关节透明软骨自我修复能力有限,其再生仍然是临床医生和研究人员面临的巨大挑战.尽管多种传统临床治疗方法显示出一定的疗效,但各自均存在其局限性和不足.近些年来,基于细胞的再生医学为软骨病损提供了一种很有希望的治疗策略.间充质干细胞作为一种多能祖细胞,具有来源广泛、多向分化及自我更新等特征,除增殖及成软骨分化外,其免疫调节、抗炎作用和旁分泌效应亦是其促进软骨修复再生的重要机制.目前,间充质干细胞在多项体内外研究中显示出较好的软骨修复效果.本文就近些年来应用于软骨修复再生的间充质干细胞生物学特征、常见组织来源、实际应用情况、各自的优缺点、最终去向和转归及促软骨修复再生机制等方面的研究进展进行综述.

局灶性关节软骨缺损在运动医学领域较为常见,通常导致关节疼痛和功能障碍,由于软骨无血管、缺乏内源性修复细胞的特点,其自愈能力有限,若不及时治疗最终将发展为骨关节炎.手术一直是临床治疗关节软骨缺损的主要手段,按照外科修复技术主要分为姑息性手术、修复性手术、重建性手术和软骨替代性手术等四大类.本文主要就近年来有关以上不同外科手段治疗局灶性软骨缺损的适应证、优势、局限性及远期疗效等方面的进展情况进行综述.

目的:通过术后CT评估,观察髁间窝外侧壁后软骨缘顶点(the apex of the deep cartilage,ADC)辅助定位前交叉韧带(anterior cruciate ligament,ACL)股骨骨道位置的解剖特点.方法:2014年7月至2016年7月,关节镜下ACL重建患者58例.术中屈膝120°,廓清髁间窝外侧壁暴露ADC.以ADC为参照,用off center为5～6 mm的股骨定位器辅助确定股骨骨道中心点的位置,继而制作股骨骨道.术后在CT上测量ADC至前骨缘的水平距离(La)以及与骨道中心点的水平距离(la),计算la/La的比值(Pa),同时测量骨道中心点与下骨缘的距离(Da).将此结果与36例残端中心定位的CT评估结果做比较(相应标记为Lb,lb,Pb及Db).符合正态分布数据采用独立样本t检验,非正态分布数据采用Mann-Whitney检验.结果:Pa平均值为44.1％(41.1％～49.5％),Pb平均值为44.9％ (40.9％～50.2％) (P=0.083).Da平均值为6.3mm(5～ 7.6 mm),Db=6.3 mm(5.1～7.5 mm)(P=0.833).结论:以ADC为标志,使用off center股骨定位器直接高屈膝位定位ACL股骨骨道,可获得与残端定位相似的位置.

Anterior cruciate ligament is the most important ligament to maintain the anterior and rotation stability of knee joint. Rupture of anterior cruciate ligament is one of the most common injuries of knee joint, and thus leads to knee instability and traumatic osteoarthritis. Anterior cruciate ligament reconstruction is usually performed to restore the anterior stability of knee joint, and is considered to reduce the secondary injury of medial meniscus, lateral meniscus and cartilage. Thus anterior cruciate ligament reconstruction can improve the function of knee joint. Traditional single bundle technique to reconstruct anterior cruciate ligament has been performed for many years. This technique can restore the anterior stability of knee joint and has excellent clini?cal results. Nearly 61%patients showed obvious radiographic osteoarthritis 20 years after anterior cruciate ligament reconstruction using bone-patella-bone graft. But, there is no agreement regarding to reduce the development of osteoarthritis after reconstruc?tion of anterior cruciate ligament. However, it has been reported that osteoarthritis would develop after reconstruction of anterior cruciate ligament in long term follow up study. Recently, with the further understanding of anatomy of biomechanics of anterior cru?ciate ligament, new techniques for anterior cruciate ligament reconstruction are developed, such as double bundle reconstruction, anatomic reconstruction and individual reconstruction. It remains controversial that whether these new technique can prevent the development of osteoarthritis after rupture of anterior cruciate ligament. Currently, no reconstruction technique for anterior cruci?ate ligament is perfect, and every technique has advantages and disadvantages. In terms of reducing the prevalence of osteoarthri?tis after reconstruction of anterior cruciate ligament, which technique is the best still remains unclear. New treatment and evalua?tion methods should be developed. In the future, not only the restoration of stability of knee joint should be considered, but also the articular cartilage contact kinematics including tibiofemoral joint and patellofemoral joint after anterior cruciate ligament recon?struction. Reduction of the development of osteoarthritis is an important topic after reconstruction of anterior cruciate ligament.

