钴铬颗粒刺激成骨细胞前体细胞可能加重假体周围的炎症反应

doi:10.3969/j.issn.2095-4344.1346

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (26): 4101-4108.doi: 10.3969/j.issn.2095-4344.1346

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 下一篇

钴铬颗粒刺激成骨细胞前体细胞可能加重假体周围的炎症反应

姜建浩¹，李朋¹，杜刚强¹，刘宏智¹，王辉¹，张锴¹，杨上游^2，3，杨淑野¹

¹滨州医学院附属医院创伤骨科，山东省滨州市256600；²美国堪萨斯州堪萨斯大学威寄托骨科中心，美国堪萨斯州威寄托市；³美国堪萨斯州威寄托州立大学生物系，美国堪萨斯州威寄托市

通讯作者: 杨淑野，博士，主治医师，山东省内滨州市滨州医学院附属医院创伤骨科，山东省滨州市 256600
作者简介:姜建浩，男，1984年生，山东省海阳市人，汉族，2015年山东大学毕业，博士，主治医师，主要从事人工关节置换假体松动方向的研究。
基金资助:
国家自然科学基金项目(81241061)，项目负责人：张锴；山东省医药卫生科技发展项目(2016WS0023)，项目负责人：杨淑野；滨州医学院科研启动资金(BY2016KYQD19)，项目负责人：杨淑野

Cobalt-chromium particles inducing preosteoblasts may aggravate periprosthetic inflammation

Jiang Jianhao¹, Li Peng¹, Du Gangqiang¹, Liu Hongzhi¹, Wang Hui¹, Zhang Kai¹, Yang Shangyou^{2, 3}, Yang Shuye¹

¹Department of Traumatic Orthopedics, Binzhou Medical University Hospital, Binzhou 256600, Shandong Province, China; ²Department of Orthopedic Surgery, the University of Kansas School of Medicine-Wichita, Wichita, KS, USA; ³Department of Biological Sciences, Wichita State University, Wichita, KS, USA

Contact: Yang Shuye, MD, Attending physician, Department of Traumatic Orthopedics, Binzhou Medical University Hopital, Binzhou 256600, Shandong Province, China
About author:Jiang Jianhao, MD, Attending physician, Department of Traumatic Orthopedics, Binzhou Medical University Hospital, Binzhou 256600, Shandong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81241061 (to ZK); the Medical and Health Technology Development Project of Shandong Province, No. 2016WS0023 (to YSY); the Science Research Startup Foundation of Binzhou Medical University, No. BY2016KYQD19 (to YSY)

摘要/Abstract

摘要：

文题释义：

无菌性假体松动：人工关节置换后，由于骨与假体磨损产生金属磨损颗粒，这些磨损颗粒被巨噬细胞吞噬，但是巨噬细胞不可能将金属颗粒清除，因而产生一系列的炎症刺激反应，导致白细胞介素1、白细胞介素6、白细胞介素12、肿瘤坏死因子、干扰素等表达增高，刺激成纤维细胞增殖，在假体周围形成炎性假膜，随着时间的延长，产生假体的微动。

成骨细胞前体细胞：在不同的微环境下，如不同浓度的钴铬颗粒刺激下，成骨细胞前体细胞分化为成骨细胞的能力下降，而分化为破骨细胞的能力增强。

背景：关节置换已经成为治疗终末期关节炎的最有效措施。然而，无菌性假体松动是关节置换的主要长期并发症。如何减缓假体松动成为研究的热点。

目的：研究在假体松动过程中，不同浓度钴铬颗粒刺激成骨细胞前体细胞的生物学反应。

方法：①体外实验：成骨细胞前体细胞以成骨细胞诱导液培养后，用0.3或1.25 g/L的钴铬颗粒进行刺激；②体内实验：将钛钉置入严重联合免疫缺陷小鼠(n=18)胫骨近端模拟膝关节置换。术后1周，将经过钴铬颗粒刺激的5×10⁵个成骨细胞前体细胞注射入膝关节腔内。稳定对照组(n=6)：小鼠胫骨仅置入钛钉，无钴铬颗粒和细胞处理；松动对照组(n=6)：小鼠置入钛钉前，向胫骨髓腔注入4×10⁴个钴铬颗粒；钴铬颗粒处理组(n=6)：小鼠置入钛钉前，向胫骨髓腔注入4×10⁴个钴铬颗粒，关节腔内注射用钴铬合金颗粒刺激的成骨细胞前体细胞。

