Journal of Hainan Medical University 2016; 22(21): 150-153 150

Journal of Hainan Medical University

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Bone microstructure characteristics in femoral intertrochanter biopsy tissue of the elderly and their correlation with the activity of osteoblast and osteoclast Wei-Jian Xie1, Chun-Hui Wu1, Yue-Bo Cai1, Nan-Sheng Yu2

1Department of Orthopaedics, Shunde Guizhou Hospital of Province, City, Guangdong Province, 528305, 2Bone Joint Department, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou City, Guangdong Province, 510120, China

ARTICLE INFO ABSTRACT

Article history: Objective: To study the bone microstructure characteristics in femoral intertrochanter biopsy Received tissue of the elderly and their correlation with the activity of osteoblast and osteoclast. Received in revised form Accepted Methods: A total of 36 elderly patients with femoral intertrochanteric fracture caused by Available online low-energy trauma who received artificial hip replacement in our hospital between May 2012 and December 2015 were selected as elderly group, and 28 young patients who were Keywords: diagnosed with pelvic fractures in our hospital during the same period were selected as Femoral intertrochanteric fracture young group. Femoral intertrochanter tissue biopsy was performed during operation, the Bone microstructure histochemical staining was used to determine trabecular number (Tb.N), trabecular width Bone trabecula (Tb.Wi) and trabecular separation (Tb.Sp), the micro-CT scan was used to determine Tb.N, Osteoblast trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th) and connection density (Conn.D), Osteoclast and the enzyme-linked immunosorbent assay was used to determine the levels of osteoblast and osteoclast marker molecules. Results: Tb.N, Tb.Wi, Tb.Th and Conn.D of elderly group were significantly lower than those of young group while Tb.Sep and Tb.Sp were significantly higher than those of youth group; ALP, OCN, OPN, Runx2 and SP7 content in femoral intertrochanter biopsy tissue of elderly group were significantly lower than those of young group, positively correlated with Tb.N, Tb.Wi, Tb.Th and Conn.D, and negatively correlated with Tb.Sep and Tb.Sp; RANK, TRAP, CTSK, CTR, NF- B and NFATc1 content in femoral intertrochanter biopsy tissue of elderly group were significantly毷 higher than those of young group, negatively correlated with Tb.N, Tb.Wi, Tb.Th and Conn.D, and positively correlated with Tb.Sep and Tb.Sp. Conclusions: Bone microstructure in femoral intertrochanter biopsy tissue of the elderly is characterized by reduced trabecular number, thinned thickness and increased spacing, and the above bone microstructure characteristics are associated with the decreased osteoblast activity and increased osteoclast activity.

the femoral neck fracture . Osteoporosis is an important factor [1-3] 1. Introduction that increases the occurrence risk of hip fracture in the elderly, the prognosis of hip fracture combined with osteoporosis is poor, and both morbidity and mortality rate are high . Osteoporosis Hip fracture is a common type of fracture in the elderly, it mostly [4,5] is characterized by bone mass loss, bone microstructure damage occurs in elderly people over 65 years old, and the most common and bone brittleness increase, and at present, dual-energy X-ray hip fracture site is femoral intertrochanteric fracture, followed by absorptiometry is mainly clinical used to detect bone mineral density (BMD) and to assess the degree of osteoporosis . However, Corresponding author: Wei-Jian Xie, Surgery Department No. 1, Shunde Guizhou [6,7] Hospital, No. 4, Shishanqian Road, Ronggui Subdistrict, Shunde , Foshan there are many influence factors of BMD test results, the organic City, Guangdong Province, 528305, China. substances decrease while mineral substances increase in the bone Tel: 0757-28385701; 015899559993 Fund project: Medical Science and Technology Key Project of Foshan City No: of elderly patients with osteoporosis, and the BMD test alone cannot 201208276. Wei-Jian Xie et al./ Journal of Hainan Medical University 2016; 22(21): 150-153 151 accurately reflect the change of bone microstructure. At present, 2.4. Imaging examination of bone microstructure the bone microstructure characteristics of femoral intertrochanter characteristics in the elderly are not fully clear. In the following study, the bone microstructure characteristics of femoral intertrochanter in the young Biopsy bone tissue fixed by 75% ethanol was collected, the tissue were used as control to study the bone microstructure characteristics under test was placed with the vertical axis perpendicular to the of femoral intertrochanter in the elderly and analyze their correlation horizontal plane, micro-CT scanner was used for scanning, and the with the activity of osteoblast and osteoclast. parameters were as follows: voltage was 70 kV, current was 114 μA, secretion level was 18 μm, and after the images were obtained, a cylindrical area 6 mm2 in floor space and 4 mm in height was selected to measure trabecular number (Tb.N), trabecular spacing 2. Materials and methods (Tb.Sp), trabecular thickness (Tb.Th) and connection density (Conn. 2.1. Research subjects D).

