ANALYSIS OF PROLIFERATIVE ACTIVITY AND APOPTOSIS OF CELLS OF FEMORAL HEAD BONE TISSUE IN VARIOUS ETIOLOGICAL FORMS OF OSTEOARTHROSIS
Novosibirsk Scientific Research Institute of Traumatology and Orthopedics named after Ya.L. Tsyvyan, Novosibirsk, Russia,
Laboratory of Molecular and Genetic Characteristics of Tumors, Altay department of Blokhin Russian Oncologic Scientific Center, Barnaul, Russia,
Regional Clinical Center of Miners’ Health Protection, Leninsk-Kuznetsky, Russia,
Institute of Molecular Pathology and Pathomorphology, Novosibirsk, Russia
During the last years the great perspectives in treatment of hip joint osteoarthrosis are associated with such low invasive surgical techniques as transplantation of mesenchymal stem cells and synthetic materials into pathologically changed structural components of the joint [1-4]. For estimation of efficiency of such treatment techniques and more proper understanding of the mechanisms of remodeling of bone tissue of the femoral head it is necessary to have the clear imagination of cell expression of the transcription factors. The various clinical and experimental studies present the good description of apoptosis of osteoblasts and osteocytes in the subchondral department of the femoral head with osteoarthrosis. One can observe some changes in balance during regulation of apoptosis of bone tissue cells under influence of various chemical and physical factors [5-7]. The examination of such marker of cellular proliferation as Ki-67 protein allows estimating the process of bone tissue remodeling in the femoral head with osteoarthrosis of various etiology. It is the fact that during the process of bone tissue remodeling Ki-67 positive osteoblasts account for 10 % of the pool of all osteoblasts. Moreover, they are widely-spaced and are alternated with significant amount of Ki-67 negative osteoblasts [8].
Despite of the significant amount of the studies dealing with the process of bone tissue remodeling from the perspective of molecular biology, many aspects of various etiologies of hip joint osteoarthrosis are not investigated to the full degree and require additional researches. Description of proliferative activity and cellular apoptosis of bone tissue with osteoarthrosis can determine more individual approach in treatment of patients with this disease.
The objective of the study – to conduct the analysis of the transcription factors of the cells of bone tissues of the femoral head in various etiological forms of osteoarthrosis.
MATERIALS AND METHODS
The object of the morphological study was 95 femoral heads resected during hip joint replacement in the patients with the clinically confirmed diagnosis “hip joint osteoarthrosis of the stage 3-4” who were admitted to the department of traumatology and orthopedics, Regional Clinical Center of Miners’ Health Protection, for hip joint replacement within the period from 2013 till 2015. The median of the age was 56 (49-64) years. The gender distribution was as follows: 51 (53.7 %) men and 44 (46.3 %) women. According to the etiological factors, hip joint osteoarthrosis demonstrated smooth distribution: dysplastic – 34 cases, ischemic – 31 cases and posttraumatic – 30 cases.
From the given materials with use of the special device we performed the vertical sawing the fragment of articular surface and subchondral bone tissue of 0.75 cm3 (the size 1.5 × 1.0 × 0.5 cm) [9]. Fixation and decalcification of the sawed fragments were made with EDTA solution (OOO ErgoProduction, Russia) owing to the common technique. After decalcification we performed the histological guiding the material followed by placement into wax. The serial histological slices (4-6 slices from one fragment) with thickness of 3-5 µm were made on the rotation histotome (Accu-Cut SRM 200, China). For initiation of histological response we used the high adhesive glasses with positively charged surface (Super Frost Plus). All stages of the immunohistochemical reactions were conducted with the automatic mode with the immunohistostainer Bench Mark XT (Ventana) with adherence to the study protocol for each antibody. For estimation of the remodeling processes we assessed the level of expression of the transcription factors (Ki-67, p53, bcl-2) in the cells of bone tissue. For estimation of proliferative activity of the cells of bone tissue we used the monoclonal antibody Ki-67 (30-9, Ventana), for registration cell apoptosis in bone tissue we used p53 antibody (DO-7, Ventana), for registration of antiapoptotic activity – bc-2 antibody (124, Ventana). The study was conducted with the light microscope (Nicon Ci-S, China) with use of the digital camera (Nicon DS-Fi2, Japan).