结果与结论：①体外实验：随着钴铬颗粒浓度的增加，碱性磷酸酶和成骨基因表达呈下降趋势；②体内实验：与松动组相比，关节腔内注射经过钴铬颗粒刺激的成骨细胞前体细胞导致假体周围炎性假膜的增厚，骨与假体界面剪切力的减少，骨密度和骨体积的降低，抗酒石酸酸性磷酸酶阳性细胞数量的增加；③结果提示钴铬颗粒抑制成骨细胞前体细胞的生长、成熟以及功能的发挥。钴铬颗粒刺激的成骨细胞前体细胞可能加重假体周围的炎症反应，促进破骨细胞的分化。

orcid: 0000-0002-3091-399X (Jiang Jianhao)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 成骨细胞前体细胞, 破骨细胞, 无菌性假体松动, 磨损颗粒, 金属离子, 全关节置换, 微型计算机断层扫描, 拔钉实验

Abstract:

BACKGROUND: Arthroplasty has been the most effective treatment for the end-stage osteoarthritis. However, aseptic loosening is the main long-term complication of arthroplasty. How to ameliorate aseptic loosening is an issue of concern.

OBJECTIVE: To investigate the biological behavior of preosteoblasts induced with cobalt-chromium particles during aseptic loosening process.

METHODS: (1) In vitro experiment: preosteoblasts were cultured in an osteoblast-induction medium and induced with different doses (0.3 or 1.25 g/L) of cobalt-chromium particles. (2) In vivo experiment: titanium screws were implanted to proximal tibia of server combined with immune-deficiency mice to simulate knee joint arthroplasty. Cobalt-chromium particles inducing MC3T3-E1 (5×10⁵) were intra-articularly injected into the implanted knee at 1 week after surgery. Stable group (n=6): screw implanted mice without local particle insertion and cells transfusion; loosening group (n=6): mice were given an intra-articular injection of cobalt-chromium particles (4×10⁴) before screw implantation; cobalt-chromium group: mice were given an intra-articular injection of preosteoblasts induced by cobalt-chromium particles and cobalt-chromium particles (4×10⁴) before screw implantation.

RESULTS AND CONCLUSION: (1) In vitro experiment: with the increasing of cobalt-chromium particles, alkaline phosphatase and osteogenic gene expression was decreasing. (2) In vivo experiment: intra-articular injection of cobalt-chromium particles inducing MC3T3-E1 cells resulted in thicker peri-implant pseudomembrane, reduced the shear strength of bone-implant, decreased bone mineral density and bone volume, and increased the number of positive cells for tartrate-resistant acid phosphatase. (3) These results indicate that cobalt-chromium particles inhibit the growth, maturation and functions of preosteoblastic cells. Cobalt-chromium particles inducing preosteoblasts may aggravate periprosthetic inflammation and promote osteoclastogenesis.

Key words: preosteoblasts, osteoclasts, aseptic loosening, wear debris, metal ion, total arthroplasty, micro-CT, pull-out test

中图分类号:

R453
R363

姜建浩，李朋，杜刚强，刘宏智，王辉，张锴，杨上游，杨淑野. 钴铬颗粒刺激成骨细胞前体细胞可能加重假体周围的炎症反应[J]. 中国组织工程研究, 2019, 23(26): 4101-4108.

Jiang Jianhao, Li Peng, Du Gangqiang, Liu Hongzhi, Wang Hui, Zhang Kai, Yang Shangyou, Yang Shuye. Cobalt-chromium particles inducing preosteoblasts may aggravate periprosthetic inflammation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(26): 4101-4108.

图/表（结果） 6

Functionality of osteoblasts induced with Co-Cr particles by ALP activity

MC3T3-E1 cells induced with Co-Cr particles exhibited significant negative influence on ALP activity in a dose-dependent manner (P < 0.001). Immunohistochemical staining revealed that only sporadic ALP⁺ cells were identified in particle-challenged pre-osteoblastic cells, significantly fewer than those in the controls (P < 0.001), which complemented the results of ALP activity assay (Figure 2).

Gene expression profile changes in MC3T3-E1 cells after Co-Cr particle induction

Real-time PCR examination suggested that the MC3T3-E1 cells with Co-Cr particle (1.25 g/L), resulted in significant elevation of chemokine MCP-1 expression (P < 0.001), simultaneously, groups with low concentrations of particles also show significant difference compared with naive cell group. Proinflammatory cytokine genes such as tumor necrosis factor-α, IL-6 and IL-8 were significantly up-regulated by Co-Cr particles. Among groups, Co-Cr particles at 1.25 g/L resulted in highest provocation on IL-6 gene expression (Figure 3).

