S. Ramesh, V. Madhuri, Paediatric Orthopaedics, Christian Medical College and Hospital, Vellore, India.
S. Ramesh, F. Zaman, L. Sävendahl, Department of Women's and Children's Health and Paediatric Endocrinology, Karolinska Institutet, Solna, Stockholm, Sweden.
Clin Orthop Relat Res. 2020 Mar;478(3):668-678. doi: 10.1097/CORR.0000000000001056.
Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors.
QUESTIONS/PURPOSES: To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis?
Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference.
The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate's cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001).
In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed.
This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.
有大量证据表明，体外冲击波疗法（rESWT）对骨形成有积极影响。然而，目前尚不清楚 rESWT 是否可以在生长板水平局部发挥作用，刺激线性骨生长。实现这一目标的一种方法是在不依赖循环系统生长因子的情况下刺激生长板中的软骨生成。我们想看看 rESWT 是否会在没有血管和相关系统生长因子的情况下刺激大鼠跖骨生长板。
问题/目的：为了研究 rESWT 对生长板软骨生成的直接影响，我们提出了以下问题：（1）rESWT 是否会刺激离体培养骨的纵向骨生长？（2）rESWT 是否会引起生长板形态的任何变化？（3）rESWT 是否会局部激活生长板软骨生成特异性的蛋白质？
从胎鼠的跖骨中未处理（对照组：n = 15）或接受低剂量 rESWT（500 次冲击，5 Hz，90 mJ；n = 15）、中剂量 rESWT（500 次冲击，5 Hz，120 mJ；n = 14）或高剂量 rESWT（500 次冲击，10 Hz，180 mJ；n = 34）单次应用，并培养 14 天。在第 0、4、7 和 14 天测量骨长度。培养 14 天后，通过组织形态计量学分析评估生长板形态，其中测量肥大细胞大小（>7 µm）和肥大区高度（n = 6 个骨）。使用 ImageJ 软件中的自动阈值方法对特定参与软骨生成的调节蛋白的免疫染色和相应染色进行数字定量（每组 n = 6 个骨）。p 值<0.05 表示差异有统计学意义。
与未处理的对照组相比，高剂量 rESWT 组的骨长度增加（4.46 mm ± 0.75 mm；95%置信区间，3.28-3.71 和对照组：3.50 mm ± 0.38 mm；95%置信区间，4.19-4.72；p = 0.01）。生长板软骨的机制研究表明，与未处理的对照组相比，高剂量 rESWT 增加了增殖性软骨细胞的数量（每骨 1363 ± 393 个免疫阳性细胞和每骨 500 ± 413 个免疫阳性细胞；p = 0.04），并增加了肥大软骨细胞的直径（18 ± 3 µm 和 13 ± 3 µm；p < 0.001）。这伴随着胰岛素样生长因子-1 的激活（每骨 1015 ± 322 个免疫阳性细胞和每骨 270 ± 121 个免疫阳性细胞；p = 0.043）和核因子-kappa beta 信号（每骨 1029 ± 262 个免疫阳性细胞和每骨 350 ± 60 个免疫阳性细胞；p = 0.01）以及抗凋亡蛋白 B 细胞淋巴瘤 2（每骨 718 ± 86 个免疫阳性细胞和每骨 35 ± 11 个免疫阳性细胞；p < 0.001）和 B 细胞淋巴瘤-特大号（每骨 107 ± 7 个免疫阳性细胞和每骨 34 ± 6 个免疫阳性细胞；p < 0.001）的水平升高。
在培养的胎鼠跖骨模型中，rESWT 通过局部诱导软骨生成增加了纵向骨生长。为了验证 rESWT 是否也可以在存在全身循环因子的情况下刺激骨生长，需要进一步的研究。
这项离体大鼠跖骨的临床前概念验证研究表明，高剂量 rESWT 可以刺激培养的胎鼠跖骨的纵向骨生长和生长板软骨生成。在进行任何临床研究之前，必须在未成熟动物中进行确认的体内研究。
