Sowmya Ramesh1,2,3, Farasat Zaman2, Vrisha Madhuri1,3, Lars Sävendahl2. 1. S. Ramesh, V. Madhuri, Paediatric Orthopaedics, Christian Medical College and Hospital, Vellore, India. 2. S. Ramesh, F. Zaman, L. Sävendahl, Department of Women's and Children's Health and Paediatric Endocrinology, Karolinska Institutet, Solna, Stockholm, Sweden. 3. S. Ramesh, V. Madhuri, Centre for Stem Cell Research, a Unit of InStem Bengaluru, Christian Medical College, Bagayam, Vellore, India.
Abstract
BACKGROUND: 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? METHODS: 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. RESULTS: 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). CONCLUSION: 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. CLINICAL RELEVANCE: 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.
BACKGROUND: 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? METHODS: 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. RESULTS: 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). CONCLUSION: 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. CLINICAL RELEVANCE: 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.