Treatment with higher concentrations of the alloy particles (1.25 g/L) p-regulated RANKL gene expression (Figure 3) and down-regulated OPG gene expression (Figure 3), which resulted in much higher RANKL/OPG ratio than controls. NFATc1 gene expression was stimulated by particles in dose-dependent pattern (P < 0.001). Among groups, the Co-Cr particle 1.25 g/L) provoked the most NFATc1 gene expression (P < 0.001) (Figure 3).

Trend of OSX and Runx2 gene expression profiles following particles changes were similar, exhibited low concentration (0.3 g/L) did not show influence on gene expressions of OSX and Runx2, although high dose of particles (1.25 g/L) significantly inhibited OSX and Runx2 gene expression (Figure 3; P < 0.001).

Effect of Co-Cr particles on inflammatory membrane thickness in the mouse joint prosthesis

Little pseudo-membrance formation was noticed only in the hematoxylin-eosin staining peri-implant sections from the stable controls (Figure 4A). Compared to the loosening group, other two groups have significant difference (P < 0.001). It appeared that there was a significantly thicker soft tissue in the particle induction group than in the loosening group (P < 0.001; Figure 4B-D).

Effect of Co-Cr particles on the BMD in the mouse joint prosthesis

The initial micro-CT scan showed that all the metal implants were well placed in position. However, the scans at sacrifice suggested that there were many focal bone resorptions or pit erosions on specimens from Co-Cr particles inducing MC3T3-E1 cell groups compared to the control group. As to the quantification of BMD and BV/TV, the Co-Cr group was significantly different from the other two groups including control group (P < 0.05, P < 0.001). In detail BMD at the peri-implant bone areas among groups indicated a much more severe BMP loss in the challenged-cell transfusion groups, compared to the loosening group (P < 0.001). Meanwhile, the result of BV/TV was similar to the data of BMD (Figure 5).

Effect of Co-Cr particles on the implant stability in the mouse joint prosthesis

When (9.40 ± 0.49) N was the pick force required on the loosening groups, significantly more force was generally needed to pull out pins in stable control group (11.38±

0.65) N, Cr-Co particle-induced MC3T3-E1 cell transfusion group (6.39±0.52) N (P < 0.05) (Figure 6).

Number of osteoclasts in the mouse joint prosthesis treated with Co-Cr particles

TRAP staining was performed to reveal the osteoclasts presence surrounding the pin implantation. The quantity of TRAP⁺ cells were significant difference in the groups compared to the loosening group (Figure 7; P < 0.01), furthermore Co-Cr groups demonstrated a higher numbers of TRAP⁺ cells than the loosening group.

参考文献

[1]Pajarinen J, Lin TH, Nabeshima A, et al. Mesenchymal stem cells in the aseptic loosening of total joint replacements. J Biomed Mater Res A. 2017;105(4): 1195-1207.

[2]Goodman SB, Gibon E, Pajarinen J, et al. Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement. J R Soc Interface. 2014;11(93):20130962.

[3]Lombardi AV Jr, Barrack RL, Berend KR, et al. The Hip Society: algorithmic approach to diagnosis and management of metal-on-metal arthroplasty. J Bone Joint Surg Br. 2012;94(11 Suppl A):14-18.

[4]Marti A. Cobalt-base alloys used in bone surgery. Injury. 2000;31 Suppl 4:18-21.

[5]Jiang Y, Jia T, Gong W, et al. Effects of Ti, PMMA, UHMWPE, and Co-Cr wear particles on differentiation and functions of bone marrow stromal cells. J Biomed Mater Res A. 2013;101(10):2817-2825.

[6]Kadoya Y, Revell PA, Kobayashi A, et al. Wear particulate species and bone loss in failed total joint arthroplasties. Clin Orthop Relat Res. 1997;(340):118-129.

[7]Yang SY, Zhang K, Bai L, et al. Polymethylmethacrylate and titanium alloy particles activate peripheral monocytes during periprosthetic inflammation and osteolysis. J Orthop Res. 2011;29(5):781-786.

[8]Zhao YP, Wei JL, Tian QY, et al. Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling. Sci Rep. 2016;6:20909.

[9]An S, Han F, Hu Y, et al. Curcumin Inhibits Polyethylene- Induced Osteolysis via Repressing NF-κB Signaling Pathway Activation. Cell Physiol Biochem. 2018;50(3): 1100-1112.

