Higher Concentration of Ascorbic Acid as a Sole Induction Factor for Osteogenesis on MC3T3-E1 Cell Model

Sains Malaysiana 51(5)(2022): 1449-1464 http://doi.org/10.17576/jsm-2022-5105-15 

ABSTRACT This research aimed to investigate the potential of ascorbic acid (Asc) to act independently as an osteogenic induction factor in a murine pre-osteoblast (MC3T3-E1) model. MC3T3-E1 cells were seeded in culture wells and hydroxyapatite scaffold for two-dimensional and three-dimensional analyses respectively. Cell morphology, viability, osteoblast differentiation, and mineralisation potentials of MC3T3-E1 cells were compared between induction of standard (50 µg/mL) and doubled (100 µg/mL) Asc concentrations in growth media. Cells with fibroblast-like morphology became confluent earlier on day 6 in the standard group compared to the doubled group on day 9. Cell viability and differentiation potential were significantly increased in the doubled group (p < 0.01). Mineralisation occurred in the doubled group after 15 days of seeding but no mineralisation was seen in the standard group. Findings were similar in 3D analysis whereby mineralized nodules were seen only in the doubled group. The relative expression of collagen 1(α) and osteocalcin genes were increased in the doubled group than the standard group. Doubling Asc concentration in a growth medium to 100 µg/mL can induce viability, differentiation, and mineralisation of MC3T3-E1 cells. Thus, higher concentration of ascorbic acid can potentially be used as the sole induction factor in osteogenic medium.

INTRODUCTION Bone grafting in dentistry is largely applied for implant and periodontal procedures in adults, and alveolar cleft repair in children (Guo et al. 2011; Jimi et al. 2012; Kumar et al. 2013). Autogenous bone is the gold standard because of its excellent immunohistocompatibility and high osteogenic cells content (Roberts et al. 2012). However, the site of harvest presents an additional area of pain and infection risk which are undesirable post-operative morbidities (Kumar et al. 2013). In addition, the volume of bone material which can be harvested may be insufficient, particularly in the paediatric population (Roberts et al. 2012). Therefore, tissue engineering-enhanced graft materials have become a popular alternative to overcome the limitations of autogenous graft (Hammoudeh et al. 2017; Mac Isaac et al. 2012). Bone regeneration via this method is achieved by inducing functional osteoblasts on a three-dimensional scaffold with biological factors (Hayrapetyan et al. 2015). The standard induction factors for osteogenic differentiation comprises a combination of dexamethasone, β- glycerophosphate (β-GP) and L-ascorbic acid (Asc) in the concentration 100 Nm, 10 Mm, and 50 µg/mL, respectively. However, osteogenesis has been shown to occur with the addition of only β-GP and Asc. β-GP acts as a source of inorganic phosphate to activate signalling pathways responsible for osteogenic gene expression (Langenbach & Handschel 2013). This is necessary for bone mineralisation (Vater et al. 2011). Nonetheless, β-GP induces matrix mineralisation at the expense of reduced cell proliferation (Ariffin et al. 2017). At a concentration above 2 mM, it can also lead to dystrophic mineralisation which causes increased false-positive results (Langenbach & Handschel 2013). Therefore, there is a need to determine if β-GP can be eliminated from an osteogenic medium without adverse effects on bone formation.

The role of Asc in osteogenic differentiation is primarily related to collagen secretion into the extracellular matrix (ECM) and its subsequent maturation. Asc stimulates pro-collagen type I messenger ribonucleic acid which is responsible for collagen synthesis and acts as a cofactor for hydroxylases for collagen maturation (Finck et al. 2014). Osteoblast differentiation requires cells to be in contact with the collagen-rich ECM before activation of downstream signalling cascade can occur (Langenbach & Handschel 2013). Specifically, the interaction of bound α2β1 integrin ligands with collagen activates the extracellular related kinase (ERK1/2) in the mitogen activated protein kinase (MAPK) signalling pathway. This leads to phosphorylation of Runx2 (osteoblast master transcriptional regulator) and accumulation of P-ERK in the nucleus which upregulate production of osteoblast marker genes like osteocalcin (OCN) and osterix (OSX) (Aghajanian et al. 2015; Langenbach & Handschel 2013). On the other hand, Asc has not been shown to induce mineralisation of MC3T3-E1 cells on its own. Two MC3T3-E1 cell studies that reported negative findings had utilized concentrations at or below 50 µg/mL Asc (Marsh et al. 2009; Quarles et al. 1992). The effects of using higher Asc concentrations towards osteogenesis is scarcely researched. Human mononuclear cells were used in one study while G292 osteosarcoma cell line was used in another (Fernandes et al. 2017; Hadzir et al. 2014). Both studies were conducted under two-dimensional culture conditions. The use of human primary cells may introduce inconsistencies as the osteoblast phenotype may differ according to location where cells were isolated. Furthermore, the proliferation profile can be affected by donor-related factors such as age. On the other hand, cell proliferation rate of G292 cells may not represent normal bone physiology due to the loss of contact inhibition property that is characteristic of malignant cell lines (Czekanska et al. 2012). In addition, the actual concentration of Asc in the experiment set up was ambiguous in the first study as the authors did not detail the type of growth medium used (Hadzir et al. 2014).

MC3T3-E1 cells are an osteogenic progenitor cell line derived from the calvaria of newborn mice (Kodama et al. 1981). They undergo the sequential processes of osteogenesis (adhesion, proliferation, differentiation, and mineralisation) when cultured in the correct osteogenic medium (Sudo et al. 1983). Unlike other malignant cell lines such as SaOs-2 and MG-63, they also demonstrate replicative senescence which is similar to human cells (Czekanska et al. 2012). As such, MC3T3-E1 cells are good candidates to study in vitro osteogenesis. Hence, the aim of this study was to determine the effects of increased Asc concentrations on the mineralisation of a well-established phenotypically stable MC3T3-E1 pre-osteoblast cell line in the absence of β-GP.

Sains Malaysiana 51(5)(2022): 1449-1464 http://doi.org/10.17576/jsm-2022-5105-15