目的:以髁间窝外侧壁后软骨缘顶点(the apex of the deep cartilage,ADC)为标志,研究术后CT与镜下评估前交叉韧带(anterior cruciate ligament,ACL)股骨骨道位置时两种方法的一致性.方法:2014年5月至2016年5月,关节镜下前交叉韧带重建患者36例纳入本研究.所有患者术中均依据完好的股骨残端进行中心点定位.屈膝90°,测量ADC至前骨缘的水平距离(L1)以及与骨道中心点的水平距离(l1),计算l1/L1比值(P1),同时测量骨道中心点与下骨缘的距离(D1).术后在CT片上测量ADC至前骨缘的水平距离(L2)以及与骨道中心点的水平距离(l2),计算l2/L2比值(P2),同时测量骨道中心点与下骨缘的距离(D2).应用线性回归、Pearson相关和Bland-Altman分析比较镜下评估与CT评估的一致性.结果:P1=44.07％±2.22％ (41.1％％～49.5％),D1=6.2±0.5 mm (5.5～7 mm). P2=44.92％±2.27％(40.9％～50.2％),D2=6.3±0.6 mm(5.1～7.5 mm).P1和P2(r=0.99,P＜0.001)、D1和D2(r=0.69,P＜0.001))分别呈正相关,且线性关系显著(P＜0.001).Bland-Ahman图显示一致性较好.结论:以ADC为标志评估ACL股骨骨道位置,术后CT与镜下评估的一致性较好.

Objective: To find the differences and disadvantages of Chinese monitoring program for residues of veterinary drugs in milk compared to U.S.and New Zealand counterparts so as to provide references for perfecting Chinese monitoring program.Methods: Data from Chinese,U.S.and New Zealand monitoring programs for veterinary drug residues in milk from 2008 through 2011 were collected and compared in terms of drug types,the number of samples and monitoring results.Results: Similar drug types but smaller amounts of samples and incomplete coverage of all pastures were observed for Chinese monitoring program compared to U.S.and New Zealand counterparts.In 2010,results of monitoring of antibiotic residues in milk in China were statistically significantly higher than those in the U.S.and New Zealand(P<0.05).Based on this analysis,we suggest that the range of Chinese monitoring program should be widened to more regions and pastures and the number of samples should be increased.

Objective To compare the biological behaviors of rats' articular chondrocytes cultured with autologous serum(AS)and fetal bovine serum(FBS)media in vitro.Methods & Materials Articular chondrocytes were isolated from 8-week male SD rats and seeded into the medium with 5% autologous serum(AS),10% autologous serum(AS),and 10% fetal bovine serum(FBS),respectively for monolayer subcultivation.Cell morphological changes were observed by light microscope and growth curves were drawn to determine cell growth rates.Biological behaviors of chondrocytes were analyzed through toluidine blue staining,typeⅠ and typeⅡ collagen immunohistochemical staining,flow cytometry for CD26,CD44,and typeⅠ and typeⅡ collagen expression.Results(1)Polygonal shape of chondrocytes of primary culture developed to spindle shape at passage 3 with no exception in the three groups;(2)Chondrocytes growth rate in 5% AS group was similar to that in 10% FBS group,whereas slower than that in 10% AS group;(3)The excretion amount of acid mucopolysaccharide protein decreased in passage 3 in all groups,and was more obvious in 10% FBS group as compared with that in 5% and 10% AS groups,whereas there was no difference between the two AS groups;(4)The expression of type Ⅱcollagen in 5% and 10% AS groups was higher than that in 10% FBS group(P < 0.05)with no significant difference between the two AS groups(P > 0.05).The expression of type Ⅱcollagen decreased gradually in the three groups and the expression of type I collagen increased gradually.(5)The expression of CD44 increased during the culture in the three groups with significant differences between primary and passage 3(P < 0.05),while the expression of CD26 showed no significant differences in the three groups under the same cultured conditions.Conclusion This study found that chondrocytes cultured in 10% autologous serum revealed a higher growth rate and better preservation of phenotype,probably indicating that there could be a possibility to utilize higher concentration of autologous culture serum for shortening the culture duration and maintaining the chondrocytes phenotype for clinical transplantation.

