Abstract
BackgroundThree-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration.MethodsPLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology.ResultsOsteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis.ConclusionDespite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications.
Highlights
Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects
Gene expression and cytokine secretion are altered in spheroid BMSC Monolayer BMSC showing characteristic morphology, immunophenotype and multi-lineage differentiation potential were expanded in human platelet lysate (HPL) supplemented growth media (GM) (Additional file 1: Figure 2); passage 2 cells were used to form 3D spheroids as previously described [17] (Fig. 1a)
After 7 days, significant upregulations of genes associated with early osteogenic [bone morphogenetic protein 2 (BMP2), 13.20-fold, p = 0.0001] and adipogenic differentiation [peroxisome proliferator-activated receptor gamma (PPARG), 2.63-fold, p = 0.0028] were observed in 3D versus 2D BMSC; upregulation of chondrogenic differentiation gene SRY-box transcription factor 9 (SOX9) was not statistically significant (1.45-fold, p > 0.05) (Fig. 1b)
Summary
Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. BTE strategies usually involve the use of adult mesenchymal stromal cells (MSC), most frequently derived from the bone marrow (BMSC) and expanded as plasticadherent monolayers [6, 7]. This expansion process can be further enhanced by replacing animal-derived supplements, e.g., fetal bovine serum (FBS), in MSC cultures with humanized or “xeno-free” alternatives such as human platelet lysate (HPL) [8–10]. This step is important to enhance the efficacy of MSC expansion and to facilitate clinical translation of cell therapies according to current regulations [11]. Several studies have demonstrated benefits of spheroid culture for promoting the differentiation [18–20], paracrine function [21] and regeneration potential of MSC [22–25]
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