Open Access Peer-reviewed Research Article
Bone Microstructure and Regional Distribution of Osteoblast and Osteoclast Activity in the Osteonecrotic Femoral Head
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To detect and compare the bone microstructure and osteoblast and osteoclast activity in different regions of human osteonecrotic femoral heads.
Osteonecrotic femoral heads were obtained from 10 patients (6 males, 4 females; Ficat IV) undergoing total hip arthroplasty between 2011 and 2013. The samples were divided into subchondral bone, necrotic, sclerotic, and healthy regions based on micro-computed tomography (CT) images. The bone microstructure, micromechanics, and osteoblast and osteoclast activity were assessed using micro-CT, pathology, immunohistochemistry, nanoindentation, reverse transcription polymerase chain reaction (RT-PCR), tartrate-resistant acid phosphatase staining and Western blotting.
(1) The spatial structure of the bone trabeculae differed markedly in the various regions of the osteonecrotic femoral heads. (2) The elastic modulus and hardness of the bone trabeculae in the healthy and necrotic regions did not differ significantly (_P_>0.05). (3) The subchondral bone and necrotic region were positive on TRAP staining, while the other regions were negative. (4) On immunohistochemical staining, RANK and RANKL staining intensities were increased significantly in the subchondral bone and necrotic region compared with the healthy region, while RUNX2 and BMP2 staining intensities were increased significantly in the sclerotic region compared with the necrotic region. (5) OPG, RANK, RANKL, RUNX2, BMP2, and BMP7 protein levels were greater in the necrotic and sclerotic region than in subchondral bone and the healthy region.
The micromechanical properties of bone trabeculae in the necrotic region did not differ significantly from the healthy region. During the progress of osteonecrosis, the bone structure changed markedly. Osteoclast activity increased in subchondral bone and the necrotic region while osteoblast activity increased in the sclerotic region. We speculate that the altered osteoblast and osteoclast activity leads to a reduction in macroscopic mechanical strength.
Citation:Wang C, Wang X, Xu X-l, Yuan X-l, Gou W-l, Wang A-y, et al. (2014) Bone Microstructure and Regional Distribution of Osteoblast and Osteoclast Activity in the Osteonecrotic Femoral Head. PLoS ONE 9(5): e96361. https://www.frankenthalerfoundation.org
Editor:Xing-Ming Shi, Georgia Regents University, United States of America
Received:December 26, 2013; Accepted:April 6, 2014; Published: May 6, 2014
Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was funded by NSFC (30930092, 31240048, 31170946), NTRDP (2012AA020502, 2012CB518106, BWS11J025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Osteonecrosis of the femoral head is a common, refractory disease in orthopedics departments. Non-traumatic femoral head necrosis, which occurs frequently in young and middle-aged patients (30 to 50 years old), progresses rapidly and has a high disability rate. Many etiologies disrupt the blood circulation to the femoral head, causing different degrees of cell death within the femoral head, affecting osteocytes, bone marrow, and hematopoietic cells. The resulting necrosis gradually decreases the macroscopic mechanical strength in the necrotic region, which leads to collapse of the femoral head and ultimately osteoarthritis of the hip[1], [2]. However, the mechanism of osteonecrosis of the femoral head is unclear. Many studies have shown that the osteonecrosis repair process requires precisely coordinated bone resorption and bone formation. Osteoblasts promote bone formation while osteoclasts give rise to bone resorption, and each regulates the other. Osteoclasts have positive and negative regulatory effects on osteoblast function [3], and the formation, differentiation, and maturation of osteoclasts are regulated by various solubility factors released by osteoblasts [4]. However, this balance is disrupted under pathological conditions, causing abnormal bone structure and function, resulting in various bone diseases, such as osteonecrosis of the femoral head.
The relationship between the decrease in mechanical strength of the femoral head and the possible restoration of the femoral head is uncertain. Therefore, it is necessary to investigate the structure of bone trabeculae and the change in osteoblast and osteoclast activity in different regions of the femoral head to explore the process of necrosis and the mechanism of femoral head collapse.
The nanoindentation technique is a new method for measuring the properties of bone [5]. It can accurately measure the elastic modulus and hardness of fine bone structures, such as trabeculae and lamellar bone, and can be used to test the toughness of bone microstructures in fracture testing.
Using pathological and immunohistochemical staining, tartrate-resistant acid phosphatase (TRAP) staining, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), micro-computed tomography (micro-CT), and Western blotting, we assessed the change in bone microstructure, micromechanical strength of bone trabeculae, and activation of osteoblasts and osteoclasts in different areas of femoral head specimens that had undergone osteonecrosis. We hypothesized that there would be significant differences in the bone microstructure in different areas of these femoral heads. The viability of osteoclasts in the subchondral bone and necrotic region was enhanced, leading to bone absorption. The viability of osteoblasts in the sclerotic regions was also enhanced. In the process of femoral head osteonecrosis, the activity of osteoblasts and osteoclasts changed, leading to a reduction in macromechanical strength. Continuous mechanical load would lead to the evetual collapse of the femoral head.
Ten femoral heads (Ficat IV) were obtained from patients with non-traumatic femoral head osteonecrosis who underwent total hip replacement in our hospital from 2011 to 2013.The study was approved by the Ethics Committee of the General Hospital of the Chinese Peoples Liberation Army, and written informed consent was obtained from all of the participants.
Micro-CT (GE Explore Locus, USA) was performed on all femoral head samples with 45-µm resolution. The scanning protocol was 80 kV and 450 mA, with an isotopic resolution of 45 × 45 × 45 mm voxel size and an integration time of 14 ms. From the micro-CT images, the femoral heads were subdivided into subchondral bone, necrotic, sclerotic, and healthy regions (Figure 1). A volume of interest (VOI) was selected from these regions for three-dimensional reconstruction and the following bone parameters were analyzed: bone volume fraction (BV/TV%), trabecular number (Tb.N, mm), trabecular separation (Tb.Sp,µm), and trabecular thickness (Tb.Th,µm).All analyses were calculated as reported previously [6].
Figure 1. Two-dimensional slices and three-dimensional reconstruction of micro-CT images of the femoral heads.
(A) Two-dimensional slices of micro-CT images of the subchondral bone (a), necrotic (b), sclerotic (c), and healthy(d) regions were distinguished according to bone mineral density. (B) The two-dimensional and three-dimensional reconstructed image of the white box regions.
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After the micro-CT scan, the fresh femoral head specimens were processed (Figure 2). Parts _a_ and _c_ were fixed in 4% paraformaldehyde for 2 weeks, and then processed to examine the pathology. Part _b_ was cut into four regions (subchondral bone, necrotic, sclerotic, and healthy regions), and stored at −80°C until RT-PCR testing.
Figure 2. Cutting method of the femoral heads.
The femoral heads were marked with an iron wire in the coronal plane and subjected to X-ray to confirm that all of the samples s
