Objective: Fibrocartilage transition zone (FC) is difficult to regenerate after surgical re-attachment of tendon to bone. Here, we investigated whether type II collagen-sponges (CII-sponges) facilitated tendon stem/progenitor cells (TSPCs) to adopt chondrogenic phenotypes and further observed if this material could increase the FC areas in bone-tendon junction (BTJ) injury model. Methods: CII-sponges were made as we previously described. The appearance and pore structure of CII-sponges were photographed by camera and microscopies. The viability, proliferation, and differentiation of TSPCs were examined by LIVE/DEAD assay, alamarBlue, and PKH67 in vitro tracking. Subsequently, TSPCs were seeded in CII-sponges, Matrigel or monolayer, and induced under chondrogenic medium for 7 or 14 days before being harvested for qPCR or being transplanted into nude mice to examine the chondrogenesis of TSPCs. Lastly, partial patellectomy (PP) was applied to establish the BTJ injury model. CII-sponges were interposed between the patellar fragment and tendon, and histological examination was used to assess the FC regeneration at BTJ after surgery at 8 weeks. Results: CII-sponges were like sponges with interconnected pores. TSPCs could adhere, proliferate, and differentiate in this CII-sponge up to 14 days at least. Both qPCR and immunostaining data showed that compared with TSPCs cultured in monolayer or Matrigel, cells in CII-sponges group adopted more chondrogenic phenotypes with an overall increase of chondrocyte-related genes and proteins. Furthermore, in PP injured model, much more new formed cartilage-like tissues could be observed in CII-sponges group, evidenced by a large amount of positive proteoglycan expression and typical oval or round chondrocytes in this area. Conclusion: Our study showed that CII-sponges facilitated the TSPCs to differentiate toward chondrocytes and increased the area of FCs, which suggests that CII-sponges are meaningful for the reconstruction of FC at bone tendon junction. However, the link between the two phenomena requires further research and validation.

BACKGROUND:Bone-tendon junction (BTJ) is a unique structure connecting tendon and bone through a fibrocartilage zone. Owing to its unique structure, the regeneration of BTJ remains a challenge. Here, we study the fibrochondrogenic differentiation of human tendon-derived stem/progenitor cells (TSPCs) both in vitro and in vivo. METHODS:TSPCs were isolated from human patellar tendon tissues and investigated for their multidifferentiation potential. TSPCs were cultured in chondrogenic medium with transforming growth factor beta 3 (TGF-β3) and BMP-2 in vitro ​and examined for the expression of fibrochondrogenic marker genes by quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence. TSPCs pretreated were also seeded in collage II sponge and then transplanted in immunocompromised nude mice to examine if the fibrochondrogenic characteristics were conserved in vivo. RESULTS:We found that TSPCs were differentiated towards fibrochondrogenic lineage, accompanied by the expression of collagen I, collagen II, SRY-box transcription factor 9 (Sox 9), and tenascin C. Furthermore, after TSPCs were seeded in collagen II sponge and transplanted in immunocompromised nude mice, they expressed fibrochondrogenic genes, including proteoglycan, collagen I, and collagen II. CONCLUSION:Taken together, this study showed that TSPCs are capable of differentiating towards fibrocartilage-like cells, and the fibrochondrogenic characteristics were conserved even in vivo, and thus might have the potential application for fibrocartilage regeneration in BTJ repair. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE:TSPCs are able to differentiate into fibrocartilage-like cells and thus might well be one potential cell source for fibrocartilage regeneration in a damaged BTJ repair.