A total of 36 elderly patients diagnosed with femoral 2.5. Detection of osteoblast and osteoclast marker molecules intertrochanteric fracture in our hospital between May 2012 and December 2015 were selected as elderly group of the study, and the Biopsy bone tissue frozen by liquid nitrogen was collected, added inclusion criteria were as follows: were > 60 years old, were with in PBS buffer and homogenized, and enzyme-linked immunosorbent low-energy trauma, were diagnosed with femoral intertrochanteric assay kits were used to detect alkaline phosphatase (ALP), fracture by imaging examination and received artificial hip osteocalcin (OCN), osteopontin (OPN), Runx2, SP7, nuclear factor- replacement. Another 28 young patients who were diagnosed B (NF- B), nuclear factor of activated T-cells (NFATc1), receptor 毷 毷 with pelvic fractures in our hospital during the same period were activator of NF- B (RANK), tartrate-resistant acid phosphatase 毷 selected as young group of the study, and the inclusion criteria were (TRAP), cathepsin K (CTSK) and calcitonin receptor (CTR) content as follows: were < 35 years old, were with high-energy trauma, in homogenate. were diagnosed with pelvic fractures by imaging examination and received surgical treatment. Elderly group included 22 male cases 2.6. Statistical methods and 14 female cases, they were (68.2±7.9) years old and the BMI was (21.5±2.8) kg/m2; young group included 17 male cases and SPSS22.0 software was used to input and statistically process data, 11 female cases, they were (32.4±5.6) years old and the BMI was measurement data analysis between two groups was performed by t (22.9±3.4) kg/m2. test and P<0.05 indicated statistical significant differences.

