Repeated In Vitro Impact Conditioning of Astrocytes Decreases the Expression and Accumulation of Extracellular Matrix.

Pathological changes to the physical and chemical properties of brain extracellular matrix (ECM) occur following injury. It is generally assumed that astrocytes play an important role in these changes. What remain unclear are the triggers that lead to changes in the regulation of ECM by astrocytes following injury. We hypothesize that mechanical stimulation triggers genotypic and phenotypic changes to astrocytes that could ultimately reshape the ECM composition of the central nervous system following injury. To explore astrocyte mechanobiology, an in vitro drop test bioreactor was employed to condition primary rat astrocytes using short duration (10 ms), high deceleration (150G) and strain (20%) impact stimuli. Experiments were designed to explore the effect of single and repeated impact (single vs. double) on mechano-sensitive behavior including cell viability; ECM gene (collagens I and IV, fibronectin, neurocan, versican) and reactivity gene [glial fibrillary acidic protein (GFAP), S100B, vimentin] expression; matrix regulatory cytokine secretion [matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP1), transforming growth factor beta 1 (TGFß1)]; and matrix accumulation [collagen and glycosaminoglycan (GAG)]. Experiments revealed that both ECM and reactive gliosis gene expression was significantly decreased in response to impact conditioning. The decreases for several genes, including collagen, versican, and GFAP were sensitive to impact number, suggesting mechano-sensitivity to repeated impact conditioning. The measured decreases in ECM gene expression were supported by longer-term in vitro experiments that demonstrated significant decreases in chondroitin sulfate proteoglycan (CSPG) and collagen accumulation within impact conditioned 3-D scaffolds accompanied by a 25% decrease in elastic modulus. Overall, the general trend across all samples was towards altered ECM and reactive gliosis gene expression in response to impact. These results suggest that the regulation of ECM production by astrocytes is sensitive to mechanical stimuli, and that repeated impact conditioning may increase this sensitivity.

Cell derived extracellular matrix fibers synthesized using sacrificial hollow fiber membranes.

The therapeutic potential of biological scaffolds as adjuncts to synthetic polymers motivates the engineering of fibers formed using the extracellular matrix (ECM) secreted by cells. To capture the ECM secreted by cells during in vitro culture, a solvent degradable hollow fiber membrane (HFM) was created and utilized as a cell culture platform. NIH/3T3 fibroblasts were injected into the narrow (0.986 ± 0.042 mm) lumina of mesoporous polysulfone HFMs and maintained in culture for up to 3 weeks. Following cell culture, HFMs were dissolved using N-methyl-2-pyrrolidone and the accumulated ECM was collected. The ECM retained the filamentous dimensions of the HFM lumen. The process yielded up to 0.89 ± 0.20 mg of ECM for every mm of HFM dissolved. Immunofluorescence, second-harmonic generation microscopy, and tandem mass spectrometry indicated the presence of an array of ECM constituents, including collagen, fibronectin, and proteoglycans, while FTIR spectra suggested thorough HFM material dissolution. Isolated ECM fibers, although fragile, were amenable to handling and exhibited an average elastic modulus of 34.6 ± 15.3 kPa, ultimate tensile strength of 5.2 ± 2.2 kPa, and elongation-at-break of 29% ± 18%. ECM fibers consisted of an interconnected yet porous (32.7% ± 5.8% open space) network which supported the attachment and in vitro proliferation of mammalian cells. ECM fibers were similarly synthesized using muscle and astrocyte cells, suggesting process robustness across different cell types. Ultimately, these ECM fibers could be utilized as an alternative to synthetics for the manufacture of woven meshes targeting wound healing or regenerative medicine applications.

Valve interstitial cell shape modulates cell contractility independent of cell phenotype.

Valve interstitial cells are dispersed throughout the heart valve and play an important role in maintaining its integrity, function, and phenotype. While prior studies have detailed the role of external mechanical and biological factors in the function of the interstitial cell, the role of cell shape in regulating contractile function, in the context of normal and diseased phenotypes, is not well understood. Thus, the aim of this study was to elucidate the link between cell shape, phenotype, and acute functional contractile output. Valve interstitial cell monolayers with defined cellular shapes were engineered via constraining cells to micropatterned protein lines (10, 20, 40, 60 or 80µm wide). Samples were cultured in either normal or osteogenic medium. Cellular shape and architecture were quantified via fluorescent imaging techniques. Cellular contractility was quantified using a valve thin film assay and phenotype analyzed via western blotting, zymography, and qRT-PCR. In all pattern widths, cells were highly aligned, with maximum cell and nuclear elongation occurring for the 10µm pattern width. Cellular contractility was highest for the most elongated cells, but was also increased in cells on the widest pattern (80µm) that also had increased CX43 expression, suggesting a role for both elongated shape and increased cell-cell contact in regulating contractility. Cells cultured in osteogenic medium had greater expression of smooth muscle markers and correspondingly increased contractile stress responses. Cell phenotype did not significantly correlate with altered cell shape, suggesting that cellular shape plays a significant role in the regulation of valve contractile function independent of phenotype.

Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells.

The performance of implantable biomaterials derived from decellularized tissue, including encouraging results with skeletal muscle, suggests that the extracellular matrix (ECM) derived from native tissue has promising regenerative potential. Yet, the supply of biomaterials derived from donated tissue will always be limited, which is why the in-vitro fabrication of ECM biomaterials that mimic the properties of tissue is an attractive alternative. Towards this end, our group has utilized a novel method to collect the ECM that skeletal muscle myoblasts secrete and form it into implantable scaffolds. The cell derived ECM contained several matrix constituents, including collagen and fibronectin that were also identified within skeletal muscle samples. The ECM was organized into a porous network that could be formed with the elongated and aligned architecture observed within muscle samples. The ECM material supported the attachment and in-vitro proliferation of cells, suggesting effectiveness for cell transplantation, and was well tolerated by the host when examined in-vivo. The results suggest that the ECM collection approach can be used to produce biomaterials with compositions and structures that are similar to muscle samples, and while the physical properties may not yet match muscle values, the in-vitro and in-vivo results indicate it may be a suitable first generation alternative to tissue derived biomaterials.