The article was designed in accordance with Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist [12].
Study Design and Ethical Considerations
The study was a cross-sectional histomorphometry study of marginal- and subarticular osteophytes in the femoral heads obtained from 25 patients with OA and 25 deceased subjects with similar sex and age distribution.
The arthritic femoral heads were obtained from patients with primary hip OA, who underwent total hip replacement surgery at the Department of Orthopaedics, Farsoe Hospital, Denmark. Prior to surgery, clinical assessment of OA based on pain, stiffness, and physical function, Western Ontario and McMaster Universities Arthritis Index (WOMAC, VAS3.1) [13] scheme was fulfilled. The WOMAC questionnaire is based on 24 component item scores, and the scores were normalised to 100 mm using the following correction factors: the sum of pain score × 0.5, the sum of stiffness score × 0.2, and the sum of physical function score × 0.059 [13]. The radiographic classification of OA in the hip joint was evaluated using the Kellgren-Lawrence grading scale, which grades OA between 0 to 4 depending on severity [14].
Macroscopically normal femoral heads from the control group were obtained at autopsy from individuals who had died suddenly from accidents or acute diseases at the Department of Forensic Medicine, Aarhus University, Denmark.
The Ethics Committee of Medical Research in Denmark Region (J. no. 10776) and The Danish Data Protection Agency (J.nr: 2003-41-3447) approved the study. Written and informed consents were obtained from the patients with OA before their hip replacement surgery.
Participant inclusion and exclusion criteria
Patients with Osteoarthritis
Inclusion criteria for the OA patients were the fulfilment of American College of Rheumatology clinical and radiographic criteria for OA [15] and a referral to total hip replacement surgery. Exclusion criteria were known bone metabolic diseases, diabetes mellitus, malignant diseases, secondary OA or other joint diseases.
Control group
Inclusion criteria were newly deceased subjects with macroscopically healthy femoral heads. The participants from the control group were excluded if they had a history of high-energy pelvic trauma, known diagnosis of bone metabolic disease, diabetes mellitus, malignant diseases or other joint diseases were also excluded.
Sample size
This is the first study to quantify subarticular osteophytes by histomorphometry. Therefore, no data is available on relevant populations for which sample size calculations can be made. We used the same study population, which we have used previously for investigating bone turnover in relation to overlying cartilage deterioration [16].
Outcome measures
Processing of tissue
Immediately after the removal, the femoral heads were fixed in 70% ethanol and then processed according to stereological sampling of vertical uniform random sections [17], previously described in detail [16, 18–20]. In brief: the entire femoral heads were rotated around a vertical axis, which was perpendicular to the anatomical top of the femoral head. After choosing a random starting point, the femoral head was sawed using a diamond precision-parallel saw (Exakt Apparatebau, Norderstedt, Germany) into 7-mm-thick parallel slices, which were halved, and alternating left and right half slices were randomly selected for the following microscopic evaluation [17, 18]. Depending on the size of the femoral head, a total of five to seven 7-mm-thick halved parallel slices were collected from both patients with OA and controls. Each of the five to seven 7-mm-thick halved parallel slices were embedded undecalcified in methylmethacrylate and cut into 7-µm-thick histological sections using a Jung model K microtome (R Jung, Heidelberg, Germany) equipped with a tungsten microtome knife. The sections were mounted and stained with Masson-Goldner trichrome [18].
Definition of tissue structures
Marginal osteophytes were defined as bony projections at the peripheral margin of the femoral head (5) (Figure 1). Subarticular osteophytes were defined as areas of bone, which expanded from the normal curvature of the femoral head into the cartilage, and which was not located in relation to the peripheral margin of the femoral head or the fovea capitis [21] (Figure 1). The fovea capitis was excluded in the present study.
Histomorphometry
Data were collected using a light microscope (Nikon Eclipse 80i, Tokyo, Japan) equipped with a motorised specimen stage (Prior Proscan 11 TM, Rockland, MA, USA), a microcator (Heidenhain MT 1201, Traunreut, Germany), and a digital video camera (OlympusDP72, Tokyo, Japan) connected to a PC running the newCAST interactive stereology software (v. 3.4.1.0, Visiopharm, Hørsholm, Denmark). Sampling regions were automatically aligned in newCAST. Test points and test lines were superimposed on the digital images of the tissue sections and viewed on the PC monitor at a total magnification of ×121.64. For each of the 7-mm-thick parallel tissue sections, the profile number of subarticular and marginal osteophytes were counted, and the area and boundary length of the femoral head, subarticular- and marginal osteophytes were estimated.
The total area (Ar) of the femoral head, subarticular- and marginal osteophytes were estimated with point counting using all 7-mm-thick halved parallel tissue section from each individual femoral head. For estimations of the area of the subarticular and marginal osteophytes, a grid with an area per point of 0.49 mm2 was used. For estimations of the area of the femoral head, a grid with an area per point of 2.93 mm2 was used.
![](data:image/png;base64,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)
Where n is the number of sections, is the total number of test points hitting the structure of interest and a(p) is the area per test point [22].
The total boundary length (Bd) of the femoral head, subarticular- and marginal osteophytes from the five to seven 7-mm-thick halved parallel tissue section, were estimated using a line probe with an area per length of 0.297 mm.
![](data:image/png;base64,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)
Where π/2 is a constant used for sine-weighted test-lines, n is the number of sections,
is the total number of intersections between the tissue surface and the sine-weighted test-line grid, and a(l) is the area per test line length of the line-grid superimposed on the tissue section [22].
Statistical methods
Data were analysed using STATA 12 (StataCorp LP, College Station, TX, USA). Normal distribution of the data was investigated with Q-Q plots and histograms. Normally distributed data are presented as mean (95% confidence interval), and differences between the two groups were tested for statistical significance using Student’s t-test. Data, which was not normally distributed, are presented as median (Interquartile range (IQR)), differences between the two groups were tested for statistical significance using the Mann-Whitney U test. Coefficient of variation (CV) was calculated as the standard deviation of the two repeated measurements divided by the subject mean in order to investigate the intraobserver reliability of the measures. The results were considered statistically significant at P < 0.05.