, RA277–281.
31. Lebrin, F. et al. TGF-beta receptor function in the endothelium.Cardiovasc Res,2005,65,599–608
32. Qu, G. and von Schroeder, H.P. Role of osterix in endothelin-1-induced downregulationof vascular endothelial growth factor in osteoblastic cells. Bone,2006,38,21–29
33. Niger, C. et al. Endothelin-1inhibits human osteoblastic celldifferentiation: influenceof connexin-43expression level. J CellBiochem,2008,103,110–122