Creating alignment and anisotropy in engineered heart tissue: role of boundary conditions in a model three-dimensional culture system

Electrical and mechanical anisotropy arise from matrix and cellular alignment in native myocardium. Generation of anisotropy in engineered heart tissue will be required to match native properties and will provide immediate opportunities to investigate the genesis and structural determinants of functional anisotropy. We investigated the influence of geometry and boundary conditions on fibroblast alignment in thin collagen gels. Consistent with previous reports, we found that human dermal fibroblasts align parallel to free edges in partially constrained gels; in contrast to at least one report, fibroblasts in fully constrained gels remained randomly aligned independent of geometry. These experiments allowed us to distinguish between two possible mechanisms for such alignment. Mean orientations that followed the shape of the free edges and stronger alignment nearest the free edges in gels with a variety of geometries suggested that cells align parallel to a local free boundary rather than to local lines of tension. These findings focus attention on the presence of voids and free surfaces such as the endocardium and epicardium, cleavage planes, and blood vessels in governing cell and fiber alignment in developing and remodeling myocardium, myocardial scar tissue, and engineered heart constructs.     

Two-photon laser scanning microscopy of epithelial cell-modulated collagen density in engineered human lung tissue

Tissue remodeling is a complex process that can occur in response to a wound or injury. In lung tissue, abnormal remodeling can lead to permanent structural changes that are characteristic of important lung diseases such as interstitial pulmonary fibrosis and bronchial asthma. Fibroblast-mediated contraction of three-dimensional collagen gels is considered an in vitro model of tissue contraction and remodeling, and the epithelium is one factor thought to modulate this process. We studied the effects of epithelium on collagen density and contraction using two-photon laser scanning microscopy (TPLSM). TPLSM was used to image autofluorescence of collagen fibers in an engineered tissue model of the human respiratory mucosa -- a three-dimensional co-culture of human lung fibroblasts (CCD-18 lu), denatured type I collagen, and a monolayer of human alveolar epithelial cell line (A549) or human bronchial epithelial cell line (16HBE14o(-)). Tissues were imaged at days 1, 8, and 15 at 10 depths within the tissue. Gel contraction was measured concurrently with TPLSM imaging. Image analysis shows that gels without an epithelium had the fastest rate of decay of fluorescent signal, corresponding to highest collagen density. Results of the gel contraction assay show that gels without an epithelium also had the highest degree of contraction (19.8% +/- 4.0%). We conclude that epithelial cells modulate collagen density and contraction of engineered human lung tissue, and TPLSM is an effective tool to investigate this phenomenon.     

Structural Mechanism for Alteration of Collagen Gel Mechanics by Glutaraldehyde Crosslinking

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.     

Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.     

Nitrite-Induced Cross-Linking Alters Remodeling and Mechanical Properties of Collagenous Engineered Tissues

Cumulative damage to long-lived connective tissue proteins play a key role in the development of age-related human diseases such as cardiovascular stiffening and age-related macular degeneration. The processes that result in the accumulation of increasingly insoluble, undigestible damaged collagen are only partially known. Nonenzymatic glycation (NEG) is one such process and has been linked to the development of diabetic-related complications and aging. An additional novel mechanism particularly relevant to smoking- and inflammation-related diseases involves the nonenzymatic nitrite (NEN) modification of connective tissue proteins. The present study was undertaken to examine the effects of NEN of fibrillar type I collagen on cell-mediated remodeling and mechanical properties of collagenous tissues. Using a modification of an in vitro fibroblast-populated collagen gel model system developed in our laboratory, we tested two hypotheses: NEN reduces the ability of primary adult cardiac fibroblasts to remodel type I collagen gels; NEN reduces the deformability of type I collagen gels subjected to mechanical testing. The results show that NEN impairs both cell-mediated remodeling and mechanical deformability in collagenous engineered tissues. Furthermore, these mechanical changes correlate with the degree of cross-linking as determined by SDS-PAGE. Thus, we concluded that NEN reactions may contribute to alterations in the biomechanical properties of collagen-containing tissues consistent with the age-related functional decline observed in human disease.     

