Difference in the three-dimensional microstructure of femoral neck specimens between osteoarthritis and rheumatoid arthritis groups

All the data were not distributed normally. Analyses of variation in the microstructural properties of different regions in the two groups are summarized in Table 1.

The patients with osteoarthritis were older than those with rheumatoid arthritis, but there was no significant difference. The entire cortical bone of the femoral neck in patients with rheumatoid arthritis had higher BV/TV, Co.Th*, Co.N* and Co.CD, lower Co.Sp, Co.DA and CSA than those with osteoarthritis, but there was no significant difference between the two groups. The entire trabecular bone in the rheumatoid arthritis group had higher BV/TV, Tb.N*, Tb.CD, Tb.DA and CSA, lower Tb.Th*, Tb.Sp than that in the osteoarthritis group, but there was no significant difference between the two groups. Properties of the femoral neck were similar to those of the trabecular bone. This decrease in bone volume in the osteoarthritis group resulted from the decreased amount of cortical and trabecular bone tissues as well as a consequent increase in dispersion degree. The osteoarthritis group was also characterized by a loss of the connectivity, an increase in DA for the cortical bone, but a decrease in DA for the cancellous bone and the entire trabecular bone when compared to the rheumatoid arthritis group.

Relations between the microstructural parameters of femoral neck specimens in osteoarthritis and rheumatoid arthritis groups

Among all the parameters, BV/TV had the strong correlation with Th*, Sp, SMI, and N* for the entire femoral head, cortical and trabecular bone in both osteoarthritis and rheumatoid arthritis groups. For all the three regions of rheumatoid arthritis and osteoarthritis patients, we found a BV/TV-related increase in Th* and a BV/TV-related decrease in Sp and SMI. But for the correlation between BV/TV and N*, there was no consistent tendency in the three regions of osteoarthritis and rheumatoid arthritis groups. The significant correlations between Sp and N* were also found in the three regions of osteoarthritis and rheumatoid arthritis groups.

Design

Histology in vitro and comparative observation.

Time and setting

The experiment was performed at the Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Odense University Hospital, University of Southern Denmark between September 2011 and September 2014.

Subjects

Human femur neck samples: human proximal femoral necks including heads were harvested from patients undergoing total hip replacements at the Department of Orthopaedic Surgery and Traumatology, Odense University Hospital. These were 7 female patients with rheumatoid arthritis (average age 67.14 years; range 52–86 years), and 10 female patients with osteoarthritis (average age 68.86 years; range 58–82 years). Prior to experiments, all written documents were obtained from patients who agreed to donate femoral necks/heads, and all written informed consents were obtained from all subjects to offer cadavers for anatomical education and research. Thus, this study included two groups: osteoarthritis and rheumatoid arthritis. All patients and donors were Caucasian. The inclusion criteria: a strict criteria for selecting samples was used, all osteoarthritis and rheumatoid arthritis samples had clinical and radiological diagnosis to confirm the diseases. They were also checked with roentgenographic, biochemical or histological evidence to rule out other bone diseases than osteoarthritis and rheumatoid arthritis. Congenital or acquired dysplasia, gout or avascular necrosis were excluded from the osteoarthritis and rheumatoid arthritis groupS. This study was approved by the Department of Orthopaedic Surgery and Traumatology, Odense University Hospital, and the Ethic Committee of the Region of Southern Denmark (ID: S-VF-20040094).

Methods

Sample preparation

According to total hip replacement procedure, the femur head was sawed off between trachantal minor and major. The femur head samples were stored in sealed plastic bag and kept in -20 ℃ for future preparation. The proximal femurs were cleaned of soft connective tissue with a scalpel. Before the specimens were harvested, the orientation of the trabeculae in the femoral neck was determined from an anterior-posterior contact radiograph. In each X-ray image, the main trabecular direction was identified by the operator and the angle between the tangent line of the inferior cortical bone of the femoral neck and the main trabecular direction was measured. A 10-mm segment of the femoral neck was cut at the base of femoral head and at the base of femoral neck, perpendicular to the main trabecular direction of each individual femur, using Exakt saw (Exakt Apparatebau GmbH & Co. KG, Norderstedt, Germany) (Figure 1).

