Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.

Effects of neurotrophin-3 intervention on on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis

Objective:To study the effects of neurotrophin-3 (NT-3) intervention on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis. Methods: Bone marrow mesenchymal stem cells were cultured and divided into control group, 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group, they were treated with different doses of NT-3 for 24 h, and then osteoblast marker gene, cell proliferation gene and apoptosis gene expression were determined.Results: RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression in 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group were significantly higher than those in control group whereas Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression were significantly lower than those in control group, and the larger the dose of NT-3, the higher the RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression whereas the lower the Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression.Conclusion: NT-3 intervention in bone marrow mesenchymal stem cells can promote osteoblast differentiation and cell proliferation and inhibit apoptosis.

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

Platelet-rich plasma-induced bone marrow mesenchymal stem cells versus autologous nerve grafting for sciatic nerve repair

Autologous nerve grafting is the gold standard of peripheral nerve repair.We previously showed that autologous platelet-rich plasma（PRP）contains high concentrations of growth factors and can induce in vitro cultured bone marrow mesenchymal stem cells（BMSCs）to differentiate into Schwann cells.Here we used PRP-induced BMSCs combined with chemically extracted acellular nerves to repair sciatic nerve defects and compared the effect with autologous nerve grafting.The BMSCs and chemically extracted acellular nerve promoted target muscle wet weight restoration,motor nerve conduction velocity,and axonal and myelin sheath regeneration,with similar effectiveness to autologous nerve grafting.This finding suggests that PRP induced BMSCs can be used to repair peripheral nerve defects.

Effects of bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery on bone metabolism and cytokines

Objective: To study the effects of bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery on bone metabolism and cytokines. Methods: Patients who were treated in our hospital due to bone nonunion after long bone fracture surgery between March 2011 and October 2017 were selected and randomly divided into two groups, combined group received bone marrow mesenchymal stem cell combined with platelet-rich plasma therapy, and control group received bone marrow mesenchymal stem cell therapy. The levels of bone metabolism markers and growth cytokines in serum as well as the expression of bone metabolism-related signal molecules in peripheral blood were determined before treatment and 1 month after treatment. Results: Compared with those of same group before treatment, serum PINP, OPG, BALP, VEGF, TGF-β1, IGF-I, IGF-II and bFGF levels as well as peripheral blood Runx2, Wnt1, Wnt3a and β-catenin expression intensity of both groups of patients significantly increased whereas serum β-CTX and RANKL levels as well as peripheral blood NOX4 and NF-κB expression intensity significantly decreased after treatment, and serum PINP, OPG, BALP, VEGF, TGF-β1, IGF-I, IGF-II and bFGF levels as well as peripheral blood Runx2, Wnt1, Wnt3a and β-catenin expression intensity of combined group after treatment were higher than those of control group whereas serum β-CTX and RANKL levels as well as peripheral blood NOX4 and NF-κB expression intensity were lower than those of control group. Conclusion: Bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery can be more effective than bone marrow mesenchymal stem cell monotherapy to improve the bone metabolism and increase the cytokines.

Platelet-rich plasma enhances adipose-derived stem cell-mediated angiogenesis in a mouse ischemic hindlimb model

AIM To evaluate the angiogenic effect of platelet-rich plasma(PRP)-preconditioned adipose-derived stem cells(ADSCs) both in vitro and in a mouse ischemic hindlimb model.METHODS ADSCs were divided based on culture medium: 2.5% PRP, 5% PRP, 7.5% PRP, and 10% PRP. Cell proliferation rate was analyzed using the MTS assay. The gene expression of CD31, vascular endothelial growth factor, hypoxia-inducible factors, and endothelial cell nitric oxide synthase was analyzed using reverse transcription polymerase chain reaction. Cell markers and structural changes were assessed through immunofluorescence staining and the tube formation assay. Subsequently, we studied the in vivo angiogenic capabilities of ADSCs by a mouse ischemic hindlimb model.RESULTS The proliferation rate of ADSCs was higher in the 2.5%, 5%, and 7.5% PRP groups. The expression of hypoxia-inducible factor, CD31, vascular endothelial growth factor, and endothelial cell nitric oxide synthase in the 5% and 7.5% PRP groups increased. The 5%, 7.5%, and 10% PRP groups showed higher abilities to promote both CD31 and vascular endothelial growth factor production and tubular structure formation in ADSCs. According to laser Doppler perfusion scan, the perfusion ratios of ischemic limb to normal limb were significantly higher in 5% PRP, 7.5% PRP, and human umbilical vein endothelial cells groups compared with the negative control and fetal bovine serum(FBS) groups(0.88 ± 0.08, 0.85 ± 0.07 and 0.81 ± 0.06 for 5%, 7.5% PRP and human umbilical vein endothelial cells compared with 0.42 ± 0.17 and 0.54 ± 0.14 for the negative control and FBS, P < 0.01).CONCLUSION PRP-preconditioned ADSCs presented endothelial cell characteristics in vitro and significantly improved neovascularization in ischemic hindlimbs. The optimal angiogenic effect occurred in 5% PRP-and 7.5% PRPpreconditioned ADSCs.

