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Objective To study the biological behavior of peripheral blood mesenchymal stem cells (PBMSCs) in 3D composite scaffolds.Methods The proliferation and chondrogenesis of rabbit PBMSCs seeded on porcine cancellous bone (DCB) scaffolds were evaluated,and bone marrow mesenchymal stem cells (BMMSCs) and articular chondrocytes (ACCs) were used as controls.Cell morphology and distribution in scaffolds were observed using scanning electron microscopy (SEM).Live/Dead staining was employed to detect cell viability,Hoechst 33258 method to measure DNA content,dimethylmethylene blue (DMMB) assay to detect glycosaminoglycan (GAG),enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to detect the content of type 2 collagen (COL 2),and RT-PCR to analyze chondrogenesis-related gene expression.Results SEM showed that three kinds of cells uniformly adhered and evenly distributed in DCB scaffolds.Live/Dead staining observed the similar viability of the three kinds of cells three days after seeding (P＞0.05).There was no significant difference in the proliferation ability and DNA content among three kinds of cells after seven days of in vitro culture.After 21 days of chondrogenic culture,both PBMSCs and BMMSCs secreted more GAGs than ACCs,while the secretion of COL 2 was similar to that of ACCs.Moreover,the gene expression of AGC,COL 2 and alkaline phosphatase (ALP) were significantly up-regulated (P＜0.05) in PBMSCs and BMMSCs but significantly down-regulated in ACCs (P＜0.05).The expression of COL 1 in MSCs groups displayed an increasing trend but a decreasing trend in ACCs group (P＞0.05).The gene expression of COL 2 and ALP,but not of AGC and COL 1,in PBMSCs and BMMSCs was higher than those in ACCs (P＜0.05).Conclusions PBMSCs and BMMSCs have similarly excellent proliferation and chondrogenesis potential in 3D porous DCB scaffolds.However,hypertrophic gene expression is still observed under in vitro culturing condition,suggesting the need to further optimize the culture system.
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Objective To explore the influence of elevating the oxygen pressure on articular chondrocytes in vitro.Method A hydrogen peroxide induced human articular chondrocyte damage model was established.Then the articular chondrocyte viability was detected using the CCK-8 kit.Collagen Ⅱ(COL Ⅱ),The expression levels of aggrecan (ACAN),matrix metalloproteinase 13 (MMP13) and adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were detected using the realtime PCR and Western blotting.Result The viability of articular chondrocytes improved at 12 h but decreased at 24 h after the stimulation of hydrogen peroxide.Twenty-four hours later,the average expression level of COL Ⅱ and ACAN decreased(P＜0.05),while that of MMP13 and ADAMTS5 elevated(P＞0.05).Conclusion Hydrogen peroxide induced elevation of the extracellular oxygen pressure can influence the synthesis and degradation of the articular chondrocyte extracellular matrix.
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Objective To study the general shape of anterior cruciate ligament (ACL) insertion in rabbits and establish an animal model of ACL reconstruction using oval tunnels.Methods Eighteen mature white New Zealand rabbits were used in this study.Eight of them were used for anatomy study and the other 10 were for building an animal model.After removal of the medial femoral condyle and other soft tissues around ACL,the morphology of the ACL insertion was examined and the diameter of ACL insertions was measured using a caliper.An oval-tunnel dilator (1.6 mm×2.5 mm) was designed to make an oval-tunnel in the right knee of the rabbits while a round tunnel was drilled using a 2 mm diameter Kirschner wire in the left knee of the rabbits.Their hamstring tendon grafts were harvested as grafts for both sides and the compatibility between the bone tunnel and graft was examined for both groups.Right after the surgery,the knees of both sides were given the three-dimensional CT scan.Results The shape of ACL insertion of rabbits was oval.In the femur side,the average major and minor diameter of the ACL insertion was 5.28 ± 0.83 mm and 2.61 ± 0.33 mm respectively.In the tibial side,the major and the minor diameter of the ACL insertion was 5.33 ± 0.40 mm and 2.68 ±0.11 mm.The bone tunnel was compatible with the graft in both groups.In the oval tunnel ACL reconstruction group,the cross sectional area of the femoral bone tunnel was 3.18 ± 0.09 mm2 and the cross sectional area of the tibial bone tunnel was 3.26 ± 0.15 mm2.In the round tunnel ACL reconstruction group,the corresponding measurements were 3.13 ± 0.10 mm2 and 3.11 ± 0.11 mm2 respectively.There was no significant difference between the two groups.Conclusion The shape of ACL insertion in rabbits is oval.Using the self-made oval tunnel dilator we have successfully built an oval tunnel ACL reconstruction animal model with a good compatibility between the bone tunnel and graft.This lays the foundation for further research in the future.
