(T0930-02-06) Co-culture of Pre-programmed Angiogenic and Osteogenic Gingival Stem Cells Enhances Osteogenesis

Purpose: Cleft lip and palatal defects are the second most common, occurring in 1 in 600-800 births annually in the United States. The reconstruction of craniofacial bone is crucial to preserve normal maxillofacial growth. Stem cell- based bone augmentation procedures offer a promising alternative for the regeneration of lost bone. However, inadequate vascularization in stem cell-based tissue engineered graft poses challenges for poor graft integration which results in failure of the engineered graft in the recipient body. Transplanting autologous stem cells for regenerating vascularized bone is a viable option in bone regenerative therapies. Our long-term goal is to regenerate vascularized bone for the repair of large defects. We intend to use gingival mesenchymal stem cells (GMSCs) due to their ease of harvesting and minimal risk of immune-rejection, high proliferation capacity in ex-vivo culture and has the property to serve as an autologous stem cell source. In this in-vitro study, we used a novel approach of co-culturing the angiogenic GMSCs and osteogenic GMSCs to determine whether the combination of angiogenic cells and osteogenic cells derived from GMSCs may lead to enhanced osteogenesis.

Methods: Cryopreserved gingiva derived mesenchymal stem cells were used for the study. GMSCs revised and cultured in growth media at 37C in 5% CO2. For all experiments third or fourth passage cells were used. To demonstrate the multi-differentiation ability of GMSCs, we conducted in-vitro differentiation assays. GMSCs at a density of 0.3 X 10⁶ cell/well three 6 well plates. Cells were treated with their respective mediums for osteogenic, adipogenic, and chondrogenic differentiation for 21 to 28 days. Cells were stained with alizarin red to determine the presence of mineral nodules which is a hallmark for osteogenic differentiation. For examining the adipogenic differentiation, cultures were characterized by Oil Red O staining and counterstained with hematoxylin. To assess chondrogenic differentiation, GMSCs were induced with chondrogenic medium, and the cultures were stained with alcian blue. For all assays, GMSCs in routine growth medium served as control. To determine angiogenic differentiation of GMSCs, they were treated with growth medium supplemented with 50ng/ml vascular endothelial growth factor (VEGF). Cells were programmed for angiogenic differentiation and osteogenic differentiation for one week. After one week of differentiation, angiogenic and osteogenic GMSCs were co-cultured together for a week. Then they were lysed and further analyzed for relative gene expression of an osteogenic marker gene, osteocalcin, by quantitative RT-PCR method. Furthermore, these co-culture’s angiogenic potential was analyzed by capillary formation using a functional tube formation assay.

Results: Our results revealed that GMSCs have multi-differentiation ability, confirming their mesenchymal stem cell property. The stimulation of GMSCs with VEGF and osteogenic medium significantly enhanced the gene expression of osteocalcin, an osteogenic marker gene (P= 0.0229), compared to GMSCs grown in osteogenic medium alone. Furthermore, the Matrigel assay demonstrated the thick capillary-like structures which indicated the functional behavior of the differentiated cells into endothelial lineage.

Conclusion: Our findings support that GMSCs as autologous stem cell source may be utilized successfully for osteogenic and endothelial lineage. This proposes a new option for developing a stem cell based bioengineered vascularized bone for the repair of craniofacial bone defects.

References: Navarro Sanchez ML, Swartz MD, Langlois PH, Canfield MA, Agopian AJ. Epidemiology of Nonsyndromic, Orofacial Clefts in Texas: Differences by Cleft Type and Presence of Additional Defects. Cleft Palate Craniofac J. 2023 Jul;60(7):789-803. doi: 10.1177/10556656221080932. Epub 2022 Feb 28. PMID: 35225696; PMCID: PMC11104489.
Jing Chen, Sangam Kanekar, Imaging of Congenital Craniofacial Anomalies and Syndromes,
Clinics in Perinatology, Volume 49, Issue 3, 2022, Pages 771-790, ISSN 0095-5108, ISBN 9780323848848
Vyas T, Gupta P, Kumar S, Gupta R, Gupta T, Singh HP. Cleft of lip and palate: A review. J Family Med Prim Care. 2020 Jun 30;9(6):2621-2625. doi: 10.4103/jfmpc.jfmpc_472_20. PMID: 32984097; PMCID: PMC7491837.
Kandalam, U., Kawai, T., Ravindran, G., Brockman, R., Romero, J., Munro, M., Ortiz, J., Heidari, A., Thomas, R., Kuriakose, S., Naglieri, C., Ejtemai, S., & Kaltman, S. I. (2021). Predifferentiated Gingival Stem Cell-Induced Bone Regeneration in Rat Alveolar Bone Defect Model. Tissue engineering. Part A, 27(5-6), 424–436.

Acknowledgements: Osteo Science Foundation


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