麝香黄芪复方滴丸含药血清促进骨髓间充质干细胞增殖分化

背景:骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)已被广泛用于治疗神经系统疾病,但由于血脑屏障的限制以及干细胞在受损部位存活率、分化率低,导致治疗效果有限。目的:探讨麝香黄芪复方滴丸含药血清对BMSCs增殖、迁移和向星形胶质细胞分化的影响。方法:SD雄性大鼠连续灌胃麝香黄芪复方滴丸5 d后进行腹主动脉取血,分离血清备用。采用CCK-8法检测体积分数5%,10%,20%含药血清对BMSCs增殖的影响;划痕实验观察体积分数10%含药血清对BMSCs横向迁移的影响;Transwell小室培养BMSCs,用结晶紫染色和DAPI核染色观察体积分数10%含药血清对BMSCs纵向迁移的影响;用含体积分数10%含药血清的诱导液或与星形胶质细胞共培养观察BMSCs向星形胶质细胞分化情况。结果与结论:①体积分数10%和20%含药血清在第2,3天促进细胞增殖更明显,且两种体积分数无统计学差异;②在划痕30,48 h时,10%含药血清组BMSCs迁移量显著高于对照组;③10%含药血清组BMSCs穿过Transwell小室滤过膜的数量高于对照组;④体积分数10%含药血清可能促进BMSCs向星形胶质细胞方向分化,但分化作用较弱,星形胶质细胞能进一步促进含药血清诱导BMSCs向星形胶质细胞方向分化;⑤结果表明,体积分数10%含药血清能促进BMSCs体外增殖、迁移;与星形胶质细胞共培养可能促进BMSCs向星形胶质细胞方向分化。

槐角提取物对颌骨骨髓间充质干细胞成骨向分化的影响

目的 :探讨槐角提取物对颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,jBMSCs)成骨分化的影响。方法:采用CCK-8法检测10、25、50、100μmol/L槐角提取物溶液对jBMSCs增殖的影响,采用BCIP/NBT碱性磷酸酯酶显色、茜素红染色检测10、25、50μmol/L槐角提取物溶液对jBMSCs成骨分化水平的影响,采用蛋白免疫印迹试验(Western blot)和实时定量荧光PCR检测槐角提取物溶液对jBMSCs成骨分化相关分子的蛋白和mRNA表达水平的影响。采用GraphPad Prism 9软件包对数据进行统计学分析。结果:100μmol/L槐角提取物溶液对jBMSCs有明显毒性作用(P<0.0001),10、25和50μmol/L浓度不影响细胞增殖(P>0.05)。与对照组相比,10、25和50μmol/L组的碱性磷酸酶活性、钙结节数量、成骨分化相关分子的蛋白和mRNA表达水平均逐渐升高。结论:10、25、50μmol/L浓度的槐角提取物能促进jBMSCs成骨向分化。

