Timing and localization of chondrocyte colony-like clusters in cartilage tissue
To observe the timing and localization of chondrocyte colony-like clusters in cartilage tissue,OARSI grade 0~6 cartilage tissue was obtained for this study (figure 1). 36 The histological sections were stained with safranin O to reveal the presence of a large number of chondrocyte colony-like clusters in the middle zone of OARSI grade 3 and grade 4 cartilage tissue (figure 1C,figure 1D).At the same time, it was found that only when the superficial cartilage tissue was severely damaged or even eliminated did more colony-like clusters appear in the middle-zone cartilage tissue. When the superficial-zone cartilage tissue was present (figure 1A,figure 1B), there was no chondrocyte colony-like cluster formation in the middle zone. The result was consistent with our previous research.37 However,the number of colony-like clusters was smaller in OARSI grade 5 and grade 6 cartilage tissues (figure 1E,figure 1F),and the middle-zone cartilage tissue of OARSI grade 5 and grade 6 cartilage tissues was severely damaged or even eliminated. We found thata few chondrocyte colony-like clusters occasionally formed in the deep zone of OARSI grade 5 and grade 6 cartilage tissue.
Biological properties of chondrocytes in colony-likeclusters
To explore the biological properties of chondrocytes in colony-like clusters, we selected OARSI grade 3 cartilage tissue as the experimental group and OARSI grade 0~1 cartilage tissue as the control group.Immunohistochemical scores of histological sections showed that the levels of collagen II and aggrecan decreased and the levels of collagen X and MMP-13 increased in chondrocytes incolony-like clusters compared to control chondrocytes (figure 2A).
After scraping the cartilage tissue of the middle zone in the two groups, PCR was used to detect the levels of collagen II mRNA, aggrecan mRNA, collagen X mRNA, and MMP-13 mRNA, and western blot analysis was used to detect the corresponding protein levels.PCR results of middle-zone cartilage tissue showed that the levels of collagen II mRNA and aggrecan mRNA decreased and the levels of collagen Ⅹ mRNA and MMP-13 mRNA increased in chondrocytes in colony-like clusters compared to control chondrocytes.Western blot results of middle-zone cartilage tissue showed that the levels of collagen II and aggrecan decreased and the levels of collagenⅩ and MMP-13 increased in chondrocytes in colony-like clusters compared to control chondrocytes(figure 2B).
The middle-zone cartilage tissue was scraped from sterile cartilage tissues, and chondrocytes were extracted for in vitro culture to generation 3.The immunohistochemical scores of cultured chondrocytes from middle-zone cartilage tissue showed that the levels of collagen II and aggrecan decreased and the levels of collagen X and MMP-13 increased in chondrocytes from colony-like clusters compared to control chondrocytes(figure 2C). PCR results of cultured chondrocytes showed that the levels of collagen II mRNA and aggrecan mRNA decreased and the levels of collagen Ⅹ mRNA and MMP-13 mRNA increased in chondrocytes from colony-like clusters compared to control chondrocytes. Western blot results of cultured chondrocytes showed that the levels of collagen II and aggrecan decreased and the levels of collagen X and MMP-13 increased in chondrocytes from colony-like clusters compared to control chondrocytes(figure 2D).
We used 5-ethynyl-2'-deoxyuridine (EDU) to detect the proliferation rate of cultured chondrocytes at 24 h and performed immunohistochemistry to detect PCNA.The EdU results showed not only a high chondrocyte proliferation rate but also a colony-like proliferation pattern in the cluster group.In addition,the level of PCNA increased in the cluster group compared to the control group (figure 2E).
Influence of colony-like cluster formation on cartilage degeneration
To observe the influence of colony-like cluster formation on cartilage degeneration, we used a cartilage tissue penetration test.OARSI grade 3 cartilage tissue was obtained, which was determined by staining of histology sections with safranin O,revealing many chondrocyte colony-like clusters in middle-zone cartilage tissue from the cluster group and no chondrocyte colony-like clusters in middle-zone cartilage tissue from the no cluster group(figure 3A,figure 3B). The results of the penetration test showed that the contrast medium did not penetrate into the main part of the cartilage tissue in the two groups at 0 h and 1 h. The contrast medium eventually penetrated into the main part of the cartilage tissue in the cluster group (figure 3B);however, there was no penetration into the main part of the cartilage tissue in the no cluster group(figure 3A). At 5 h, the contrast medium completely penetrated the cartilage tissue in the cluster group;however, the contrast medium had not yet fully penetrated the entire cartilage layer in the no cluster group at this time point(figure 3A,figure 3B). To make a quantitative comparison ofthe penetration rate between the two groups, we performed micro-CT with the same resolution to generate images of the same size and selected a specificarea of the analysis map on the imageto ensure that the analysis maps hadsimilarnumbers of pixels.We specified that a change in pixels from yellow to red on the false-colour image is a positive pixel.The total numbers of positive pixels at 0, 1, 3, and 5 h were counted by a computer and divided by the interval time (seconds); thus, and "positive penetration pixels per second" values ofthe two groups were calculated and compared(figure 3C, table 1).
Pathological stages of chondrocyte colony-like clusters
We found that the number of chondrocytes in the colony-like clusters increased to a certain number, then gradually decreased and eventually became zero,leaving large chondrocyte lacunae. To better understand the biological properties of chondrocytes in clusters with different chondrocyte densities, we used the ratio of the total chondrocyte area to the chondrocyte lacunae area to stage the chondrocyte cluster(figure 4A). First, we drew the outline of the chondrocyte lacunae;then,we drew the long axis of the contour line across the centre of the chondrocyte lacunae.We divided the long axis into 4 equal parts and drew a line perpendicular to the long axis through the equal points. In this way, the chondrocyte lacunae were divided into 8 parts, and the ratio of the chondrocyte area to the chondrocyte lacunae area for each part was visually measured.The fraction corresponding to the ratio of each part was calculated, and these values were added to gether to generatethe total score of the 8 parts.We assigned the following scores based onthese ratios: 0 if the relative area was 0%, 1 if the relative area was 1% to 25%, 2 if the relative area was 26% to 50%, and 3 if the relative area was 51% to 100%.Finally, we divided the chondrocyte clusters into 4 stages according to the total score:Grade I if the score was 17~24, Grade II if the score was 9~16, Grade III if the score was 1~8,and Grade IV if the score was 0(figure 4B).The results were assessed by 2 experienced pathologists in a blinded manner.
To examine the biological properties of chondrocytes in colon-like clusters at different stages, immunohistochemistry was used to detect the levels of collagen II, PCNA,MMP-13 and caspase-3.The results showed that there was little collagen II, abundant PCNA,little MMP-13, and little caspase-3 expressed in chondrocytes of stage I colony-like clusters and that the cell density in cartilage lacunae was very high. There was little collagen II, little PCNA,a moderate amount of MMP-13, and a moderate amount of caspase-3 expressed in chondrocytes of stage II colon-like clusters, and the cell density in cartilage lacunae was sparse.There was little collagen II, little PCNA,abundant MMP-13, and abundant caspase-3 expressed in chondrocytes of stage III colony-like clusters, and only a few remaining chondrocytes were in the cartilage lacunae.There were no chondrocytes in the stage IV colony-like clusters, which are empty cartilage lacunae(figure 4C,figure 5).