Percutaneous CO2Treatment Accelerates Bone Generation During Distraction Osteogenesis in Rabbits
Percutaneous CO2Treatment Accelerates Bone Generation During Distraction Osteogenesis in Rabbits
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DOI：
10.1097/corr.0000000000001288
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发表时间：
2020-08-01
影响因子：
4.2
通讯作者：
Niikura, Takahiro
中科院分区：
文献类型：
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作者：
Kumabe, Yohei;Fukui, Tomoaki;Niikura, Takahiro
背景牵引成骨已广泛用于治疗各种结构性骨畸形和缺陷。然而，延长的愈合时间仍然是一个主要问题。人们已经研究了各种方法来缩短治疗周期，包括使用低强度脉冲超声、甲状旁腺激素和骨形态发生蛋白（BMP），但效果有限。我们之前对大鼠的研究报道，经皮应用 CO(2) 可通过促进血管生成、血流和软骨内骨化来加速大鼠骨折修复和骨缺损愈合。这种疗法还可能加速牵张成骨过程中的骨生成，但据我们所知，尚未有研究调查 CO(2) 疗法对牵张成骨的影响。问题/目的 我们的目的是研究经皮 CO(2) 在兔子牵引成骨过程中的作用，就肢体尺寸而言，兔子是最适合作为延长器牵引成骨模型的动物。我们询问：牵引成骨过程中经皮 CO(2) 是否会改变愈合过程中牵引间隙中的 (1) 放射线骨密度？ (2)骨痂参数，包括骨痂骨矿物质含量、体积骨矿物质密度、骨体积分数； (3)新生骨面积、软骨面积及血管生成情况，以及白细胞介素6(IL-6)、BMP-2、BMP-7、缺氧诱导因子(HIF)-1α、血管内皮生长因子(VEGF)的表达情况； (4) 三点弯曲生物力学强度、刚度和能量？方法 根据我们机构伦理委员会批准的研究方案，使用 40 只 24 周龄雌性新西兰白兔。如前所述创建牵引成骨兔胫骨模型。简而言之，将外部延长器应用于右胫骨，并在中轴处进行横向截骨术。通过调整固定器连接截骨残端，使其无间隙。经过 7 天的潜伏期后，继续以每天 1 毫米的距离分散注意力，持续 10 天。从截骨术后第二天开始，CO(2) 组每周使用 CO(2) 吸收增强水凝胶在手术腿上经皮应用 CO(2) 20 分钟（n = 20）。对照组 (n = 20) 进行空气假治疗。在分心期后（n = 10）、分心完成后2周（n = 10）和4周（n = 20）立即对动物实施安乐死。我们对平片进行骨密度量化，以使用图像分析软件评估牵引间隙的巩固情况。使用显微 CT 测量愈伤组织参数以评估愈伤组织微观结构。用番红O/固绿染色对新形成的骨面积和软骨面积进行组织学测量，以评估骨化的进展。我们还使用荧光素标记的异凝集素 B4 对内皮细胞进行免疫组织化学染色，并检查毛细血管密度以评估血管生成。通过实时聚合酶链式反应分析新产生的愈伤组织中的基因表达。通过三点弯曲测试确定生物力学强度、刚度和能量，以评估愈伤组织的机械强度。结果 X 线照片显示，在巩固阶段第 4 周，CO(2) 组分散区域的像素值高于对照组（0.98 +/- 0.11 [95% 置信区间 0.89 至 1.06] 对比 1.19 +/- 0.23 [95% CI 1.05 至 1.34]；p = 0.013）。Micro-CT 表明，骨体积分数CO(2)组高于 第 4 周对照组的结果（5.56 +/- 3.21 % [95% CI 4.32 至 6.12 %] 对比 11.90 +/- 3.33 % [95% CI 9.63 至 14.25 %]；p = 0.035）。任何其他参数（即第 2 周和第 4 周的愈伤组织骨矿物质含量；第 2 周和第 4 周的体积骨矿物质密度；第 2 周的骨体积分数）没有差异。第2周时，CO(2)组兔子的软骨面积比对照组更大（2.09 +/- 1.34 mm(2)[95% CI 1.26 to 2.92 mm(2)] vs 5.10 +/- 3.91 mm(2)[95% CI 2.68 to 7.52 mm(2)]；p = 0.011）。第 4 周时，CO(2) 组比对照组观察到更多新形成的骨（68.31 +/- 16.32 mm(2)[95% CI 58.19 至 78.44 mm(2)] 对比 96.26 +/- 19.37 mm(2)[95% CI 84.25 至 108.26 mm(2)]；p < 0.001）。任何其他参数均无差异（第 0 周和第 4 周的软骨面积；第 0 周和第 2 周的新形成骨面积）。免疫组织化学同凝素 B4 染色显示，CO(2) 组兔子在第 0 周的牵引区域以及第 0 周和第 2 周的周围组织中毛细血管密度高于对照组（第 0 周的牵引区域为 286.54 +/- 61.55 /mm(2)[95% CI 232.58 至 340.49] 对比 410.24 +/- 55.29 /mm(2)[95% CI 361.78 至 458.71]； p < 0.001；第 0 周周围组织 395.09 +/- 68.16/mm(2)[95% CI 335.34 至 454.83] 对比 589.75 +/- 174.42/mm(2)[95% CI 436.86 至 742.64]； p = 0.003；第 2 周时为 271.22 +/- 169.42 /mm(2)[95% CI 122.71 至 419.73] 对比 508.46 +/- 49.06/mm(2)[95% CI 465.45 至 551.47]； p < 0.001 分别）。第2周的分散区域没有差异。CO(2)组第2周的BMP -2、第2周的HIF1-α以及第0周和第2周的VEGF的表达高于对照组（第2周的BMP -2为3.84 +/- 0.83倍[95% CI 3.11至4.58]对比7.32 +/- 1.63倍[95% CI 5.88至8.75]； p < 0.001；第 2 周时的 HIF1-α 为 10.49 +/- 2.93 倍 [95% CI 7.91 至 13.06]，而第 2 周时为 20.74 +/- 11.01 倍 [95% CI 11.09 至 30.40]； p < 0.001；第 0 周时的 VEGF 为 4.80 +/- 1.56 倍 [95% CI 3.43 至 6.18] 对比 11.36 +/- 4.82 倍 [95% CI 7.13 至 15.59]； p < 0.001；第 2 周为 31.52 +/- 8.26 倍 [95% CI 24.27 至 38.76] 对比 51.05 +/- 15.52 倍 [95% CI 37.44 至 64.66]； p = 0.034，分别）。任何其他参数均无差异（第0周和第4周的BMP-2；第0周、第2周和第4周的BMP -7；第0周和第4周的HIF-1α；第0周、第2周和第4周的IL-6；第4周的VEGF）。在生物力学评估中，第 4 周时 CO(2) 组的极限应力和失效能量高于对照组（极限应力为 259.96 +/- 74.33 N [95% CI 167.66 至 352.25] 对比 422.45 +/- 99.32 N [95% CI 299.13 至 545.77]；p < 0.001，失效能量311.32+/- 99.01 Nmm [95% CI 188.37 至 434.25] 对比 954.97 +/- 484.39 Nmm [95% CI 353.51 至 1556.42]； p = 0.003，分别）。刚度没有差异（216.77 +/- 143.39 N/mm [95% CI 38.73 至 394.81] 与 223.68 +/- 122.17 N/mm [95% CI 71.99 至 375.37]；p = 0.92）。结论 经皮应用CO(2)可加速兔胫骨牵张成骨模型中的骨生成。正如先前的研究表明，CO(2)治疗可能通过促进血管生成、血流和软骨内骨化来影响牵张成骨中的骨再生。