2.2. Femoral intertrochanter tissue biopsy methods 3. Results The two groups of patients received femoral intertrochanter tissue biopsy during operation, the tissue biopsy was conducted in the 3.1. Bone microstructure characteristics of femoral femoral intertrochanter area, the equipment was the trephine 1 cm in intertrochanter biopsy tissue of two groups diameter, 3 pieces of cylindrical bone tissues 1cm in diameter and 2 Tb.N and Tb.Wi of elderly group were significantly lower than cm in height were drilled, the attached soft tissue was removed, then those of young group while Tb.Sep was significantly higher than the tissues were washed with normal saline for 2-3 times, one was that of youth group (Table 1) (P<0.05); Tb.N, Tb.Th and Conn.D of fixed in 4% paraformaldehyde and used for histochemical staining, elderly group were significantly lower than those of young group one was fixed in in 75% ethanol and used for micro-CT scan, and while Tb.Sp was significantly higher than that of youth group (Table one was frozen in liquid nitrogen and used for enzyme-linked 2) (P<0.05). immunosorbent assay. Table 1 Histochemical staining parameters of bone microstructure characteristics of 2.3. Histochemical staining of bone microstructure characteristics femoral intertrochanter biopsy tissue of two groups. Groups Case No. Tb.N (/mm) Tb.Wi (μm) Tb.Sep (μm) Biopsy bone tissue fixed by 4% paraformaldehyde was collected, Elderly group 36 1.03±0.15 89.35±10.38 924.55±113.48 embedded in paraffin and then made into thin sections 5 μm in Young group 28 1.67±0.22 125.63±16.58 562.35±76.39 t 7.598 6.647 9.147 thickness and thick sections 9 μm in thickness. Thin sections were P ＜0.05 ＜0.05 ＜0.05 stained by the masson-goldner trichrome staining kits, the sclerotin was in green and the bone marrow was in red; thick sections were 3.2. Osteoblast marker protein expression stained by AgNO3 staining kits, the sclerotin was in black, and then the trabecular number (Tb.N), trabecular width (Tb.Wi) and ALP, OCN and OPN content in femoral intertrochanter biopsy trabecular separation (Tb.Sep) were observed under microscope. tissue of elderly group were significantly lower than those of young group; Runx2 and SP7 content in femoral intertrochanter biopsy tissue of elderly group were significantly lower than those of young Wei-Jian Xie et al./ Journal of Hainan Medical University 2016; 22(21): 150-153 152 group (P<0.05) (Table 3). marrow in serial sections, and then continuous two-dimensional image information was used to derive the three-dimensional 3.3. Osteoclast marker protein expression characteristics of bone microstructure and calculate Tb.N, Tb.Sep, Tb.Wi and other related parameters; micro-CT scan can obtain RANK, TRAP, CTSK, CTR, NF- B and NFATc1 content the femoral intertrochanter form through thin slice scanning and in femoral intertrochanter biopsy tissue毷 of elderly group were three-dimensional reconstruction, and then calculate the Tb.N, significantly higher than those of young group (P<0.05) (Table 4). Tb.Sp, Tb.Th, Conn.D and other related parameters. In the study, comparison of bone microstructure characteristics-related parameters between elderly patients and young patients showed that Tb.N, 4. Discussion Tb.Wi and Tb.Th of elderly group were significantly lower than those of young group while Tb.Sep and Tb.Sp were significantly Femoral intertrochanteric fracture is a common type of fracture higher than those of youth group. Tb.N can directly reflect the in the elderly, and low-energy trauma caused by fall is the main trabecular number, Tb.Wi can directly reflect the trabecular width, cause of femoral intertrochanteric fracture. The elderly are mostly Tb.Th can directly reflect the trabecular thickness, and the more complicated with osteoporosis or osteopenia, and the bone strength the number and the greater the width and thickness, the more dense reduces correspondingly, so they are more prone to fractures after the bone microstructure; Tb.Sep and Tb.Sp reflect the trabecular low-energy trauma. DXA is the gold standard for clinical detection spacing, and the smaller the spacing and the greater the trabecular of BMD and judgment of osteoporosis, and has a certain predictive density, the more dense the bone microstructure. Combined with the value for the occurrence risk of femoral intertrochanteric fracture. analysis results in the study, it can show that the bone microstructure However, BMD test results can only simply reflect the bone mass of the elderly is characterized by reduced number of trabecular bone, in the bone, and have limited effect on the evaluation of bone thinned thickness and increased spacing. microstructure. The amount of bone mass and characteristics of bone Osteoblasts and osteoclasts are the important functional cells that microstructure are the important factors that affect bone strength, mediate bone resorption and bone formation in bone tissue, and the and although DXA testing alone can reflect the amount of bone weakened osteoblast activity and enhanced osteoclast activity can mass, it cannot reflect the characteristics of bone microstructure. cause bone resorption process exceeding bone formation process, Therefore, BMD testing by DXA alone will ignore the effect of bone which results in a decreased bone density. The RANK on the microstructure characteristics on bone strength. osteoclast precursor cell membrane surface is the key molecule that In the study, femoral intertrochanter biopsy was carried out during adjusts the osteoclast differentiation and maturation . RANK [8,9] operation, and the immunohistochemical staining and micro-CT can be combined with RANKL on cell membrane and then activate scan were used to measure and calculate the bone microstructure the NF- B, NFATc1 and other transcription factors, promote characteristics of femoral intertrochanter biopsy tissue. osteoclast毷 precursor cells to differentiate into mature and active Immunohistochemical staining can stain the sclerotin and bone osteoclasts, synthesize and secrete the TRAP, CTSK, CTR and so

Table 2 Imaging parameters of bone microstructure characteristics of femoral intertrochanter biopsy tissue of two groups. Groups Case No. Tb.N (μm) Tb.Sp (μm) Tb.Th (μm) Conn.D (mm3) Elderly group 36 813.57±96.48 1158.35±134.47 83.51±9.58 1.78±0.23 Young group 28 1332.56±173.94 794.56±93.42 145.47±17.57 2.95±0.37 t 7.918 6.782 8.418 7.527 P ＜0.05 ＜0.05 ＜0.05 ＜0.05

Table 3 Osteoblast marker protein expression in femoral intertrochanter biopsy tissue (ng/mL). Secretory proteins Transcription factors Groups Case No. ALP OCN OPN Runx2 SP7 Elderly group 36 8.69±0.95 5.72±0.71 11.25±1.75 2.59±0.42 1.84±0.22 Young group 28 17.57±2.46 13.46±1.86 25.42±4.29 5.83±0.81 3.47±0.46 t 9.389 14.482 16.183 13.289 9.115 P ＜0.05 ＜0.05 ＜0.05 ＜0.05 ＜0.05

