Histomorphometric study of femoral heads in hip osteoarthritis and osteoporosis

During the period from 2000 to 2003, ninety eight samples of femoral heads were collected. In these pieces, two zones were analyzed: a high-load zone (the hard core of the head) and a low-load zone (the round ligamentum teres zone). As control group, 6 femoral heads (3 of women and 3 of men), proceeding from autopsy in peoples without pathological antecedents and youngs, were studied. After the samples had been embedded in methylmethacrylate and stained, they were subjected to an histomorphometric study. By means of histomorphometry, trabecular bone volume (TBV) and osteoid substance (OSV) was determined. Statistically significant differences were found as for peripheral osteoid volume (low-load zone) (p=0.036) and trabecular bone volume, both peripheral and central. Both volumes decreased in osteoporotic samples and in those from women (p=0.000), in comparison with control group. Regarding the relationship between the high-load and low-load zone, significant data were obtained. The high-load zone had a greater trabecular bone volume than the low-load zone, regardless of the pathology and sex, but this increase was more pronounced in the arthrosic samples and in those from men. Additionally, this trabecular bone volume in the high-load zone decreased with increasing age of the donor (p=0.037), when the control group is compared. In sum, we observed a reduction in the formation of TBV and OSV in osteoporosis but also a decrease in the arthrosic, in samples from older subjects, in women, and in the low-load zone of the samples, when the control group is compared. These data suggest the coexistence of both pathologies, which is more pronounced in older subjects and women.     

Adaptation of subchondral bone in osteoarthritis

Osteoarthritis is a chronic joint disease with pathological changes in the articulating cartilage and all other tissues that occupy the joint. Radin and coworkers have suggested the involvement of subchondral bone in the disease process. However, evidence for an essential role in the etiology has never been proven. Recent studies showing reduced chemical and mechanical properties of subchondral bone in various stages of the disease have invigorated interest in the role of subchondral bone in the development and progression of the disease. The current study showed that the concept of bone adaptation might explain subchondral stiffening, a process where subchondral bone becomes typically sclerotic in osteoarthritis. In addition, we report reduced mechanical matrix tissue properties as well as an increase in denatured collagen content. In conclusion, although osteoarthritic bone tissue contains increased denatured collagen and has reduced matrix mechanical properties, the widely accepted concept of subchondral stiffening is compatible with the process of normal bone adaptation.     