The statistical analysis of the results was made with Statistica 6.0 software. The normalcy of data distribution was tested with Shapiro-Wilk test (W). The measure of central tendency was the median (Me), and the measure of scatter – interquartile range – the range between 25 % and 75 % of data in the sample. For identification of intergroup differences we used non-parametrical Mann-Whitney test. The differences were statistically significant, if p value was less than 0.05. The correlation analysis was conducted with Spearman's rank correlation coefficient [10].
RESULTS AND DISCUSSION
The level of expression of Ki-67 protein allows estimating the proliferative activity of the cells. So, Ki-67 protein was registered only in bone tissue of the subchondral departments of the femoral head with osteoarthrosis, but osteoblasts and osteocytes showed only the negative immunohistochemical response with Ki-67 protein in all cases of the study. Ki-67 positive osteoblasts were one after one or in view of the groups of 7-10 cells on the surface of bone rods. These areas alternate with the high amount of Ki-67 negative osteoblasts (Fig. 1).
Figure 1
The positive immunohistochemical response of osteoblasts (indicated with an arrow) in subchondral bone tissue of the femoral head with antibodies Ki-67 in dysplastic osteoarthrosis of the hip joint. 400-fold amplification
The specific nuclear staining of osteoblasts with Ki-67 marker was registered in 84 cases (88.4 %) among 95, and the negative responses were registered in 11 cases (11.6 %). In the group of dysplastic osteoarthrosis (n = 34) the expression of Ki-67 protein was realized with osteoblasts in all studied cases. The median of the expression level of this marker by osteoblasts was 7 (5-8) % of the cells. Ki-67 expression was identified in 26 of 31 cases (83.9 %) in the group of osteoarthrosis of ischemic origin. The median of expression was 6 (4-6) % of osteoblasts. Ki-67 expression was registered in 24 of 30 cases (80 %) with posttraumatic osteoarthrosis of the hip joint. The median of expression of this marker in the subchondral bone tissue of the femora head was 3 (2.5-4) % of osteoblasts.
The table 1 shows the findings of the level of expression of Ki-67 protein by osteoblasts of subchondral bone tissue of the femoral head with consideration of the etiological type of osteoarthrosis.
| Table 1 |
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| Level of expression of protein Ki-67 by osteoblasts in subchondral bone tissue of the femoral head with consideration of an etiological form of osteoarthrosis, Ìå (LQ – UQ) | ||
| Etiological form of osteoarthrosis, (n = 95) | Expression of Ki-67 protein by osteoblasts | |
| Proportion of positive cells, % | Number of positive cases, n | |
| Dysplastic, (n = 34) | 7 (5-8) 1, 2 | 34 |
| Ischemic, (n = 31) | 6 (4-6) 3 | 26 |
| Posttraumatic, (n = 30) | 3 (2.5-4) | 24 |
| Note: statistically significant differences: | ||
| 1 - with level of expression of Ki-67 by osteoblasts in ischemic osteoarthrosis (p = 0.0105); | ||
| 2 - with level of expression of Ki-67 by osteoblasts in posttraumatic osteoarthrosis (p < 0.0001); | ||
| 3 - with level of expression of Ki-67 by osteoblasts in posttraumatic osteoarthrosis (p = 0.0013). | ||
The statistical analysis showed the presence of maximal expression of Ki-67 by osteoblasts: it is 1.2 times higher than in ischemic osteoarthrosis (p = 0.0105) and 2.3 times higher than in posttraumatic osteoarthrosis (p < 0.0001). Also there were some statistically significant differences in the levels of expression of this marker by osteoblasts of the femoral head tissue in ischemic and posttraumatic osteoarthrosis. So, the amount of Ki-67 positive osteoblasts is 2 times higher in ischemic osteoarthrosis as compared with posttraumatic origin of osteoarthrosis (p = 0.0013).
The examination of expression of such inhibitor of apoptosis as bcl-2 protein is an important aspect in estimation of processes of remodeling in the femoral head bone tissue in hip joint osteoarthrosis. The conducted analysis shows the cytoplasmic pattern of expression of this marker.
Protein bcl-2 was identified in cytoplasm of osteocytes and osteoblasts. Osteoclasts expressing this protein were not identified. Bcl-2 positive osteoblasts were located on the surface of the bone rod with the groups of 10-12 cells, alternating with the groups of bcl-2 negative osteoblasts. Osteocytes with expression of this protein demonstrate the focal positioning and were visible in some fields (Fig. 2).
