Analysis Of Spontaneous Calcium Signals In In-situ Chondrocytes Of Human Knee Osteoarthritis

Objective To explore the association between the spontaneous intracellular free calcium concentration([Ca²⁺]_i)signal and the degree of cartilage degeneration by comparing the difference of spontaneous [Ca²⁺]_i signal in in-suit chondrocytes of human normal and osteoarthritis?OA?articular cartilage,and provide a theoretical basis for the diagnosis of early OA.Methods Using human knee cartilage samples as the research object,the severity of cartilage degradation using International Cartilage Repair Society?ICRS?Visual Assessment Scale,divided into ICRS 0,ICRS 1,ICRS 2and ICRS 3 groups.Spontaneous[Ca²⁺]_i signal was recorded with Fluo-8 AM calcium indicator and fluorescence microscope.The fold increment,peak number and percentage of responding cells were selected as the indexes to evaluate the spontaneous[Ca²⁺]_i signal characteristics of cells.Data was analyzed with image processing software and One-way ANOVA analysis.Results 1.Both normal?ICRS 0?and OA?ICRS 1-3? chondrocytes were able to display spontaneous[Ca²⁺]_i signal,which usually propagated to neighboring chondrocytes.2.In ICRS 0 samples,[Ca²⁺]_i intensity oscillation increased with the depth of cartilage.While a significant larger portion of chondrocytes ?percentage of responding cells:47.17�4.01%vs.13.33�13.33%,P<0.05?located in single lacunas in deep zone demonstrated repetitive?peak number:0.60�0.07 vs.0.13�0.09,P<0.05?and stronger[Ca²⁺]_i peaks?fold increment:2.17�0.08,P<0.05?during the same recording period.3.In deep zone of articular cartilage,compared with ICRS 0 samples,chondrocytes in ICRS 2?fold increment:1.83�0.06 vs.2.17�0.08,P<0.05?and ICRS 3?fold increment:1.64�0.04 vs.2.17�0.08,P<0.05,peak number:0.26�0.04 vs.0.60�0.07,P<0.05,percentage of responding cells:24.65�5.14%vs.47.17�4.01%,P<0.05?samples demonstrated significant weaker[Ca²⁺]_i intensity oscillation.4.In deep zone of articular cartilage,spontaneous[Ca²⁺]_i signal calcium peak magnitude?r=-0.33,P<0.05?,percentage of responding cells?r=-0.382,P<0.05?and number of peaks?r=-0.205,P<0.05?were negatively correlated with ICRS grade.Conclusions 1.Our results suggest parameters of[Ca²⁺]_i intensity oscillation in in-situ chondrocytes differ with layers and environmental Ca²⁺concentrations.2.There were significant differences in the spontaneous[Ca²⁺]_i signal characteristics of deep articular cartilage cells in different ICRS grades,there was no difference between ICRS 0 and ICRS 1.3.The spontaneous[Ca²⁺]_i signal of deep in-situ chondrocytes was negatively correlated with the degree of cartilage degeneration.