[10]Man K, Jiang LH, Foster R, et al. Immunological Responses to Total Hip Arthroplasty. J Funct Biomater. 2017;8(3):E33.

[11]Dyskova T, Gallo J, Kriegova E. The Role of the Chemokine System in Tissue Response to Prosthetic By-products Leading to Periprosthetic Osteolysis and Aseptic Loosening. Front Immunol. 2017;8:1026.

[12]Wang Z, Liu N, Liu K, et al. Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis. Autophagy. 2015;11(12):2358-2369.

[13]Pajarinen J, Nabeshima A, Lin TH, et al. Murine Model of Progressive Orthopedic Wear Particle-Induced Chronic Inflammation and Osteolysis. Tissue Eng Part C Methods. 2017;23(12):1003-1011.

[14]Terkawi MA, Hamasaki M, Takahashi D, et al. Transcriptional profile of human macrophages stimulated by ultra-high molecular weight polyethylene particulate debris of orthopedic implants uncovers a common gene expression signature of rheumatoid arthritis. Acta Biomater. 2018;65:417-425.

[15]Ollivere B, Wimhurst JA, Clark IM, et al. Current concepts in osteolysis. J Bone Joint Surg Br. 2012;94(1):10-15.

[16]Wooley PH, Morren R, Andary J, et al. Inflammatory responses to orthopaedic biomaterials in the murine air pouch. Biomaterials. 2002;23(2):517-526.

[17]Zhang K, Jia TH, McQueen D, et al. Circulating blood monocytes traffic to and participate in the periprosthetic tissue inflammation. Inflamm Res. 2009;58(12):837-844.

[18]Yang SY, Nasser S, Markel DC, et al. Human periprosthetic tissues implanted in severe combined immunodeficient mice respond to gene transfer of a cytokine inhibitor. J Bone Joint Surg Am. 2005;87(5):1088-1097.

[19]Thomas P, Thomsen M. Allergy diagnostics in implant intolerance. Orthopade. 2008;37(2):131-135.

[20]Nakamura-Ota M, Hamanaka R, Yano H, et al. A new murine osteoblastic cell line immortalized with the SV40 large T antigen. Cell Tissue Bank. 2014;15(3):373-380.

[21]Bauer TW. Particles and periimplant bone resorption. Clin Orthop Relat Res. 2002;(405):138-143.

[22]Au A, Ha J, Hernandez M, et al. Nickel and vanadium metal ions induce apoptosis of T-lymphocyte Jurkat cells. J Biomed Mater Res A. 2006;79(3):512-521.

[23]Cadosch D, Sutanto M, Chan E, et al. Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes. J Orthop Res. 2010; 28(3):341-347.

[24]Jiang Y, Jia T, Gong W, et al. Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis. Acta Biomater. 2013;9(7):7564-7572.

[25]Jacobs JJ, Gilbert JL, Urban RM. Corrosion of metal orthopaedic implants. J Bone Joint Surg Am. 1998;80(2): 268-282.

[26]Sunderman FW Jr, Hopfer SM, Swift T, et al. Cobalt, chromium, and nickel concentrations in body fluids of patients with porous-coated knee or hip prostheses. J Orthop Res. 1989;7(3):307-315.

[27]Purdue PE, Koulouvaris P, Potter HG, et al. The cellular and molecular biology of periprosthetic osteolysis. Clin Orthop Relat Res. 2007;454:251-261.

[28]Hirakawa K, Jacobs JJ, Urban R, et al. Mechanisms of failure of total hip replacements: lessons learned from retrieval studies. Clin Orthop Relat Res. 2004;(420):10-17.

[29]Tucci M, Tsao A, Hughes J Jr. Analysis of capsular tissue from patients undergoing primary and revision total hip arthroplasty. Biomed Sci Instrum. 1996;32:119-125.

[30]Chang YS, Kobayashi M, Li ZL, et al. Significance of peak value and duration of the interfacial shear load in evaluation of the bone-implant interface. Clin Biomech (Bristol, Avon). 2003;18(8):773-779.

[31]Goodman S, Ma T, Trindade M, et al. COX-2 selective NSAID decreases bone ingrowth in vivo. J Orthop Res. 2002;20(6):1164-1169.

[32]El-Warrak AO, Olmstead M, Schneider R, et al. An experimental animal model of aseptic loosening of hip prostheses in sheep to study early biochemical changes at the interface membrane. BMC Musculoskelet Disord. 2004;5:7.

