永生化下颌骨髁突软骨细胞的微囊化研究

为了探讨微囊包裹软骨细胞在软骨组织工程中的适用性 ,根据气流切割原理采用海藻酸钠 -多聚赖氨酸 -海藻酸钠 ( APA)对永生化下颌骨髁突软骨细胞 ( Im mortalized mandibular condylar chondrocyte,IMCC)进行微囊包裹。用倒置显微镜观察、台盼蓝染色、细胞记数、HE染色、免疫组化等方法检测微囊的大小、细胞的生长及微囊内组织的软骨特性等情况。研究发现 ,IMCC可在微囊内存活 ,活细胞率 >80 % ,微囊直径平均 779μm。细胞数量随着培养时间的延长逐渐增多 ,约 2 0 d左右达到平台期 ,细胞在囊内呈簇样生长 ,高表达软骨特异的蛋白多糖和 型胶原。提示 IMCC可在微囊内形成类软骨组织样结构 。

软骨母细胞瘤的CT诊断和鉴别诊断

目的探讨软骨母细胞瘤(CB)的CT表现,观察CT对该病的诊断及鉴别诊断价值。方法对经手术病理证实的34例CB患者的CT资料进行回顾性分析,分析其病灶位置、形态、密度及增强表现等特征。结果 28例(82.35%)患者病灶位于长骨;19例(55.88%)病灶边缘可见硬化边;21例(61.76%)病灶内可见点状或沙粒状钙化;全部21例接受增强扫描者均表现为轻中度强化。结论 CB有较为典型的CT表现;CT对CB的诊断和鉴别诊断具有重要价值。

血管内皮生长因子及其受体在大鼠下颌骨髁突软骨细胞中的表达和意义

目的 :研究VEGF及其受体 (Flt- 1和Flk - 1 )在大鼠下颌骨髁突软骨细胞中的表达 ,探讨其对大鼠下颌骨髁突软骨生长发育的影响。方法 :用免疫组化方法 ,对VEGF及其受体 (Flt - 1和Flk - 1 )在大鼠下颌骨髁突软骨细胞中的表达进行检测。结果 :大鼠下颌骨髁突软骨的增殖层、肥大层、矿化和钙化层均有表达 ,而纤维层没有表达。结论 :大鼠下颌骨髁突软骨的增殖层、肥大层及矿化和钙化层软骨细胞可产生并分泌VEGF及其受体 (Flt- 1和Flk - 1 ) ,VEGF可能在大鼠下颌骨髁突软骨生长发育中发挥作用。

脱细胞骨软骨支架的细胞相容性测定及与软骨细胞体外的复合

目的测定脱细胞和脱钙处理的脱细胞骨软骨支架的细胞相容性及该支架与软骨细胞体外复合培养的生物学特性和黏附关系,以探讨脱细胞骨软骨支架作为软骨组织工程支架的可行性。方法采用自制脱细胞骨软骨支架与软骨细胞体外复合培养,用细胞增殖度法测定脱细胞骨软骨支架的细胞相容性,并行电镜观察其相互黏附关系。结果脱细胞骨软骨支架的细胞相容性良好;体外复合实验中,软骨细胞可贴附于材料上生长增殖,并分泌细胞外基质。结论细胞增殖实验显示脱细胞骨软骨支架的细胞相容性良好;体外复合实验显示脱细胞骨软骨支架可与软骨细胞良好复合,适于软骨细胞贴附生长。

脱细胞骨软骨支架的制备及形态学观察

目的:探讨脱细胞骨软骨支架制备的可行性,为关节软骨组织工程提供新的支架材料。方法:3月龄雄性新西兰大白兔,取膝关节髌股关节面的股骨侧,切取4mm×4mm×3mm大小(长×宽×深)骨软骨块,用去垢剂-酶法对骨软骨组织块进行脱细胞脱钙处理,冻干机冻干,制备脱细胞骨软骨支架,并对其进行形态学观察。结果:脱细胞骨软骨支架呈半透明状,弹性良好,组织学评价提示细胞成分已完全祛去,保留了纤维支架。结论:去垢剂-酶法可以祛去骨软骨块中的细胞成分,成为带有空隙的支架材料。

