Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels (original) (raw)
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Acta Biomaterialia, 2010
This work investigated the delivery of marrow mesenchymal stem cells (MSCs), with or without the growth factor transforming growth factor-β1 (TGF-β1), from biodegradable hydrogel composites on the repair of osteochondral defects in a rabbit model. Three formulations of oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites containing gelatin microparticles (GMPs) and MSCs were implanted in osteochondral defects, including (1) OPF/GMP hydrogel composites; (2) OPF/ GMP hydrogel composites encapsulating MSCs; and (3) OPF hydrogel composites containing TGF-β1 loaded GMPs and MSCs. At 12 weeks, the quality of new tissue formed in chondral and subchondral regions of defects was evaluated based on subjective and quantitative histological analysis. OPF hydrogel composites were partially degraded and the defects were filled with newly formed tissue at 12 weeks with no sign of persistent inflammation. With the implantation of scaffolds alone, newly formed chondral tissue had an appearance of hyaline cartilage with zonal organization and intense staining for glycosaminoglycans, while in the subchondral region hypertrophic cartilage with some extent of bone formation was often observed. The addition of MSCs, especially with TGF-β1 loaded GMPs, facilitated subchondral bone formation, as evidenced by more trabecular bone appearance. However, the delivery of MSCs with or without TGF-β1 at the dosage investigated did not improve cartilage morphology. While OPF-based hydrogel composites supported osteochondral tissue generation, further investigations are necessary to elucidate the effects of MSC seeding density and differentiation stage on new tissue formation and regeneration.
In vitro generation of an osteochondral interface from mesenchymal stem cell–collagen microspheres
Biomaterials, 2011
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 cellecollagen 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.
Tissue Engineering Part A, 2013
Current surgical techniques for osteochondral injuries in young active patients are inadequate clinically. Novel strategies in tissue engineering are continuously explored in this area. Despite numerous animal studies that have shown encouraging results, very few large-scale clinical trials have been done to address this area of interest. To facilitate the eventual translation from rabbit to human subjects, we have performed a study using bone marrowderived mesenchymal stem cell (BMSC)-oligo[poly(ethylene glycol) fumarate] (OPF) hydrogel scaffold in a porcine model. Our objective was to analyze the morphology of BMSCs seeded into rehydrated freeze-dried OPF hydrogel and in vivo gross morphological and histological outcome of defects implanted with the BMSCs-OPF scaffold in a porcine model. The analyses were based on magnified histologic sections for different types of cartilage repair tissues, the outcome of the subchondral bone, scaffold, and statistical assessment of neotissue-filling percentage, cartilage phenotype, and Wakitani scores. The morphology of the BMSCs seeded into the rehydrated freeze-dried OPF scaffold was observed 24 h after cell seeding, through the phase-contrast microscope. The three-dimensional and cross-sectional structure of the fabrication was analyzed through confocal microscopy and histological methods, respectively. The BMSCs remained viable and were condensed into many pellet-like cell masses with a diameter ranging from 28.5 to 298.4 (113.5-47.9) mm in the OPF scaffold. In vivo osteochondral defect repair was tested in 12 defects created in six 8-month-old Prestige World Genetics micropigs. The implantation of scaffold alone was used for control. Gross morphological, histological, and statistical analyses were performed at 4 months postoperatively. The scaffold-MSC treatment led to 99% defect filling, with 84% hyaline-like cartilage at 4 months, which was significantly (p < 0.0001) more than the 54% neotissue filling and 39% hyaline-like cartilage obtained in the scaffold-only group. The implantation of BMSCs in freeze-dried OPF hydrogel scaffold, which created a conducive environment for cell infiltration and clustering, could fully repair chondral defects with hyaline-like cartilage. This protocol provides a clinically feasible procedure for osteochondral defect treatment.
Osteoarthritis and Cartilage, 2010
Objective: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. Design: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. Results: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. Conclusions: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the boneecartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).
Cartilage tissue engineering: From hydrogel to mesenchymal stem cells
2010
Articular cartilage does not repair itself spontaneously. To promote its repair, the transfer of stem cells from adipose tissue (ATSC) using an injectable self-setting cellulosic-hydrogel (Si-HPMC) appears promising. In this context, the objective of this work was to investigate the influence of in vitro chondrogenic differentiation of ATSC on the in vivo cartilage formation when combined with Si-HPMC. In a first set of experiments, we characterized ATSC for their ability to proliferate, self renew and express typical mesenchymal stem cell surface markers. Then, the potential of ATSC to differentiate towards the chondrogenic lineage and the optimal culture conditions to drive this differentiation were evaluated. Real-time RT-PCR and histological analysis for sulphated glycosaminoglycans and type II collagen revealed that 3-dimensional culture and hypoxic condition favored ATSC chondrogenesis regarding mRNA expression level and the corresponding proteins production. In order to assess the phenotypic stability of chondrogenically-differentiated ATSC, real-time RT-PCR for specific terminal chondrogenic markers and alkaline phosphatase activity assay were performed. In addition to promote chondrogenesis, our culture conditions seem to prevent the terminal differentiation of ATSC. Histological examination of ATSC/Si-HPMC implants suggested that the in vitro chondrogenic pre-commitment of ATSC in monolayer is sufficient to obtain cartilaginous tissue in vivo.