Table 4 Osteoclast marker protein expression in femoral intertrochanter biopsy tissue (ng/mL). Groups Case No. RANK TRAP CTSK CTR NF-κB NFATc1 Elderly group 36 11.58±1.49 3.58±0.62 5.52±0.78 2.24±0.36 7.79±0.93 1.29±0.16 Young group 28 5.82±0.71 1.47±0.19 3.28±0.49 1.09±0.15 3.62±0.52 0.58±0.07 t 11.592 16.283 8.584 12.209 10.498 13.427 P ＜0.05 ＜0.05 ＜0.05 ＜0.05 ＜0.05 ＜0.05 Wei-Jian Xie et al./ Journal of Hainan Medical University 2016; 22(21): 150-153 153 on and mediate bone degradation . In the study, analysis of of postoperative peri-prosthetic fractures in octogenarians with hip [10,11] osteoclast marker molecules in femoral intertrochanter biopsy tissue fractures operated with a cemented, polished tapered stem rather than an showed that RANK, TRAP, CTSK, CTR, NF- B and NFATc1 anatomic stem. Acta Orthop 2016; 87(3): 257-261. content in femoral intertrochanter biopsy tissue毷 of elderly group [4] Erhart S, Zegg M, Kralinger F, et al. Fast and easy preoperative were significantly higher than those of young group. Further analysis estimation of cancellous bone mineral density in patients with proximal of the correlation between osteoclast maker molecules and bone microstructure characteristics showed that RANK, TRAP, CTSK, femur fractures. Arch Orthop Trauma Surg 2015; 135(12): 1683-1689. CTR, NF- B and NFATc1 content in biopsy bone tissue were [5] Liu LK, Lee WJ, Chen LY, et al. Association between frailty, negatively 毷correlated with Tb.N, Tb.Wi, Tb.Th and Conn.D, and osteoporosis, falls and hip fractures among community-dwelling people positively correlated with Tb.Sep and Tb.Sp. This means that the aged 50 years and older in Taiwan: Results from I-Lan longitudinal aging osteoclast activity is enhanced in the elderly and it is associated with study. PLoS One 2015; 10(9): e0136968. the changes in bone microstructure of femoral intertrochanter. [6] Baum T, Karampinos DC, Seifert-Klauss V, et al. Use of MR- A variety of marker molecules will be secreted in osteoblast based trabecular bone microstructure analysis at the distal radius for differentiation and maturation process, ALP and OCN are the marker molecules of early differentiation and middle differentiation osteoporosis diagnostics: a study in post-menopausal women with breast respectively, and their secretion significantly increases in the early cancer and treated with aromatase inhibitor. Clin Cases Miner Bone Metab and middle differentiation ; OPN is closely related to the 2016; 13(1): 29-32. [12,13] osteoblast activity, and participates in the bone mineralization and [7] Munemoto M, Kido A, Sakamoto Y, et al. Analysis of trabecular bone bone reconstruction process . In the study, analysis of osteoblast [14] microstructure in osteoporotic femoral heads in human patients: in vivo marker secretory proteins in femoral intertrochanter biopsy tissue study using multidetector row computed tomography. BMC Musculoskelet showed that ALP, OCN and OPN content in femoral intertrochanter 2016; (17): 13. biopsy tissue of elderly group were significantly lower than those of Disord 12 young group. Osteoblast differentiation and maturation are regulated [8] Park SY, Lee SW, Kim HY, et al. Suppression of RANKL-induced by a variety of transcription factors, Runx2 is a specific transcription osteoclast differentiation by cilostazol via SIRT1-induced RANK factor of osteoblasts, and it is the common downstream molecule inhibition. Biochim Biophys Acta 2015; 1852(10 Pt A): 2137-2144. of multiple pro-osteogenesis signaling pathways. In the early [9] Zhang J, Zhu L, Yan P, et al. Effect of bioaggregate on receptor activator osteoblast differentiation, increasingly expressed Runx2 promotes of nuclear factor-kappa b ligand-induced osteoclastogenesis from murine osteoblast maturation through SP7, and the Runx2 and SP7 are also macrophage cell line in vitro. J Endod 2015; 41(8): 1265-1271. regarded as the specific transcription factors of osteoblasts . [15,16] In the study, analysis of osteoblast marker transcription factors in [10]Tian J, Qi W, Zhang Y, et al. Bioaggregate inhibits osteoclast femoral intertrochanter biopsy tissue showed that Runx2 and SP7 differentiation, fusion, and bone resorption in vitro. J Endod 2015; 41(9): content in femoral intertrochanter biopsy tissue of elderly group 1500-1506. were significantly lower than those of young group. Further analysis [11]Guha M, Srinivasan S, Koenigstein A, et al. Enhanced osteoclastogenesis of the correlation between osteoblast maker molecules and bone by mitochondrial retrograde signaling through transcriptional activation microstructure characteristics showed that ALP, OCN, OPN, Runx2 of the cathepsin K gene. Ann N Y Acad Sci 2016; 1364(1): 52-61. and SP7 content in biopsy bone tissue were positively correlated [12]Jain KG, Mohanty S, Ray AR, et al. 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[3] Mukka S, Mellner C, Knutsson B, et al. Substantially higher prevalence