Bone Tissue Engineering with Multilayered Scaffolds-Part II: Combining Vascularization with Bone Formation in Critical-Sized Bone Defect.

Our previous in vivo study showed that multilayered scaffolds made of an angiogenic layer embedded between an osteogenic layer and an osteoconductive layer, with layer thickness in the 100-400 μm range, resulted in through-the-thickness vascularization of the construct even in the absence of exogenous endothelial cells. The angiogenic layer was a collagen-fibronectin gel, and the osteogenic layer was made from nanofibrous polycaprolactone while the osteoconductive layer was made either from microporous hydroxyapatite or microfibrous polycaprolactone. In this follow-up study, we implanted these acellular and cellular multilayered constructs in critical-sized rat calvarial defects and evaluated their vascularization and bone formation potential.

Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model.

There exists a strong clinical need for a more capable and robust method to achieve bone augmentation, and a system with fine-tuned delivery of demineralized bone matrix (DBM) has the potential to meet that need. As such, the objective of the present study was to investigate a synthetic biodegradable hydrogel for the delivery of DBM for bone augmentation in a rat model. Oligo(poly(ethylene glycol) fumarate) (OPF) constructs were designed and fabricated by varying the content of rat-derived DBM particles (either 1:3, 1:1 or 3:1 DBM:OPF weight ratio on a dry basis) and using two DBM particle size ranges (50-150 or 150-250 μm).

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model.

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(ɛ-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated.

Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2.

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)-hyaluronic acid (bPEI-HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI-HA complexes.

Enhancing chondrogenic phenotype for cartilage tissue engineering: monoculture and coculture of articular chondrocytes and mesenchymal stem cells.

Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage.

Chondrogenic phenotype of articular chondrocytes in monoculture and co-culture with mesenchymal stem cells in flow perfusion.

This work investigated the effect of flow perfusion bioreactor culture with and without transforming growth factor-β3 (TGF-β3) supplementation on the proliferation, extracellular matrix (ECM) production, and chondrogenic gene expression of chondrocytes both in monoculture and in co-culture with bone marrow-derived mesenchymal stem cells (MSCs). Both cell populations were cultured on electrospun poly(ɛ-caprolactone) scaffolds for 2 weeks in static or flow perfusion culture with and without TGF-β3. Overall, it was observed that without growth factors, flow perfusion culture resulted in increased cell proliferation and ECM with a more cartilage-like composition.

Articular chondrocyte redifferentiation in 3D co-cultures with mesenchymal stem cells.

In this work, we evaluated the ability of 3D co-cultures with mesenchymal stem cells (MSCs) to redifferentiate monolayer expanded articular chondrocytes (ACs) and produce cartilaginous extracellular matrix at varying stages of the dedifferentiation process and further examined the dependency of this effect on the culture medium composition. Primary bovine ACs were expanded in monolayers for up to nine population doublings to obtain seven cell stocks with gradually increasing levels of dedifferentiation. Culture expanded ACs were then seeded as monocultures and co-cultures with rabbit bone marrow-derived MSCs (30:70 ratio of ACs-to-MSCs) on porous scaffolds.

TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds.

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(ε-caprolactone) scaffolds.

Tissue response to composite hydrogels for vertical bone augmentation in the rat.

The objective of the present study was to develop a preclinical animal model for evaluating bone augmentation and to examine the level of bone augmentation induced by hydrogel composites. Design criteria outlined for the development of the animal model included rigid immobilization of bilateral implants apposed to the parietal bone of the rat, while avoiding the calvarial sutures. The animal model was evaluated through the implantation of hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles releasing bone morphogenetic protein-2 (BMP-2).

Flow perfusion co-culture of human mesenchymal stem cells and endothelial cells on biodegradable polymer scaffolds.

In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Osteogenically precultured hMSCs were seeded onto electrospun scaffolds in monoculture or a 1:1 ratio with HUVECs, cultured for 7 or 14 days in osteogenic medium under static or flow perfusion conditions, and the resulting constructs were analyzed for cellularity, alkaline phosphatase (ALP) activity and calcium content. In flow perfusion, constructs with monocultures of hMSCs demonstrated higher cellularity and calcium content, but lower ALP activity compared to corresponding static controls.

Enhanced osteogenesis in cocultures with human mesenchymal stem cells and endothelial cells on polymeric microfiber scaffolds.

In this work, human mesenchymal stem cells (hMSCs) and their osteogenically precultured derivatives were directly cocultured with human umbilical vein endothelial cells (HUVECs) on electrospun three-dimensional poly(ɛ-caprolactone) microfiber scaffolds to evaluate the coculture's effect on the generation of osteogenic constructs. Specifically, cells were cultured on scaffolds for up to 3 weeks, and the cellularity, alkaline phosphatase (ALP) activity, and bone-like matrix formation were assessed. Constructs with cocultures and monocultures had almost identical cellularity after the first week, however, lower cellularity was observed in cocultures compared to monocultures during the subsequent 2 weeks of culture.

The effect of hypoxia on the chondrogenic differentiation of co-cultured articular chondrocytes and mesenchymal stem cells in scaffolds.

In this work, we investigated the effects of lowered oxygen tension (20% and 5% O2) on the chondrogenesis and hypertrophy of articular chondrocytes (ACs), mesenchymal stem cells (MSCs) and their co-cultures with a 30:70 AC:MSC ratio. Cells were cultured for six weeks within porous scaffolds, and their cellularity, cartilaginous matrix production (collagen II/I expression ratio, hydroxyproline and GAG content) and hypertrophy markers (collagen X expression, ALP activity, calcium accumulation) were analyzed. After two weeks, hypoxic culture conditions had expedited chondrogenesis with all cell types by increasing collagen II/I expression ratio and matrix synthesis by ~2.

Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells.

In this work, articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) with 1:1 and 1:3 cell ratios were co-cultured in order to evaluate if a majority of primary ACs can be replaced with MSCs without detrimental effects on in vitro chondrogenesis. We further used a xenogeneic culture model to study if such co-cultures can result in redifferentiation of passaged ACs. Cells were cultured in porous scaffolds for four weeks and their cellularity, cartilage-like matrix formation and chondrogenic gene expression levels (collagen I and II, aggrecan) were measured.

Design of a high-throughput flow perfusion bioreactor system for tissue engineering.

Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering.

Polymeric nanofibers in tissue engineering.

Polymeric nanofibers can be produced using methods such as electrospinning, phase separation, and self-assembly, and the fiber composition, diameter, alignment, degradation, and mechanical properties can be tailored to the intended application. Nanofibers possess unique advantages for tissue engineering. The small diameter closely matches that of extracellular matrix fibers, and the relatively large surface area is beneficial for cell attachment and bioactive factor loading.