Targeting subchondral bone in osteoporotic osteoarthritis

The identification of well-defined phenotypes along the course of the disease may open new avenues for personalized management in osteoarthritis (OA). In vivo research carried out in various animal models as well as epidemiological and clinical data support the existence of a particular phenotype – osteoporotic OA. In fact, subchondral bone has become a potential therapeutic target in OA. Depending on the ratio between formation and resorption, subchondral bone remodeling can culminate in either a sclerotic or an osteoporotic phenotype. Patients with osteoporotic OA may thus achieve clinical and structural benefit from treatment with bone-targeted interventions.Subchondral bone has become a potential therapeutic target in osteoarthritis (OA). In a previous issue of Arthritis Research & Therapy, Wang and colleagues demonstrate that osteoporosis aggravates cartilage damage in an experimental model of knee OA in rats [1]. Interestingly, the authors also describe that extracorporeal shockwave therapy (ESWT), a mechanical therapeutic intervention probably acting at subchondral bone, may reduce OA progression [1]. The significance of these findings in experimental osteoporotic OA relates to the search for well-defined phenotypes in human OA that will lead to personalized therapy.The controversy regarding the relationship between subchondral bone quality and cartilage integrity originates from the complex biological and mechanical nature of the osteochondral junction [2]. OA progression is often accompanied by increased subchondral bone remodeling that enables mechanical forces to dynamically modify its structure. Depending on the ratio between formation and resorption, subchondral bone can exhibit either a sclerotic or an osteoporotic phenotype [3]. These phenotypes may represent up to 70% and 30% of patients in daily practice, respectively [4]. Furthermore, OA in females can display a different pathogenic profile from OA in males. In this sense, it is reasonable to underline the consequences of estrogen deficiency during menopause [5]. A low estrogen state could induce a deleterious effect on all articular tissues of the knee joint, the subchondral bone being particularly affected due to its capacity for high bone turnover. Thus, during early post menopause, estrogen deficiency may be a risk factor for the development of knee OA. Taking all these facts into consideration, the characterization of patients with either sclerotic or osteoporotic OA phenotypes may enable individualized targeted therapy [3].The effects of estrogen deficiency on the knee joint have been reported in various experimental animal models of OA. The findings obtained by Wang and colleagues on subchondral bone quality and articular cartilage damage support previous research carried out in rabbits, in which osteoporosis aggravated instability-induced OA [6]. In this combined model, the induction of systemic and subchondral osteoporosis associated with increased bone remodeling resulted in worse cartilage damage compared with control animals. Greater fragility of the subchondral bone was suggested to account for the aggravation of cartilage damage when early OA and osteoporosis coexist [7]. In a further study carried out in the same model, the intermittent administration of parathyroid hormone 1-34, a bone-forming agent, was used to increase subchondral bone density and quality [8]. As a consequence, the improvement of subchondral bone integrity was associated with reduced progression of cartilage damage in OA preceded by osteoporosis. In a similar approach, the inhibition of bone resorption by pamidronate in osteoporotic mice alleviated the instability-induced OA histological score with a reduction in the expression of aggrecanases [9]. Several experimental models therefore indicate that osteopenia/osteoporosis induces an accelerated progression of knee OA that can be reversed not only by bone-forming agents but also by antiresorptive drugs.These findings in animal models could be translated to humans, and together with epidemiological and clinical data they support the existence of a particular phenotype – osteoporotic OA [10]. Indeed, this phenotype characterized by decreased density and high remodeling at subchondral bone defines a subgroup of patients treatable with specific agents. In fact, beneficial effects of bone-acting drugs in OA are increasingly reported, but reliable conclusions regarding their efficacy are hindered by methodological drawbacks in study design [10]. Identifying patients with osteoporotic OA may improve the success of bone-directed agents.The original approach of using ESWT in OA by Wang and colleagues remains intriguing. These authors have reported previously that the application of ESWT to subchondral bone of the proximal tibia showed a chondroprotective effect in the initiation of knee OA and regression of established OA of the knee in rats. These effects were attributed to the ESWT multifunctional actions on cartilage and bone. Yet achieving such beneficial effects in this osteoporotic OA model suggests that the main mechanism of action of ESWT may be improving subchondral bone structure [1]. However, some limitations on the study design and the lack of adequate standardization of dosages and optimal frequency, as well as little information regarding the molecular mechanisms underlying the effects of ESWT, hold back the achievement of solid results. In any case, this study points out the potential benefit of nonpharmacological interventions aiming to improve mechanical properties of articular tissues in OA.In summary, the study by Wang and colleagues further supports the existence of the osteoporotic OA subtype and the potential benefit of bone-acting therapeutic interventions. Consequently, the identification of patient phenotypes along with the discovery of specific therapeutic interventions targeting relevant pathogenic mechanisms during the course of the disease could lead to a personalized approach to the management of OA.     

Biochemical and mechanical properties of subchondral bone in osteoarthritis

The subchondral bone has long been known to thicken in osteoarthritis. However, recent evidence has demonstrated that the turnover of the bone is increased several fold, and further suggests that the thickening occurs prior to degradation of the articular cartilage, indicating that it plays a role in the pathogenesis of osteoarthritis. The mechanical and biochemical properties of the subchondral bone are therefore of particular interest in any attempt to determine the nature of the factors initiating osteoarthritis. We have shown that the subchondral bone collagen of the femoral head possessed a 20-fold increase in turnover, as assessed by procollagen rate of synthesis and metalloproteinase degradation, and a 25% decrease in mineralisation. This increased metabolism and high lysyl hydroxylation leads to narrower and weaker fibres. Additionally the phenotypic expression of the osteoblasts is modified to produce increasing proportions of type I homotrimer in addition to the normal type I heterotrimer, which further reduces the mechanical strength of the bone. Overall, the narrow immature collagen fibres, the reduction in pyrrole cross-linking, decreased mineralisation, and increased amounts of type I homotrimer, all contribute to a weakening of the mechanical properties of the subchondral bone.     

Fundamental subchondral bone changes in spontaneous knee osteoarthritis

Osteoarthritis has an unknown aetiology, and tissue samples from early stage human osteoarthritis tissue cannot be reliably obtained. Therefore understanding the development of OA relies on using animal models: such as the spontaneous changes seen in the Dunkin-Hartley guinea pig strain, which are biochemically, histologically and radiologically similar to human OA. We investigated the role of bone change in early OA development using the non-OA developing Bristol strain-2 as control from 3 to 36 weeks by standard microfocal X-ray imaging and histological techniques. The patella, tibia and femur epiphyseal region and immediate subchondral area were analysed for bone density at all ages. We found that both radiological and histological osteoarthritis scores increased progressively for the Dunkin-Hartley, but not for the BS2 demonstrating its value as a control. The Dunkin-Hartley had a higher bone density and greater subchondral bone thickness from 24 weeks of age. We conclude that prior to any gross osteoarthritis pathology the Dunkin-Hartley are undergoing subchondral bone remodelling, thus demonstrating the fundamental role of early bone remodelling in the development of osteoarthritis.     