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<p><b>BACKGROUND</b>The tibial plateau is asymmetric with a larger medial plateau. We observed from clinical practice that the shape of the tibial plateau does not always present a larger medial plateau. Tibial plateau also showed other shapes. The purpose of this study was to analyze the anthropometric data of the proximal tibia in a large group of Chinese patients undergoing total knee arthroplasty and to investigate the morphology of the resected proximal tibial surface and its gender differences.</p><p><b>METHODS</b>A total of 822 knees (164 males, 658 females) from the Chinese population were measured intraoperatively for medial anteroposterior (MAP) and lateral anteroposterior (LAP) dimensions of the resected proximal tibial surface. The difference of MAP and LAP (DML) was also calculated as MAP minus LAP. We then classified the data into three groups based on the DML (<-2, -2 to 2, and >2 mm) to analyze the morphology of the proximal tibia and its distribution between male and female.</p><p><b>RESULTS</b>The shape of proximal tibial plateau was of three types: larger medial plateau type, symmetric type, and larger lateral plateau type. There were significant differences between males and females in relation to the shape distribution of the proximal tibial plateau (P < 0.05). Most of the proximal tibial plateau was asymmetric, with 517 of 822 (62.9%) tibia having a DML >2 mm and 120 of 822 (14.6%) tibia having a DML<-2 mm. Only 185 of 822 (22.5%) tibia had a DML between -2 and 2 mm.</p><p><b>CONCLUSION</b>The results of this study can be used as a guideline to design tibial components with different DMLs to better match the different anthropometry of the resected tibial surface.</p>
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Aged , Female , Humans , Male , Middle Aged , Anthropometry , Methods , Arthroplasty, Replacement, Knee , Methods , Asian People , Knee Joint , General Surgery , Sex Factors , Tibia , General SurgeryABSTRACT
Objective:To analyse the biological function of anti-IL-6Rβ(gp130) monoclonal antibody and its regulatory effect on IL-6 signaling.Methods:Biological characteristics of anti-IL-6Rβ(gp130) mAb were assessed by Western blot analysis, capture ELISA and peptide ELISA .The phosphorylation of STAT 3 was tested by Western blot analysis in IL-6-stimulated U266/RA-FLS/RA-PBMC with or without anti-IL-6Rβ(gp130) mAb treatment.Results:3 strains of mouse anti-human gp130 mAb were with high affini-ty and different binding epitopes , the kaff of 10A1 was 2.62E-10.In U266, RA-PBMC and RA-SFMC, IL-6 signaling highly activated STAT3 which could be inhibited by anti-gp130 mAb.Conclusion: Anti-IL-6Rβ( gp130 ) mAb might have different binding epitopes and could affect IL-6 stimulated phosphorylation of STAT3, which provides a preliminary experiment for analyse the correlation of IL-6 signaling and RA .
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Objective: To investigate the variation of γδ T cells from healthy human peripheral blood(PB)and neonatus cord blood (CB)in proliferation and subtypes with isopentenyl pyrophosphate(IPP), and to acquire enough γδ T cells possessing distinct characteristics for experimental study.Methods: Mononuclear-cells from peripheral blood and cord blood induced by IPP were stained separately with monoclonal antibodies,which were fluorescein-labeled,and then used for assaying the expressing condition of surfaco molecules,as well as to evaluate the variation of γδ T cells on the percentage, subtypes and pbenotypes by FCM.Results:γδ T cells only account for a small proportion in both PB and CB.However,there was a significant difference in the heterogeneity between peripheral blood and cord blood γδ T cells.Vγ9Vδ2 T cells were dominant in peripheral blood γδ T cells.Most of Vγ9Vδ2 T cells in fresh isolated PBMC were central memory-type(CD27~+ CD45RA~-)and effector memory-type(CD27~-CD45RA~-)with IPP, PB γδ T cells proliferated strongly;The effector memory-typo(CD27~-CD45RA~-)turned into the main subtype in all Vγ9Vδ2 T cells,and HLA-DR and B7 molecules were highly expressed on the populations.But the cord blood γδ T cells showed rather complex subgroup heterogeneity,and Vγ9Vδ2 T cells were almost na(i)ve-type(CD27~+ CD45RA~+); Though γδ T cells were expanded(the percent of γδ T cells was increased),and Vγ9Vδ2 T cells were differentiated towards central memory-type and effector memory-type on day 14 with IPP,most of γδ T celLs still remained in the phase of na(i)ve-type(CD27~+ CD45RA~+).Conclusion:Tbere lies great differences of γδ T cells in quantity and subtypes between healthy person peripheral blood and neonatus cord blood.Peripheral blood γδ T cells can be activated and proliferated with IPP, while cord blood γδ T cells have the potential to deferentiate into director memory-type which can be used for experimental and clinical study with the synergy of corresponding cytokines.The immuno-regulation and effector function will be reported in other papers.
