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Objective To investigate the optimal centrifugation conditions for preparation of rat and mouse platelet rich plasma (PRP) by single centrifugation.Methods Arterial blood of rats and mice by femoral artery cannulation and cardiac puncture were obtained respectively, anticoagulation with 14％CPDA-1, while white blood cells in the blood were filtered out.Then the blood was divided into sterile EP tubes, while PRP was prepared by centrifugation in different conditions (the centrifugal force was 300×g-600×g, and the centrifugal time was 4-12min).The number of blood cells of the anticoagulant whole blood, the leukocyte-depleted blood sample and PRP were counted by hematology analyzer, and platelet recovery rates were compared between different methods.Results The platelet recovery rate was highest when the blood samples of rats and mice were centrifuged at 400×g and 300×g for 8min respectively.Conclusion It is a key to prepare PRP by single centrifugation that selecting the appropriate centrifugal force and time and reaching a critical state before the formation of the buffy coat.
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Objective To evaluate the repeatability and stability of double antibody sandwich ELISA kit for micro-quantitative soluble complement receptor 1(sCR1) in human serum and to understand its practical application effect .Methods 50 patients with middle and advanced liver cirrhosis and 50 individuals undergoing physical examination served as the liver disease group and normal control group respectively .The mouse anti-human CD35 monoclone antibody ,rabbit anti-human sCR1 polyclonal antibody and goat anti-rabbit IgG labeled by horseradish peroxidase served as the envelope antibody ,sandwich antibody and detection antiboby .The purified recombinant human sCR1 protein served as the standard substance .The human serum micro-quantitative double antibody sandwich CR1 ELISA kit was established .Then the repeatability and stability tests were performed .Then the sCR1 protein level of two group of serum was detected by this kit .Results The linear range of double antibody sandwich ELISA for detecting human se-rum micro-quantitative sCR1 protein was 15 .60 -250 .00 ng/mL ;the regression equation of sCR1 protein concentration to absor-bance value was Y=112 .10X2 +18 .21X+1 .694(r2 =0 .998);in the repeatability test ,the intra-batch relative standard deviation (RSD) in high and low concentrations of standard substance detection value was 6 .20% and 7 .40% respectively ,the inter-batch RSD was 6 .70% and 7 .90% respectively ;in the stability test ,RSD was not more than 0 .01;the serum sCR1 expression level in the liver disease group was significantly higher than that in the normal control group (P<0 .01) .Conclusion The human serum double antibody sandwich ELISA kit for detecting human sCR1 has wide linear range ,good repeatability ,is easy to be stored and suitable for clini-cal and scientific research detection work .
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Objective To explore the histological properties of isolated chondrons and chondrocytes from rabbit knee cartilage,and to determine if these properties vary with age.Methods Three groups of rabbit knees were evaluated according to different age:(1) young (2 months,n=10);(2) adult (8 months,n=10);and (3) old (31 months,n=10).The cartilage structure,proteoglycan,collagen-2,and collagen-6 content were determined by light microscopic using hematoxylin-eosin (HE),Toluidine Blue,and col-2,6 staining.The chondrons were enzymatically isolated using 0.3 g/L dispase and 0.2 g/L collagenase-2 by shaking for 3 hours.The morphology and composition of isolated chondrons were observed by HE and collagen-6 immunostaining staining after overnight coverslip monolayer culture under a microscopy.Results The chondrocytes became sparser and the total content of proteoglycans and collagen-2 were decreased in the articular cartilage with age.Compared to the chondrocytes,the surrounding rim or capsule was more obvious in the isolated chondrons,and they exhibited obvious differences in shape.The cells within one cluster from different age groups were similar to the morphology observed in cartilage in situ.The adult and old chondrons generally possessed a thicker pericellular matrix with more enclosed cells,and the chondrons contained more cells can reach 47％.Conclusion These findings further suggest that the properties of the chondrons and pericellular matrix have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with age.
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Objective To characterize the biomechanical behavior and properties of the chondrons enzymatically isolated from rabbit knee articular cartilage in virto. Methods Eight months old New Zealand white rabbits were randomly divided into chondroctye and chondron groups (4 rabbits in each group). In chondrocyte groups, the full articular cartilages from both knees were enzymatically isolated to chondrocytes by 0.4% pronase and 0.025% collagenase type-Ⅱ in turn. In chondron groups, chondrons were obtained from articular cartilage using the mixture of 0.3% dispase (a neutral protease) and 0.2% collagenase type-Ⅱin at 37C for 3 h. The micropipette aspiration was used to quantify changes in biomechanical properties of chondrons and chondrocytes and the viscoelastic parameters, including K1, K2, E∞ (equilibrium modulus), E0(instantaneous modulus), and μ (apparent viscosity), were calculated coupled with standard linear half-space viscoelastic solid model. Results In response to a constant negative pressure of 0.2-0.4 kPa, the chondrocytes exhibited standard linear viscoelastic solid properties. Namely, the cells showed an initial elastic response followed by a viscoelastic creep response. then cells continued to enter into the micropipette with a monotonically decreasing rate of deformation, until reaching equilibrium within about (110±18) s. Comparing with chondrocytes, the chondrons exhibited significant viscoelasticity under a greater negative pressure of 1.0-1.2 kPa. But the instantaneous length deformed into the micropipette significantly reduced, and the equilibrium time reduced to (36.5±4.5) s. The equilibrium modulus (E∞), the instantaneous modulus (E0) and the apparent viscosity (μ) of chondrons were significantly higher than the those of chondrocytes. Conclusion Comparing with chondrocytes, the chondrons exhibited significant viscoelastic properties, and viscoelastic properties of chondrons have increased in vitro.
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BACKGROUND: Chondron is a basic microanatomical unit of articular cartilage. lncreasing evidence suggests that the pericellular matrix (PCM) is a distinct functional compartment in articular cartilage, influencing the metabolic, micromechanical environment, and degeneration of chondrocytes. But the precise functions and action mechanism need further investigation.OBJECTIVE: To review the literature pertinent to the morphology, function, isolation of the chondron in articular cartilage, and its degenerative events during the progression of osteoarthritis (OA).METHODS: This review summarized the articles published in the PubMed database before July 2009. In addition, recent data and figure of our laboratory on the morphology and biomechanics of chondron and chondrocyte were supplemented. RESULTS AND CONCLUSION: The PCM is primarily characterized by the presence of type Ⅵ collagen, and these components are widespread in the expansive extracellular matrix (ECM) in newborns, while in mature the components are mainly localized to a narrow pericellular zone. The three-dimensional morphology of chondron has been recently quantified in situ with fluorescence confocal microscopy, and the mechanical properties of the isolated individual chondrons and their PCM are measured using the micropipette technique and atomic force microscopy. More studies have shown that the presence of the PCM in chondrons has a profound influence on chondrocyte gene expression. At the same time, structural, histochemical and biomechanical studies indicate the chondron and their PCM may undergo degenerative processes with osteoarthritis, similar to those occurring in the ECM, Although the precise function of the PCM is unknown, increasing evidence in vivo or in vitro suggests that the PCM is a basic microanatomical unit in articular cartilage, influencing the metabolic and micromechanical environment of chondrocytes.Department of Orthopedics, the Second Hospital of Shanxi Medical University, Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair,Taiyuan 030001,Shanxi Province,China