The Effect of Secretome, Xenogenic Bone Marrow-Derived Mesenchymal Stem Cells, Bone Morphogenetic Protein-2, Hydroxyapatite Granule and Mechanical Fixation in Critical-Size Defects of Rat Models

Document Type : RESEARCH PAPER

Authors

1 1 Department of Orthopedic and Traumatology, Faculty of Medicine, Universitas Indonesia-Dr Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia 2 Stem Cell and Tissue Engineering Research Cluster, Indonesia Medical Education and Research Institute, Faculty of Medicine Universitas Indonesia 3 Stem Cell Medical Technology Integrated Service Unit, Dr Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia

2 Department of Orthopedic and Traumatology, Faculty of Medicine, Universitas Indonesia-Dr Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia

3 2 Stem Cell and Tissue Engineering Research Cluster, Indonesia Medical Education and Research Institute, Faculty of Medicine Universitas Indonesia 3 Stem Cell Medical Technology Integrated Service Unit, Dr Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia 4 Department of Histology, Faculty of Medicine Universitas Indonesia

4 Stem Cell and Tissue Engineering Research Cluster, Indonesia Medical Education and Research Institute, Faculty of Medicine Universitas Indonesia

Abstract

Background: Recent studies have shown that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have several drawbacks in treating critical-sized bone defect (CSD). Secretome may offer considerable advantages over living cells in terms of potency, manufacturing and storing easiness, and potential as a ready-to-go osteoinductive agent. However, thus far, there are no studies regarding the efficacy of secretome in bone healing. The objective of this study is to investigate the effect of the secretome in rat models with CSD. Methods: This was an experimental study with post-test only control group design using 60 skeletally mature Sprague Dawley rat which was divided evenly into 5 treatment groups (MSC only, Secretome only, MSC + Secretome, MSC + Secretome + BMP-2, Control group using Normal Saline). We used Bone Marrow derived MSC in this research. The critical-sized bone defect was created by performing osteotomy and defect was treated according to the groups. Rats were sacrificed on 2nd and 4th week and we measured the radiological outcome using Radiographic Union Score for Tibia (RUST) and histomorphometric (callus, osseous, cartilage, fibrous, and void area) evaluation using Image J. Results: There was no difference in the weight of rats between groups before and after the intervention. RUST score in all intervention group is significantly higher than the control group, however, the MSC-only group was not statistically significant higher than the control group. There is no statistically significant difference in RUST Score between intervention groups. Histomorphometric evaluation showed that total callus formation is the widest in the MSC+Secretome+BMP-2 combination group while the osseous area is found highest on the secretome-only group. Conclusion: Secretome, whether used solely or combined with BM-MSC and BMP-2, is a novel, potent bone-healing agent for CSD in rat models. Level of evidence: V

Keywords


1. Nauth A, Schemitsch E, Norris B, Nollin Z, Watson JT. Critical-Size Bone Defects: Is there a consensus for diagnosis and treatment? J Orthop Trauma. 2018 ;32(3):S7-11.
2. Roddy E, DeBaun MR, Daoud-Gray A, Yang YP, Gardner MJ. Treatment of critical-sized bone defects: clinical and tissue
engineering perspectives. Eur J Orthop Surg Traumatol. 2018 ;28(3):351-62.
3. Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: The diamond concept. Injury. 2007 ;38(4):3-6.
4. Linero I, Chaparro O. Paracrine effect of mesenchymal stem cells derived from human adipose tissue in bone regeneration Plos One. 2014 ;10(3):e0119262.
5. Pawitan JA. Prospect of Adipose Tissue Derived Mesenchymal Stem Cells in Regenerative Medicine. Cell & Tissue Transplantation & Therapy 2009 ;2:7-9.
6. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal stem cell secretome: toward cell free therapeutic strategies in regenerative medicine. Int. J Mol. Sci. 2017 ;18(9):1852.
7. Manassero M, Decambron A, Thong BTH, Viateau V, Bensidhoum M, Petite H. Establishment of a segmental femoral critical-size defect model in mice stabilized by plate osteosynthesis. J Vis Exp. 2016 ;10(116): 1-11.
8. Decambron A, Fournet A, Bensidhoum M, Manassero M, Sailhan F, Petite H, et al. Low-dose BMP-2 and SPM dual delivery onto coral scaffold for critical-size bone defect regeneration in sheep. J Orthop Res. 2017 ;35(12):2637-45.
9. Kasman D, Kurniawan A. Histomorphometric analysis of fracture healing using ImageJ software in Sprague-Dawley rat models of fractures with mechanical force to the bone only and to the bone and periosteum. Journal of Physics: Conf. Series 1073. 2018 ;4:042036.
10. Holstein JH, Garcia P, Histing T, et al. Advances in the establishment of defined mouse models for the study of
fracture healing and bone regeneration. J Orthop Trauma 2009 ;23(5):31–38
11. Kilborn SH, Trudel G, Uhthoff H. Review of growth plate closure compared with age at sexual maturity and lifespan in laboratory animals. Contemp Top Lab Anim Sci 2002 ;41(5):21–26.
12. Marcucio RS, Nauth A, Giannoudis PV, et al. Stem cell therapies in orthopaedic trauma. Journal of Orthopaedic Trauma. 2015 ;29(12):S24-S27.
13. Kooistra B.W., Dijkman B.G., Busse J.W., Sprague S., Schemitsch E.H., Bhandari M. The radiographic union scale in tibial fractures: reliability and validity. J Orthop Trauma. 2010 ;24(3):81–6
14. Niemeyer P, Szalay K, Luginbuhl R, Sudkamp NP, Kasten P. Transplantation of human mesenchymal stem cells in a non-autogeneous setting for bone regeneration in a rabbit critical-size defect model. Acta Biomater. 2010 ;6(3):900-8.
15. Perri B, Cooper M, Lauryssen C, Anand N. Adverse swelling associated withuse of rh-BMP-2 in anterior cervical discectomy and fusion: a case study. Spine J. 2007 ;7(2):235.
16. Kawasaki K, Aihara M, Honmo J, Sakurai S, Fujimaki Y, Sakamoto K, et. Effects of recombinant human bone morphogenetic protein-2 on differentiation of cells isolated from human bone, muscle, and skin. Bone. 1998 ;23(3):223-31.