Drilling Combined with Adipose-derived Stem Cells and Bone Morphogenetic Protein-2 to Treat Femoral Head Epiphyseal Necrosis in Juvenile Rabbits

Abstract

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine (BrdU)-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Two-month-old, healthy male rabbits (1.2 to 1.4 kg, n=45) underwent ischemic induction and were randomly divided into five groups (group A: animal model control; group B: drilling; group C: drilling & ADSCs; group D: drilling & BMP-2; and group E: drilling & ADSCs & BMP-2). Magnetic resonance imaging (MRI), X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and 10 weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and 10. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P<0.01). Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P<0.05). In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

Keywords

adipose-derived stem cells / bone morphogenetic protein-2 / bone regeneration / drilling / Perthes’ disease

References

[1] MaisuradzeTG. Complex conservative treatment of legg-calve-perthes disease with dona-glucosamine sulfate (sachet). Georgian Med News (Russian), 2012, 205(205): 58-67

[2] JohanssonT, LindbladM, BladhM, et al.. Incidence of Perthes’ disease in children born between 1973 and 1993. Acta Orthop, 2017, 88(1): 96-100

[3] GueradoE, CasoE. The physiopathology of avascular necrosis of the femoral head: an update. Injury, 2016, 47(6): S16-S26

[4] SalterRB. Legg-Perthes disease: the scientific basis for the methods of treatment and their indications. Clin Orthop Relat Res, 1980, 150(150): 8-11

[5] AruwajoyeOO, PatelMK, AllenMR, et al.. Microcrack density and nanomechanical properties in the subchondral region of the immature piglet femoral head following ischemic osteonecrosis. Bone, 2013, 52(2): 632-639

[6] StanitskiCL. Hip range of motion in Perthes’ disease: comparison of pre-operative and intra-operative values. J Child Orthop, 2007, 1(1): 33-35

[7] MehtaJS, ConybeareME, HinvesBL, et al.. Protein C levels in patients with Legg-Calve-Perthes disease: is it a true deficiency. J Pediatr Orthop, 2006, 26(2): 200-203

[8] NakamuraN, InabaY, MachidaJ, et al.. Rotational open-wedge osteotomy improves treatment outcomes for patients older than eight years with Legg-Calve-Perthes disease in the modified lateral pillar B/C border or C group. International Orthopaedics (SICOT), 2015, 39(7): 1359-1364

[9] YoungML, LittleDG, KimHK. Evidence for using bisphosphonate to treat Legg-Calvé-Perthes disease. Clin Orthop Relat Res, 2012, 470(9): 2462-2475

[10] LittleDG, KimHK. Future biologic treatments for Perthes disease. Orthop Clin North Am, 2011, 42(3): 423-427

[11] MurpheyMD, RobertsCC, BencardinoJT, et al.. ACR appropriateness criteria osteonecrosis of the hip. J Am Coll Radiol, 2016, 13(2): 147-155

[12] KimSS, LeeCW, KimHJ, et al.. Treatment of lateonset Legg-Calve-Perthes disease by Arthrodiastasis. Clin Orthop Surg, 2016, 8(4): 452-457

[13] ElzohairyMM. Short follow-up evaluation of proximal femoral varus osteotomy for treatment of Legg-Calvé-Perthes disease. J Orthop Traumatol, 2016, 17(4): 345-351

[14] IbrahimT, LittleDG. The pathogenesis and treatment of Legg-Calvé-Perthes Disease. JBJS Rev, 2016, 4(7): e4

[15] ShohatN, CopeliovitchL, SmorgickY, et al.. The long-term outcome after varus derotational osteotomy for Legg-Calvé-Perthes disease: a mean follow-up of 42 years. J Bone Joint Surg Am, 2016, 98(15): 1277-1285

[16] KongSY, GongSY, KimHW, et al.. Effects of multiple drilling on the ischemic capital femoral epiphysis of immature piglets. Yonsei Med J, 2011, 52(5): 809-817

[17] HanN, LiZ, CaiZ, et al.. P-glycoprotein overexpression in bone marrow-derived multipotent stromal cells decreases the risk of steroid-induced osteonecrosis in the femoral head. J Cell Mol Med, 2016, 20(11): 2173-2182

[18] RubessaM, PolkoffK, BionazM, et al.. Use of pig as a model for mesenchymal stem cell therapies for bone regeneration. Anim Biotechnol, 2017, 2: 1-13

[19] GuoSC, TaoSC, YinWJ, et al.. Exosomes from human synovial-derived mesenchymal stem cells prevent glucocorticoid-induced osteonecrosis of the femoral head in the rat. Int J Biol Sci, 2016, 12(10): 1262-1272

[20] ZouY, FisherPD, HorstmannJK, et al.. Synergistic local drug delivery in a piglet model of ischemic osteonecrosis. Journal of Pediatric Orthopaedics B, 2015, 24(6): 483-492

[21] YangXF, HeX, HeJ, et al.. High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. J Biomed Sci, 2011, 18(1): 1-9

[22] XiaoC, ZhouH, LiuG, et al.. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration. Biomed Mater, 2011, 6(1): 015013