[33]Warme BA, Epstein NJ, Trindade MC, et al. Proinflammatory mediator expression in a novel murine model of titanium-particle-induced intramedullary inflammation. J Biomed Mater Res B Appl Biomater. 2004;71(2):360-366.

引言

材料方法

Design

Cytological experiment and randomized controlled animal experiment.

Time and setting

This study was completed at Clinical Laboratory, Binzhou Medical College and Wichita State University in May, 2018.

Materials

MC3T3-E1 cells were provided by ATCC, Manassas, VA, USA.

Eighteen female server combined with immune-deficiency mice, aged 8 to 10 weeks, SPF level, were provided by Shandong Experimental Animal Center, license No. SCXK (Lu) 20140007. Animal experiment was approved by the Ethics Committee of the Binzhou Medical University Hospital, approval number: 2018-003-01.

Related information of Co-Cr particles is shown in Table 1.

Methods

Cell culture and induction

MC3T3-E1 preosteoblasts were cultured in α-MEM with ribonucleosides, deoxyribonucleosides, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate, without ascorbic acid supplemented with 5% FBS, 100 U/mL penicillin and 100 g/L streptomycin at 37% and 5% CO₂ atmosphere. For induction of osteoblast differentiation, MC3T3-E1 cells were cultured for 3 days in α-MEM culture medium supplemented with 5% FBS, 10 mmol/L β-glycerolphosphate, 50 μg/mL L-ascorbicacid, and

100 nmol/L dexamethasone, 100 U/mL penicillin, and

100 g/L streptomycin.

Biomaterial particles

The particulate and ion forms of Co-Cr alloy were used to induce MC3T3-E1 cells. Co-Cr particles were washed 3 times in 70% ethanol and sterile PBS, then confirmed endotoxin-free using a Limulus assay^[7,^16-18].

ALP staining assay

Pre-osteoblastic cells at 6×10⁴ cells per well of a 48-well plate were cultured with Co-Cr particles (0.3 or 1.25 g/L) in osteoblast induction medium for 72 hours. Cell lysates were assayed using the ALP assay kit. Based on the colorimetric reaction with hydrolysis of p-nitrophenyl phosphate, ALP activity was determined by optical density values at 405 nm wave length. The enzyme activity was normalized against each sample’s protein concentration. Briefly, alkaline-dye mixture was prepared to dissolve Fast Violet B capsule and Naphthol AS_MX Alkaline Phosphate in double distilled water. After fixationin citrate buffered acetone for 30 seconds, cells were incubated in alkaline-dye mixture for 30 minutes at room temperature and in Mayer’s Hematoxylin for 10 minutes (counterstain). Digital images of ALP stained sections were captured and analyzed by the Image-Pro Plus software. The quantity of these positive cells were evaluated in six different fields and expressed as ALP positive cells/mm².

Real-time PCR

RT-PCR was performed to examine the gene expression profiles of monocyte chemo-attractant protein-1 (MCP-1), tumor necrosis factor-α, interleukin-6 (IL-6), interleukin-8 (IL-8), receptor activator of nuclear factor kappa-B ligand (RANKL), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), osteoprotegerin, osterix (OSX) and runt-related transcription factor 2 (Runx2) on cells challenged with Co-Cr particles. As we know, they stand for inflammatory response and osteogenesis gene marker. Briefly, RNA from the cell homogenates was extracted by a commercial kit. cDNA was reverse transcribed from 0.5 μg of total RNA in a 40 μL reaction mixture containing 1× PCR buffer, MgCl₂ 5.5 mmol/L, each deoxynucleotide triphosphates 500 μmol/L, RNase inhibitor 0.5 U/μL, random hexamers 2.5 μmol/L, and reverse transcriptase. 1.25 U/μL with use of Veriti 96-well Thermal Cycler at 25 ^oC for 10 minutes, 48 ^oC for 25 minutes, followed by 95 ^oC for 5 minutes. RT-PCR was performed according to the manufacturer’s instructions. Reaction mixtures of 25 μL included 12.5 μL of 2× SYBR Green PCR Master Mix and 0.5μL each of 400 nM target gene primer pairs and 2 μL cDNA. The PCR run in a MicroAmp optical 96-well reaction plates with MicroAmp optical caps for 40 cycles (denaturing at 94 ^oC for 1 minute, annealing at 60 ^oC for 55 seconds, and elongation at 72 ^oC for 60 seconds) in a StepOnePlus Real-Time PCR System. The fluorescent signals were recorded dynamically. The values of the threshold cycle (Ct) at which a substantial increase in reporter-dye signals was first detected were normalized against expression of a housekeeping gene (18S), and the comparative quantification of the target gene expressionsin the biomaterial-challenged samples were calculated according to the formula given in the manufacturer’s manual.