[Objective] To investigate the clinical outcomes of minimally invasive percutaneous plate osteosynthesis (MIPPO) versus open reduction and internal fixation (ORIF) in the treatment of proximal humerus fracture.[Methods]We retrospectively analyzed 57 patients with proximal humerus fracture who were followed up after their surgeries from January to December,2014.Among these patients,27 (9 males and 18 females) with a mean age of 70.5 (51.4 and 89.5 for males and females,respectively) were treated with MIPPO,and based on the Neer classification,there were 14,11,and 2 cases with two,three,and four-part fractures of proximal humerus,respectively.The remaining 30 cases (11 males and 19 females) with a mean age of 69.5 (52.3 and 86.8 for males and females,respectively) were treated with ORIF,and there were 13,14,and 3 cases with two,three,and four-part fractures of the proximal humerus,respectively.We compared the operation-related indices,postoperative complications,and patients' shoulder functions assessed by Neer score after surgery.[Results] Fifty-sevenpatients were followed up for 13 to 24 months (mean 17.8 months).Infection,axillary nerve paralysis,and avascular necrosis of the humeral head was each identified in 1 case,and 3 cases had ineffective internal plate fixation post-operatively.The MIPPO group had shorter incision length (P＜0.05),less blood loss (P＜0.05),shorter hospital stay (P＜0.05),and faster fracture healing (P＜0.05) compared with the ORIF group.There was no difference in the operation time,the incidence of post operational complication,or the Neer score of shoulder function at 12 months after surgery between the two groups (P ＞0.05).[Conclusions] MIPPO generates small invasion and faster facture healing.

目的 比较股骨近端防旋髓内钉与骨水泥型人工股骨头置换术治疗高龄不稳定股骨转子间骨折患者术后短期活动功能的疗效.方法 选取2010年1月~2013年12月在我院接受治疗的117例70岁以上不稳定股骨转子间骨折患者,其中69例接受PFNA闭合复位内固定术,48例接受人工股骨头置换术.比较两组术中情况和术后髋关节功能恢复情况.结果 人工股骨头置换组手术时间、术中出血量、下床活动时间均明显少于PFNA组(P<0.05),两组患者术后引流量差异不显著(P>0.05).术后1、3和6个月时,人工股骨头置换组患者Harris评分均显著高于PFNA组(P<0.05),但在12个月时两组差异不显著.两组共出现4例并发症,3例PFNA的螺旋刀头切割,1例人工股骨头置换术患者发生假体松动.结论 骨水泥型人工股骨头置换术治疗伤前活动功能良好的高龄不稳定股骨转子间骨折患者的短期步行活动功能较好.

目的：对比观察镍钛聚髌器(NT-PC)与AO张力带治疗髌骨骨折的临床效果。方法74例髌骨骨折患者，随机分为A组(32例)与B组(42例)， A组采用镍钛聚髌器内固定， B组采用AO张力带内固定，根据患者骨折愈合时间、膝关节活动度及膝关节功能评价两种固定方法的临床效果。结果A组和B组患者骨折愈合时间分别为(14.1±2.1)周和(13.8±2.2)周，组间比较差异无统计学意义(P>0.05)；A、B两组患者膝关节活动度优良率分别为87.5%和81.0%，膝关节功能的优良率分别为93.8%和85.7%，差异均无统计学意义(P>0.05)。结论NT-PC与AO张力带治疗髌骨骨折，均取得了良好的治疗效果， AO张力带操作相对复杂， NT-PC在粉碎性髌骨骨折的治疗表现出较多的优势，因此本文为临床应用的手术方式选择提供了参考。

p 62/SQSTM 1是一种多功能泛素结合蛋白及信号转导途径中的支架和适配子蛋白,其分子结构中的多个功能结构域可与其它蛋白质相互作用,介导多种细胞功能.在破骨细胞中,p 62/SQSTM 1信号传导通路对破骨细胞的激活及分化等发挥重要作用.因此,p 62/SQSTM 1与骨质疏松、Paget骨病等的发病机制密切相关.本文主要从p 62/SQSTM 1的概述及在破骨细胞中的相关信号传导通路等做一综述,旨在为骨质疏松、Paget骨病等相关骨骼疾病的研究提供参考.