机械牵张通过Piezo1调控骨髓间充质干细胞成骨分化的作用机制

目的探讨机械牵张通过Piezo1促进小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用及机制。方法将BMSCs分为0%、3%、6%和12%机械牵张强度组,CCK-8检测细胞增殖,PCR检测成骨因子(Runx2、Osterix、ALP和OPN)mRNA,筛选出最佳牵张强度。再分别设置对照、机械牵张、Piezo1抑制剂、机械牵张+Piezo1抑制剂组。干预结束后,进行ALP染色和ALP活性检测。茜素红染色观察钙结节形成。Western blot检测Piezo1、TGF-β1、p-Smad2和Runx2蛋白水平。结果与0%机械牵张相比,3%和6%机械牵张促进了细胞增殖(P<0.05),且6%机械牵张组Runx2、Osterix、ALP和OPN的mRNA均显著升高(P<0.05)。另外,与对照组相比,机械牵张组ALP染色较深,ALP活性显著升高,钙结节数量增加,Piezo1、TGF-β1、p-Smad2和Runx2蛋白也显著升高(P均<0.05);Piezo1抑制剂组ALP染色较浅,ALP活性显著降低,钙结节数量减少,Piezo1、TGF-β1、p-Smad2和Runx2蛋白也显著降低(P均<0.05)。与机械牵张组相比,机械牵张+Piezo1抑制剂组ALP染色较浅,ALP活性显著降低,钙结节数量减少,Piezo1、TGF-β1、p-Smad2和Runx2蛋白也显著降低(P均<0.05)。与Piezo1抑制剂组相比,机械牵张+Piezo1抑制剂组ALP染色较深,ALP活性显著升高,钙结节数量增加,Piezo1、TGF-β1、p-Smad2和Runx2蛋白也显著升高(P均<0.05)。结论机械牵张可能通过Piezo1上调了TGF-β1/Smad2信号通路表达,促进BMSCs的增殖和成骨分化。

MYSM1对大鼠颌骨骨髓间充质干细胞增殖和成骨分化能力的影响

背景去泛素化酶能够调控骨骼生长发育,MYSM1基因缺失会造成全身骨骼疏松,当前并无MYSM1对颌骨发育影响的研究。目的探讨MYSM1对颌骨来源的骨髓间充质干细胞成骨分化能力的影响。方法分离10只8周Wistar大鼠下颌骨,利用组织消化法分离培养颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,JBMMSCs)。取生长良好的第3~5代JBMMSCs进行成骨诱导,利用qPCR、Western blot技术检测成骨标志物OCN、Runx2、ALP及去泛素化酶MYSM1的变化。分别利用空转染慢病毒和MYSM1敲低的慢病毒转染JBMMSCs,设置对照组和敲低组细胞,qPCR检测敲低效率。CCK-8、细胞克隆形成实验检测其敲低MYSM1对细胞的增殖能力影响,细胞划痕实验检测细胞迁移能力。空转染慢病毒和敲低MYSM1的JBMMSCs成骨诱导7 d后利用qPCR技术、ALP、茜素红染色检测成骨的表达变化。结果JBMMSCs成骨诱导成功后,MYSM1的mRNA和蛋白表达水平升高(P<0.05)。CCK-8显示对照组和敲低组的细胞增殖能力无统计学差异(P>0.05),两组间细胞集落形成实验和细胞迁移能力有统计学差异(P<0.05)。敲低组JBMMSCs在成骨诱导7 d后OCN、Runx2、ALP的mRNA表达水平降低(P<0.05),ALP、茜素红染色浅于对照组。结论敲低MYSM1并未对JBMMSs的增殖和迁移能力造成显著影响,而敲低MYSM1的JBMMSCs成骨分化能力降低。

黄连素在高糖环境下促进骨髓间充质干细胞的成骨分化

背景:糖尿病患者的口腔种植体骨结合率低下,失败率高,严重影响了生活质量,亟需有效手段改善糖尿病患者种植体骨结合以提高成功率。探究黄连素在高糖环境下对骨髓间充质干细胞成骨分化的影响及具体机制,将能为解决上述问题提供有效的理论支撑。目的:探讨天然提取物黄连素在高糖微环境下对大鼠骨髓间充质干细胞成骨分化的影响。方法:通过全骨髓贴壁法培养SD大鼠骨髓间充质干细胞。CCK-8法检测在高糖生理环境下添加不同浓度黄连素对骨髓间充质干细胞增殖的影响并筛选出最适黄连素浓度。通过碱性磷酸酶活性、茜素红染色以及PCR检测Runx2、Osx表达,判断黄连素在高糖环境下对骨髓间充质干细胞成骨分化的影响。为进一步探索作用机制,添加AMPK特异性抑制剂Dorsomorphin,使用DCFH-DA活性氧荧光探针检测活性氧水平,Western blot检测p-AMPK的表达。结果与结论:①10μmol/L为黄连素促进骨髓间充质干细胞增殖的最适浓度;②黄连素在高糖微环境下促进骨髓间充质干细胞矿化结节形成并提高碱性磷酸酶活性;③黄连素在高糖微环境下促进Runx2和OSx基因表达;④黄连素可有效抑制高糖环境下骨髓间充质干细胞的活性氧水平;⑤黄连素促进骨髓间充质干细胞成骨及抑制活性氧的作用被AMPK抑制剂逆转;⑥黄连素可激活AMPK,促进p-AMPK表达;⑦上述结果表明,黄连素(10μmol/L)能够通过激活AMPK并降低细胞内活性氧水平,从而促进高糖环境下骨髓间充质干细胞的成骨分化。

