Xeno-free 3D Culture of Mesenchymal Stromal Cells For Bone Tissue Engineering

Abstract

Clinical translation of innovative regenerative approaches using mesenchymal stromal cells (MSCs) is urgently needed for the treatment of challenging bone defects. The overall aim of this thesis, comprising of one systematic review and four original studies, was to optimize a xeno-free three-dimensional (3D) culture system of MSCs, as a clinically relevant strategy for bone tissue engineering (BTE). Secondary aims were to identify a minimally invasive source for MSCs, and to promote angiogenesis within the xeno-free 3D cultures.

Human platelet lysate (HPL) represents a favourable supplement for xeno-free expansion of MSCs (Study I). To standardize HPL production, the storage time of platelet concentrates was optimized in terms of HPL cytokine content and biological efficacy on MSCs. Advantages of HPL culture (vs. bovine serum) were observed in relation to all relevant in vitro aspects of MSCs, i.e., growth, immunophenotype and osteogenic differentiation (Studies II and III).

Progenitor cells showing a characteristic MSC-like phenotype and multipotency were isolated from human gingiva (GPCs) and periodontal ligament (PDLCs). Both GPCs and PDLCs demonstrated superior growth and osteogenic differentiation in HPL vs. FBS; a subset of GPCs also showed potent neurogenic differentiation (Study III). Given their relative ease of isolation and minimally invasive tissue harvesting, GPCs were prioritized in subsequent experiments.

To overcome the limitations of traditional monolayer (2D) culture, 3D spheroid cultures were established in HPL. Both GPCs and BMSCs demonstrated significantly increased expression of stemness- and osteogenesis-related genes in spheroids vs. monolayers, confirmed at the protein level by immunocytochemistry. Moreover, the cytokine release profile of GPC and BMSC spheroids was considerably enhanced compared to monolayers. Under osteogenic conditions, GPC spheroids showed in vitro mineralization comparable to that of BMSCs (Study III). When implanted in vivo, xeno-free GPCs and BMSCs showed ectopic mineralization after 4 and 8 weeks based on micro-CT and histology; implanted human cells were identified at the mineralization sites via in situ hybridization. In the case of BMSCs, significantly greater mineralization was observed in constructs containing spheroids vs. single cells (Study V).

To enhance angiogenesis, a coculture strategy was tested using a xeno-free spheroid coculture model of GPCs and human umbilical vein ECs (HUVECs) embedded in an HPL-hydrogel (HPLG). When cultured as spheroids, HUVECs showed characteristic in vitro sprouting angiogenesis in HPLG. A trend for increased in vitro HUVEC-sprouting was observed in co-culture with GPCs. Constructs of coculture and HUVEC spheroids in HPLG comparably supported in vivo neoangiogenesis in a chorioallantoic membrane (CAM) assay (Study IV).

Clinically relevant BTE constructs were designed combining BMSCs (as spheroids or single cells) encapsulated in HPLG and 3D printed copolymer scaffolds. Viability and osteogenic differentiation of cells within the constructs was confirmed up to 21 days in vitro; greater mineralization was observed in constructs containing spheroids vs. single cells. When implanted in rats’ calvarial defects, constructs of both spheroids and single cells revealed abundant in vivo bone regeneration for up to 12 weeks (Study V).

The results herein suggest clear advantages of xeno-free 3D cultures of MSCs for BTE. GPCs represent a promising alternative to BMSCs with osteogenic and proangiogenic potential, and further work is needed to facilitate clinical translation. In particular, the constructs of xeno-free MSCs, HPLG and 3D printed scaffolds developed herein, represent a clinically relevant strategy for BTE.