| The distribution of oxygen tension is heterogeneous in cartilage,and the morphology and biology of chondrocytes are in varying along the zones,in vitro experiments have confirmed that changes in oxygen tensions significantly affect the proliferation,differentiation and phenotype of chondrocytes,but the effect of physiological oxygen tensions on in situ chondrocytes has not yet clearly.Studies have shown that oxygen tensions are able to affect the concentration of intracellular calcium,and calcium levels affect the extracellular matrix secretion of cartilage cells,cartilage aging,mechanical conduction and other physiological functions.Therefore,this study focus on the effect of physiological oxygen tensions on in situ cartilage and matrix deposition by intracellular calcium signaling.Secondly,this study sought methods for the deposition of extracellular matrix from the oxygen tensions and calcium signaling.In addition,because the data processing of the existing calcium signaling detection technology is cumbersome and time consuming,this study improved the data processing of calcium signaling detection technology based on the deep learning method.The results of this study have inspired that the influence of histological structures and oxygen tentions should be taken into full consideration in the key scientific issues of cartilage development.In researches,physiological and pathological status of chondrocytes and cartilage are discussed in different layers as much as possible.And this study is helpful to guide future clinical applications,such as providing theoretical basis of calcium signaling and oxygen tentions for cartilage tissue repair,in vitro culture of chondrocytes,construction of cartilage tissue by 3D printing,etc.,and detection indicators of calcium signaling;affecting the cartilage development by simulation and regulation of optimal oxygen tentions.In addition,the improvement of calcium signaling detection technology based on deep learning overcomed difficulties of cell recognition due to thicker cartilage tissue and dynamically changing cell outlines,as well as the disadvantages of irregular calcium calcium signaling curves in living cells that are not easily identifiable,and is conducive to improving the detection efficiency,popularizing calcium signaling detection technology,and reducing the threshold of calcium signaling detection.Therefore,this study has important significance in enriching theoretical knowledge and clinical application.Based on the above,the main research contents and results of this paper are as follows:?1?Establishing detection methods for spontaneous calcium response test in in situ chondrocytes and extracellular matrix depositionIn order to the calcium signaling measurement is used widely,we reconstructed the calcium signaling measurement method:in situ chondrocytes were labeled with a fluorescent Ca2+probe,and the calcium signalings were recorded and calculated within10 minutes under an ordinary fluorescence microscope.It needs to be emphasized that we used the calcium response peak shape to define the fast and slow peaks,and calculated the occurrence rate of fast peaks;the other hand the total calcium amount was calculated using the area of calcium response peaks.Extracellular matrix anabolic experiments:COLL-II and GAG contents were evaluated by a standard immunohistochemical and Alcian blue staining techniques followed digitization of images,and semi-quantitative measurement using a method of oval-shaped circles choosing extracellular matrix.The results showed that the self-constructed measurement methods for calcium signalings in in situ chondrocytes and extracellular matrix deposition were feasible.According to traditional histological structure of cartilage and our observations during calcium response and extracellular matrix deposition tests,we divided the hyaline cartilage of one explant into six zones from synovium surface to deep area.According to results,calcium signaling and matrix deposition were affected by histological zones significantly.?2?The effect of oxygen tensions on spontaneous calcium responseCartilage explants were treated with different oxygen tensions and calcium sources for 3 and 24 hours.The results showed that excluding the total amount of calcium in the normal hypoxia,other calcium signalings were more obvious under hyperoxia;the calcium signaling was weaken with the increase of culture time;the results from the mimic hypoxia cultured cartilage here could not represent those from the low oxygen tension;and we provided optimal oxygen tension for all zones of in situ cartilage or chondrocytes in names of spontaneous calcium response firstly.In addition,the the main contribution of spontaneous calcium response in in situ chondrocytes are from both the external and internal calcium sources,in spontaneous calcium response of in situ chondrocytes for without any of them,the calcium response was significantly lowered;a short of both external and internal ER calcium,chondrocytes in all zones still maintained capability to utilize calcium source under hypoxia,but only chondrocytes in middle and deep areas could utilize calcium at higher oxygen tensions.And the calcium release from intracellular ER might be faster,while the calcium entry from extracellular medium might be relatively slower.?3?The effect of oxygen tensions on extracellular matrix depositionCartilage tissue was treated with different oxygen tensions for 3 and 7 days.It was found that the normal hypoxia promoted COLL-II deposition,while hyperoxia promoted GAG deposition;hyperoxia reduced the contents of COLL-II and GAG during the long culture period,while COLL-II contents increased but GAG contents decaresd at normal hypoxia during the long culture period;the results from the mimic hypoxia cultured cartilage here could not represent those from the low oxygen tension;we find optimal oxygen tensions for all zones of in situ cartilage or chondrocytes in names of COLL-II and GAG,which provided a theoretical basis for cartilage or artificial cartilage maintenance.?4?Relationship between calcium signaling and extracellular matrix deposition in in situ chondrocytesIt was found that the total calcium amount was related to COLL-II,and the occurrence rate of fast peaks was related to the GAG content among all of calcium signaling indicators.Because calcium sources are related to calcium signaling closely,so they were traced using EGTA and thapsigargin?TG?to investigating the effect of calcium sources on extracellular matrix deposition,which contributed to understanding the relationship between calcium signaling and matrix deposition.Cytoplasm calcium was conducive to synthesize COLL-II,and both extracellular calcium and intracellular ER calcium stores were required to contributing the synthesis of COLL-II at the same time;when the chondrocytes were short of external and internal ER calcium,the synthesis of COLL-II increases,which may initiate another signaling pathwayw;cytoplasm calcium was detrimental to synthesize GAG,when the intracellular calcium signaling decreases due to the reduction of calcium sources,the GAG content increase significantly.?5?Improved detection mathods of intracellular calcium signaling based on deep learningThrough literature investigation and analysis of calcium signaling measurement characteristics,a novel intracellular calcium signaling measurement technology was constructed using full convolutional neural network?FCN?and recurrent neural network?LSTM-F model?.The results show that the FCN semantic segmentation method with three times adjusted was able to accurately recognise the in situ chondrocytes in the process of spontaneous calcium response;restoring the cells location of the overlay image to the original video could get the calcium signaling time series information,and the calcium signaling curve could not be affected by the color of the original image;it can be confirmed that the LSTM-F model had a faster convergence speed,a higher accuracy,and a batter stability,in addition,it can accurately identify the number of peaks,the start and end points of a calcium response peak. |
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