Physeal histological morphology after thermal epiphysiodesis using radiofrequency ablation
© The Author(s) 2016
Received: 26 October 2015
Accepted: 22 September 2016
Published: 5 October 2016
Several treatments have been described for leg length discrepancy. Epiphysiodesis is the most commonly used because of its effectiveness. Thermal epiphysiodesis using radiofrequency ablation (RFA) alters the growth plate morphology without damaging the adjacent articular cartilage; it is a minimally invasive method that has shown excellent results in animal models. This study describes the macro and micro morphology after the procedure.
Materials and methods
Epiphysiodesis using RFA was performed in vivo for 8 min (92–98 °C) at two ablation sites (medial and lateral) in one randomly-selected tibia in eight growing pigs. The contralateral tibia was used as control. After 12 weeks, the pigs were killed and the tibiae harvested. The specimens were studied macroscopically and histology samples were obtained. Physeal morphology, thickness and characteristics were then described.
Macroscopically, the articular cartilage was normal in all the treated tibiae. Microscopically, the physis was detected as a discontinuous line on the treated tibiae while it was continuous in all controls. In the control specimens, the mean thickness of the physis was 625 µm (606–639, SD = 14). All the physeal layers were organized. In the ablated specimens, disorganized layers in a heterogeneous line were observed. Bone bridges were identified at the ablation sites. The central part of the physis looked normal. Next to the bone bridge, the physis was thicker and presented fibrosis. The mean thickness was 820 µm (628–949, SD = 130). No abnormalities in the articular cartilage were observed.
Thermal epiphysiodesis with RFA disrupts the physeal morphology and causes the formation of bone bridges at the ablation sites. This procedure does not damage the adjacent articular cartilage. The damaged tissue, next to the bone bridges, is characterized by disorganization and fibrosis.
KeywordsRadiofrequency ablation Epiphysiodesis Physeal morphology Histology
Materials and methods
One tibia was selected and treated using RFA in eight healthy, skeletally immature, 10-week-old female Danish Landrace pigs, average 38 kg in weight (36.5–40.8). These were followed for 12 weeks. After this period, the animals were killed and both tibiae harvested for analysis. Untreated tibiae served as control specimens
Anesthesia and medication
All the procedures were done under general anesthesia. For premedication the animals received an intramuscular injection of ketaminol (5 mg/kg, S-ketamine, Pfizer, Berlin, Germany) and midazolam (0.5 mg/kg, Hypnomidate, Janssen-Cilag, Beerse, Belgium). Anesthesia was maintained with an intravenous infusion of propofol (5 mg/kg/h, Fresenius Kabi AB, Uppsala, Sweden) and fentanyl (0.025 mg/kg/h, Haldid, Janssen-Cilag). Before the ablation, the animals received one prophylactic intramuscular penicillin dose (1 ml/10 kg, procaine penicillin G 150 000 IU/1 ml), and then repeatedly daily for 3 days. The animals were given intramuscular flunixin (2.2 mg/kg, Finadyne Vet, Schering-Plough, Skovlunde, Denmark) for 3 days after the procedure.
Animals were housed in individual pens at Aarhus University Påskehøjgaard Research Center, Trige, Denmark. They were fed standard Danish diet to which they had free access together with water. Animal-care staff took daily care of the animals.
At the end of the study, under general anesthesia, the animals received a lethal intravenous injection of pentobarbital 0.5 ml/kg.
Growth plate continuity The growth plate is seen as a continuous line . After RFA epiphysiodesis, it is expected that the growth plate will be disrupted at the ablation sites.
Growth plate thickness Along a normal growth plate, no thickness variations are expected to be found. Edema and injury can affect the growth plate volume . Changes in growth plate thickness are expected to be found at the ablation sites.
Growth plate columnar organization The growth plate is a columnar-arranged structure . This arrangement is expected to be affected after RFA epiphysiodesis.
Possibility of distinguishing organized layers A sandwich-like arrangement in layers is described in normal growth plates. This arrangement is affected by injury . RFA epiphysiodesis is expected to disarrange the normal organization of the growth plate.
Presence or absence of bone bridges Bone bridge formation can be caused by growth plate injury or fracture . Bone bridges are expected to be evident after RFA epiphysiodesis.
The data obtained from the treated tibiae were compared to those from the non-treated, which were considered as control.
Non-treated specimens Histological sections from controls showed a continuous and organized growth plate. Layers could be identified and columnar organization was observed (Fig. 5). The mean thickness was 625 µm (606–639, SD = 14). No bone bridges were observed. All the control specimens were classified as normal.
Growth plate thickness The central part of the physis looked normal. Next to the bone bridge, the physis looked thicker (Fig. 6b). The mean thickness of the treated physis was 820 µm (628–949, SD = 130).
Growth plate columnar organization At the ablation sites, it was observed that the normal columnar organization of the growth plate was lost. Torsion and angulation of the columns was found (Fig. 6c).
Possibility of distinguishing organized layers At the ablation sites and next to them, disorganized layers in a heterogeneous line were observed (Fig. 6c).