关节软骨与软骨下骨改变在不同骨关节炎动物模型中的特点

目的 探讨不同骨关节炎(osteoarthritis,OA)动物模型中软骨下骨和关节软骨的病理改变特征。方法 采用三种SD大鼠骨关节炎模型,将24只6月龄雌性大鼠随机分为4组:假手术对照组(Sham,n=6),前交叉韧带切除手术组(ACLT,n=6),木瓜蛋白酶关节腔注射组(Papain,n=6),以及卵巢切除术组(OVX,n=6)。造模后8周取膝关节,胫骨平台行Micro-CT扫描分析,关节软骨行甲苯胺蓝染色、Mankin评分,比较软骨下骨和关节软骨改变情况。结果 造模操作后8周,不同OA模型的软骨破坏程度有所不同。ACLT和Papain组软骨破坏比较严重,OVX组软骨变化较轻。所有OA模型中的软骨下松质骨均发生改变,OVX组相对于Sham对照组,软骨下骨微结构显著疏松,而ACLT组与Papain组相对于Sham对照组,软骨下骨微结构没有显著改变,但相对于OVX组,有显著性差异。三种OA模型的软骨下骨板厚度都较Sham组减少。结论 三种动物模型软骨下骨和关节软骨都发生明显病理改变,并且改变有所不同。不同OA模型代表不同病理,预示着软骨下骨所发挥的作用有所不同,这为进一步研究不同类型OA发生发展的机制,以及将软骨下骨作为OA治疗的可能靶点提供了更多的依据。     

Gene expression analyses of subchondral bone in early experimental osteoarthritis by microarray

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA.     

Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT

In the past few years there has been a considerable interest in the role of bone in osteoarthritis. Despite the increasing evidence of the involvement of bone in osteoarthritis, it remains very difficult to attribute the cause or effect of changes in subchondral bone to the process of osteoarthritis. Although osteoarthritis in mice provides a useful model to study changes in the subchondral bone, detailed quantification of these changes is lacking. Therefore, the goal of this study was to quantify subchondral bone changes in a murine osteoarthritis model by use of micro-computed tomography (micro-CT). We induced osteoarthritis-like characteristics in the knee joints of mice using collagenase injections, and after four weeks we calculated various 3D morphometric parameters in the epiphysis of the proximal tibia. The collagenase injections caused cartilage damage, visible in histological sections, particularly on the medial tibial plateau. Micro-CT analysis revealed that the thickness of the subchondral bone plate was decreased both at the lateral and the medial side. The trabecular compartment demonstrated a small but significant reduction in bone volume fraction compared to the contralateral control joints. Trabeculae in the collagenase-injected joints were thinner but their shape remained rod-like. Furthermore, the connectivity between trabeculae was reduced and the trabecular spacing was increased. In conclusion, four weeks after induction of osteoarthritis in the murine knee subtle but significant changes in subchondral bone architecture could be detected and quantified in 3D with micro-CT analysis.     

Identification of gene expression profiles and key genes in subchondral bone of osteoarthritis using weighted gene coexpression network analysis

Osteoarthritis (OA) significantly influences the quality life of people around the world. It is urgent to find an effective way to understand the genetic etiology of OA. We used weighted gene coexpression network analysis (WGCNA) to explore the key genes involved in the subchondral bone pathological process of OA. Fifty gene expression profiles of GSE51588 were downloaded from the Gene Expression Omnibus database. The OA‐associated genes and gene ontologies were acquired from JuniorDoc. Weighted gene coexpression network analysis was used to find disease‐related networks based on 21756 gene expression correlation coefficients, hub‐genes with the highest connectivity in each module were selected, and the correlation between module eigengene and clinical traits was calculated. The genes in the traits‐related gene coexpression modules were subject to functional annotation and pathway enrichment analysis using ClusterProfiler. A total of 73 gene modules were identified, of which, 12 modules were found with high connectivity with clinical traits. Five modules were found with enriched OA‐associated genes. Moreover, 310 OA‐associated genes were found, and 34 of them were among hub‐genes in each module. Consequently, enrichment results indicated some key metabolic pathways, such as extracellular matrix (ECM)‐receptor interaction (hsa04512), focal adhesion (hsa04510), the phosphatidylinositol 3'‐kinase (PI3K)‐Akt signaling pathway (PI3K‐AKT) (hsa04151), transforming growth factor beta pathway, and Wnt pathway. We intended to identify some core genes, collagen (COL)6A3, COL6A1, ITGA11, BAMBI, and HCK, which could influence downstream signaling pathways once they were activated. In this study, we identified important genes within key coexpression modules, which associate with a pathological process of subchondral bone in OA. Functional analysis results could provide important information to understand the mechanism of OA.     