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Objective To establish the type Ⅱ collagen specific T cell line of Wistar rat and observe its effect on transferring arthritis.Methods The Wistar rats were immunized with emulsified chicken type Ⅱ collagen (CCⅡ) in complete Freund′s adjuvant by intradermal injection to induce the rat model of collagen induced arthritis (CIA).The lymphocytes were obtained from mesenteric lymph nodes of CIA rats,and the type Ⅱ collagen reactive T cell line was selected and propagated by CCⅡ stimulating in vitro .The proliferation response and phenotype were analyzed by 3 H TdR incorporation and fluorescence activated cell sorter (FACS).The onset of arthritis and pathological characteristic in ankle joints of recipient rats were observed with naked eye and histochemical examination.Anti CCⅡ antibody in serum was assayed by enzyme linked immunosorbent assay (ELISA).Results A T cell line was successfully established.The results of FACS labeled with fluorescent antibodies showed that 98 2% of the line cells were T cells,of which 89 7% were CD4 + T cells.The results of adoptive transfer showed that the incidence of arthritis was 50% when the injected cell number was 5?10 7,meanwhile the level of anti CCⅡ antibody in serum was elevated more than that of the control.Conclusion A cell line has been successfully established.The result of arthritis transferring by T cell line shows that the T cell plays a great role in the pathogenesis of CIA and provides a research datum for rheumatoid arthritis therapy with T cell vaccine.
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Objective To study the prognosis of the implanted xenogenic and allogenic menisci, and the implanted menisci's protection effects on articular cartilage. Methods 30 adult New Zealand white rabbits were divided equally into two groups and the medial meniscus defection models were established by medial meniscectomy. Allogenic medial menisci implantation were done in group A. Small pieces of menisci tissue were taken from pig meniscus and moulded them as the same shape and size as rabbit's medial menisci,then, xenogenic menisci implantation were done in group B with these moulded grafts. 6, 12 and 24 weeks after the operations, rabbits were sacrificed to observe the implanted menisci and the articular cartilage on medial tibial plateau, medial femoral condylus and femoral trochlea with naked eye and histological method. Results After allogenic meniscus transplantation, the state of implanted meniscus was good, and healed well around the capsula. 24 weeks post allogenic meniscus transplantation, the injury of the articular cartilage was not in evidence. In short time post xenogenic meniscus transplantation, the state of meniscus and cartilage was good and after 24 weeks the xenogenic meniscus was dissolved and absorbed, the articular cartilage also showed degeneration and injury. Conclusion With xenogenic meniscus moulded from pig meniscus tissue,the implanted meniscus was resolved and absorbed and the cartilage degeneration also appeared after half a year. Post allogenic meniscus transplantation, the meniscus structure and function were reconstructed well and the articular cartilage was also well protected.
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Objective To explore the difference between T cells in the synovial fluid and peripheral blood in patients with rheumatoid arthritis(RA). Method Samples from 22 patients were studied. The differentiation and activation markers expressed on T cell surface were detected by immunofluorscence using flow cytometer. The specific proliferation of collagen Ⅱ and heat shock protein 70 was analyzed using standard 3H-TdR incorporation method. Restricted V beta usage of these T cell was analyzed by semi-quantitied RT-PCR. Results The majority of the T cell subsets in the synovial fluid were demonstrated to be CD4 and CD8 positive cells in which (40?10)% were CD4 positive and (36?16)% were CD8 T cells respectively. The ratio between CD4 and CD8 was much lower than that found in the PBL of RA patients. The percentage of CD3+/CD25+ T cells was (16?6)%. The specific proliferation of collagen Ⅱ and HSP70 to CD3+/CD25+ T cell was higher than that of CD3+/CD25+ negative T cells. The T cell receptor expressed on the T cells from both peripheral blood and synovial fluid were tested for ?? TCR (70?26)%. However, the T cells in the synovial fluid showed V?14,16 and 17 restriction. Conclusion The data here reported indicates that T cell subsets in the synovial fluid and peripheral blood circulation in patients with rheumatoid arthritis are different. The T cells in the synovial fluid demonstrates more activation and higher reactivation to collagen Ⅱ and HSP70. The TCR of T cells showes V?14,16 and 17 restriction.