The mechanism of endochondral bone growth is incompletely understood and continues to be an active and dynamic area of research . To date, the exact post-fractural reactions of the growth plate are poorly understood. Previous studies have indicated that bone bridge formation does not involve endochondral ossification, and also that there is an initial inflammatory response involving infiltration of inflammatory cells at the growth plate injury site . Epiphysiodesis using RFA was reported as successful but histology was not described . Our results are in accordance to previous published studies. In a rabbit model, using percutaneous drilling, Kömür et al. reported, at the treated sites, significant losses of the physis zones in the treated group after the second post-operative week, a normal columnar arrangement at the non-treated physeal regions, and presence of bone bridges . In addition, in a RFA epiphysiodesis study using rabbits, Ghanem et al. reported that treated specimens revealed cellular anarchy, loss of columnar stratification, and loss of the physeal height, while the medial counterpart remained normal . Sgariglia et al. reported that injured physes are characterized by disorganization of the proliferative, pre-hypertropic and hypertrophic zones . Changes in the growth plate can be observed after 7 days of injury. After 14 days they are evident, and after 4 weeks evidence in growth can be observed . Bone bridges observed in our samples may have caused growth arrest; our animals were followed for 12 weeks. Previous histological studies were done in small species (rabbits and rodents), and it is important to emphasize that similar results were observed in our study which used a large species (pig) where the growth plate volume is higher and the bone structure more similar to human . We used H&E and toluidine blue staining which allows an accurate morphological analysis. It has been reported H&E staining is the best for measurements, except when metabolic parameters are studied, which would require special staining [1, 11, 12, 14]. Light microscopy is been reported to be adequate for analyzing growth plate morphology . In conclusion, epiphysiodesis performed using RFA disrupts the growth plate morphology and causes the formation of bone bridges at the ablation sites. The damaged tissue, next to the bone bridges, is characterized by disorganized structures of the physis and presence of fibrosis.
Compliance with ethical standards
Conflict of interest
All the authors declare they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Musumeci G, Castrogiovanni P, Loreto C, Castorina S, Pichler K, Weinberg AM (2013) Post-traumatic caspase-3 expression in the adjacent areas of growth plat injury site: a morphological study. Int J Mol Sci 14:15756–15784. doi:10.3390/ijms140815767 View ArticleGoogle Scholar
- Pichler K, Musumeci G, Vielgut I, Martinelli E, Castrogiovanni P, Loreto C, Weinberg AM (2013) Towards a better understanding of bone bridge formation in the growth plate—an immunohistochemical approach. Connect Tissue Res. 54(6):408–415. doi:10.3109/03008207.2013.828715
- Gottliebsen M, Rahbek O, Poulsen HD, Møller-Madsen B (2013) Similar growth plate morphology in stapling and tension band plating hemiepiphysiodesis: a porcine experimental histomorphometric study. J Orthop Res 31(4):574–579. doi:10.1002/jor.22276 View ArticlePubMedGoogle Scholar
- Kömür B, Coşkun M, Kömür AA, Oral A (2013) Permanent and temporary epiphysiodesis: an experimental study in a rabbit model. Acta Orthop Traumatol Turc 47(1):48–54. doi:10.3944/AOTT.2013.2949 View ArticlePubMedGoogle Scholar
- Ghanem I, El Hage S, Diag M, Saliba E, Khazzaka A, Aftimos G et al (2009) Radiofrequency application to the growth plate in the rabbit: a new potential approach to epiphysiodesis. J Pediatr Orthop 29:629–635View ArticlePubMedGoogle Scholar
- Widmann RF, Amaral TD, Yildiz C, Yang X, Bostrom M (2010) Percutaneous radiofrequency epiphysiodesis in a rabbit model. A pilot study. Clin Orthop Relat Res 468:1943–1948. doi:10.1007/s1.1999-010-1286-8 View ArticlePubMedPubMed CentralGoogle Scholar
- Heisterkamp J, van Hillegersberg R, IJzermans JN (1999) Critical temperature and heating time for coagulation damage: implications for interstitial laser coagulation (ILC) of tumors. Lasers Surg Med 25(3):257–262View ArticlePubMedGoogle Scholar
- Shiguetomi-Medina JM, Rahbek O, Abood AA, Stødkilde-Jørgensen H, Møller-Madsen B (2014) Thermal epiphysiodesis performed with radio frequency in a porcine model. Acta Orthop 85(5):538–542. doi:10.3109/17453674.2014.939014 View ArticlePubMedPubMed CentralGoogle Scholar
- Delgado-Martos MJ, Fernández AT, Canillas F, Quintana-Villamandos BQ, Santos del Riego S, Delgado-Martos E et al (2013) Does the epiphyseal cartilage of the long bones have one or two ossification fronts? Med Hypotheses 81(4):695–700. doi:10.1016/j.mehy.2013.07.029 View ArticlePubMedGoogle Scholar
- Burdan F, Szumilo J, Krorobowicz A, Farooquee R, Patel S, Patel A et al (2009) Morphology and physiology of the epiphyseal growth plate. Folia Histochem Cytobiol 47(1):5–6. doi:10.2478/v10042-009-0007-1 View ArticlePubMedGoogle Scholar
- Marino R (2011) Growth plate biology: new insights. Curr Opin Endocrinol Diabetes Obes 18(1):9–13. doi:10.1097/MED.0b013e3283423df9 View ArticlePubMedGoogle Scholar
- Sgariglia F, Candela ME, Huegel J, Jacenko O, Koyama E, Yamaguchi Y et al (2013) Epiphyseal abnormalities, trabecular bone loss and articular chondrocyte hypertrophy develop in the long bones of postnatal Ext1-deficient mice. Bone 57(1):220–231. doi:10.1016/j.bone.2013.08.012 View ArticlePubMedPubMed CentralGoogle Scholar
- Aerssens J, Boonen S, Lowet G, Dequeker J (1998) Interspecies differences in bone composition, density, and quality: potential implications for in vivo bone research. Endocrinology 139(2):663–670View ArticlePubMedGoogle Scholar
- Yang L, Lawson KA, Teteak CJ, Zou J, Hacquebord J, Patterson D et al (2013) ESE histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates. Dev Biol 380(1):99–101. doi:10.1016/j.ydbio.2013.04.031 View ArticlePubMedGoogle Scholar