Micro-CT scanning and three-dimensional microstructural analysis

The specimens were analyzed by cone-beam micro-CT system (vivaCT 40, Scanco Medical AG., Zurich, Switzerland). The entire cortical and cancellous bone was scanned continuously with a tube voltage of 70 kV, tube current of 84 μA. Data from each three-dimensional image consisted of approximately micro-CT slide images (512× 512 pixels) with 16-bit grey levels.

After scanning, the micro-CT image data were transferred to a workstation, and the entire cortical and trabecular region of the femoral head were selected and segmented using an individual density threshold value. The cortical bone was defined by removing the osteophyte at the periosteal surface 

operator. We binarized the image to separate pure bone from background or pores, using a single threshold.

of the cortex and the pores at the endosteal surface of the cortex by the same Based on the three-dimensional reconstructions of the total cortical (Co) and trabecular (Tb) bone, mean bone density (MBD, mg HA/ccm), bone surface (BS, mm²), bone volume (BV, mm³), total volume (TV, mm³), thickness (Th*, mm), number (N*, mm^-1), separation (Sp,mm), were analyzed. MBD, BS, BV, and TV were calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface. BS/BV, BS/TV, BV/TV were calculated. Th*, N*, and Sp of the cortical and trabecular bone were based on direct measurement by a distance transformation method. The structure model index (SMI) is a parameter that quantified the characteristic form of a three-dimensional described structure in terms of the plate-like or rod-like nature of the complete structure. Connectivity density (CD, mm^-3) is a topological parameter that estimate the number of trabecular connections per cubic millimeter. The degree of anisotropy (DA) defines the direction and magnitude of the preferred orientation of trabeculae and uses the ratio between the maximum and minimum radii of the mean intercept length ellipsoid. All the above parameters were computed in three-dimensional without model assumptions required for two-dimensional analysis. Cross sectional area (CSA) was calculated according to two-dimensional reconstruction data, and the value was the mean value of three different cross sections (top, middle and bottom).

Main outcome measures

The entire microstructure of the femoral neck and measurement indicators.

Statistical analysis

All statistical analyses were completed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Data were presented as mean±SD. Normality of the distributions was assessed using the Kolmogorov-Smirnov test with the significance level set at 0.05. Differences of these properties between the two groups were analyzed using independent-samples t test. The data were not normally distributed, so non-parametric tests (Mann-Whitney U test) were performed. Bivariate correlation analysis and linear regression analysis were conducted to assess the association of different properties between the groups. A value of P < 0.05 was considered significant.

This study investigated the bone microarchitectural changes of the femoral neck in patients with osteoarthritis and rheumatoid arthritis. It was an interesting observation that there were no significant differences in the microarchitectural parameters of the entire femoral neck between osteoarthritis and rheumatoid arthritis groups. These results did not support our hypothesis that the microarchitecture was significantly different between osteoarthritis and rheumatoid arthritis patients. Thus, our data might suggest that osteoarthritis and rheumatoid arthritis had a similar trend of global bone microarchitectural degeneration in the femoral neck, despite marked erosion in rheumatoid arthritis, but the local difference could not be eliminated.

We did not find significant differences in bone quality and trabecular microarchitecture between patients with osteoarthritis and rheumatoid arthritis in integrity, cortical and trabecular bone properties of the femoral neck. To our knowledge, this is the first study evaluating the three-dimensional structure of the femoral neck in patients with rheumatoid arthritis using micro-CT.

It is a surprising finding that the global microarchitecture of the cancellous bone in the femoral neck did not differ between osteoarthritis and rheumatoid arthritis patients, despite apparent erosion in rheumatoid arthritis bone. As it is generally believed that rheumatoid arthritis is a major cause of secondary osteoporosis, and thus the deteriorated microarchitecture of cancellous bone should be typically characterized as decreased bone density (volume fraction), transformation of cancellous bone structure into extremely rod-like. However, our data did not support this assumption. The global changes in cancellous bone and cortical bone were similar in the femoral necks of osteoarthritis and rheumatoid arthritis patients, but this did not propose that local changes were similar as well. Given the fact that rheumatoid arthritis-induced erosion particularly close to the joint surface was apparent, there were significant differences in local microarchitecture of bone tissues. Nevertheless, our current data suggest a similar trend of global microarchitectural degeneration in osteoarthritis and rheumatoid arthritis patients.

