Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids

Abstract

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and β-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Dual functional construct containing kartogenin releasing microtissues and curcumin for cartilage regeneration

“…SEM images of the MPs showed that they had a regular and smooth spherical morphology. According to the SEM images, the particles had an average diameter of 11 ± 5.5 μm, which was in the range of the previously reported MPs for incorporation within cell spheroids [24,25].…”

“…MPs, as a scaffold, mimic the micro-environment of the cell and provide an active surface for cell attachment. Eventually, they can be used as a carrier for controlled release of differentiation factors [25,[29][30][31][32][33][34].…”

3D-microtissue derived secretome as a cell-free approach for enhanced mineralization of scaffolds in the chorioallantoic membrane model

“…1), a recent in vitro study showed that that the formation of human cardiopoietic stem cells into 3D-MTs substantially increases their overall angiogenic potential and functional neovascularization capacity 24 . Furthermore, MSC-based 3D-MTs with incorporated calcium phosphate microparticles significantly promoted blood vessel reconstruction and bone formation 8 weeks after transplantation into a critical-sized defect rat model 25 . The process of mineralization of hard collagenous tissue is influenced by amorphous calcium phosphate (a-CaP), non-collagenous proteins and collagen (COL) itself 26 .…”

Functional tissue-engineered microtissue formed by self-aggregation of cells for peripheral nerve regeneration

“…Microtissues can be directly injected into target tissues [ 19 , 24 , 25 ], or can replace traditional 2D-cultured cells combined with 3D scaffolds [ 26 ] or be mixed into bio-ink for 3D printing [ 27 , 28 ], all of them have achieved promising repair effects. The microtissue strategy has made breakthroughs in bone tissue engineering [ 26 , 29 – 33 ], cartilage tissue engineering [ 19 , 24 , 34 – 36 ], adipose tissue engineering [ 22 , 28 ], myocardial tissue engineering [ 37 – 40 ], vascular tissue engineering [ 41 , 42 ], oncology [ 43 , 44 ], etc. However, to the best of our knowledge, microtissues are rarely used in neural tissue engineering; thus, we intended to use microtissues to repair PNI.…”