OBJECTIVE:To isolate the tendon stem cells (TSCs) from rat patellar tendon and to investigate the effect of mechanical stretching on the expression of Sox-9.METHODS:TSCs were isolated from Sprague Dawley rat (12 weeks old) patellar tendon by collagenase digestion and low density culture. The cell colony morphology and number were observed by crystal violet staining; the cell morphology was observed by inverted phase contrast microscope, and the immunophenotypes of mesenchymal stem cells (MSCs) were determined by flow cytometry. The TSCs at passage 3 was given the mechanical stretching at 4%, 0.17 Hz for 4 hours and 24 hours in the experimental group, and cells without stretching was used as control. The Sox-9 gene and protein expressions were detected by real-time fluorescence quantitative PCR and Western blot.RESULTS:Primary cells showed clonal growth and star shape; after subculture, cells at passage 1 showed fibroblast-like shape. The cells formed cell colonies after 7 days; the expressions were positive for CD29, CD44, and CD90 and negative for CD45. The result of real-time fluorescence quantitative PCR showed that Sox-9 gene was down-regulated at 4 hours after mechanical stretching compared with control (P < 0.05), and up-regulated at 24 hours after mechanical stretching when compared with control group (P < 0.05). The result of Western blot showed that Sox-9 protein expression was lower at 4 hours after stretching, but higher at 24 hours after mechanical stretching than that in control group (P < 0.05).CONCLUSION:The rat patellar TSCs can be isolated successfully, and mechanical stretching inhibits the Sox-9 expression, but the inhibited effect might stimulate the Sox-9 expression after the mechanical stretching effect disappears.

Summary Degeneration of the intervertebral disc ( IVD ) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus ( AF ) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2‐D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver‐stained 2‐D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71‐ kD a protein, glucose‐6‐phosphate 1‐dehydrogenase, and protocadherin‐23, whereas seven proteins were increased, including guanine nucleotide‐binding protein G(i) subunit α ‐2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase‐related protein, and fatty acyl‐crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD .

Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.

Objective To observe the expression changes of inflammatory cytokines of human tendon-derived stem cells (TDSCs) under uniaxial stretching.Methods TDSCs were isolated from human gracilis tendon by collagenase digestion at very low density.Cells were detected for surface markers by flow cytometry,and tested for multi-differentiation potential in vitro.Repetitive uniaxial stretching was applied on the cells at 0％,4％,8％ or 10％ strain.Under the microscopy,cell alignment was observed after mechanical stretching.Expressions of inflammation factors COX-2 and MMP-1 were detected by qPCR and western blotting.Results TDSCs were successfully isolated from human gracilis tendon.Inflammatory cytokines CD29,CD44 and CD105 were positive,but CD45 and CD14 were negative.TDSCs could differentiate into osteocytes,adipocytes and chondrocytes in vitro.Cells were not realigned4 hours after mechanical stretching.MMP-1 mRNA expression was significantly down-regulated at 4％ strain (0.090 ± 0.007) compared to that at 0％ strain (0.247 ± 0.032,P ＜ 0.05).No significant difference was found in COX-2 mRNA expression at 4％ and 8％ strain (both was 0.005 ±0.001,P ＞0.05).MMP-1 and COX-2 mRNA expressions at 8％ strain (0.168 ± 0.040 and 0.007 ± 0.001)revealed no significant differences from those at 0％ strain (0.134 ±0.075 and 0.006 ±0.003) (P ＞0.05),whereas at 10％ strain MMP-1 and COX-2 mRNA expressions were significantly up-regulated (0.047 ± 0.003 and 0.496 ± 0.036) compared to those at 0％ strain (0.011 ± 0.003 and 0.005 ± 0.003)(P ＜ 0.05).Changes in MMP-1 and COX-2 protein expressions revealed similar trend as their mRNA expressions.In contrast to the setting of 0％ strain,4％ strain induced down-regulated MMP-1 and COX-2 proteins,8％ strain induced no significant changes in MMP-1 and COX-2 proteins,and 10％ strain induced up-regulated COX-2 protein despite minor increase in MMP-1 protein.Conclusions Mechanical stretching can affect the secretion of inflammatory cytokines.Low tensile stretch is associated with decreased expression of inflammatory cytokines while high tensile stretch promotes secretion of inflammatory cytokines.

