Abstract
Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based on in vitro proliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271+ GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, and in vitro mineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.
Highlights
Adult mesenchymal stromal cells (MSCs) are increasingly being used in bone tissue engineering (BTE) for the reconstruction of clinically challenging bone defects
GPCs demonstrating characteristic plastic adherence and fibroblastic morphology were isolated from gingiva explants in both human platelet lysate (HPL)- and fetal bovine serum (FBS)-media
GPCs in HPL appeared smaller and more spindle-shaped, especially in early passages (Figure 1A), and demonstrated a higher proliferation rate (p < 0.05) (Figure 1B). Both HPL- and FBS-expanded GPCs demonstrated a characteristic MSC phenotype, i.e., > 95% of the cells were positive for CD73, CD90 and CD105, and < 5% of the cells expressed the hematopoietic markers CD34 and CD45; HLA-DR expression was < 8% (Figure 1C)
Summary
Adult mesenchymal stromal cells (MSCs) are increasingly being used in bone tissue engineering (BTE) for the reconstruction of clinically challenging bone defects. MSCs were originally identified in the bone marrow (BMSCs), and these are still the most frequently tested cells in clinical studies (Friedenstein et al, 1968; Pittenger et al, 2019). Considerable donor-related variations in BMSCs, in addition to the morbidity associated with bone marrow harvesting, have prompted the investigation of ‘MSC-like’ cells from other, relatively less invasive, tissue sources (Mohamed-Ahmed et al, 2018; Wilson et al, 2019). Oral tissues, such as dental pulp, mucosa, periodontal ligament (PDL) and gingiva, represent alternative sources of ‘MSC-like’ progenitor cells (Sharpe, 2016). In all of these studies, GPCs were cultured in xenogeneic media
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