Abstract
Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4–8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC (p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications.
Highlights
Adult mesenchymal stromal cells (MSC) are increasingly being used in bone tissue engineering (BTE) for the reconstruction of clinically challenging bone defects, and to overcome the limitations of existing bone-substitute materials (Shanbhag et al, 2019)
We have recently reported that human platelet lysate (HPL) cultured Gingiva-derived progenitor cells (GPC) and bone marrow MSC (BMSC) demonstrate superior proliferation, osteogenic gene expression and in vitro mineralization vs. corresponding FBSbased cultures (Shanbhag et al, 2020a; Shanbhag et al, 2020b)
We have recently reported that the expressions of several genes associated with self-renewal and osteogenic differentiation were significantly enhanced in xenofree 3D spheroid vs. 2D monolayer cultures of GPC and BMSC, independent of osteogenic induction via media supplements (Shanbhag et al, 2020b)
Summary
Adult mesenchymal stromal cells (MSC) are increasingly being used in bone tissue engineering (BTE) for the reconstruction of clinically challenging bone defects, and to overcome the limitations of existing bone-substitute materials (Shanbhag et al, 2019). In particular, can be harvested with minimal morbidity and contains a subpopulation of multipotent progenitor cells (GPCs), which demonstrate an MSC-like phenotype, immunomodulatory properties, and osteogenic potential both in vitro and in vivo (Fournier et al, 2010; Mitrano et al, 2010; Tomar et al, 2010). Exclusion of animal-derived supplements, e.g., fetal bovine serum (FBS), in ex vivo culture systems is considered important to facilitate clinical translation of cell therapies and is a recommendation by regulatory health authorities (Bieback et al, 2019). Pooled human platelet lysate (HPL) has been identified as the optimal “xeno-free” supplement for MSC culture, with particular benefits for BTE by promoting MSC osteogenic differentiation (Fekete et al, 2012; Shanbhag et al, 2017). We have recently reported that HPL cultured GPC and BMSC demonstrate superior proliferation, osteogenic gene expression and in vitro mineralization vs. corresponding FBSbased cultures (Shanbhag et al, 2020a; Shanbhag et al, 2020b)
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