[目的]观察白藜芦醇对人肌腱干细胞成骨分化的作用及其细胞信号通路.[方法]低密度接种法和胶原酶消化法分离培养人肌腱干细胞,结晶紫染色观察分离出的细胞形成的单克隆,体外诱导检测分离细胞的多向分化能力;在/无成骨诱导因子下,不同浓度白藜芦醇刺激人肌腱干细胞荧光定量PCR检测骨相关基因(Run x2和OCN)表达,免疫印迹检测Run x2蛋白表达,茜素红S染色观察钙盐沉积,免疫印迹检测β-catenin和NK-KB表达以检测相关细胞信号通路.[结果]成功从人髌腱组织中分离培养出肌腱干细胞,体外可被诱导成骨细胞和脂肪细胞;在/无成骨诱导因子影响下,白藜芦醇均促进骨相关因子Run x2和其下游因子OCN表达,且随着白藜芦醇的浓度升高而上调;无成骨诱导因子影响下,Run x2蛋白表达随着白藜芦醇浓度升高而增多,而在成骨诱导因子存在下,其蛋白表达变化不明显.在/无成骨诱导因子影响下,茜素红S染色显示白藜芦醇促进入肌腱干细胞的钙盐沉积.随着白藜芦醇浓度的提高,β-catenin表达减少,而NF-KB表达无明显变化.[结论]白藜芦醇可以体外诱导并促进人肌腱干细胞的成骨分化,可能通过调节β-catenin信号通路来调控.

INTRODUCTION:Structural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known.METHODS:Histological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone-cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice.RESULTS:We uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone-cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production.CONCLUSIONS:Our data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.

Objective To investigate curative effect of proximal femoral nail antirotation ( PFNA) combined with ca-ble in treatment of Russell-Taylor typeⅡfemoral subtrochanteric fracture. Methods 21 cases of Russell-Taylor typeⅡ femoral subtrochanteric fractures were treated with PFNA combined with cable. Results The operation time was 62~127 min, the blood loss was 150~420 ml. Good fracture reduction and reliable fixation were obtained. 21 cases were followed up for 7~20 months, and fracture healing time was 5~9 months, with Harris score of 79~93, there were 15 excellent cases, 5 good, and 1 fair. Conclusions PFNA combined with cable for the treatment of Russell-Taylor type Ⅱ femoral subtrochanteric fracture is an effective methrod.

Objective To observe the effect of sustained release type Ⅰ collagen-vascular endothelial growth factor (VEGF) on healing of bone-tendon junction injuries.Methods Partial patellectomy was conducted in 72 rabbits divided equally into control group,type Ⅰ collagen group,and collagen type Ⅰ-VEGF group.The scaffold was planted into the bone-tendon interface.Animals were sacrificed at 4,8 and 12 weeks.New bone formation into the patella-patella tendon surface was detected using X-ray films and histological observations.Quality of bone healing was assayed using biomechanical testing.Results At postoperative 4,8 and 12 weeks,X-ray films showed bone formation of type Ⅰ collagen group [(4.1 ± 0.4) mm2,(12.1 ± 0.5) mm2,(13.0 ± 1.2) mm2 respectively] and of collagen type Ⅰ-VEGF group [(3.8 ± 0.4) mm2,(11.0 ± 0.5) mm2,(13.1 ± 1.0) mm2 respectively] were more than that of control group [(2.1 ± 0.6) mm2,(4.1 ± 0.3) mm2,(6.6 ± 0.6) mm2 respectively] (P ＜ 0.05).Histology identified few new bone,massive fibrocyte accumulation and disrupted alignment of tendon fiber in control group,massive new bone formation,neat and orderly alignment of collagen fiber tissues and massive aggrecan expression at postoperative 4 and 8 weeks (fibrous cartage repair in largely) in collagen type Ⅰ-VEGF group,and massive new bone formation but worse alignment of tendon collagen fibers and less aggrecan expression (fibrous repair in largely) in type Ⅰ collagen group.Biomechanical test showed the ultimate tensile strength increased over time in all groups,with significantly higher value at 12 weeks than that at 4 and 8 weeks.At the same time point,ultimate tensile strength ranged in an order as follows:collagen type Ⅰ-VEGF group ＞ collagen type Ⅰ group ＞ control group (P ＜ 0.05).Conclusion Sustained release type Ⅰ collagen-VEGF can accelerate early healing of bone-tendon junction injury and improve the histological and mechanical properties.

