52 research outputs found
Temperature Control During Fibrillogenesis Allows for Improved Magnetic Alignment of Collagen
Bone Tissue Engineering: Scalability and Optimization of Densified Collagen-Fibril Bone Graft Substitute Materials
Over 240 million people missing teeth worldwide experience lingering problems such as difficulty speaking and eating, undesirable aesthetics, and resorption of bone supporting neighboring teeth. The gold standard of treatment utilizes grafts to attach a function-restoring implant to supporting bone. Current graft materials suffer from problems including autologous donor site morbidity, long resorption time, incomplete integration with the maxillae or mandible, and structural weakness. Patient-specific, cellularized bone grafts may be a solution to these issues by accelerating and improving the quality of regenerated bone. Recently, encapsulation of mesenchymal stem cells within self-assembling type I collagen oligomer matrices has been shown to support rapid mineralization of small-scale bone constructs (cylinders with diameter and height of 6mm and 1mm, respectively) in vitro. However, this method’s volume and geometric constraints for nutrient transport and cell viability are still unknown. In this study, the effects of construct size and medium formulation on mineralization were investigated using conventional static culture methods. To create constructs, human adipose stem cells (hASCs) were embedded in oligomer matrices, allowed to polymerize, and compressed to final cell and fibril densities of 3x107 cells/mL and 50 mg/mL, respectively. Varying construct sizes (maximum diameter and thickness of 11 mm and 0.81 mm) were cultured for 1 week in growth medium or osteogenic medium with varying calcium concentrations. Alizarin red staining was used to detect calcium deposits indicative of cell-induced mineralization. Preliminary data suggests that culture in osteogenic medium supplemented with both 8 mM and 16 mM calcium may induce rapid, uniform mineralization across all sizes tested, and 16 mM calcium supplementation induces greater mineralization. However, additional validation by direct measurement of cell viability and osteogenic differentiation will be needed to better compare bone regeneration as a function of scale
Tissue Engineering: Applications in Developmental Toxicology
In vivo toxicology assays are expensive, low-throughput, and often not predictive of a human response. Three-dimensional in vitro human cell-based tissue systems incorporating cell-cell and cell-matrix interactions have promise to provide high-throughput, physiologically-relevant information on the mechanism of the toxin and a more accurate assessment of the toxicity of a chemical before progression to human trials. Quantification of the disruption of vasculogenesis, the de novo formation of blood vessels from endothelial progenitor cells, can serve as an appropriate indicator of developmental toxicity since vasculogenesis is critical to the early development of the circulatory system. The current routinely used in vitro angiogenesis assays are 2D and thus not physiologically-relevant and analyzed semi-quantitatively. Recently, a 3D in vitro model of vasculogenesis, involving a type I collagen oligomer matrix and endothelial colony forming cells, was described to be capable of producing and maintaining lumenized blood vessel networks up to 14 days. Here we utilized this model to develop a 3D vasculogenesis assay. We quantified the performance of this assay with a set of known angiogenesis modulators (imazamox, thalidomide, 2-methoxyestradiol, levamisole) through cytotoxicity testing and 3D image analysis. Preliminary data suggests a difference in the drug sensitivity and response when measured with the 3D vasculogenesis assay and compared to a well-established 2D angiogenesis assay. We expect our model to more closely mimic results from in vivo studies, but further validation is needed. This new 3D vasculogenesis model can provide a physiologically-relevant, high-throughput screening and mechanistic assay for applications in pharmaceutical development and developmental toxicology
Local, three-dimensional strain measurements within largely deformed extracellular matrix constructs
The ability to create extracellular matrix (ECM
Injectable Highly Tunable Oligomeric Collagen Matrices for Dental Tissue Regeneration
Current stem cell transplantation approaches lack efficacy, because they limit cell survival and retention and, more importantly, lack a suitable cellular niche to modulate lineage-specific differentiation. Here, we evaluate the intrinsic ability of type I oligomeric collagen matrices to modulate dental pulp stem cells (DPSCs) endothelial and odontogenic differentiation as a potential stem cell-based therapy for regenerative endodontics. DPSCs were encapsulated in low-stiffness (235 Pa) and high-stiffness (800 Pa) oligomeric collagen matrices and then evaluated for long-term cell survival, as well as endothelial and odontogenic differentiation following in vitro cell culture. Moreover, the effect of growth factor incorporation, i.e., vascular endothelial growth factor (VEGF) into 235 Pa oligomeric collagen or bone morphogenetic protein (BMP2) into the 800 Pa oligomeric collagen counterpart on endothelial or odontogenic differentiation of encapsulated DPSCs was investigated. DPSCs-laden oligomeric collagen matrices allowed long-term cell survival. Real time polymerase chain reaction (RT-PCR) data showed that the DPSCs cultured in 235 Pa matrices demonstrated an increased expression of endothelial markers after 28 days, and the effect was enhanced upon VEGF incorporation. There was a significant increase in alkaline phosphatase (ALP) activity at Day 14 in the 800 Pa DPSCs-laden oligomeric collagen matrices, regardless of BMP2 incorporation. However, Alizarin S data demonstrated higher mineralization by Day 21 and the effect was amplified in BMP2-modified matrices. Herein, we present key data that strongly support future research aimed at clinical translation of an injectable oligomeric collagen system for delivery and fate regulation of DPSCs to enable pulp and dentin regeneration at specific locations of the root canal system
Monitoring focal adhesion kinase phosphorylation dynamics in live cells
Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging
Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells
Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs
Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices
Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC
Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis
Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression
Design and biofabrication of dermal regeneration scaffolds: role of oligomeric collagen fibril density and architecture
Aim: To evaluate dermal regeneration scaffolds custom-fabricated from fibril-forming oligomeric collagen where the total content and spatial gradient of collagen fibrils was specified. Materials & methods: Microstructural and mechanical features were verified by electron microscopy and tensile testing. The ability of dermal scaffolds to induce regeneration of rat full-thickness skin wounds was determined and compared with no fill control, autograft skin and a commercial collagen dressing. Results: Increasing fibril content of oligomer scaffolds inhibited wound contraction and decreased myofibroblast marker expression. Cellular and vascular infiltration of scaffolds over the 14-day period varied with the graded density and orientation of fibrils. Conclusion: Fibril content, spatial gradient and orientation are important collagen scaffold design considerations for promoting vascularization and dermal regeneration while reducing wound contraction
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