Hypoxia Effects in Intervertebral Disc-Derived Stem Cells and Discus Secretomes: An in vitro Study

Romaniyanto,1– 3 Ferdiansyah Mahyudin,4,5 Cita Rosita Sigit Prakoeswa,5,6 Hari Basuki Notobroto,7 Damayanti Tinduh,5,8 Ryan Ausrin,2,3 Fedik Abdul Rantam,9,10 Heri Suroto,4,5 Dwikora Novembri Utomo,4,5 Sholahuddin Rhatomy11,12 1Doctoral Program, Faculty of Medicine, Airlangga University, Surabaya, Indonesia; 2Department of Orthopedic and Traumatology, Prof. Dr. R. Soeharso Orthopedic Hospital, Surakarta, Indonesia; 3Faculty of Medicine, Sebelas Maret University, Surakarta, Indonesia; 4Department of Orthopedic and Traumatology, Dr. Soetomo General Hospital, Surabaya, Indonesia; 5Faculty of Medicine, Airlangga University, Surabaya, Indonesia; 6Department of Dermatology and Venereology, Dr. Soetomo General Hospital, Surabaya, Indonesia; 7Faculty of Public Health, Airlangga University, Surabaya, Indonesia; 8Department of Physical Medicine and Medical Rehabilitation, Dr. Soetomo General Hospital, Surabaya, Indonesia; 9Virology and Immunology Laboratory, Microbiology Department, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia; 10Stem Cell Research and Development Center, Airlangga University, Surabaya, Indonesia; 11Department of Orthopaedics and Traumatology, Dr. Soeradji Tirtonegoro General Hospital, Klaten, Indonesia; 12Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, IndonesiaCorrespondence: Sholahuddin Rhatomy, Department of Orthopaedics and Traumatology, Dr. Soeradji Tirtonegoro General Hospital, Klaten, Indonesia, Email sholahuddin.rhatomy@ugm.ac.idBackground: This study aimed to investigate the effects of hypoxia and normoxia preconditioning in rabbit intervertebral disc-derived stem cells (IVDSCs) and discus-derived conditioned medium (DD-CM)/secretomes in vitro. Transforming growth factor (TGF)-β 1, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) have a role in the proliferation, development, differentiation, and migration of MSCs.Materials and Methods: Intervertebral discs were isolated from rabbit and incubated in normoxia and hypoxia 1%, 3%, and 5% (hypoxia groups) condition. Cell counting was performed after 24 hours of manipulation, then analyzed using one-way ANOVA. TGF-β 1, PDGF, FGF, and VEGF were measured using the ELISA.Results: The highest number of cells was in the hypoxia 3% preconditioning compared to the normoxia, hypoxia 1%, and hypoxia 5% groups. Hypoxia 3% also had the highest increase in PDGF protein production compared to normoxia, with hypoxia 1% and 5%. Among hypoxia groups, the highest secretions of VEGF and FGF proteins were in the hypoxia 3% group. Based on TGF-β 1 protein measurement, the hypoxia 1% group was the highest increase in this protein compared to other groups.Conclusion: Oxygen level in hypoxia preconditioning has a role in the preparation of IVDSCs and secretome preparation in vitro. The highest cell numbers were found in the treatment group with 3% hypoxia, and 3% hypoxia was significantly related to support IVDSCs preparation. Preconditioning with 3% hypoxia had higher PDGF and VEGF levels than other hypoxia groups.Keywords: intervertebral disc-derived stem cells, secretomes, growth factors, hypoxia, normoxia