Growth Plate Chondrocytes Were Cultured In Three Dimensional Porous Scaffolds And The Possible Mechanisms Of Chondrocytes Responding To Mechanical Loading

Objective1. To observe the biological behavior of cultured growth plate chondrocytes and precartilaginous stem cells (PSCs) combined with chitosan-alginate composite scaffold, and investigate the feasibility of them for tissue engineering.2. To study the effect of dynamic compressive force on mRNA expression of parathyroid hormone-related peptide (PTHrP) and chondrogenic extracellular matrix in rat growth plate chondrocytes in vitro.3. To investigate the effect of dynamic compressive force on the Sox9 expression in rat growth plate chondrocytes in vitro, and to explore the possible mechanotransduction mechanisms of the chondrocytes responding to loading.4. To investigate whether short-term cyclic mechanical strain regulates the PTHrP expression in postnatal growth plate chondrocytes, and to explore whether the cytoskeleton signal pathway is involved in this process.Methods1. The composite scaffolds were prepared by a freeze-drying technique. Chondrocytes were isolated from rat tibial and femoral growth plate. The PSCs were isolated from La Croix ring and purified using immunomagnetic microbeads, and were induced into chondrogenic differentiation. Collagenâ…¡were detected by immunocytochemistry, and the extracellular matrix was detected by Toluidine Blue and Safranin O staining. Then the cells were seeded onto the composite scaffolds and the effects of adhesion and morphological changes were observed by scanning electron microscopy (SEM). The proliferation behavior of the cells was analyzed by MTT assay. The glycosaminoglycan (GAG) content synthesized by growth plate chondrocytes and PSCs in the culture medium was measured by histochemistry of alcian blue.2. The rat growth plate chondrocytes were isolated from rat tibial and femoral growth plate and cultured in vitro. The subcultured cells were divided into different groups and dynamic compressive forces were exerted onto them. The time course for expression pattern and level for cartilaginous specific markers, typeâ…¡collagen, typeâ…©collagen and aggrecan, and PTHrP were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and compared with non-compressed control group.3. The rat growth plate chondrocytes were isolated from rat tibial and femoral growth plate and cultured in vitro. The subcultured cells were then subjected to a short-term cyclic loading (at 12 kPa a frequency of 0.1 Hz) for 24 h. The mRNA and protein expressions of Sox9 were examined by the reverse transcription polymerase chain reaction (RT-PCR) and the Western blot analysis, respectively.4. The growth plate chondrocytes were obtained from 2-week-old Sprague-Dawley rats, and sorted prehypertrophic and hypertrophic chondrocytes by immunomagnetic beads coated with anti-CD200 antibody. Sorted chondrocytes were subsequently subjected to cyclic mechanical strain in different magnitudes (3%,6% and 12% elongation) at a frequency of 1Hz for 24 hours. The organization of F-actin micro filaments was observed by laser scanning confocal microscope (LSCM).Results1. The growth plate chondrocytes and PSCs have been successfully cultured in vitro. The induced PSCs showed positive expression of Collagenâ…¡and positive staining of extracellar matrix. Cells were adhered to the surface of the scaffolds and proliferated well. Like to growth plate chondrocytes, the induced PSCs cultured in scaffolds underwent chondrogenic differentiation, as evidenced by higher expression of typeâ…¡collagen and Sox9, and the difference of GAG content between the growth plate chondrocytes group and the PSCs group had no statistic significance.2. The expression levels of typeâ…¡collagen, aggrecan and PTHrP markedly exceeded the control groups at different intensity and durations of compressive force (P<0.05), whereas the expression levels of typeâ…©collagen increased insignificantly (P>0.05). The expression levels of typeâ…¡collagen, aggrecan mRNA reached the peak after stimulation for 6 hours, and the expression levels of PTHrP mRNA increased continuously with durations of compressive force in this study.3. The short-term cyclic load (at 12 kPa a frequency of 0.1Hz) for 24 h significantly increased Sox9 mRNA and protein expression levels. There was no difference in fluorescence intensity between the loaded group and control group. Incubation of chondrocytes with nifedipine, a Ca2+ channel blocker, inhibited partly the induction of Sox9 expression in response to strain.4. The cyclic strain regulated PTHrP expression in magnitude- and time-dependent. Cyclic strain at 6% elongation for 24 h significantly increased PTHrP mRNA and protein expression levels, and induced remodeling of F-actin microfilaments. Incubation of chondrocytes with cytochalasin D, an actin microfilament disrupting reagent, blocked the induction of PTHrP expression in response to strain.Conclusion1. The results suggest that the PSCs may be a potential cell source for cartilage tissue engineering, and the chitosan-alginate composite 3-D porous scaffold could provide a favorable microenvironment for supporting proliferation and chondrogenic differentiation of cells.2. Moderate dynamic compressive forces promote the expression levels of typeâ…¡collagen, aggrecan mRNA in rat growth plate chondrocytes in vitro, and propose that the PTHrP may be an important mediator in signal transduction from biomechanical stimuli.3. Moderate dynamic compressive forces promote the Sox9 mRNA and protein expression in rat growth plate chondrocytes in vitro, and propose that the Ca2+ may be an important mediator in signal transduction.4. The results suggest that short-term cyclic mechanical strain induced the PTHrP expression in postnatal growth plate prehypertrophic and hypertrophic chondrocytes that may subsequently affect growth plate development, and also support that actin microfilament plays an important role in mechanotransduction.