Establishment of the mouse model joint prosthesis

Eighteen female server combined immune-deficiency mice were quarantined in our facility for 2 weeks before experimentation. Titanium screws were 0.8 mm in diameter and 5 mm in length with a flat large top of

1.2 mm diameter (Figure 1). Briefly, after anesthetized with Xylazine and Ketamine, under aseptic condition a medial parapatellar incision of 5 mm was made on left hind limb to expose the proximal tibia condyle, a part of articulate cartilage astern of patellar ligament were removed using a #11 scalpel followed by reaming a intramedullary cavity at least 5 mm deep with a dental drill (diameter: 0.7 mm). The Ti-pin was pushed into the canal in a manner such that the surface of the pin-head was flush with the cartilaginous surface of the tibial plateau and contiguous with the joint surface. The knee was rinsed with sterile PBS containing antibiotics (100 U/mL penicillin + 100 g/L streptomycin), and the wound was sutured in layers. In order to simulate prosthetic wear, 10 μL of a Co-Cr particle suspension (4×10⁴ particles) was pipetted into the tibia canal.

Intra-articular transfusion of particle-induced preosteoblasts

MC3T3-E1 cells (6x10⁴ cells/well) of a 48-well plate were cultured with Co-Cr particles (1.25 g/L) in osteoblast induction medium for 72 hours. Before collecting the cells, the medium was changed three times to remove the particles.

At 24 hours after the Ti-pin implantation surgery, the mice were randomly divided into three groups: (1) stable group: pin implanted mice without local particle insertion and cells transfusion (n=6); (2) loosening group: mice were given an intra-articular injection of 10 μL medium containing Co-Cr particle suspension (4×10⁴ particles) during surgery; (3) Co-Cr group: mice were given an intra-articular injection of 10 μL medium containing 5×10⁵ MC3T3-E1 cells induced by Co-Cr particles (1.25 g/L) and Co-Cr particle suspension (4×10⁴ particles). All animals were sacrificed at 5 weeks after operation, and the knee joints were collected for mechanical (pin-pullout) test and histological evaluation.

Micro-CT scan

The micro-CT scanning was performed on a SCANCO Micro-CT System immediately after the pin implantation and at sacrifice. The parameters of 114 μA current, 70 kW voltages, 200 ms exposure time, 400 projections and

30 μm isotropic voxel size were set for the scans. The image of plain scan and reconstruction of the limbs and its bone mineral density (BMD), bone and total volume (BV and TV) were analyzed by micro-CT evaluation program V6.5-1 software.

Shear strength test

After sacrifice, the mouse limbs with the intact implant were immediately removed by disarticulating the knee joint. The pin-implant and the tibia were prepared by removing all soft tissue, and the limb was cemented into a potting box as reported previously for the pin pullout test, utilized a Bose 3200 ElectroForce^TM load frame which extracting the pin implant at a rate of 1 mm/min. The extracting force was recorded as a function of time using Bose WinTest^® software.

Histological evaluation

The peri-implant proximal tibiae were fixed in formalin, decalcified in 12% EDTA, and embedded in paraffin for routine histology process and hematoxylin-eosin staining. A computerized image analysis system with the Image-Pro+ software was used to examine for evidence of inflammation, bone formation or resorption around pin and the thickness of the soft-tissue membrane at the bone-pin interface. In addition, histochemical tartrate-resistant acid phosphatase (TRAP) staining on paraffin-sectioned prosthetic joints was performed to detect osteoclasts. The positive stains were evaluated on six randomly selected microscopic fields of the each peri-implant tissue section and recorded as integrated optical densities or the counts of positive-stained cells.

Main outcome measures

Different concentrations of Co-Cr particles stimulating the biological response of osteoblast precursor cells.

Statistical analysis

Mean ± SEM express the results. For statistical analysis of data, one-way analysis of variance and t-tests were performed using the SPSS 19.0 software package. Significance was set at P < 0.05.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

讨论

Aseptic loosening has been one of the most common long-term complication following MOM total arthroplasty^[19]. Previous studies have suggested that hypersensitivity and inflammatory responses to metals debris may closely associated with this osteolytic process^[20-23]. However, there is still lack of information on effects of Co-Cr particle on osteoblastic progenitors while most studies focused on other types of cells (macrophages/monocytes, mesenchymal stem cells, T-lymphocytes and osteoclasts) and other metal ions: nickel (II) and vanadium (V) and titanium (IV)^[24-26]. This study may offer a new method to find the underlying mechanism of aseptic loosening.