淫羊藿苷含药血清促进3种细胞共培养体系中软骨细胞增殖和干细胞成软骨分化

背景:关节软骨损伤修复能力十分有限,组织工程技术为修复受损软骨提供了新的治疗方案,其中软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞之间的相互影响和诱导作用是自体软骨损伤愈合的基础。目的:构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系用以模拟软骨细胞体内微环境,并探究其最佳细胞接种比例,同时观察淫羊藿苷含药血清对该体系中软骨细胞增殖和干细胞成软骨分化的影响。方法:提取、培养、鉴定大鼠膝关节软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞,按不同细胞接种比例构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞非接触共培养体系,共培养72 h后观察软骨细胞增殖活性和表型能力,选择综合效应最佳的共培养体系;用淫羊藿苷溶液(0.25 mg/m L)灌胃新西兰大白兔制备淫羊藿苷含药血清,对照组共培养体系用含体积分数为10%胎牛血清、1%双抗的高糖DMEM培养液培养,实验组共培养体系在此基础之上加入体积分数为10%淫羊藿苷含药血清进行干预,24,48 h后检测两组软骨细胞增殖活性和Ⅱ型胶原表达,14 d后采用免疫荧光染色检测骨髓间充质干细胞、滑膜间充质干细胞成软骨分化情况。结果与结论:(1)3种细胞以不同比例共培养时均可正常贴壁生长,当软骨细胞、骨髓间充质干细胞、滑膜间充质干细胞接种比例为2∶1∶1时,共培养体系中软骨细胞表现出最佳增殖活性和表型能力;(2)与对照组相比,实验组培养24 h后软骨细胞增殖活性和Ⅱ型胶原表达显著升高(P<0.01),48 h后两组仍有差异(P<0.05),培养14 d后两组骨髓间充质干细胞和滑膜间充质干细胞出现明显的软骨分化,部分细胞呈现圆形或椭圆形,胞浆Ⅱ型胶原免疫荧光染色为阳性,实验组荧光强度明显高于对照组(P<0.01);(3)结果表明,以非接触共培养方法可以成功建立软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系且细胞比例为2∶1∶1时软骨细胞增殖活性和表型能力最佳;同时淫羊藿苷含药血清具有促进该体系中软骨细胞增殖及骨髓间充质干细胞、滑膜间充质干细胞成软骨分化的作用。

