R-3 Repository :: Browsing by Author "Dahlin, Rebecca L."

Browsing by Author "Dahlin, Rebecca L."

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Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model
(Elsevier, 2014) Dahlin, Rebecca L.; Kinard, Lucas A.; Lam, Johnny; Needham, Clark J.; Lu, Steven; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.
Co-Cultures of Articular Chondrocytes and Mesenchymal Stem Cells for Cartilage Tissue Engineering
(2014-04-24) Dahlin, Rebecca L.; Mikos, Antonios G.; Kasper, Kurt; Ludwig, Joseph A.; Zygourakis, Kyriacos
Articular cartilage lines the surfaces of synovial joints to protect underlying bone and provide a smooth surface for articulation. Damage to articular cartilage typically leads to long-term pain and disability, as current treatments are unable to fully restore the functional tissue. Thus, tissue engineers seek to develop technologies to enhance cartilage repair. This thesis investigated two strategies for cartilage engineering: flow perfusion bioreactor culture and co-cultures of chondrocytes with mesenchymal stem cells (MSCs). First, we designed a novel bioreactor and then investigated the effect of flow perfusion on chondrocytes when combined with chondrogenic stimuli, including hypoxia and transforming growth factor-β3 (TGF-β3). We demonstrated that the combination of flow perfusion and hypoxic conditions enhanced proliferation, cartilage-like extracellular matrix production, and chondrogenic gene expression compared to perfusion alone. However, these results also demonstrated the need for a more potent chondrogenic stimulus, and thus the effect of perfusion with TGF-β3 was investigated on both chondrocytes and co-cultures of chondrocytes and MSCs. Here, we described the advantages of using exogenous growth factors in flow perfusion cultures, and the utility of flow perfusion for creating large tissue-engineered constructs. The second part of this thesis investigated co-cultures of chondrocytes and MSCs having the potential to reduce the demand for chondrocytes, which overcomes a significant challenge to current approaches toward cartilage repair. We first investigated the sensitivity of this cell population to TGF-β3 and then investigated the stability of the cell phenotype resulting from growth factor supplementation. The results demonstrated that co-cultures of chondrocytes and MSCs enable a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to chondrocyte or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3. The final section of this thesis investigated the ability of such co-cultures to repair cartilage in a rat osteochondral defect model. Here, it was demonstrated that co-cultures achieved equivalent cartilage repair compared to the chondrocytes, thus demonstrating the potential use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.
Design of a High-Throughput Flow Perfusion Bioreactor System for Tissue Engineering
(Mary Ann Liebert, Inc., 2012) Dahlin, Rebecca L.; Meretoja, Ville V.; Ni, Mengwei; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.
The effect of hypoxia on the chondrogenic differentiation of cocultured articular chondrocytes and mesenchymal stem cells in scaffolds
(Elsevier, 2013) Meretoja, Ville V.; Dahlin, Rebecca L.; Wright, Sarah; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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 ∼2.5–11 and ∼1.5–3.0 fold, respectively. At later times, hypoxia decreased cellularity but had little effect on matrix synthesis. ACs and co-cultures showed similarly high collagen II/I expression ratio and GAG rich matrix formation, whereas MSCs produced the least hyaline cartilage-like matrix and obtained a hypertrophic phenotype with eventual calcification. MSC hypertrophy was further emphasized in hypoxic conditions. We conclude that the most promising cell source for cartilage engineering was co-cultures, as they have a potential to decrease the need for primary chondrocyte harvest and expansion while obtaining a stable highly chondrogenic phenotype independent of the oxygen tension in the cultures.
Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells
(Elsevier, 2012) Meretoja, Ville V.; Dahlin, Rebecca L.; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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. Constructs with primary bovine ACs had ∼1.6 and 5.5 times higher final DNA and glycosaminoglycan contents, respectively, in comparison to those with culture expanded chondrocytes or MSCs harvested from the same animals. Equally robust chondrogenesis was also observed in co-cultures, even when up to 75% of primary ACs were initially replaced with MSCs. Furthermore, species-specific RT-PCR analysis indicated a gradual loss of MSCs in bovine-rabbit co-cultures. Finally, co-cultures using primary and culture expanded ACs resulted in similar outcomes. We conclude that the most promising cell source for cartilage engineering was the co-cultures, as the trophic effect of MSCs may highly increase the chondrogenic potential of ACs thus diminishing the problems with primary chondrocyte harvest and expansion.
Enhanced osteogenesis in co-cultures with human mesenchymal stem cells and endothelial cells on polymeric microfiber scaffolds
(Mary Ann Liebert, Inc., 2013) Gershovich, Julia G.; Dahlin, Rebecca L.; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
In this work, human mesenchymal stem cells (hMSCs) and their osteogenically precultured derivatives were directly co-cultured with human umbilical vein endothelial cells (HUVECs) on electrospun 3D poly(-caprolactone) microfiber scaffolds in order to evaluate the co-culture’s effect on the generation of osteogenic constructs. Specifically, cells were cultured on scaffolds for up to three weeks, and the cellularity, alkaline phosphatase activity (ALP), and bone-like matrix formation were assessed. Constructs with co-cultures and monocultures had almost identical cellularity after the first week, however lower cellularity was observed in co-cultures compared to monocultures during the subsequent two weeks of culture. Scaffolds with co-cultures showed significantly higher ALP activity, glycosaminoglycan and collagen production, as well as greater calcium deposition over the course of study compared to monocultures of hMSCs. Furthermore, the osteogenic outcome was equally robust in co-cultures containing osteogenically precultured and non-precultured hMSCs. The results demonstrate that the combination of MSC and HUVEC populations within a porous scaffold material under osteogenic culture conditions is an effective strategy to promote osteogenesis.
Flow Perfusion Co-culture of Human Mesenchymal Stem Cells and Endothelial Cells on Biodegradable Polymer Scaffolds
(Springer, 2014) Dahlin, Rebecca L.; Gershovich, Julia G.; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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. ALP activity was enhanced in co-cultures under flow perfusion conditions, compared to hMSCs alone; however unlike the static controls, the calcium content of the co-cultures in flow perfusion was not different from the corresponding hMSC monocultures. The data suggest that co-cultures of hMSCs and HUVECs did not contribute to enhanced mineralization compared to hMSCs alone under the flow perfusion conditions investigated in this study. However, flow perfusion culture resulted in an enhanced spatial distribution of cells and matrix compared to static cultures, which were limited to a thin surface layer.
Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2
(Elsevier, 2014) Needham, Clark J.; Shah, Sarita R.; Dahlin, Rebecca L.; Kinard, Lucas A.; Lam, Johnny; Watson, Brendan M.; Lu, Steven; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI–HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6 weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.
Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model
(Elsevier, 2014) Kinard, Lucas A.; Dahlin, Rebecca L.; Lam, Johnny; Lu, Steven; Lee, Esther J.; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering; Chemical and Biomolecular Engineering
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). The physical properties of the constructs and the bioactivity of the DBM were evaluated. Selected formulations (1:1 and 3:1 with 50–150 μm DBM) were evaluated in vivo compared to an empty control to investigate the effect of DBM dose and construct properties on bone augmentation. Overall, 3:1 constructs with higher DBM content achieved the greatest volume of bone augmentation, exceeding 1:1 constructs and empty implants by 3- and 5-fold, respectively. As such, we have established that a synthetic, biodegradable hydrogel can function as a carrier for DBM, and that the volume of bone augmentation achieved by the constructs correlates directly to the DBM dose.
TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds
(Elsevier, 2014) Dahlin, Rebecca L.; Ni, Mengwei; Meretoja, Ville V.; Kasper, F. Kurtis; Mikos, Antonios G.; Bioengineering
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(ɛ-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.

Browsing by Author "Dahlin, Rebecca L."

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