Objective To study the effect of Smad4-specific siRNA and Runx2-specific siRNA on the formation of heterotopic ossification in rats.Methods(1)Ambion's siRNA target design online tool was utilized to design three sequences of DNA oligonucleotides for targeting rat Smad4 mRNA,and another three sequences of DNA oligonucleotides for targeting rat Runx2 mRNA.They were synthesized in vitro through transcription.The most optimal siRNA and transfection condition were determined by mRNA levels in cultured primary osteoblastic cells through RT-PCR.(2)Ambion's siRNA target design online tool was utilized to design two DNA oligonucleotides which encoded two hairpin siRNA templates for the gene of Smad4 and Runx2,the DNA oligonucleotides were ligated into shuttle vectors.The siRNA cassettes were then copy out with AscI and inserted into the AscI site of pBac(3xP3-EGFPafm).The non-viral vecotrs were transfected into primary osteoblastic cells and then the silencing effects on Smad4 and Runx2 were tested with RT-PCR and Western blot.(3)The evidence of heterotopic ossification after transfer of gene for inhibition of either Smad4 or Runx2 was observed.Results(1)Of the 3 Smad4-siRNA and 3 Runx2-siRNA,the inhibition of siRNA139-159 for Smad4,and the inhibition of siRNA1057-1077 for Runx2 appeared to be most evident.(2)Rat primary osteoblast cells were infected with the Smad4-siRNA,and the expression of Smad4 was reduced evidently.Rat primary osteoblast cells were infected with the Runx2-siRNA,and the expression of Runx2 was reduced evidently.(3)The formation of heterotopic ossification induced by tenotomy of Achilles tendon was significantly inhibited.Heterotopic ossification was inhibited with Smad4-specific siRNA by approximately 92%(P < 0.05)and with Runx2-specific siRNA by 93%(P < 0.05).The inhibition effects between Smad4-specific siRNA and Runx2-specific siRNA was statistical insignificance(P > 0.05).The blocking way of Smad4 or Runx2 was identical in vivo.Conclusion Non-virus-mediated transfering siRNA against Smad4 or Runx2 was able to inhibit heterotopic ossification induced by Achilles tenotomy in rats without different change in the way of blocking heterotopic ossification between Smad4 and Runx2.

Objective To measure the morphology of posterior cruciate ligament(PCL) insertion in human cadaver knee,and to search the optimized tunnel diameter for PCL reconstruction.Methods Twenty human cadaver knees were dissected,the periphery of PCL and boundary of anterolateral(AL)bundle and poteriomedial(PM)bundle in femoral and tibial attachment were marked,and the major axis and length of PCL,AL and PM bundles were confirmed and measured.The vertical line was drawn through the midpoint of the major axis,and the width of the footprint was measured along the vertical line to represent the maximum diameter of tunnel.The values were expressed as mean(from 25% to 75% percentile).Results The width of PCL,and AL and PM bundles in tibial attachment was 10.64(9.52 ~ 11.51)mm,7.54(6.50 ~ 8.58)mm and 6.85(6.37 ~ 7.56)mm,respectively.and in femoral attachment was 11.01(10.27 ~ 12.04)mm,9.56(8.91 ~ 10.52)mm and 9.47(8.85 ~ 10.28)mm,respectively.Conclusion According to principle of anatomic PCL reconstruction and consistency of femoral and tibial tunnel,the diameter of tunnel should be determined by intraoperative measurement.The mean diameter of tunnel for single-bundle PCL reconstruction and for double-bundle PCL reconstruction was 10.5 mm(from 9.5 mm to 11.5 mm)and 6.5 mm(from 6.0 mm to 7.5 mm) respectively.

Lin Lin, Henrik Gårdsvoll, Qing Huai, Mingdong Huang, and Michael Ploug, From the Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215, USA; Finsen Laboratory, Rigshospitalet section 3735, Copenhagen Biocenter room 3.3.31, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark; State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 155 Yang Qiao West Road, Fuzhou, Fujian 350002, China; Center for Proteases and Cancer Running Title: Crystal structure of the mouse ATFuPAR complex To whom correspondence should be addressed: Michael Ploug (m-ploug@finsenlab.dk) or Mingdong Huang (mhuang1@bidmc.harvard.edu; mhuang@fjirsm.ac.cn)

Objective To Find an optimal reaction condition for Runx2 specific siRNA(small interfering RNA)for the purpose of inhibiting osteoblast differentiation. Methods The Runx2 specific siRNA was synthesized in vitro transcription and then transfected into MC3T3-E1 cells with liposome to screen out an efficacious siRNA. The efficacious siRNA was transfected into cells,and total mRNA and protein were extracted. The expression of Runx2 and osteogenic genes was assessed by RT-PCR and Western blot. The alkaline phosphatase activity analysis was investigated. Results Our results show that the runx2/Cbfa1-specific siRNA inhibits the expression of Runx2 at the level of mRNA and protein. The expression levels of osteoblast maturation genes,type I collagen,osteopontin,bone sialoprotein,and osteocalcin are significantly decreased. The alkaline phosphatase activity in MC3T3-E1 cells is inhibited. Conclusion These studies show Runx2 specific siRNA inhibits differentiation in cultured MC3T3-E1 cells.It is likely that the inhibition of Runx2 by RNAi could be developed as a powerful approach to prevent or treat heterotopic ossification.