[23] TachiK, TakamiM, ZhaoB, et al.. Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-?B ligand expression in osteoblasts. Cell Tissue Res, 2010, 342(2): 213-220

[24] BossJH, MisselevichI. Osteonecrosis of the femoral head of laboratory animals: the lessons learned from a comparative study of osteonecrosis in man and experimental animals. Vet Pathol, 2003, 40(4): 345-354

[25] TevesME, SundaresanG, CohenDJ, et al.. Spag17 deficiency results in skeletal malformations and bone abnormalities. PLoS One, 2015, 10(5): e0125936

[26] YuZ, ZhuT, LiC, et al.. Improvement of intertrochanteric bone quality in osteoporotic female rats after injection of polylactic acid-polyglycolic acid copolymer/collagen type I microspheres combined with bone mesenchymal stem cells. Int Orthop, 2012, 36(10): 2163-2171

[27] ZhangP, LiangY, KimH, et al.. Evaluation of a pig femoral head osteonecrosis model. J Orthop Surg Res, 2010, 5(1): 1-7

[28] Martínez-ÁlvarezS, Epeldegui-TorreT, Manso-DíazG, et al.. Experimental induction of Perthes disease in lambs. Rev Esp Cir Ortop Traumatol, 2014, 58(2): 68-77

[29] NormanD, ReisD, ZinmanC, et al.. Vascular deprivation-induced necrosis of the femoral head of the rat. An experimental model of avascular osteonecrosis in the skeletally immature individual or Legg-Perthes disease. Int J Exp Pathol, 1998, 79(3): 173-181

[30] KimHK, SuPH. Development of flattening and apparent fragmentation following ischemic necrosis of the capital femoral epiphysis in a piglet model. J Bone Joint Surg Am, 2002, 84: 1329-1334

[31] AtsumiT, YamanoK, MurakiM, et al.. The blood supply of the lateral epiphyseal arteries in Perthes’ disease. J Bone Joint Surg Br, 2000, 82(3): 392-398

[32] KimHK, Morgan-BagleyS, KostenuikP. RANKL inhibition: a novel strategy to decrease femoral head deformity after ischemic osteonecrosis. J Bone Miner Res, 2006, 21(12): 1946-1954

[33] DahnersLE, HillsgroveDC. The effects of drilling on revascularization and new bone formation in canine femoral heads with avascular necrosis: an initial study. J Orthop Trauma, 1989, 3(4): 309-312

[34] ShiF, ChenQ, LiuW. Different diameters injury of epiphyseal plate impact on the development of growth. Chin J Pediatic Surg (Chinese), 2002, 23: 53-55

[35] HoudekMT, WylesCC, MartinJR, et al.. Stem cell treatment for avascular necrosis of the femoral head: current perspectives. Stem Cells Cloning, 2014, 7: 65-70

[36] TanzerM, BobynJD, KrygierJJ, et al.. Histopathologic retrieval analysis of clinically failed porous tantalum osteonecrosis implants. J Bone Joint Surg Am, 2008, 90(6): 1282-1289

[37] FennemaEM, TchangLAH, YuanH, et al.. Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: a comparative study. J Tissue Eng Regen Med, 2017

[38] HacobianAR, Posa-MarkaryanK, SpergerS, et al.. Improved osteogenic vector for non-viral gene therapy. Eur Cells Mater, 2016, 31(31): 191-204

[39] VanhatupaS, OjansivuM, AutioR, et al.. Bone morphogenetic protein-2 induces donor-dependent osteogenic and adipogenic differentiation in human adipose stem cells. Stem Cells Transl Med, 2015, 4(12): 1391-1402

[40] QingW, Guang-XingC, LinG, et al.. The osteogenic study of tissue engineering bone with BMP2 and BMP7 gene-modified rat adipose-derived stem cell. J Biomed Biotechnol, 2012, 2012: 410879

[41] JainA, KebaishKM, SponsellerPD. Factors associated with use of bone morphogenetic protein during pediatric spinal fusion surgery: an analysis of 4817 patients. J Bone Joint Surg Am, 2013, 95(14): 1265-1270

[42] GressotLV, PatelAJ, HwangSW, et al.. Rh-BMP-2 for L5-S1 arthrodesis in long fusions to the pelvis for neuromuscular spinal deformity in the pediatric age group: analysis of 11 patients. Childs Nerv Syst, 2014, 30(2): 249-255

[43] VandermeerJS, KamiyaN, Aya-ayJ, et al.. Local administration of ibandronate and bone morphogenetic protein-2 after ischemic osteonecrosis of the immature femoral head: a combined therapy that stimulates bone formation and decreases femoral head deformity. J Bone Joint Surg Am, 2011, 93(10): 905-913

[44] KimHK, AruwajoyeO, DuJ, et al.. Local administration of bone morphogenetic protein-2 and bisphosphonate during non-weight-bearing treatment of ischemic osteonecrosis of the femoral head: an experimental investigation in immature pigs. J Bone Joint Surg Am, 2014, 96(18): 1515-1524

[45] AçilY, ZhangX, NitscheT, et al.. Effects of different scaffolds on rat adipose tissue derived stroma cells. J Craniomaxillofac Surg, 2014, 42(6): 825-834