Background Distraction osteogenesis has been broadly used to treat various structural bone deformities and defects. However, prolonged healing time remains a major problem. Various approaches including the use of low-intensity pulsed ultrasound, parathyroid hormone, and bone morphogenetic proteins (BMPs) have been studied to shorten the treatment period with limited success. Our previous studies of rats have reported that the transcutaneous application of CO(2)accelerates fracture repair and bone-defect healing in rats by promoting angiogenesis, blood flow, and endochondral ossification. This therapy may also accelerate bone generation during distraction osteogenesis, but, to our knowledge, no study investigating CO(2)therapy on distraction osteogenesis has been reported. Questions/purposes We aimed to investigate the effect of transcutaneous CO(2)during distraction osteogenesis in rabbits, which are the most suitable animal as a distraction osteogenesis model for a lengthener in terms of limb size. We asked: Does transcutaneous CO(2)during distraction osteogenesis alter (1) radiographic bone density in the distraction gap during healing; (2) callus parameters, including callus bone mineral content, volumetric bone mineral density, and bone volume fraction; (3) the newly formed bone area, cartilage area, and angiogenesis, as well as the expression of interleukin-6 (IL-6), BMP-2, BMP-7, hypoxia-inducible factor (HIF) -1 alpha, and vascular endothelial growth factor (VEGF); and (4) three-point bend biomechanical strength, stiffness, and energy? Methods Forty 24-week-old female New Zealand white rabbits were used according to a research protocol approved by our institutional ethical committee. A distraction osteogenesis rabbit tibia model was created as previously described. Briefly, an external lengthener was applied to the right tibia, and a transverse osteotomy was performed at the mid-shaft. The osteotomy stumps were connected by adjusting the fixator to make no gap. After a 7-day latency phase, distraction was continued at 1 mm per day for 10 days. Beginning the day after the osteotomy, a 20-minute transcutaneous application of CO(2)on the operated leg using a CO(2)absorption-enhancing hydrogel was performed five times per week in the CO(2)group (n = 20). Sham treatment with air was administered in the control group (n = 20). Animals were euthanized immediately after the distraction period (n = 10), 2 weeks (n = 10), and 4 weeks (n = 20) after completion of distraction. We performed bone density quantification on the plain radiographs to evaluate consolidation in the distraction gap with image analyzing software. Callus parameters were measured with micro-CT to assess callus microstructure. The newly formed bone area and cartilage area were measured histologically with safranin O/fast green staining to assess the progress of ossification. We also performed immunohistochemical staining of endothelial cells with fluorescein-labeled isolectin B4 and examined capillary density to evaluate angiogenesis. Gene expressions in newly generated callus were analyzed by real-time polymerase chain reaction. Biomechanical strength, stiffness, and energy were determined from a three-point bend test to assess the mechanical strength of the callus. Results Radiographs showed higher pixel values in the distracted area in the CO(2)group than the control