Objective To observe the effect of rabbit anterior cruciate ligament (ACL) reconstruction with irradiated Achilles tendon allograft, which was added with PDGF-BB、VEGF165 or TGFβ1 genes transduced mesenchymal stem cells(MSCs). Methods Forty male New Zealand white rabbits were divided randomly into five groups with eight animals for each:(1)control group, (2) MSCs group, (3) PDGF-BB transfected MSCs group, (4) VEGF165 transfected MSCs group, (5) TGFβ1 transfected MSCs group. The ACL was replaced with a Achilles tendon allograft. The 2×106 cells with 100 μL fibrin sealant was applied on the Achilles allograft. All animals were sacrificed 12 weeks after ACL reconstruction. Results Histologic findings showed that newly formed vessels and infiltrative fibroblasts were more abundant in VEGF165 group than other guoups. Biomechanical properties of PDGF-BB or TGFα1 groups were significantly better than B control on the max load, the stiffness, the absorbed energy and the maximum tensile strength (P0.05). The max load, the stiffness and the absorbed energy of MSCs or VEGF165 groups were higher than that of control group,while there were no significant differences (P0.05). The snap displacement of VEGF165 group was significantly higher than that of control group (P0.05). The rupture site was all at the body part of the graft, normal ACL and Achilles. Conclusion VEGF165 gene transfected MSCs could significantly promote angiogenesis in the reconstructed ligament, but it did not enhance the mechanical properties of the reconstructed ligament in the rabbit model. Both PDGF-BB gene and TGFβ1 gene transfected MSCs could accelerate the remoedling of the reconstructed ligament.

Objective To construct a recombinant adenovirus carrying BMP4 gene which is transfected into NIH3T3 cells and expressed.Methods BMP4 gene was cloned into the shuttle vector pAdTrack-CMV;the bacterium BJ5183 was contransfected with the shuttle and the pAdEasy vector and the recombinant adenoviral plasmid was produced by homologous.Recombinant adenovirus was packaged in HEK293 cells.NIH3T3 cells were transfected with recombinant adenovirus and BMP4 expression was confirmed by reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Modified Gomori method was performed on 3th and 6th day after transfection with BMP4 adenovirus.von kossa staining was used to evaluate the osteoblast differentiation.Results PCR and digesting demonstrated that the shuttle plasmid pAdTrack-BMP4 and the recombinant adenoviral plasmid were obtained.The recombinant adenovirus was packaged in HEK293 cells.The results of RT-PCR and western blot revealed that BMP4 was expressed in NIH3T3 cells.ALP expression was increased significantly in the NIH3T3 cells transfected with BMP4 adenovirus.von kossa staining confirmed the osteoblast differentiation in the cells transfected with BMP4 adenovirus.Conclusion Construction of BMP4 recombinant adenovirus and its expression in NIH3T3 laid the basis for BMP4 gene therapy for bone repair.

目的:验证核心结合因子(Cbfa1/Runx2)的重组腺病毒载体可高效感染脂肪组织来源干细胞并表达蛋白.方法:分离、扩增脂肪组织来源干细胞并诱导其向骨细胞、脂肪细胞分化.将Cbfa1/Runx2 cDNA的全长序列亚克隆到穿梭质粒pAdTrack上,在BJ5183细菌内和pAdEasy1同源重组,筛选阳性克隆,命名为Ad-Cb-fa1/Runx2.经酶切、PCR鉴定,线性化后以脂质体法转染293T细胞进行包装、扩增,利用报告基因GFP对病毒滴度和感染效率进行监测.通过RT-PCR、间接免疫荧光检测Ad-Cbfa1/Runx2感染脂肪组织来源干细胞后Cbfa1/Runx2基因的表达.结果:脂肪组织来源干细胞细胞数目两天增长一倍.在特定诱导条件下,可向成脂细胞、成骨细胞分化.酶切及PCR鉴定证实Cbfa1/Runx2基因重组腺病毒载体构建成功.Ad-Cbfa1/Runx2对脂肪组织来源干细胞的转染效率为81.39%.转染3天后,PCR、间接免疫荧光检测到脂肪组织来源干细胞中Cbfa1/Runx2蛋白的表达.结论:成功分离、培养了脂肪组织来源干细胞,构建了含有小鼠Cbfa1/Runx2基因的重组腺病毒载体,证明了Ad-Cbfa1/Runx可高效感染脂肪组织来源干细胞并表达Cbfa1/Runx2蛋白.