Bone Regeneration in a Large Animal Model Featuring a Modular Off-the-Shelf Soft Callus Mimetic.

Implantation of engineered cartilage with soft callus features triggers remodeling to bone tissue via endochondral bone regeneration (EBR). Thus far, EBR has not progressed to the level of large animals on the axis of clinical translation. Herein, the feasibility of EBR is aimed for a critical-sized defect in a large animal model. Chondrogenesis is first induced in goat-derived multipotent mesenchymal stromal cells (MSCs) by fine-tuning the cellular differentiation process. Through a unique devitalization process, two off-the-shelf constructs aimed to recapitulate the different stages of the transient cartilaginous soft callus template in EBR are generated. To evaluate bone regeneration, the materials are implanted in an adapted bilateral iliac crest defect model in goats, featuring a novel titanium star-shaped spacer. After 3 months, the group at the more advanced differentiation stage shows remarkable regenerative capacity, with comparable amounts of bone regeneration as the autograft group. In contrast, while the biomaterial mimicking the earlier stages of chondrogenesis shows improved regeneration compared to the negative controls, this is subpar compared to the more advanced material. Concluding, EBR is attainable in large animals with a soft callus mimetic material that leads to fast conversion into centimeter-scale bone, which prospects successful implementation in the human clinics.

Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues.

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. STATEMENT OF SIGNIFICANCE: In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.