The purpose of this study was to characterize the bone quality features of patients receiving long-term BP therapy in the chronic stage of GC treatment. To achieve this, we analyzed the trabecular microarchitecture, bone metabolism, and material strength of bone biopsy samples from the iliac crest of female patients with rheumatoid arthritis (RA) receiving long-term BP therapy for GIO with reference to those of age- and bone mineral density (BMD)-matched non-RA postmenopausal women.
Patients
This study was approved by the Hokkaido University Hospital Institutional Review Board (#012-0023). Written informed consent was obtained from all participants. For this observational study, 10 female Japanese patients with RA receiving GC and BP therapies for longer than 3 years and undergoing spine surgery with autologous iliac bone graft were recruited (BP therapy for GIO [GIOBP] group). All patients fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA [18, 19] and were ambulatory at the time of the study.
The exclusion criteria were as follows: history of metabolic disorder that could affect bone metabolism (such as severe renal impairment defined as a creatinine clearance of less than 30 mL/min), thyroid or parathyroid disease, and malignancy. Ten age- and BMD-matched female patients who underwent spine surgery with an autologous iliac bone graft were recruited (reference group). All eligible participants in the reference group were as follows: 1) ambulatory, 2) without a history of autoimmune, thyroid or parathyroid disease, malignancy, or any other significant medical problems that required long-term treatment, with the exception of hypertension, type 2 diabetes mellitus (T2D), and dyslipidemia (under control with or without treatment), and 3) without a history of osteoporosis medication use.
Demographic data
The following data were collected from all patients: age, height, and weight (from which body mass index [BMI] was calculated); a complete medical history, including age at menopause, parental history of hip fracture, alcohol use, GC use, conventional and biological disease-modifying antirheumatic drug use, smoking, and current medication use. The 10-year probabilities for major osteoporotic and hip fractures were calculated using the World Health Organization Fracture Risk Assessment Tool (FRAX) [20].
Serologic examination, dual-energy X-ray absorptiometry (DXA) testing, and assessment of existing vertebral fractures
Blood sampling was performed preoperatively and included renal function, electrolytes, and bone metabolic markers, including bone-specific alkaline phosphatase (BAP) and tartrate-resistant acid phosphatase 5b (TRACP-5b). BMD (g/cm2) was measured at the spine (L2–L4) and left femoral neck by DXA (Discovery A, Hologic Japan, Inc, Tokyo, Japan). The number and severity of vertebral fractures were evaluated by a semiquantitative technique using lateral plain radiographs of the whole spine[15].
The vertebral fracture severity index, which we defined as an integrated value of semiquantitative grades of vertebral fractures, was calculated for each patient by two orthopedic surgeons[16]. For example, if a patient had two mild vertebral fractures (grade 1), one moderate vertebral fracture (grade 2), and one severe fracture (grade 3), the fracture severity was calculated as 2 × 1 + 1 × 2 + 1 × 3 = 7.
Bone specimen collection
Bone biopsy was performed during the autologous bone graft harvest from the posterior iliac crest. Bone specimens (10 × 10 × 10 mm) were harvested and subjected to radiographic analysis. After collecting three-dimensional structure data by micro-computed tomography (micro-CT), the specimens were divided into two pieces and subjected to mechanical testing and histological observation.
Micro-CT bone structure analysis
The specimens were scanned using micro-CT (R_mCT2; Rigaku, Tokyo, Japan) at a 20-µm isotropic resolution. Trabecular bone parameters, including the volume bone mineral density (vBMD), tissue mineral density (TMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), structural model index (SMI), and connectivity density (Conn.D), were determined using TRI/3D-BON (Ratoc System Engineering Co., Tokyo, Japan) in accordance with the guidelines described by Bouxsein et al. [17].
Micro-indentation testing for evaluation of bone strength
Bone material strength was evaluated using micro-indentation[16, 18]. The specimens for mechanical analysis were embedded in epoxy resin, and 5-mm thick sections were cut. The bone-exposed surfaces of the resin-embedded specimens were polished and mirror-finished. The specimens were set on an indentation test machine equipped with a combination of a universal mechanical testing machine (Model 4411, Instron Corp., Norwood, MA, USA) and a microscope (VH5000, KEYENCE, Japan). The mechanical strength of three points in the cortical bone region of each specimen, the sites of which were confirmed under a microscope, was examined using a spherical indenter with a 500-µm diameter.
An indentation test was carried out under the following conditions: indentation speed of 10 µm/min, maximum indentation depth of 10 µm, and sampling time of 100 ms. The samples were kelp in a most condition during the mechanical testing. After the load-displacement relationship was recorded, the contact stiffness was determined by calculating the slope during loading.
Histology, histochemistry, and immunohistochemistry
The specimens were fixed in paraformaldehyde and decalcified in 5% ethylenediaminetetraacetic acid. Paraffin-embedded sections (5-µm thick) were assessed using hematoxylin and eosin staining. To evaluate osteoblast and osteoclastic activity, paraffin sections were examined for tissue non-specific alkaline phosphatase (TNAP) and tartrate-resistant acid phosphatase (TRAP) activity. To evaluate osteocyte activity, dewaxed paraffin sections were incubated with rabbit polyclonal antibody against dentin matrix protein 1 (DMP-1) (Code No. M176; TaKaRa Bio Inc., Otsu, Japan), followed by incubation with horse radish peroxidase-conjugated anti-rabbit IgG (Chemicon International Inc., Temecula, CA). For visualization of immunoreactions, diaminobenzidine tetrahydrochloride was used as a substrate. To evaluate osteocyte apoptosis, the ratio of empty lacunae was determined in a region of interest measuring 3200 × 2300 µm. All sections were counterstained with methyl green and observed under a light microscope (Eclipse E800, Nikon Instruments Inc., Tokyo, Japan).
A part of each specimen was immersed in a half-strength Karnovsky fixative, dehydrated with ascending concentrations of acetone and embedded in epoxy resin (TAAB, Berkshire, UK). Undecalcified semi-thin sections were stained with toluidine blue and observed under a light microscope (Merck, Darmstadt, Germany). Von Kossa staining was also performed on semi-thin sections to observe bone mineralization on the trabecular bone surface[19].
Statistical analysis
Comparisons of the between-group data were performed using Student’s t-test and Fisher’s exact test, as appropriate. A P-value < 0.05 was considered significant for all comparisons. Data are presented as means ± standard deviation (SD). Linear regression was used to calculate correlations between contact stiffness and other variables using Pearson’s product-moment correlation coefficient, with the significance level set at P < 0.05. All statistical analyses were performed using JMP Pro version 15 (SAS Institute Inc., Cary, NC, USA).