骨软骨前体细胞分离鉴定及转化生长因子β3对其成软骨分化的影响

背景:关节软骨中存在着前软骨干细胞,可能成为软骨组织工程潜在的种子细胞,转化生长因子β3对前软骨干细胞的增殖及成软骨分化具有正向调节作用。目的:分析转化生长因子β3对骨软骨前体细胞成软骨的分化作用。方法:分离筛选出晚期骨关节炎患者软骨组织CD146^+软骨细胞并鉴定。培养CD146^+软骨细胞团块分为4组:为普通培养基组,转化生长因子β3-诱导组,转化生长因子β3+诱导组(含2.5μg/L重组人转化生长因子β3)和转化生长因子β3++诱导组(含10μg/L重组人转化生长因子β3)。培养4周后行组织块Ⅱ型胶原、Aggrecan免疫组织化学及相关基因实时荧光定量PCR检测。结果与结论:(1)成软骨诱导培养时,转化生长因子β3++诱导组所形成软骨块>转化生长因子β3+诱导组>转化生长因子β3-诱导组;(2)免疫组织化学结果显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan的表达明显高于转化生长因子β3+诱导组(P<0.05),转化生长因子β3+诱导组表达明显高于转化生长因子β3-诱导组(P<0.05);(3)实时荧光定量PCR检测显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan m RNA的表达明显强于转化生长因子β3+诱导组(P<0.05),转化生长因子β3+诱导组2指标表达明显强于转化生长因子β3-诱导组(P<0.05);转化生长因子β3++诱导组和转化生长因子β3+诱导组性别决定区Y框蛋白-9的表达均明显强于转化生长因子β3-诱导组(P<0.05);(4)结果表明,晚期骨关节炎患者残余关节软骨中存在着具有干细胞特性的骨软骨前体细胞;转化生长因子β3具有较强促骨软骨前体细胞成软骨分化的能力,其有可能成为软骨组织工程中理想的细胞因子。

珍骨胶囊对兔关节退变软骨细胞增殖的影响

目的研究珍骨胶囊含药血清对新西兰大白兔退变软骨细胞的调节作用。方法用含药血清、空白血清对大白兔退变软骨细胞干预,用MTT法检测含药血清组、空白组对软骨细胞增殖的影响。结果含药血清组细胞随血清浓度的增加而加快增殖速度,与同浓度空白血清组比较有显著性差异(P<0.05)。结论珍骨胶囊含药血清能促进体外培养退变软骨细胞迅速进入增殖周期,促进软骨细胞的增殖。

足跟骨软骨母细胞瘤的影像诊断及鉴别诊断

目的分析足跟骨软骨母细胞瘤(CB)的影响和鉴别诊断。方法选取2015年3月-2019年3月足跟骨CB患者20例,均进行MRI、CT、X线诊断。对其表现进行观察。结果20例足跟CB都有肿块,表现为膨胀感不明显、偏心性生长、伴有硬边化、边界清晰;7例X线呈现絮状钙化,6例呈现砂砾状钙化;1例呈现网格状分离;2例CT呈现不完整硬化边,毛刺;MRI显示11例T1W1较脂肪信号低,较高肌肉信号,明显硬化边,不均匀T2W1信号,高信号坏死囊变和钙化。骺软骨板增厚和化膜囊积液共2例。结论足跟骨CB经影像学表现一定特征,按照MRI、CT、X线表现,诊断和鉴别此病有促进作用。

“双相”组织工程软骨修复兔关节骨软骨缺损

目的探讨“双相”异体骨基质明胶(bonematrixgelatin,BMG)作为组织工程软骨载体,与同体骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)结合,构建组织工程软骨修复兔关节骨软骨缺损的效果。方法4月龄新西兰兔32只,雌雄不限,体重2～3kg。1体外实验:取5只新西兰兔,处死后取髂骨和四肢骨,制备一侧松质骨,一侧皮质骨的“双相”异体BMG载体,扫描电镜观察。另取新西兰兔18只,抽取骨髓,分离MSCs并诱导成软骨分化;将诱导而来的软骨前体细胞与“双相”BMG载体复合构建组织工程软骨,分别于1、3和5周取材行Masson、PAS染色和扫描电镜观察。2体内实验:将抽取骨髓的18只及余下的9只新西兰兔制成双侧股骨内髁骨软骨缺损模型,将前期制备的组织工程软骨同体植入18只兔的右股骨内髁骨软骨缺损(A组),左侧缺损移植异体BMG(B组),其余9只双侧软骨缺损未予处理作为空白对照(C组),分别于术后1、3和6个月取材,行大体、组织学和型胶原mRNA原位杂交观察,改良Wakitani法评分,比较各组修复效果差异。结果1体外实验:“双相”BMG松质骨面孔隙大小100～800μm,细胞于其中增生,形成富含细胞的软骨层;皮质骨面孔隙大小10～40μm,细胞层状覆盖于其表面,可作为起支撑作用的软骨下骨。2体内实验:A组术后1个月即可重建关节骨软骨缺损;修复软骨在观察期内逐渐变薄,但在6个月内始终保持关节面及软骨下骨结构完整。B、C组未能修复缺损,缺损周边软骨磨损加剧。改良Wakitani评分显示A组在3个时间点的各项评分结果,除6个月软骨厚度外,其它指标均优于B、C组,且差异有统计学意义(P<0.01)。型胶原mRNA原位杂交显示,A组缺损区修复组织中细胞阳性染色率明显高于B、C组,且差异有统计学意义(P<0.01)。结论“双相”异体BMG可作为组织工程软骨载体材料,其结合自体MSCs诱导的软骨前体细胞制备的组织工程软骨,可修复兔关节软骨和软骨下骨。