Figure 2
The positive immunohistochemical response of osteoblasts (indicated with a black arrow) and osteocytes (indicated with a red arrow) in subchondral bone tissue of the femoral head with antibodies bcl-2 in ischemic osteoarthrosis of the hip joint. 400-fold amplification
Among 95 cases of hip joint osteoarthrosis the protein bcl-2 was identified in 71 cases (74.7 %) in osteoblasts, and in 70 cases (73.7 %) – in osteocytes. Expression of this protein by osteoblasts was registered in 24 of 34 (70.6 %) cases. The expression level was 3 (2-5) % of the cells. Expression of bcl-2 by osteocytes was identified in 24 cases (70.6 %). Its value was 13.5 (10-15.5) % of the cells. In ischemic osteoarthrosis of the hip joint the expression of bcl-2 by osteoblasts was registered in 26 of 31 cases (83.9 %). Its level was 7 (3-12) % of the cells. Bcl-2 expression by osteocytes was identified in 26 cases (83.9 %). Its level was 10 (5-16) % of the cells. In posttraumatic osteoarthrosis the expression of this protein by osteoblasts was registered in 21 of 30 cases (70 %), and its value was 4 (3-6) % of the cells. Bcl-2 expression by osteocytes was identified in 20 cases (66.6 %); its value was 7.5 (5-12.5 %) of the cells.
The table 2 shows the summary data of expression of bcl-2 by osteoblasts and osteocytes of subchondral tissue of the femoral head with consideration of the etiological types of hip joint osteoarthrosis.
| Table 2 | Level of expression of bcl-2 protein by osteoblasts and osteocytes in subchondral bone tissue of the femoral head with consideration of an etiological form of osteoarthrosis of the hip joint, Ìå (LQ – UQ) | |||
| Etiological form of osteoarthrosis, (n = 95) | Expression of bcl-2 protein by osteoblasts | Expression of bcl-2 protein by osteocytes | ||
| proportion of positive cells, % | number of positive cases, n | proportion of positive cells, % | number of positive cases, n | |
| Dysplastic, (n = 34) | 3 (2-5) 4 | 24 | 13.5 (10-15.5) 3 | 24 |
| Ischemic, (n = 31) | 7 (3-12) 1, 2 | 26 | 10 (5-16) | 26 |
| Posttraumatic, (n = 30) | 4 (3-6) 5 | 21 | 7.5 (5-12.5) | 20 |
| Note: statistically significant differences: | ||||
| 1 - with level of expression of bcl-2 by osteoblasts in posttraumatic osteoarthrosis (p = 0.025); | ||||
| 2 - with level of expression of bcl-2 by osteoblasts in dysplastic osteoarthrosis (p = 0.0289); | ||||
| 3 - with level of expression of bcl-2 by osteocytes in posttraumatic osteoarthrosis (p = 0.0088); | ||||
| 4 - with level of expression of bcl-2 by osteocytes in dysplastic osteoarthrosis (p < 0.0001); | ||||
| 5 - with level of expression of bcl-2 by osteocytes in posttrumatic osteoarthrosis (p < 0.000451). | ||||
The conducted statistical analysis showed the maximal value of cytoplasmic expression of bcl-2 by osteoblasts in the femoral head bone tissue in the group with ischemic osteoarthrosis and it was 1.75 times higher than in the group of posttraumatic osteoarthrosis (p = 0.025) and 2.3 times higher than in the group of dysplastic osteoarthrosis (p = 0.0289). There were no statistically significant differences in the levels of expression of this protein in dysplastic and posttraumatic hip joint osteoarthrosis (p = 0.7243). The maximal value of cytoplasmic expression of bcl-2 by osteocytes in the femoral head tissue was identified in the group of dysplastic osteoarthrosis and it was 1.8 times higher than in the group of osteoarthrosis of posttraumatic origin (p = 0.0088). There were no statistically significant differences in the levels of expression of this protein in dysplastic and ischemic osteoarthrosis (p = 0.2175). No statistically reliable differences were found after comparison of the level of cytoplasmic expression of bcl-2 by osteocytes in the groups of osteoarthrosis of ischemic and posttraumatic origins (p = 0.6656). In dysplastic osteoarthrosis the proportion of bcl-2 positive osteocytes was 4.5 times higher than osteoblasts (p < 0.0001), but in posttraumatic osteoarthrosis – 1.9 times higher (p = 0.000451). The exclusion is hip joint osteoarthrosis of ischemic origin, when the differences were not statistically significant (p = 0.088).
The research of such initiator of apoptosis as p53 protein is an important aspect in estimation of the processes of the femoral head tissue remodeling in hip joint osteoarthrosis. The conducted analysis shows the nuclear pattern of expression of this marker. The study showed that nuclear expression of p53 protein is common exclusively for single osteocytes (Fig. 3).