The model applied in the aseptic loosening research should have characters as follows: 1) Simulating aseptic loosening process; 2) Providing basis process for living model; 3) Guiding how to reduce the aseptic loosening occurrence^[27-29]. Our existing animal models of aseptic loosening in knee prosthesis include large animals such as rabbits, dogs, sheep and horses^[30-32]. The shape and size of their joints are similar to those of human beings and can be studied for a long time. But the disadvantages are high cost, difficult feeding and management, and the inability to carry out large-scale experimental studies, which limits the wide use. So that, how to select a proper animal to be an experimental model becomes a difficulty. At present, rodents are becoming more and more experimental research choices because they are easy to be managed and breed, and are not limited by the number of samples, furthermore have homology with human genes. So mice have been widely used in the research. At present researchers have implanted simulated prosthetic materials into the distal femur to study the mechanism of aseptic loosening^[33]. Ceramic prostheses were also implanted into the proximal tibia for study^[34], Millett et al. had improved the rat model^[35]. But these models are short-term research models, and lack of mechanical stimulation.

Our laboratory has recently established a mouse model of knee implantation with a metal pin in proximal tibia to mimic human weight-bearing knee arthroplasty that can sustain for up to 6 months without signs of loosening. The current study established the pin-implantation model in mice with metal particle and cell introductions to mimic aseptic implant loosening condition, and evaluated using bio-mechanical, micro-CT, and histological approaches. The implanted pin pullout test was performed to evaluate interfacial strength, which also reflects implant stability. It was required averagely 9.40 N to pull out of the pin from surrounding bone for mice in loosening group. Transfusion of stimulative preosteoblasts to the metal particle challenged failing prosthesis, resulting in significantly loosening effects and the average of 6.39 N force was required to dissociate the pin implant in particle challenged cell-introduction groups. The data suggested that the particle challenged preosteoblasts lost their ability of bone formation. The results from µCT scans were consistent with pullout test. The stimulated preosteoblasts can result in bone resorption around the pins, according to the data of BMD and BV/TV, which is confirmed by the quantity of TRAP⁺ cells.

It had been well-known that the generation of wear-debris at the interface played a crucial role in the formation of periprosthetic inflammatory tissue and many of them were engulfed by or in close contact with macrophages, MSCs, osteoblasts, lymphocytes and other inflammatory cells^[6]. Figure 2 showed particles promoted preosteoblasts to upgrade inflammatory gene expressions such as MCP-1, tumor necrosis factor and IL-6 which may explain the thicker pseudo-membranes at bone-implant interface following particle-challenged cell transfusion. The data complement a previous report that orthopedic implant cobalt-alloy particles resulted in greater inflammatory cytokines than titanium and zirconium alloy-based particles on human osteoblasts, fibroblasts and macrophages.

It is known that preosteoblast induced by wear debris resulted in increased osteoclastogenesis and decreased osteoblastogenesis. In the current study, particles inhibited the proliferation of preosteoblasts in vitro at dose-dependent pattern. Runx2 contributes to inducing the preosteoblasts differentiation into immature osteoblasts, as well as promotes expression of several key proteins that maintain osteoblast differentiation and bone matrix production. So, the Runx2 is pivotal to the proliferation of osteoblasts and the bone formation. In this study, the PCR data demonstrated that particle form of metal debris prohibits the Runx2 gene expression at dose-dependent pattern, which was in accordance with IHC results.

It is postulated that the particles impaired bone formation by reducing the quantity of the osteoblastic precursor and retarding osteoblastogenesis. RANKL/OPG ratio plays a critical role in the differentiation of osteoclast from macrophage or osteoclast precursor, which indirectly dictates the quantity of bone resorption and the kinetics of remodeling. From RT-PCR data, in the particle-challenging group, the OPG gene expression was inhibited, which was relative to the RANKL/OPG ratio. The low RANKL/OPG ratio complemented the chemical staining data of significantly more TRAP⁺ cells in the Co-Cr group.

Limitations of this study include the short co-culture time used to challenge the cells with particles. The reason that the experiment in vivo chose the concentrations of Co-Cr particle (1.25 g/L) based on the result of the MTT in preliminary study^[36]. Aseptic loosening is a complicated process which is a cascade biological response through multiple cell pathways. However, this study does not find the mechanism of pre-osteoblasts how to differentiate into osteoclasts.