Wnt/β-catenin信号通路与骨关节炎

近年大量研究证实Wnt/β-catenin信号通路通过调节胚胎期软骨发育及出生后软骨发生、成骨细胞和破骨细胞生成、软骨内成骨、骨重塑、骨折修复等过程,调控骨骼系统相关疾病和骨代谢,对骨关节炎(OA)发生发展、骨软骨组织诱导和修复起着至关重要的作用。基因敲除小鼠模型常用于Wnt/β-catenin信号通路与OA关系研究,全基因组扫描已发现一些与OA密切相关的单核苷酸多态性位点。目前在多个水平进行Wnt/β-catenin信号通路靶向治疗OA研究,重点考虑药物安全性。该文就Wnt/β-catenin与OA关系的研究进展作一综述。

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

干细胞研究文章在国内外医学期刊发表的相关记载

1998年11月，美国威斯康星大学的汤姆生和约翰霍普金斯大学的吉尔哈特教授分别在《科学》和《美国科学院论文集》上报道了他们用不同方法获得了具有无限增殖和全能分化潜力的人胚胎干细胞。 这条消息引起了国际医学界的关注，并由此引发了全球性干细胞研究的热潮…… 干细胞的用途可涉及到医学界的多个领域。目前，科学家们已经完成了胚胎干细胞的体外鉴别、分离、纯化、扩增和培养，并以此为“种子”，培育出了人体的一些组织器官。

炎性预处理的脂肪干细胞条件培养基对骨性关节炎软骨细胞表型的调节作用

目的探讨炎性因子TNF-α、IL-1β和IFN-γ预处理脂肪间充质干细胞(Adipose mesenchymal stem cells,ADMSCs)是否可以改变其分泌组的生物学特性并分析其对人骨性关节炎软骨细胞的表型产生影响。方法获取并培养AD-MSCs和人骨性关节炎软骨细胞,分别使用工作浓度为10 ng/mL的TNF-α、IL-1β、IFN-γ的条件培养液和对照培养液(DMEM-F12+2%FBS)预处理AD-MSCs并收集对应的条件培养基(CM-1组、CM-2组、CM-3组和CM-0组),各组分别培养骨性关节炎软骨细胞72 h后,通过免疫荧光染色、RT-PCR和Western Blot法检测软骨细胞SOX-9、COL2、RUNX2、MMP13的表达情况,所有细胞实验均重复3次,比较4组SOX9、COL2、MMP13、RUNX2表达水平。结果CM-1组、CM-2组、CM-3组AD-MSCs较CM-0组细胞明显变得扁平、粗大。CM-1组、CM-2组、CM-3组SOX9、COL2的表达水平较CM-0组高,MMP13表达水平较CM-0组低,差异有统计学意义(P<0.05)。在基因水平上4组RUNX2的表达水平差异无统计学意义(P>0.05)。结论采用炎性因子预处理MSCs,形成的条件培养基可以明显增强骨性关节炎软骨细胞的成软骨能力,并降低骨性关节炎软骨细胞的肥大化和降解代谢水平。

Altered Gene Expression in Articular Chondrocytes of Smad3^(ex8/ex8) Mice, Revealed by Gene Profiling Using Microarrays

It has been previously reported that small mother against decapentaplegic 3 （Smad3） gene knockout （Smad3^ex8/ex8） mice displays phenotypes similar to human osteoarthritis, as characterized by abnormal hypertrophic differentiation of articular chondrocytes. To further clarify the crucial target genes that mediate transformation growth factor-β （TGF-β）/Smad3 signals on articular chondrocytes differentiation and investigate the underlying molecular mechanism of osteoarthritis, microarrays were used to perform comparative transcriptional profiling in the articular cartilage between Smad3^ex8/ex8and wild-type mice on day five after birth. The gene profding results showed that the activity of bone morphogenetic protein （BMP） and TGF-β/cell division cycle 42 （Cdc42） signaling pathways were enhanced in Smad3^ex8/ex8 chondrocytes. Moreover, there was altered gene expression in growth hormone/insulin-like growth factor 1 （Igfl） axis and fibroblast growth factor （Fgf） signaling pathway. Notably, protein synthesis related genes and electron transport chain related genes were upregulated in Smad3^ex8/ex8 chondrocytes, implying that accelerated protein synthesis and enhanced cellular respiration might contribute to hypertrophic differentiation of articular chondrocytes and the pathogenesis of osteoarthritis.