Figure 3
The positive immunohistochemical response with antibody p53 of an osteocyte (indicated with an arrow) in subchondral bone tissue of the femoral head in dysplastic osteoarthrosis. 400-fold amplification
Among 95 examined cases p53 protein was registered in osteocytes of bone tissue of the femoral head in 37 patients (38.9 %). This protein could not be determined in 61.1 % of the cases. In the group of dysplastic osteoarthrosis the expression of p53 by osteocytes was registered in 14 of 34 cases (41.2 %). The median of the level of expression of this marker was 0.2 (0.2-0.3) % of osteocytes. In the group of ischemic osteoarthrosis the expression of p53 was determined in 13 of 30 cases (43.3 %). The median of its expression was 0.2 (0.2-0.3) % of osteocytes.
The summary data of expression of p53 by osteocytes in bone tissue of the femoral head (with consideration of the etiological types of osteoarthrosis) are presented in the table 3.
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Table 3 |
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| Level of expression of p53 protein by osteocytes in bone tissue of the femoral head with consideration of an etiological form of osteoarthrosis, Ìå (LQ – UQ) | ||
| Etiological form of osteoarthrosis, (n = 95) | Expression of p53 protein by osteocytes | |
| Proportion of positive cells, % | Number of positive cases, n | |
| Dysplastic, (n = 34) | 0.2 (0.2-0.3) | 14 |
| Postischemic, (n = 31) | 0.2 (0.2-0.3) | 10 |
| Posttraumatic, (n = 30) | 0.2 (0.2-0.3) | 13 |
The statistical analysis did not show any reliable findings in the level of expression of p53 protein by osteocytes in various etiological types of hip joint osteoarthrosis. Therefore, it was found that hip joint osteoarthrosis was accompanied by exclusively insignificant amounts of osteocytes.
The correlation analysis was conducted for identification of statistically significant relationships between expression of the transcription factors by the cells of the femoral head tissue. So, the strong inverse correlation relationship was found between expression of p53 and bcl-2 by osteocytes (r = -0.712; ð = 0.0043) in the femoral head with osteoarthrosis of dysplastic origin. Also the strong direct correlation relationship was found between expression of bcl-2 by osteocytes and osteoblasts (r = 0.886; ð = 0.0001). In the femoral head tissue with ischemic osteoarthrosis the strong inverse correlation relationship was found between expression of bcl-2 by osteocytes and p53 protein by osteocytes (r = -0.888; ð = 0.0439), as well as the strong correlation relationships with bcl-2 of osteoblasts (r = 0.965; ð = 0.0001) and Ki-67 of osteoblasts (r = 0.838; ð = 0.000002). Moreover, the strong correlation relationship was found between the level of expression of p53 by osteocytes and Ki-67 by osteoblasts (r = 0.754; ð = 0.0117), and between the level of expression of bcl-2 and Ki-67 by osteoblasts (r = 0.772; ð = 0.000041). In bone tissue of the femoral head with posttraumatic osteoarthrosis we found the strong correlation relationship between the levels of expression of bcl-2 by osteoblasts and osteocytes (r = 0.877; ð = 0.000004).
CONCLUSION
The conducted analysis of the molecular and biological values of bone tissue of the femoral bone identified the range of the specific features of osteoarthrosis of dysplastic, ischemic and posttraumatic origin. In dysplastic hip joint osteoarthrosis in the femoral head the proliferative activity of osteoblasts and the level of expression of bcl-2 in osteocytes show the maximal expression. Moreover, increase in levels of this protein in osteocytes and osteoblasts is accompanied by additional decrease in the level of expression of the protein p53, which activates apoptosis in osteocytes. Posttraumatic hip joint osteoarthrosis shows the minimal proliferative activity of osteoblasts. The cell composition of the femoral head is mainly supported by increasing levels of expression of antiapoptotic protein bcl-2 in osteocytes and osteoblasts. In ischemic hip joint osteoarthrosis the bone tissue of the femoral head is characterized by the maximal level of expression of bcl-2 by osteoblasts. Therefore, one can suppose that improvement in bone tissue synthesis in dysplastic osteoarthrosis requires the search of therapy with orientation to increasing antiapoptotic activity of osteoblasts, and in postischemic osteoarthrosis – for increasing proliferative activity of osteoblasts and antiapoptotic activity of osteocytes.