聚己内酯-聚多巴胺-AOPDM1支架在高糖环境下的促成骨性能

背景:口腔颌面骨组织缺损会严重影响患者的身心健康,当糖尿病患者发生骨缺损时血糖异常导致的骨代谢紊乱使修复治疗更加困难。目的:试图将一种具有改善肥胖潜在生物活性的多肽AOPDM1应用于糖尿病患者的骨损伤修复。方法:在正常或高糖环境下,分别采用不同浓度的AOPDM1干预小鼠骨髓间充质干细胞,检测细胞增殖、碱性磷酸酶活性、矿化结节形成及成骨分化基因的表达。利用静电纺丝技术制备聚己内酯支架,以聚多巴胺对支架进行表面改性,制备聚己内酯-聚多巴胺复合支架;最后将支架置于AOPDM1溶液中,制备聚己内酯-聚多巴胺-AOPDM1支架,检测3组支架的水接触角及力学性能。在正常或高糖环境下,将3组支架分别与小鼠骨髓间充质干细胞共培养,检测细胞黏附、碱性磷酸酶活性及骨桥蛋白的表达。结果与结论:①与正常环境相比,高糖环境抑制了骨髓间充质干细胞的增殖;相同环境下,AOPDM1可促进小鼠骨髓间充质干细胞的增殖。在AOPDM1浓度相同的情况下,与正常环境相比,高糖环境下骨髓间充质干细胞的碱性磷酸酶活性、矿化能力及Ⅰ型胶原、骨桥蛋白、碱性磷酸酶、Runx2 mRNA表达均降低;相同环境下,AOPDM1可提高骨髓间充质干细胞的碱性磷酸酶活性、矿化能力及Ⅰ型胶原、骨桥蛋白、碱性磷酸酶、Runx2 mRNA表达。②聚己内酯-聚多巴胺支架、聚己内酯-聚多巴胺-AOPDM1支架的亲水性高于聚己内酯支架(P<0.001),3组支架的抗拉强度与弹性模量比较差异均无显著性意义(P>0.05)。与其他两组支架相比,聚己内酯-聚多巴胺-AOPDM1支架上的细胞具有更好的黏附形态。当支架相同时,与正常环境相比,高糖环境抑制了骨髓间充质干细胞的碱性磷酸酶活性及骨桥蛋白表达;当环境相同时,聚己内酯-聚多巴胺-AOPDM1支架上骨髓间充质干细胞的碱性磷酸酶活性及骨桥蛋白表达高于其他两组支架。③结果表明,聚己内酯-聚多巴胺-AOPDM支架可促进高糖环境下骨髓间充质干细胞的成骨性能。

Does progress in microfracture techniques necessarily translate into clinical effectiveness?

BACKGROUND Multitudinous advancements have been made to the traditional microfracture(MFx)technique,which have involved delivery of various acellular 2nd generation MFx and cellular MFx-III components to the area of cartilage defect.The relative benefits and pitfalls of these diverse modifications of MFx technique are still not widely understood.AIM To comparatively analyze the functional,radiological,and histological outcomes,and complications of various generations of MFx available for the treatment of cartilage defects.METHODS A systematic review was performed using PubMed,EMBASE,Web of Science,Cochrane,and Scopus.Patients of any age and sex with cartilage defects undergoing any form of MFx were considered for analysis.We included only randomized controlled trials(RCTs)reporting functional,radiological,histological outcomes or complications of various generations of MFx for the management of cartilage defects.Network meta-analysis(NMA)was conducted in Stata and Cochrane’s Confidence in NMA approach was utilized for appraisal of evidence.RESULTS Forty-four RCTs were included in the analysis with patients of mean age of 39.40(±9.46)years.Upon comparing the results of the other generations with MFX-I as a constant comparator,we noted a trend towards better pain control and functional outcome(KOOS,IKDC,and Cincinnati scores)at the end of 1-,2-,and 5-year time points with MFx-III,although the differences were not statistically significant(P>0.05).We also noted statistically significant Magnetic resonance observation of cartilage repair tissue score in the higher generations of microfracture(weighted mean difference:17.44,95%confidence interval:0.72,34.16,P=0.025;without significant heterogeneity)at 1 year.However,the difference was not maintained at 2 years.There was a trend towards better defect filling on MRI with the second and third generation MFx,although the difference was not statistically significant(P>0.05).CONCLUSION The higher generations of traditional MFx technique utilizing acellular and cellular components to augment its potential in the management of cartilage defects has shown only marginal improvement in the clinical and radiological outcomes.

牵张应力调控Notch1信号通路促进大鼠BMSCs增殖和成骨分化

目的 探讨牵张应力调控Notch1促进骨髓间充质干细胞(BMSCs)增殖和成骨分化的机制。方法 制备并鉴定大鼠BMSCs。细胞分组设置为:0%拉伸幅度组、0%+DAPT组、5%拉伸幅度组、5%+DAPT组。运用CCK-8法测定BMSCs增殖;采用碱性磷酸酶、茜素红和油红O染色法检测细胞成骨和成脂分化;通过Western bloting检测Notch1和Jagged1蛋白表达。结果 5%拉伸幅度牵张应力可显著提高BMSCs增殖、碱性磷酸酶表达及钙化结节形成,抑制BMSCs成脂分化(P<0.05或P<0.01),同时显著提高Notch1和Jagged1蛋白表达(P<0.01);DAPT可显著逆转牵张应力对BMSCs增殖、碱性磷酸酶表达、钙化结节形成、成脂分化及Notch1和Jagged1蛋白表达的影响(P<0.05或P<0.01)。结论 5%拉伸幅度的牵张应力促进了BMSCs增殖和成骨分化,Notch1信号通路可能是牵张应力促进BMSCs增殖和成骨分化的靶点。

