THE LEVEL OF EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND TARTRATE-RESISTANT ACID PHOSPHATASE (TRAcP) IN FEMORAL HEAD BONE TISSUE IN COXARTHROSIS
Regional Clinical Center of Miners’ Health Protection,
Leninsk-Kuznetsky, Russia
Laboratory of molecular and genetic characteristics of tumors, Altay department of Blokhin Russian Cancer Research Center,
Barnaul, Russia
Institute of Molecular Pathology and Pathologic Morphology,
Novosibirsk, Russia
Deforming arthrosis of the hip joint relates to the group of degenerative dystrophic diseases [4]. Progression of the pathologic state results in changes in the balance of the system of bone tissue remodeling with further disarrangement of supporting functioning of the bone and the joint.
At the present time, one of the available methods for estimation of the processes of bone remodeling is immunohistochemistry [8].
Tartrate-resistant acid phosphatase (TRAcP) is a lysosomal enzyme of osteoclasts influencing on bone tissue resorption. Osteoclasts create an isolated cavity of resorption on the surface of the bone. In this cavity, secretion of acid phosphatase is realized by means of exocytosis. Determination of the level of TRAcP expression is a classic method for identification of osteoclasts, as well as for estimation of the resorption activity [2]. It is noted that the level of TRAcP expression in osteoclasts changes both under physiologic conditions and under the influence of pharmaceuticals [5, 6]. The available scientific studies of resorption activity of osteoclasts in the femoral head bone tissue in various nosologic types of coxarthrosis are considerably controversial and do not reflect any well-defined estimation of this process [12].
Progression of coxarthrosis is also influenced by inevitable hypoxia in bone tissue of the femoral head as result of hemodynamic disorders [3, 10]. One of the promoters of osteogenesis is vascular endothelial growth factor. Estimation of its expression in various pathologic processes is a quite objective method for estimation of tissue responses to hypoxia [7, 9].
The study objective – to make the analysis of expression of VEGF and TRAcP in bone tissue of the femoral head with deforming arthrosis of the hip joint.
MATERIALS AND METHODS
The morphological study included 95 femoral heads removed during hip endoprosthetics in the patients with diagnosed coxarthrosis of the stage 3-4. The patients were treated in Regional Clinical Center of Miners’ Health Protection. The age of the patients was 56 (49-64). There were 51 men (53.7 %) and 44 women (46.3 %). According to the nosologic types, coxarthrosis showed equal distribution: dysplastic type (34 cases), postischemic type (31 cases), posttraumatic type (30 cases).
A fragment of the articular surface and subchondral bone tissue (1.5×1.0×0.5 cm) was carved out of the samples. Fixation and decalcification of the carved fragments was made with EDTA solution (Ergoproduction, Russia) according to the instruction of the manufacturer.
The standard procedure of production of histological specimen with the following hematoxylin-eosin and hematoxylin-picrofuxin staining was conducted after decalcification. The examination was conducted with the optical microscope (Nikon Eclipse E200) and the digital camera (Nikon DS-Fi2).
The arrangement of immunohistochemical reactions was realized in automatic mode with the immunohistostainer Bench Mark XT (Ventana) with adherence to the examination protocol for each antibody. After conducting the reactions, the sections were dehydrated and placed under cover glass.
For identification of the vessels of microcirculatory bed we used CD34 antibody (QBEnd/10, Ventana). Vascular endothelial growth factor was estimated with the antibody to VEGF (SP28, Spring Bio). The resorption activity of osteoclasts was conducted with the antibody to acid phosphatase – TRAcP (9C5, Cell Marque). The special software NIS-Elements (version BR 4.30.00) was used for measurement of optical density (conventional units) of cytoplasmic expression of VEGF in osteoblasts and osteoclasts, and TRAcP in osteoclasts, as well as for estimation of the square of the vessels of microcirculatory bed (µm2).
Statistica 6.0 was used for the statistical analysis.
Shapiro-Wilk test (W) was used for testing the normalcy of distribution of quantitative (continuous and ordinal) data. The null hypothesis about correspondence of the analyzed data to normal probability law was rejected in case of significant values of the test. The data was presented as Me (LQ – UQ), where Me – median, (LQ – UQ) – interquartile range. Kruskal-Wallis test and Mann-Whitney test were used for identification of intergroup differences according to quantitative values. P value < 0.05 was related to statistically significant differences.
The correlation analysis was conducted with Spearman's rank correlation coefficient. The value of rank correlation coefficient was used for estimation of the neighboring relationship between the signs: the values of 0.3 and lower indicated the weak neighboring relationship, the values of more than 0.4 but lower than 0.7 – the moderate neighboring relationship, the values of 0.7 and more – the strong neighboring relationship.
RESULTS AND DISCUSSION
The histologic examination of the femoral heads with posttraumatic coxarthrosis showed the interchange of regions of unaltered hyaline cartilage and regions of disordered architectonics of chondrocytes. The interchange of regions of spinal cord edema and fibrosis was observed in the subchondral departments of bone tissue.