A Large scale of full-thickness skin defects is lack of auto-grafts and which requires the engineered skin substitutes for repair and regeneration. One major obstacle in skin tissue engineering is to expand epidermal stem cells (ESCs) and develop functional substitutes. The other one is the scaffold of the ESCs. Here, we applied type I collagen-modified chitin membrane to form collagen-chitin biomimetic membrane (C-CBM), which has been proved to have a great biocompatibility and degraded totally when it was subcutaneously transplanted into rat skin. ESCs were cultured, and the resulting biofilm was used to cover full-thickness skin defects in nude mice. The transplantation of ESCs- collagen- chitn biomimetic membrane (ESCs-C-CBM) has achieved in situ skin regeneration. In nude mice, compared to controls with collagen-chitin biomimetic membrane (C-CBM) only, the ESCs-C-CBM group had significantly more dermatoglyphs on the skin wound 10 w after surgery, and the new skin was relatively thick, red and elastic. In vivo experiments showed obvious hair follicle cell proliferation in the full-thickness skin defect. Stem cell markers examination showed active ESCs in repair and regeneration of skin. The results indicate that the collagen-modified chitin membrane carry with ESCs has successfully regenerated the whole skin with all the skin appendages and function.

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase beta (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase beta mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5'-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.

[目的]研究从人的髌腱组织中分离出肌腱干细胞,并进行体外培养扩增鉴定.[方法]通过胶原酶消化法和低密度接种培养法从人的髌腱组织中分离出肌腱干细胞.通过三期分化鉴定,分离出的肌腱干细胞具有向骨细胞、脂肪细胞和软骨细胞方向分化的能力.通过流式细胞术,鉴定分离出细胞的干细胞表面标记因子的表达.通过免疫组织化学检测其肌腱细胞和软骨细胞相关蛋白的表达.[结果]从人的髌腱组织中成功分离出肌腱干细胞,大约有1.2％ ～1.4％分离出的细胞形成单细胞克隆.分离出的细胞表达CD44+,CD105+,CD29+,CD45-,和CD14-,并具有向不同细胞分化的能力;另外,肌腱干细胞不仅表达肌腱细胞相关蛋白,如Ⅰ型胶原和tenascin C,也同时会表达软骨细胞相关的蛋白,如Sox 9和Ⅱ型胶原,但其并不表达糖氨多糖.[结论]成功分离鉴定人髌腱干细胞,虽然与间充质干细胞有相同的干细胞特性,是不同于其他间充质干细胞的一种特殊的间充质干细胞.肌腱干细胞的成功分离鉴定有利于以后干细胞相关的肌腱组织修复的研究,并有助于更好的理解肌腱干细胞在肌腱相关疾病和修复的作用.

Objective To investigate the histocompatibility of the type Ⅰ collagen (CI)/vascular endothelial growth factor (VEGF) slow-release material.Methods Bone marrow-derived mesenchymal stem cells (BMSCs) were obtained with passage isolation,and then seeded on cross-linked CI,CI + VEGF materials and cell culture plate.Cytocompatibility was detected by using scanning electron microscopy (SEM) at day 5 and cell counting kit-8 (CCK-8) test at every day during 10 days after seeding.The CI and CI + VEGF materials were subcutaneously implanted into 8 New Zealand albino rabbits.The rabbits were sacrificed at 7,14,21 and 28 days and the scaffolds and the surrounding tissues were extracted for hematoxylin-eosin (HE) staining.Results CCK-8 test revealed that the cell proliferation in 3 groups was as follows:CI + VEGF group ＞ CI group ＞ common culture group,and the cell viability in CI + VEGF group was significantly higher than that in controls (day 4:2.77 ±0.21 ＞2.21 ±0.17 ＞ 1.86 ±0.13 P ＜ 0.05 ; day 8:2.47 ± 0.19 ＞ 1.89 ± 0.15 ＞ 1.16 ± 0.11,P ＜ 0.05).SEM image of the scaffolds showed that BMSCs spread well and homogenously distributed throughout the entire scaffolds.Histological HE staining showed that the immune rejection of CI + VEGF sponge material was similar to CI,and CI + VEGF sponge material could be degradated at 28 days in animals.Conclusion CI + VEGF sponge materials have well histocompatibility. Key words: Type Ⅰ collagen;  Bone marrow-derived mesenchymal stem cells;  Vascular endothelial growth factor;  Histocompatibility