不同牵张应力对大鼠BMSCs增殖和成骨分化作用

目的:考察牵张应力大小和作用时间对骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。方法:SPF级SD大鼠10只,分离培养并采用成骨和成脂分化法鉴定BMSCs。细胞分组设置为牵张应力加载大小(0.5%拉伸幅度,5%拉伸幅度,10%拉伸幅度、15%拉伸幅度)和作用时间(0.5h,6h,12h,24h,48h,72h)。CCK-8法测定BMSCs增殖。BCIP/NBT法测定细胞碱性磷酸酶活性。茜素红和油红O染色法检测细胞成骨和成脂分化。结果:10%拉伸幅度的牵张应力作用12h,可最大提高BMSCs增殖速度1.53倍,高于5%和15%拉伸幅度组,差异具有统计学意义(P<0.01)。5%拉伸幅度的牵张应力作用48h,可最大提高BMSCs碱性磷酸酶表达4.2倍,提高BMSCs矿化结节形成65%,减少BMSCs形成脂滴32%,高于10%和15%拉伸幅度组,差异具有统计学意义(P<0.05或P<0.01)。结论:牵张应力拉伸幅度为10%作用时间12h促进BMSCs增殖效果最佳。牵张应力拉伸幅度为5%作用时间48h提高BMSCs碱性磷酸酶和矿化结节形成最显著,进而对BMSCs发挥促进成骨分化作用。牵张应力促进BMSCs增殖和成骨分化所需牵张应力大小及作用时间不同。

单味中药提取物及有效成分对骨髓间充质干细胞增殖和分化的研究进展

骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)为骨髓中的多能干细胞,在一定条件下在诱导因子的诱导下可分化为多种细胞。近年来BMSCs成为各项生命科学研究的重点,临床上对于很多疾病的诊治提供新的思路与方向,具有广泛的应用前景。近年来大量研究表明单味中药提取物或有效成分对BMSCs的增殖、分化有显著促进作用,现做一综述。

神经生长因子对小鼠颌骨骨髓间充质干细胞增殖、迁移、分化及凋亡的影响

背景神经生长因子(nerve growth factor,NGF)是神经营养因子中的一类,能够促进中枢及外周神经元加速生长,修复损伤的神经系统,有研究证实NGF能促进中胚层来源的长骨骨髓间充质干细胞的成骨分化及骨形成,而NGF对外胚层来源的颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,JBMMSCs)生物学特性的影响尚不明确。目的探讨NGF对JBMMSCs增殖、迁移、分化及凋亡特性的影响。方法原代培养小鼠JBMMSCs并鉴定。取第二代JBMMSCs加入含50 ng/mL NGF培养液孵育72h后,通过CCK-8、qRT-PCR、Western blot及免疫荧光(immunofluorescence,IF)等方法检测细胞增殖及其相关标志物MCM-2、Ki67的表达;qRT-PCR、Western blot检测凋亡相关标志物Bcl-3、Bax的表达;划痕实验检测JBMMSCs迁移能力;碱性磷酸酶(alkaline phosphatase,ALP)染色、qRT-PCR、Western blot及IF检测成骨相关标志物表达。结果CCK-8结果显示,50 ng/mL NGF可显著增强JBMMSCs第3天及第5天的OD值,Western blot及IF结果显示,50 ng/mL NGF可显著增强JBMMSCs中增殖标志物Ki67、MCM-2的蛋白水平;划痕实验结果显示,50 ng/mL NGF可显著提高JBMMSCs的迁移率,有效缩小各个时间点的划痕面积;50 ng/mL NGF可有效增强JBMMSCs的碱性磷酸酶活性,上调ALP、OCN、RUNX2、BMP2的mRNA表达水平及ALP、OCN、BMP2、OPN、RUNX2的蛋白水平;此外,50 ng/mL NGF还可上调Bcl-2的mRNA及蛋白表达水平,下调Bax的mRNA及蛋白表达水平。结论50 ng/mL NGF能有效提高JBMMSCs增殖、迁移及成骨分化能力,同时抑制其凋亡。