Biology Properties Of Chondrons Isolated From Age-relatd Normal Rabbit Knee

Background: The pericellular matrix (PCM) characterized by the presence of type VI collagenis a narrow tissue region surrounding chondrocytes in articular cartilage, which together with theenclosed cell(s), has been termed the"chondron". As the local mechanical environment of thecell, the PCM is thought to play an important role in transmitting both biochemical andbiomechanical cues from the ECM to the cell through''outside-in"signaling mechanisms. Weand others have previously examined the mechanical properties of individual chondrocytes insuspension using micropipette aspiration. And, in osteoarthritis (OA) cartilage, chondrocyteswere more stiff than normal cartilage. However, how the biological properties of isolatedchondrons compared with chondrocytes vary with aging have not hitherto been analysed andquantified. Understanding the biological properties of chondrocytes and chondrons and how thePCM influences the cell response to mechanical stimulation will be vital to functionalengineering cartilage.Objectives: The aims of this study are to measure the biological properties of isolated chondronsand chondrocytes from rabbit knee cartilage, and to determine if these properties vary withaging.Methods:The research has done by 3 serial experiments.①. Histomorphometric analysis of the chondrons islated from age-related normal rabbit kneearticular cartilage: Three aging groups of rabbit were used for this study: Young (2-month-old,N=10), Adult (8-month-old, N=10), and Old group (31-month-old, N=10). The cartilagestructure and proteoglycan, Collagen-2 content were determined by light microscopic usingH&E, Toluidine Blue, and Col-2 staining. The chondrocytes were isolated from right kneescartilage and chondrons from left knees using enzymatic methods. Chondrons wereenzymatically isolated using 0.3% dispase (Sigma) and 0.2% collagenase-2 in DMEM-F12 at37°C with shaking for 3 hours. The cells and chondrons were seeded in 6 Well Cell CultureCluster at a density of 2×105 cells in 3 mL culture medium per well at 37°C and 5% CO2. Themorphology and composition of isolated different age chondrons were observed byhematoxylin-eosin (HE) and type VI collagen immunostaining staining after overnight coverslip monolayer culture under a microscopy.②.Viscoelastic properties of chondrons and chondrocytes isolated from age-related normal rabbit knee:The chondrocytes and chondrons were isolated from full thickness of the young (N=16), adult (N=16), and old(N=16) rabbit knees cartilage as described above. The viscoelastic properties of all aging cells and chondrons were quantified using micropipette aspiration technique combined with a standard linear viscoelastic solid model within 2hrs after isolation. Meanwhile, the changes of the cytoskeleton components and network within cells were visualized qualitatively by confocal microscopy using immunofluorescence staining and real time PCR, Western bloting.③. Biology properties of the isolated chondrons during monolayer cultivation in vitro. The chondrocytes and chondrons were isolated from full thickness of the young (N=18) rabbit knees cartilage as described above.The cells and chondrons were seeded in 6 Well Cell Culture Cluster at a density of 2×105 cells in 3 mL culture medium per well at 37°C and 5% CO2. The morphology and composition were observed by hematoxylin-eosin (HE) and type VI collagen immunostaining staining after 1,3,6, and 9 days coverslip monolayer culture under a microscopy. The proliferation of the cells and chondrons were analysed by MTT, and the glycosaminoglycans(GAG), collagen-typeⅡ(Col-2), matrix metalloproteinase (MMP-3), and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA expression were analysed by real time PCR after cells and chondrons 1,3,6, and 9 days coverslip monolayer culture. Meanwhile, the metabolize of the GAG, Col-2, MMP-3, and TIMP-1 in liquid supernatant were analysed by Elisa.Results and conclusions:①In old group, the chondrocytes were more sparse and the total content of proteoglycans and collagen-2 were decreased in the articular cartilage with aging. Meanwhile, TEM have shown that the cell membranes were not clear, organelles were fewer and the nuclears were deform or shrink in old cells. Compared to the chondrocytes, the surrounding rim or capsule was evident with many of the isolated chondrons, they exhibited major differences in shape, and the contained cells within one cluster from different age groups similar to the morphology observed in cartilage in situ.②All aging chondrocytes in suspension showed significantly viscoelastic creep behavior, but the deformation rate and amplitude in old chondrocytes were increased under the same negative pressure. Data have shown that the viscoelastic properties in old cells, including equilibrium modulus (E∞), the instantaneous modulus (E0) and the apparent viscosity ( ) were significantly lower than the young and adult ones (P < 0.001), no significant difference was found between young and adult chondrocytes (P >0.05). Moreover, we found that the cytoskeletal network were more sparsely, and the components content were reduced in old cells by immunohistochemistry staning, LSCM, real time PCR and western bloting. Meanwhile, the young and adult chondrons exhibited the same viscoelastic creep behavior only under a greater applied pressure of 1.0～1.1 kPa, no deformation as observed in old chondrons. The viscoelastic properties (E∞, E0 and ) of young and adult chondrons were significantly greater than young and adult cells respectively (P<0.001), and the adult chondrons were stiffer than the young chondrons under micropipette aspiration (P< 0.001).③After the PCM rim were degradated in chondrons monolayer culture 3 days, the chondrons were started to proliferate as the cells invtro. MTT data have shown that the cells in chondrons number were more decreased than the cells at 3, 6, and 9 days, in which the initiative number were same in 1 days for chondrons and cells. The GAG, and Col-2 mRNA expression in chondrons were increased than cells, these findings indicated that the chondrons were preponderant in Tissue engineering cartilage as seeding cells.In this paper, base on the enzymatic methods for isolated chondrons was successfully established, further analysed the availability for isolated chondrons from different aging rabbit knee cartilage by this enzymatic methods. Meanwhile, the viscoelastic properties of all aging cells and chondrons were quantified using micropipette aspiration technique, and the changes of the cytoskeleton components and network within cells were visualized qualitatively by confocal microscopy using immunofluorescence staining and real time PCR, Western bloting. Further suggest that the alteration of viscoelastic properties in the aged-chondrocytes is associated with the changes of the cell structure and the cytoskeleton elements. Meanwhile, compared with the chondrocytes, the Biology properties of the isolated chondrons in vitro were analysed, incluing proliferation, and metabolize.