In summary, Co-Cr particles reduced cell proliferation to some extent. Co-Cr particles participate in the regulation of differentiation and function of preosteoblasts, which in turn will promote osteoclastogenesis and bone resorption. However, preosteoblasts may respond differently to different forms of Co-Cr alloys in the process of aseptic loosening.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

钴铬颗粒刺激成骨细胞前体细胞可能加重假体周围的炎症反应

Cobalt-chromium particles inducing preosteoblasts may aggravate periprosthetic inflammation

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表（结果） 6

参考文献

相关文章 15

引言

材料方法

讨论

文章快阅

延伸阅读

编辑推荐

Metrics

本文评价

[1]	王秋霏, 顾叶, 彭育沁, 薛峰, 巨荣, 朱锋, 王熠军, 耿德春, 徐耀增. 人工假体磨损颗粒作用下Wnt/β-catenin信号通路对成骨细胞的影响[J]. 中国组织工程研究, 2021, 25(24): 3894-3901.
[2]	杨彩会, 刘启成, 董明, 王丽娜, 左美娜, 陆颖, 牛卫东. 丝氨酸/苏氨酸蛋白激酶促进慢性根尖周炎模型小鼠的骨破坏[J]. 中国组织工程研究, 2021, 25(23): 3654-3659.
[3]	霍花, 程余婷, 周倩, 齐昱晗, 伍超, 石前会, 杨童景, 廖健, 洪伟. 种植体表面药物涂层对骨结合的影响[J]. 中国组织工程研究, 2021, 25(22): 3558-3564.
[4]	蒋昇源, 李丹, 姜建浩, 杨上游, 杨淑野. 假体无菌性松动过程中Co2+对成骨前体细胞的生物学反应[J]. 中国组织工程研究, 2021, 25(21): 3292-3299.
[5]	李啸群, 徐凯航, 纪方. 补骨脂异黄酮抑制破骨细胞分化缓解小鼠去卵巢骨质疏松[J]. 中国组织工程研究, 2021, 25(2): 186-190.
[6]	辛鹏飞, 柯梦楠 , 张海涛, 乌日莎娜, 李子祺, 庄至坤, 魏秋实, 何伟. 活血化瘀中药治疗股骨头坏死共同作用机制的网络药理学数据[J]. 中国组织工程研究, 2021, 25(17): 2727-2733.
[7]	张为, 崔帅帅, 周志超, 胡小华, 杨晓红. 微小RNA调控组蛋白去乙酰化酶治疗骨相关疾病的发展现状[J]. 中国组织工程研究, 2021, 25(17): 2767-2774.
[8]	范思奇, 曾平, 农焦, 刘金富, 钱晓芬. 通络生骨胶囊含药血清对破骨细胞及Toll样受体4/核因子κB信号通路的影响[J]. 中国组织工程研究, 2021, 25(14): 2155-2160.
[9]	吴钰坤, 韩杰, 温帅波. 骨折愈合过程中Runx2基因的作用机制[J]. 中国组织工程研究, 2021, 25(14): 2274-2279.
[10]	李冬冬, 廖红兵. miR-214参与骨组织代谢的调控[J]. 中国组织工程研究, 2021, 25(11): 1779-1784.
[11]	李啸群, 徐凯航, 纪方. 补骨脂异黄酮抑制破骨细胞分化缓解小鼠去卵巢骨质疏松[J]. 中国组织工程研究, 2020, 24(在线): 4-.
[12]	许诺, 曹蓁, 李晓杰, 史春. MiR-21可调控牙周炎破骨细胞的增殖[J]. 中国组织工程研究, 2020, 24(8): 1225-1230.
[13]	葛均成, 马金辉, 王佰亮, 岳德波, 孙伟, 王卫国, 郭万首, 李子荣. 股骨头缺血性坏死中双膦酸盐的应用[J]. 中国组织工程研究, 2020, 24(5): 753-759.
[14]	张亚楠, 严霞, 孟增东. Zn、Mg增强羟基磷灰石骨修复材料临床应用与机制：生物活性及成骨诱导的研究进展[J]. 中国组织工程研究, 2020, 24(4): 606-611.
[15]	宋世雷, 陈跃平, 章晓云. PI3K/AKT信号通路调控股骨头坏死的相关机制[J]. 中国组织工程研究, 2020, 24(3): 408-415.