group at Week 4 of the consolidation phase (0.98 +/- 0.11 [95% confidence interval 0.89 to 1.06] versus 1.19 +/- 0.23 [95% CI 1.05 to 1.34]; p = 0.013).Micro-CT demonstrated that bone volume fraction in the CO(2)group was higher than that in the control group at Week 4 (5.56 +/- 3.21 % [95% CI 4.32 to 6.12 %] versus 11.90 +/- 3.33 % [95% CI 9.63 to 14.25 %]; p = 0.035). There were no differences in any other parameters (that is, callus bone mineral content at Weeks 2 and 4; volumetric bone mineral density at Weeks 2 and 4; bone volume fraction at Week 2). At Week 2, rabbits in the CO(2)group had a larger cartilage area compared with those in the control group (2.09 +/- 1.34 mm(2)[95% CI 1.26 to 2.92 mm(2)] versus 5.10 +/- 3.91 mm(2)[95% CI 2.68 to 7.52 mm(2)]; p = 0.011). More newly formed bone was observed in the CO(2)group than the control group at Week 4 (68.31 +/- 16.32 mm(2)[95% CI 58.19 to 78.44 mm(2)] versus 96.26 +/- 19.37 mm(2)[95% CI 84.25 to 108.26 mm(2)]; p < 0.001). There were no differences in any other parameters (cartilage area at Weeks 0 and 4; newly formed bone area at Weeks 0 and 2). Immunohistochemical isolectin B4 staining showed greater capillary densities in rabbits in the CO(2)group than the control group in the distraction area at Week 0 and surrounding tissue at Weeks 0 and 2 (distraction area at Week 0, 286.54 +/- 61.55 /mm(2)[95% CI 232.58 to 340.49] versus 410.24 +/- 55.29 /mm(2)[95% CI 361.78 to 458.71]; p < 0.001; surrounding tissue at Week 0 395.09 +/- 68.16/mm(2)[95% CI 335.34 to 454.83] versus 589.75 +/- 174.42/mm(2)[95% CI 436.86 to 742.64]; p = 0.003; at Week 2 271.22 +/- 169.42 /mm(2)[95% CI 122.71 to 419.73] versus 508.46 +/- 49.06/mm(2)[95% CI 465.45 to 551.47]; p < 0.001 respectively). There was no difference in the distraction area at Week 2. The expressions of BMP -2 at Week 2, HIF1-alpha at Week 2 and VEGF at Week 0 and 2 were greater in the CO(2)group than in the control group (BMP -2 at Week 2 3.84 +/- 0.83 fold [95% CI 3.11 to 4.58] versus 7.32 +/- 1.63 fold [95% CI 5.88 to 8.75]; p < 0.001; HIF1-alpha at Week 2, 10.49 +/- 2.93 fold [95% CI 7.91 to 13.06] versus 20.74 +/- 11.01 fold [95% CI 11.09 to 30.40]; p < 0.001; VEGF at Week 0 4.80 +/- 1.56 fold [95% CI 3.43 to 6.18] versus 11.36 +/- 4.82 fold [95% CI 7.13 to 15.59]; p < 0.001; at Week 2 31.52 +/- 8.26 fold [95% CI 24.27 to 38.76] versus 51.05 +/- 15.52 fold [95% CI 37.44 to 64.66]; p = 0.034, respectively). There were no differences in any other parameters (BMP-2 at Week 0 and 4; BMP -7 at Weeks 0, 2 and 4; HIF-1 alpha at Weeks 0 and 4; IL-6 at Weeks 0, 2 and 4; VEGF at Week 4). In the biomechanical assessment, ultimate stress and failure energy were greater in the CO(2)group than in the control group at Week 4 (ultimate stress 259.96 +/- 74.33 N [95% CI 167.66 to 352.25] versus 422.45 +/- 99.32 N [95% CI 299.13 to 545.77]; p < 0.001, failure energy 311.32 +/- 99.01 Nmm [95% CI 188.37 to 434.25] versus 954.97 +/- 484.39 Nmm [95% CI 353.51 to 1556.42]; p = 0.003, respectively). There was no difference in stiffness (216.77 +/- 143.39 N/mm [95% CI 38.73 to 394.81] versus 223.68 +/- 122.17 N/mm [95% CI 71.99 to 375.37]; p = 0.92). Conclusion Transcutaneous application of CO(2)accelerated bone generation in a distraction osteogenesis model of rabbit tibias.As demonstrated in previous studies, CO(2)treatment might affect bone regeneration in distraction osteogenesis by promoting angiogenesis, blood flow, and endochondral ossification.