A total of 16 consecutive osteoarthritis patients (7 men and 9 women; mean age 59.8 ± 7.2 years, range 52-78 years) and 35 femoral neck fracture patients (18 men and 17 women; mean age 70.8 ± 12.4 years, range 45-89 years) undergoing primary THA in the posterolateral Moore approach (lateral position) performed by the same team of 2 experienced surgeons.
Standardization was conducted as previously described by Lebherz et al. , wherein the accuracy of DEXA was confirmed by controlling hip rotation. The current studies were approved by the Institutional Ethics Committee of the Renji Hospital, Shanghai JiaoTong University School of Medicine, and written informed consent was obtained from all patients for their participation.
Inclusion criteria were: 1) diagnosis of end-stage osteoarthritis or femoral neck fracture; 2) age >18 years; 3) a surgical candidate for uncemented stems THA (Dorr ≥ 0.75); 4) underwent primary THA with the Smith & Nephew Synergy™ Hip System (Smith & Nephew Advanced Orthopedic Devices, Memphis, TN, USA); and 5) underwent surgery using uncemented stems (Porous Plus HA and a grit blasted) and Reflection™ cup press-fit acetabular components (Smith & Nephew Advanced Orthopedic Devices, Memphis, TN, USA). Exclusion criteria were: 1) symptoms or signs of inflammation and infection, rheumatoid arthritis, or another autoimmune disease; or 2) took non-steroidal anti-inflammatory drugs (NSAIDs) within 1 month prior to surgery. Preoperative pain management was done using other types of drugs. This study was approved by the institutional review board of the Renji Hospital (Shanghai, China), and written informed consent was obtained from each participant.
All patients had prosthetic hip replacements. During the surgical procedure, the soft tissues surrounding the femoral head were displaced, after which the femoral head and neck were extracted from the acetabulum manually in all patients to avoid any metal contamination. Then, the sample was cleaned using 1,000 mL of physiological saline and stored at -70°C for biochemical and radiological examinations.
Human bone marrow stromal cells (BMSC)
Bone marrow aspirates (5 mL) were obtained from the discarded metaphysis region of the femoral head during THA. The bone marrow aspirates were diluted 1:4 with phosphate buffered saline (PBS) and layered on Histopaques (Sigma Aldrich, St. Louis, MO, USA) density gradient. Mononuclear cells were isolated by density gradient centrifugation at 600 g for 30 minutes and washed in PBS. The supernatant (bone marrow aspirate washout) was collected and stored frozen at -70°C for the measurement of BMPs. BMSC collected were then culture-expanded in alpha-modified Eagle’s medium (MEM)/10% fetal bovine serum (FBS) medium. The medium was changed initially at day four and then every other day thereafter until the cultures reached confluence. At day 14, the cell was digested with TrypLE Express (Gibco, Grand Island, NY, USA) and collected by centrifugation at 200 g for ten minutes. Harvested BMSCs were fixed in 2% (w/v) paraformaldehyde (in PBS) for 15 minutes, and then used for the following analysis.
Flow cytometry analysis
Detection of Strol-1+, the cell surface marker, was performed by trained technicians blinded to patient identity using Becton Dickinson FACS Calibur Flow Cytometry System (Becton Dickinson, Beckman Coulter, Brea, CA, USA) equipped with Cell Quest software (Beckman Coulter). BMSC cell suspension was incubated with primary antibody for one hour at 4°C. Unbound antibodies were removed by washing with PBS. The secondary monoclonal antibodies conjugated with allophycocyanin (APC) were used to detect Stro-1+ (BD Pharmingen, San Diego, CA, USA) (1:100 dilution). After incubation, cells were washed and resuspended in 500 L of wash buffer and measured by FACS. The signals corresponding to debris and cell aggregates were first gated out by using the forward light scatter (FSC) and side light scatters (SSC) display. Furthermore, absolute counts of Stro-1+ positive cells in BMSC were determined using BD TruCOUNT Tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. During analysis, the absolute number of Stro-1+ positive cells in cultured BMSC was manually calculated using the following equation: (events of Stro-1+ positive cells/events of beads) * (number of beads per test/test volume).
Enzyme-Linked Immunosorbent Assay (ELISA)
Concentrations of BMP2 and BMP7 in the samples of bone marrow aspirates were determined by commercial ELISA kits (R&D System, Minneapolis, MN, USA), following the manufacturer's instructions. The levels of TRACP5b in the patient’s serum was determined by human tartrate-resistant acid phosphatase 5b (TRACP5b) ELISA kit (Wuhan Saipei Biotechnology Co., LTD., Hubei, China) according to the manufacturer’s instructions. A standard curve was generated and the concentrations (pg/mL; IU/L) of the samples were calculated from the standard curve.
Slides were fixed by Fixative solution for 30 s at room temperature, and then thoroughly rinsed in deionized water pre-warmed to 37°C. The prepared dye solution was added to the dye jar and warmed to 37°C in a water bath. Slides were added to dye jars and incubated 1 h in 37℃ water bath protected from light. Slides were rinsed thoroughly in deionized water then counterstained 2 min in Hematoxylin Solution. Slides were rinsed several minutes in alkaline tap water to blue nuclei. After air drying, the slides were sealed with glycerine gelatin and evaluated microscopically.
Assessment of trace elements
Levels of Ca2+, Mg2+, and Zn2+ in the bone tissue were determined using an atomic absorption spectrophotometer device (Varian AA240FS model; Varian Inc., Belrose, Australia). The measurements were conducted twice for each sample, using light at 2,139 nm wavelength according to flame atomization method.
Measurement of PTH and vitamin D
The measurement of PTH and VD was performed by radio-immune assay (RIA) method with Architect c8000 Clinical Chemistry Analyzer device (Abbott Laboratories. Abbott Park, Illinois, USA).
DEXA analysis of bone mineral density
DEXA scans were performed using a HOLOGIC Discovery W (Hologic Inc., Waltham, MA, USA) scanner at 1 week and at 3, 6, and 12 months. Patients were placed in supine position with the affected leg at 10° internal rotation (patella up) with the foot secured in the Hologic foot positioning device to obtain reproducible rotation and thereby limiting measurement errors, as previously described . The femoral stem component and cortical bone were excluded manually during DEXA analysis. Regions of interest (ROI) for each patient were saved using the Hologic image analysis software system (Hologic, Inc., USA) and used for all subsequent measurements. DEXA precision was assessed for all subjects. BMD (g/cm2) was determined in the proximal femur regions R1 (greater trochanter region) and R7 zones (calcar region) for each patient using the Gruen zone partition method  (Figure 1).
Patients were stratified as younger (<70 years) or older (≥70 years), or according to gender. All data were analyzed using SPSS 18.0 (SPSS, Inc. Chicago, IL, USA) and expressed as mean ± SD. Variables were compared using one-way analysis of variance (ANOVA) with Newman-Keuls post hoc test (normally distributed) or Mann-Whitney U test (non-normally distributed). Correlation analysis was assessed using Spearman’s tests. P values < 0.05 were considered to be statistically significant.