Repair of articular cartilage defects in minipigs by microfracture surgery and BMSCs transplantation

Objective:To investigate the feasibility of minimal invasive repair of cartilage defect by arthroscope-aided microfracture surgery and autologous transplantation of mesenchymal stem cells. Methods: Bone marrow of minipigs was taken out and the bone marrow derived mesenchymal stem cells (BMSCs) were isolated and cultured to passage 3. Then 6 minipigs were randomly divided into 2 groups with 6 knees in each group. After the articular cartilage defect was induced in each knee, the left defect received microfracture surgery and was injected with 2.5 ml BMSCs cells at a concentration of 3×107 cells/ml into the articular cavity; while right knee got single microfracture or served as blank control group. The animals were killed at 8 or 16 weeks, and the repair tissue was histologically and immunohistochemically examined for the presence of type Ⅱ collagen and glycosaminoglycans (GAGs) at 8 and 16 weeks. Results: Eight weeks after the surgery, the overlying articular surface of the cartilage defect showed normal color and integrated to adjacent cartilage. And 16 weeks after surgery, hyaline cartilage was observed at the repairing tissues and immunostaining indicated the diffuse presence of this type Ⅱ collagen and GAGs throughout the repair cartilage in the treated defects. Single microfracture group had the repairing of fibrocartilage, while during the treatment, the defects of blank group were covered with fewer fiber tissues, and no blood capillary growth or any immunological rejection was observed. Conclusion: Microfracture technique and BMSCs transplantation to repair cartilage defect is characterized with minimal invasion and easy operation, and it will greatly promote the regeneration repair of articular cartilage defect.

Chondrogenic differentiation of rat bone marrow mesenchymal stem cells induced by puerarin and tetrandrine

Objective: This study aims to clarify the effect of the active components puerarin and tetrandrine on the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: Using network pharmacology, protein targets of puerarin and tetrandrine were predicted, and a database of cartilage formation targets was established. The protein target information related to disease was then collected, and the drug-targeting network was constructed by analyzing the protein–protein interactions. Genes related to chondrogenesis induced by puerarin and tetrandrine and chondroblast differentiation signaling pathways were searched. Finally, potential drug-and disease-related genes,as well as proteins, were screened and verified using real-time RT-PCR and western blotting.Results: Network pharmacological studies have shown that puerarin and tetrandrine are involved in BMSCs cartilage differentiation. The experimental results showed that puerarin and tetrandrine could regulate the expression of cartilage differentiation-related genes and proteins. Puerarin increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9,as well as the gene expression of Col2 a1, Mmp13, Tgfb1, and Sox-9. Tetrandrine increased the protein expression of COL2 A1,COL10 A1, MMP13, and SOX-9, as well as the gene expression of Col10 a1, Tgfb1, Sox-9, and Acan. The combination of puerarin and tetrandrine increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9 and the gene expression of Col2 a1,Col10 a1, Sox-9, and Acan.Conclusions: Puerarin, tetrandrine, and their combination can promote the proliferation of BMSCs and induce their differentiation into chondrocytes, and they are thus expected to be inducers of chondrogenic differentiation. These results suggest that puerarin and tetrandrine have potential therapeutic effects on osteoarthritis.

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

4-Axis printing microfibrous tubular scaffold and tracheal cartilage application

Long-segment defects remain a major problem in clinical treatment of tubular tissue reconstruction.The design of tubular scaffold with desired structure and functional properties suitable for tubular tissue regeneration remains a great challenge in regenerative medicine.Here,we present a reliable method to rapidly fabricate tissueengineered tubular scaffold with hierarchical structure via 4-axis printing system.The fabrication process can be adapted to various biomaterials including hydrogels,thermoplastic materials and thermosetting materials.Using polycaprolactone(PCL)as an example,we successfully fabricated the scaffolds with tunable tubular architecture,controllable mesh structure,radial elasticity,good flexibility,and luminal patency.As a preliminary demonstration of the applications of this technology,we prepared a hybrid tubular scaffold via the combination of the 4-axis printed elastic poly(glycerol sebacate)(PGS)bio-spring and electrospun gelatin nanofibers.The scaffolds seeded with chondrocytes formed tubular mature cartilage-like tissue both via in vitro culture and subcutaneous implantation in the nude mouse,which showed great potential for tracheal cartilage reconstruction.