Baicalein alleviates osteoarthritis by protecting subchondral bone,inhibiting angiogenesis and synovial proliferation

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.     

Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis

Introduction  

Oestrogen depletion may influence onset and/or progression of osteoarthritis. We investigated in an ovariectomized mouse model the impact of oestrogen loss and oestrogen supplementation on articular cartilage and subchondral bone in tibia and patella, and assessed bone changes in osteoarthritis development.     

Specific inhibition of FAK signaling attenuates subchondral bone deterioration and articular cartilage degeneration during osteoarthritis pathogenesis

Osteoarthritis (OA), a disease of the entire joint, is characterized by abnormal bone remodeling and coalescent degradation of articular cartilage. We have previously found that elevated levels of H-type vessels in subchondral bone correlate with OA and that focal adhesion kinase (FAK) is critical for H-type vessel formation in osteoporosis. However, the potential role of FAK in OA remains unexplored. Here, we demonstrate that the p-FAK level was dramatically elevated in subchondral bone following anterior cruciate ligament transection (ACLT) in rats. Specific inhibition of FAK signaling with Y15 in subchondral bone resulted in the suppression of subchondral bone deterioration and this effect was mediated by H-type vessel-induced ectopic bone formation. Further, articular cartilage degeneration was also alleviated after Y15 treatment. In vitro, the p-FAK level was significantly elevated in mesenchymal stem cells (MSCs) from vehicle-treated ACLT rats as compared to that in MSCs from sham controls and Y15-treated ACLT rats. Elevated p-FAK level in MSCs promoted vascular endothelial growth factor (VEGF) expression, as demonstrated from the high VEGF level in the blood, subchondral bone, and conditioned medium (CM) of MSCs from vehicle-treated ACLT rats. The CM of MSCs from vehicle-treated ACLT rats might promote the angiogenesis of endothelial cells and the catabolic response of chondrocytes through the FAK-growth factor receptor-bound protein 2-mitogen-activated protein kinase-mediated expression of VEGF. The effect of the CM from MSCs of Y15-treated ACLT rats or that treated with a VEGF-neutralizing antibody on vessel formation and the catabolic response was lowered. Thus, the specific inhibition of FAK signaling may be a promising avenue for the prevention or early treatment of OA.     

A micro-computed tomography study of the trabecular bone structure in the femoral head

The goal of this study was to characterize the trabecular microarchitecture of the femoral head using micro-computed tomography (ICT). Femoral head specimens were obtained from subjects following total hip replacement. Cylindrical cores from the specimens were scanned to obtain 3-D images with an isotropic resolution of 26 Im. Bone structural parameters were evaluated on a per millimeter basis: relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th) and separation (Tb.Sp), structure model index (SMI), and connectivity (Conn.D). The ICT data show that the first two millimeters, starting at the joint surface, are characterized by more plate-like trabeculae, and are significantly denser than the underlying trabecular bone. Regional differences in the trabecular architecture reveal that the superior pole has significantly higher BV/TV, Tb.N and Tb.Th values, with lower Tb.Sp compared to the inferior and side poles. Because subchondral bone is essential in the load attenuation of joints, the difference in bone structure between the subchondral and trabecular bone might arise from the different functions each have within joint-forming bones. The denser trabecular structure of the superior pole as compared to the inferior pole can be interpreted as a functional adaptation to higher loading in this area.     

Investigation of elemental content distribution in femoral head slice with osteoporosis by SRXRF microprobe

A synchrotron radiation X-ray fluorescence microprobe analysis technique was used to scan a slice of the femoral head from its periphery to its center, via cartilage, compact, and spongy zones in order to determine the distribution and the way inorganic substances are lost in bone tissue. The sample preparation and experimental apparatus are described in detail. The quantitative computerized tomography of elemental distribution, such as Ca, P, K, Fe, Zn, Sr, and Pb in bone slice tissue, including cartilage, substantial compact, and substantial spongy, is investigated. Combined with the correlation among, P, K, Zn, Sr, and Ca, the route of loss of minerals and the physiological functions of some metal elements in bone are also discussed.