The histologic analysis of the femoral heads with postischemic coxarthrosis indicated aseptic necrosis of fat tissue, bone marrow and bone rods. The necrotic regions were presented as eosinophilic masses without inflammatory feedback. A great amount of osteoclasts was identified on the nonuniformly thin trabecular surface.
As for dysplastic coxarthrosis, the surface of the femoral head was in view of sclerotic compact subchondral bone plate. Interrod spaces included fibrous tissue. Bone rods of spongy substance included some events of osteosclerosis.
The estimation of such molecular biologic values as the level of VEGF and TRAcP expression indicated some common features of bone tissue in the above mentioned nosologic types of coxarthrosis. The findings are shown in the table and in the figure.
| Table | |||||||||||||
| The level of VEGF and TRAP expression in femoral head bone tissue with various nosologic types of coxarthrosis, Ме (LQ – UQ). | |||||||||||||
| Notes: | |||||||||
| a - statistically significant differences between osteoblast VEGF expression level in the femoral head with coxarthrosis of dysplastic and traumatic origin (p = 0.03); | |||||||||
| b - statistically significant differences between osteoclast VEGF expression level in the femoral head with coxarthrosis of postischemic and dysplastic origin (p < 0.0001); | |||||||||
| c - statistically significant differences between mean square of vessels in the femoral head with coxarthrosis of postischemic and posttraumatic origin (p = 0.001); | |||||||||
| d - statistically significant differences between osteoclast TRAcP expression level in the femoral head with coxarthrosis of postischemic and dysplastic origin (p < 0.0001); | |||||||||
| e - statistically significant differences between amount of osteoclasts in the femoral head with coxarthrosis of postischemic and dysplastic origin (p < 0.0001). | |||||||||
The figure
The molecular and biological features of femoral head tissue with coxarthrosis, 100-fold magnification.
a – osteoblast VEGF expression (indicated with the arrow) in dysplastic coxarthrosis; b – osteoclast VEGF expression (indicated with the arrow) in postischemic coxarthrosis; c – vessels of microcirculatory bed (CD34+ endothelium) of femoral head bone tissue in postischemic coxarthrosis; d – osteoclast TRAcPexpression (indicated with the arrow) in posttraumatic coxarthrosis.
The results testify that the maximal level of VEGF expression in osteoblasts is registered in coxarthrosis of dysplastic origin. At the same time, the levels of VEGF and TRAcP in osteoclasts, and the amount of osteoclasts are minimal in such pathology. The similar results were obtained in other studies [1, 11].
The lowest level of VEGF expression in osteoblasts was found in posttraumatic coxarthrosis. A specific feature was the presence of the smallest vessels of microcirculatory bed in bone tissue in the above mentioned nosologic type of coxarthrosis.
Postischemic coxarthrosis is characterized by maximal values of VEGF expression in osteoclasts, square of microcirculatory bed, TRAcP expression and amount of osteoclasts.
The correlation analysis identified some relationships between the examined indices. So, dysplastic coxarthrosis showed the strong direct correlation between VEGF expression in osteoblasts and the square of vessels (r = 0.7; ð < 0.00001). The presence of the moderate inverse correlation between VEGF expression in osteoblasts and VEGF expression in osteoclasts, and the amount of osteoclasts and osteoblasts (r = -0.4; ð = 0.02 and r = -0.4; ð = 0.03 correspondingly) indicates the greater role of osteoclasts in formation of the vessels of microcirculatory bed in such pathologic process.
The moderate direct correlation between the level of VEGF expression in osteoblasts and the square of the vessels was in posttraumatic coxarthrosis (r = 0.4; p < 0.027).
CONCLUSION
Therefore, the analysis of VEGF and TRAcP expression in the femoral head bone tissue indicates some specific features, which are common for each nosologic type of coxarthrosis.
Coxarthrosis of dysplastic origin is characterized by maximal expression of VEGF in osteoblasts – 0.66 (0.62-0.69) c.u. and minimal expression of VEGF and TRAcP in osteoclasts – 0.48 (0.38-0.53) c.u. correspondingly. The presence of the strong correlation between the level of VEGF expression in osteoblasts and the square of the vessels indicates the higher contribution of osteoblasts to formation of the vessels of microcirculatory bed in dysplastic coxarthrosis.
VEGF expression in osteoblasts and the square of the vessels of microcirculatory bed are minimal in posttraumatic coxarthrosis – 0.55 (0.5-0.69) c.u. and 1,539.5 (1,104-4,242) µm correspondingly.
Postischemic coxarthrosis is characterized by maximal VEGF expression in osteoclasts, the maximal square of the vessels of microcirculatory bed, the highest TRAcP expression and the maximal amount of osteoclasts. Such values are the common signs of postischemic coxarthrosis.