Femoral neck fracture is one of the most common fractures in osteoporosis patients. Rheumatoid arthritis is a major cause of secondary osteoporosis and is frequently associated with both paraarticular and generalized osteoporosis^[12]. Femoral neck fracture is attributed to the loss of both cortical and trabecular bone mass^[30-33]. Bone mass in osteoarthritis patients is higher than that in normal subjects^[1]. It is demonstrated that the femoral neck in patients with osteoarthritis has increased cancellous bone area, connectivity and trabecular thickness which may all protect the neck against fractures^{[9, 29]}. Although several studies have found that the microstructural changes of the femoral neck not only exist in the trabecular bone but also in the cortical bone which determine that the bone strength of femoral neck play the crucial role in prevalence of femoral neck fracture, these studies did not investigate the integrity structure of femoral neck^{[1, 2, 4, 8-10, 20, 29]}. This study was the first to measure the three-dimensional microstructural parameters of the entire femoral neck instead of partial specimens from some part of the femoral neck. We think that it is of great significance for assessing the effects of changes in the bone properties on femoral neck fracture.

In this study, the microarchitectural parameters measured were not different between the two groups. The bone volume fraction of the aging control was similar to that of the aging tibial cancellous bone. The structure type of the control group was typical rod-like, and was similar to what was reported earlier in human aging tibial cancellous bone^[5]. In general, the decreased bone tissues in the osteoarthritis and rheumatoid arthritis groups were compensated during aging and disease processes, resulting in severe bone loss with aging. According to our data, the entire cortical bone of the femoral neck in patients with rheumatoid arthritis increased by 2.19% in BV/TV, 2.89% in Co.Th*, 2.07% in Co.N* and 26.55% in Co.CD, respectively; declined by 4.16% in Co.Sp, 0.7% in Co.DA compared to osteoarthritis patients. The bone properties of the trabecular bone and the entire femoral neck were similar to those of the cortical bone. The change of BV/TV was associated with change in Co.Th, Co.N, and Co.Sp in our findings which are in line with previous studies^{[1-2, 5-10]}.

It seems our findings are not in agreement with the results of previous studies that there are more bone quality in osteoarthritis patients that osteoporosis patients^{[9, 20, 29]}. We think the discrepancy of our findings can be interpreted by several causes. First, in all almost previous studies, for assessing cortical and trabecular bone structures and their possible regional variability in the femoral neck, the osteoporosis specimen was taken from osteoporotic hip fracture (primary osteoporosis) instead of secondary osteoporosis^{[1-2, 4, 7, 20, 29]}. Rheumatoid arthritis is a major cause of secondary osteoporosis and is frequently associated with both paraarticular and generalized osteoporosis. Rheumatoid arthritis is an autoimmune disorder of unknown etiology characterized by progressive damage of synovial-lined joints and variable extra-articular manifestations. Detailed microarchitecture of the cancellous bone and cortical bone in rheumatoid arthritis patients is still not well known^{[12, 18, 34-35]}. The factors of rheumatoid arthritis-induced osteoporosis are more complex than primary osteoporosis. Generalized bone loss may be influenced by immobility, the inflammatory process and treatments such as steroids, while paraarticular loss is probably due to local release of inflammatory agents such as cytokines from the rheumatoid synovium and articular immobility. Determinants of bone mass in rheumatoid arthritis are multifactorial such as sex and menopausal status, disease duration, disease activity, and reduced mobility and function^[18-36]. Due to this, it is possibly inapposite that the changes of bone microstructure in rheumatoid arthritis-induced secondary osteoporosis are similar to those in primary osteoporosis. Although there are many studies demonstrating that rheumatoid arthritis shows high bone turnover and the same decreased bone mineral density^[12-17], we did not find any studies addressing the difference in three-dimensional bone microstructure between primary osteoporosis and rheumatoid arthritis using micro-CT. Hence, we think if there is another study group of primary osteoporosis as control group, it is very helpful to detect the difference. But it is regretful that it is very difficult to perfectly obtain the entire femoral neck from osteoporotic hip fracture patients due to the operation or fracture damage of the specimen. Secondly, it is well accepted that age-related bone loss is an important factor leading to enhanced bone fragility and fracture risk in the elderly^[37-39]. Age-related changes of trabecular bone include a decrease in BV/TV, Tb.N and Conn.D, an increase in Tb.Sp, a shift from plate-like trabeculae to rod-like structure. With normal aging, this thin cortical zone in the femoral neck becomes substantially thinner^[40-43]. Although rheumatoid arthritis patients possibly have osteoporotic changes of bone microstructure, patients with rheumatoid arthritis have a mean age of 67.1 years, who are younger than those with primary osteoarthritis (an average age of 68.2 years).