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<p><b>OBJECTIVE</b>To investigate the effect of rupture and reconstruction of the anterior cruciate ligament (ACL) on the degeneration of rabbit knee articular cartilage.</p><p><b>METHODS</b>14 mature New Zealand white rabbits were divided into four groups. In group I, the ACL of the right knees in 7 rabbits was resected and immediately reconstructed, and the contralateral ACL was resected only in controll f group I. In group II, the ACL of the right knees in 7 rabbits was reconstructed 3 weeks after the ACL was resected and the contralateral joints in control group II, in which only a medial arthrotomy was performed. The rabbits were killed 8 weeks after the operation. The methods of ink straining, histology and SEM were used to analyze the changes in articular cartilage of the joints.</p><p><b>RESULTS</b>The results of ink method and HE straining were analyzed quantitatively. The degeneration of knee articular cartilage in group I was significantly weaker than that in control group I (Hc = 5.9889, P = 0.0144). The degeneration of knee articular cartilage in group II was as serious as that in control group I (Hc = 0.7143, P = 0.785).</p><p><b>CONCLUSIONS</b>Immediate reconstruction of the ACL can effectively prevent articular cartilage from degeneration. Once the articular cartilage damaged moderately, delayed reconstruction of the ACL could not effectively reduce the development of degeneration. So once the ACL is ruptured, reconstruction should be performed in the early stage to restore the stability of knee joint to prevent the articular cartilage from degeneration.</p>
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Animals , Rabbits , Anterior Cruciate Ligament Injuries , Cartilage, Articular , Pathology , Disease Models, Animal , Knee JointABSTRACT
Using HB55 McAb, we detected HLA-DR antigens on Daudi, H18, PBL and K562 cells by dot-ELISA. Results corresponding to situation of the antigen expressions on various cell lines were obtained: colour reaction in Daudi, H18 cells and PBL was positive in different degree and K562 sample presented negative reaction. Chromatogram peaks were printed by Dual-Wavelength Flying-Spot scanner CS-9000 (SHIMADZU). Areas and high values of peaks, meaning size and colour of spot respectively, were increased with cell concentration. Both cell concentration and volume of cell suspension did not influence the diffusion of sample. An analysis for the antigen expression on cell surface and its dynamic change can be supplied by the technique.The study on quantitative assay of MHC class Ⅱ antigens on cell surface are still going on.
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Objective To study the type Ⅰ,Ⅱ,Ⅲ and Ⅹcollag e n expression in meniscus and articular cartilage and immunoreactions post xenoge nic and allogenic meniscus transplantation. Methods After men iscectomy,30 adult New Zealand white rabbits were divded into two groups:group A undertook allogenic medial meniscus transplantation and group B undertook xenog enic meniscus transplantation with fitted meniscus tissues harvested from swine. 6, 12 and 24 weeks post transplantation, the histological and immunohistochemical analysis of type Ⅰ ,Ⅱ,Ⅲ and Ⅹ collagens monoclonal antibody were investigated in menisci and a rticular cartilage. Peripheral blood were obtained for immunoreactions study thr o ugh the methods of CDMT (Complement Dependent Microlymphocytotoxicity Test) and RIA (Radioimmunoassay)of IL-2 and IL-6. Results The meniscu s and articular cartilage were good post allogenic meniscus transplantation, but the transplanted xenogenic meniscus appeared to be dissolved and absorbed and c art ilage lesions were observed in 24 week post operation. No significant difference was found between two groups in type Ⅰ,Ⅱ and Ⅲ collagen expression during e xperiment. From 1～12 weeks, no significant diffe rence was found among type Ⅰ,Ⅱ and Ⅲ collagen expression in articular cartil age in two groups. However,24 weeks post operation, type Ⅹ collagen's expressio n in cartilage was abnormally increased. Neither allogenic nor xenogenic meniscu s transplantation caused fatal immunoreaction during the whole experiment. Con clusion The transplantation meniscus began to be dissolved and absorbed after half a year, but allogenic meniscus transplantation achieved good results . Type Ⅰ,Ⅱ and Ⅲ collagen expression in transplanted meniscus and articular cartilage showed no difference between two groups, but the expression of type Ⅹ collagen was abnormally increased in xenogenic group 24 weeks post operation. No fatal immunoreaction was found in both groups.