Differentiation of equine mesenchymal stem cells into cells of osteochondral lineage: potential for osteochondral tissue engineering.

Damage to the hyaline cartilage of the joint surface and osteochondral fractures are key factors leading to the development of osteoarthritis in racehorses, representing a significant cause of racehorse retirement. To tissue-engineer an osteochondral unit that is suitable for joint repair, incorporation of a zone of calcified cartilage should be considered so as to mimic itsin vivocounterpart. To date, equine mesenchymal stem cells (eMSCs) have been reported to have multilineage differentiation potential. Yet the generation of a zone of calcified cartilage using eMSCs has not been reported. This work is an initial attempt to generate a zone of calcified cartilage using eMSCs as the single source of cells and collagen as the scaffolding material. Main advantages of using eMSCs over equine deep zone chondrocytes for the generation of a zone of calcified cartilage include no donor site morbidity and their ease of expansion in culture. Initially, we fabricated cartilage-like tissues and bone-like tissuesin vitroby differentiating eMSCs toward chondrogenic and osteogenic lineages for 21 d, respectively. We then aggregated the cartilage-like and bone-like tissues together with a layer of undifferentiated eMSCs-collagen gel in between to generate a 3-layer osteochondral unit. A zone of calcified cartilage was found between the cartilage-like and bone-like layers after a 14-day culture in chondrogenic differentiation medium. These results provide a solution toward tissue engineering of equine osteochondral units with interfacial zone without using chondrocytes harvested from the deep zone of healthy articular cartilage, and contribute to the future development of osteochondral tissue engineering strategies for human cartilage injuries in the long run.

Bioengineering a multicomponent spinal motion segment construct--a 3D model for complex tissue engineering.

Intervertebral disc degeneration is an important clinical problem but existing treatments have significant drawbacks. The ability to bioengineer the entire spinal motion segment (SMS) offers hope for better motion preservation strategies but is extremely challenging. Here, fabrication of a multicomponent SMS construct with complex hierarchical organization from mesenchymal stem cells and collagen-based biomaterials, using a module-based integrative approach, is reported. The construct consists of two osteochondral subunits, a nucleus pulposus (NP-)-like core and a multi-lamellae annulus fibrosus (AF-)-like component. Chondrogenic medium is crucial for stabilizing the osteochondral subunits, which are shown to allow passive nutrient diffusion, while cyclic compression is necessary for better fiber matrix organization. Cells adhere, survive, and interact with the NP-like core. Cyclic torsional loading stimulates cell alignment in the AF-like lamellae and the number of lamellae affects the mechanical properties of the construct. This work represents an important milestone in SMS tissue engineering and provides a 3D model for studying tissue maturation and functional remodeling.

In vitro generation of an osteochondral interface from mesenchymal stem cell-collagen microspheres.

Creating biological interfaces between mechanically dissimilar tissues is a key challenge in complex tissue engineering. An osteochondral interface is essential in preventing mechanical failure and maintaining normal function of cartilage. Despite tremendous efforts in developing osteochondral plugs, formation of the osteochondral interface with proper zonal organization has not yet been reported. Here, we present a mesenchymal stem cell-collagen microsphere-based approach for complex tissue engineering and demonstrate in vitro formation of a stem cell-derived osteochondral interface with calcified cartilage interface separating a non-calcified cartilage layer and an underlying bone layer. Cells at the interface region are hypertrophic chondrocytes while the extracellular matrix in this region contains collagen type II and X, calcium deposits and vertically running fibers. The simultaneous presence of appropriate medium and configuration during co-culture is necessary for the interface formation.

Decellularization of chondrocyte-encapsulated collagen microspheres: a three-dimensional model to study the effects of acellular matrix on stem cell fate.

Extracellular matrix (ECM) partially constitutes the stem cell niche. Reconstituting the ECM niche in a three-dimensional (3D) configuration will significantly enhance our understanding of how stem cells interact with and respond to the ECM niche. In this study, we aimed to reconstitute a glycosaminoglycan (GAG)-rich ECM using a microencapsulation technology, produce acellular matrix using a decellularization technique, and investigate the effect of acellular matrix on stem cell fate by repopulating the matrix with human mesenchymal stem cells (hMSCs). We demonstrated that porcine chondrocytes were able to deposit a GAG-rich ECM within the 3D collagen microsphere. All decellularization treatment groups resulted in significant removal of chondrocyte nuclei, but acellular matrix was only achieved using 2% sodium deoxycholate. Nevertheless, decellularization resulted in significant loss in GAG content in almost all treatment groups, and the 2% sodium deoxycholate group was able to preserve about 40% of the GAGs compared with the control group. We further demonstrated that hMSCs seeded onto the decellularized microspheres were able to survive and penetrate into the centre, while hMSCs seeded in the acellular matrix showed positive immunostaining against sox9, indicating that they may be differentiating toward the chondrogenic lineage without the need to supplement the chondrogenic differentiation medium.