There are several limitations needed to be discussed. Firstly, there was a small sample-size in each group. Apparently, if more samples were included in the study, the results were possibly more convincing. Secondly, no mechanical test was performed, since these valuable samples were used for another study to investigate ultrastructure of bone tissues. The relative contributions of cortical and trabecular bone is important to maintain the bone strength at the femoral neck^{[23, 31-32]}. Hence, our study was the first to scan the entire femoral neck once, and calculated the three-dimensional parameters of whole cortical and trabecular part in every specimen. According to our method not applied in previous studies, we could easily assess the global properties of the femoral neck, cortical bone and trabecular bone, and analyze the difference between osteoarthritis and rheumatoid arthritis. The relative importance of cortical and trabecular bone to the femoral neck strength has been reported in studies that have used several experimental methods^[44-47]. All the studies have taken one part of the femoral neck (cortical or trabecular bone) as interesting specimen, which cannot veritably reflect the general change in the femoral neck in human body and produce bias. In human beings, trabecular bone density of the femoral neck declines twice as much as does cortical thickness or cortical bone density^[48]. Moreover, an analytical study found that, at the mid-femoral neck, 50% of the applied load, either during gait or during a sideways fall, was supported by the trabecular bone^[32]. But in our study there was no difference in the tendency of changes in cortical, trabecular and entire femoral neck both in osteoarthritis and rheumatoid arthritis patients. We think both cortical and trabecular bones play the same important role in the ability of femoral neck to sustain stresses produced by falls. If with mechanical test, we possibly get the association between microarchitectrual properties and mechanical properties in osteoarthritis and rheumatoid arthritis, and know which part of femoral neck play its different role in change of bone strength while with tension or torsion by falls.

In conclusion, this study demonstrated that there were no significant differences in the global microarchitectural parameters of the femoral neck between osteoarthritis and rheumatoid arthritis. These results might suggest that osteoarthritis and rheumatoid arthritis have a similar trend of global microarchitectural degeneration in the femoral neck, despite marked erosion in the bone of rheumatoid arthritis and osteophyte formation in the bone of osteoarthritis, but the local difference cannot be eliminated. The bone loss with aging in the osteoarthritis and rheumatoid arthritis was not as serious as that of osteoporosis according to literature reports, suggesting a compensation effect of the diseases that increase 

1 对于骨关节炎和类风湿性关节炎股骨近端骨组织(包括皮质骨与松质骨)的三维微观结构观察的研究国内外均未见报道。
2 文章的目的是对比研究骨性关节炎与类风湿性关节炎间的骨微观结构差异性。
3 实验的创新性在于首次采用高分辨micro-CT技术分析股骨颈部位骨组织的整体、松质骨和皮质骨的微观结构。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

原发性、继发性骨质疏松症-此病是骨代谢失衡造成的，会有过量的骨丢失，通常伴有骨微观结构破坏。类风湿性关节炎往往造成继发性骨质疏松，经常伴有关节周围及全身的骨质疏松。大量研究证实类风湿关节炎有更高的骨转换，与原发骨质疏松症相同的骨密度下降。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程