Nitrite-induced cross-linking alters remodeling and mechanical properties of collagenous engineered tissues

Cumulative damage to long-lived connective tissue proteins play a key role in the development of age-related human diseases such as cardiovascular stiffening and age-related macular degeneration. The processes that result in the accumulation of increasingly insoluble, undigestible damaged collagen are only partially known. Nonenzymatic glycation (NEG) is one such process and has been linked to the development of diabetic-related complications and aging. An additional novel mechanism particularly relevant to smoking- and inflammation-related diseases involves the nonenzymatic nitrite (NEN) modification of connective tissue proteins. The present study was undertaken to examine the effects of NEN of fibrillar type I collagen on cell-mediated remodeling and mechanical properties of collagenous tissues. Using a modification of an in vitro fibroblast-populated collagen gel model system developed in our laboratory, we tested two hypotheses: NEN reduces the ability of primary adult cardiac fibroblasts to remodel type I collagen gels; NEN reduces the deformability of type I collagen gels subjected to mechanical testing. The results show that NEN impairs both cell-mediated remodeling and mechanical deformability in collagenous engineered tissues. Furthermore, these mechanical changes correlate with the degree of cross-linking as determined by SDS-PAGE. Thus, we concluded that NEN reactions may contribute to alterations in the biomechanical properties of collagen-containing tissues consistent with the age-related functional decline observed in human disease.     

Thrombin and TNF-alpha/IL-1beta synergistically induce fibroblast-mediated collagen gel degradation

Degradation of preexisting and newly synthesized extracellular matrix is thought to play an important role in tissue remodeling. The current study evaluated whether thrombin and TNF-alpha/IL-1beta could collaboratively induce collagen degradation by human fetal lung fibroblasts (HFL-1) and adult bronchial fibroblasts cultured in three-dimensional collagen gels. TNF-alpha/IL-1beta alone induced production of matrix metalloproteinases (MMPs)-1, -3, and -9, which were released in latent form. With the addition of thrombin, the latent MMPs were converted into active forms and this resulted in collagen gel degradation. Part of the activation of MMPs by thrombin resulted from direct activation of MMP-1, MMP-2, MMP-3, and MMP-9 in the absence of cells. In addition, tissue inhibitor of metalloproteinase-1 production was inhibited by the combination of thrombin and TNF-alpha/IL-1beta. These results suggest that thrombin and TNF-alpha/IL-1beta synergize to induce degradation of three-dimensional collagen gels through increasing the production and activation of MMPs, and that this effect is mediated through both direct activation of MMPs by thrombin and indirectly by thrombin activation of fibroblasts. Through such mechanisms, thrombin could contribute to many chronic lung disorders characterized by tissue remodeling.     

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α-smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.     

Compressed collagen gel as the scaffold for skin engineering

Collagen gel scaffolds can potentially be utilized as cell seeded systems for skin tissue engineering. However, its dramatic contraction after being mixed with cells and its mechanical weakness are the drawbacks for its application to skin engineering. In this study, a compressed collagen gel scaffold was fabricated through the rapid expulsion of liquid from reconstituted gels by the application of ‘plastic compression’(PC) technique. Both compressed and uncompressed gels were characterized with their gel contraction rate, morphology, the viability of seeded cells, their mechanical properties and the feasibility as a scaffold for constructing tissue-engineered skin. The results showed that the compression could significantly reduce the contraction of the collagen gel and improve its mechanical property. In addition, seeded dermal fibroblasts survived well in the compressed gel and seeded epidermal cells gradually developed into a stratified epidermal layer, and thus formed tissue engineered skin. This study reveals the potential of using compressed collagen gel as a scaffold for skin engineering.     

Evaluation of Lateral Mechanical Tension in Thin-Film Tissue Constructs

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.     

Influence of cyclic strain and decorin deficiency on 3D cellularized collagen matrices

Cyclic strain evokes the expression of the small leucine-rich proteoglycans decorin and biglycan in 2D cultures and native tissues. However, strain-dependent expression of these proteoglycans has not been demonstrated in engineered tissues. We hypothesized that the absence of decorin may compromise the effect of cyclic strain on the development of engineered tissues. Thus, we investigated the contribution of decorin to tissue organization in cyclically strained collagen gels relative to statically cultured controls. Decorin null (Dcn(-/-)) and wild-type murine embryonic fibroblasts were seeded within collagen gels and mechanically conditioned using a Flexcell Tissue Train culture system. After 8 days, the cyclically strained samples demonstrated greater collagen fibril density, proteoglycan content, and material strength for both cell types. On the other hand, increases in cell density, collagen fibril diameter, and biglycan expression were observed only in the cyclically strained gels seeded with Dcn(-/-) cells. Although cyclic strain caused an elevation in proteoglycan expression regardless of cell type, the type of proteoglycan differed between groups: the Dcn(-/-) cell-seeded gels produced an excess of biglycan not found in the wild-type controls. These results suggest that decorin-mediated tissue organization is strongly dependent upon tissue type and mechanical environment.     

Production of a novel fibroblast-populated platelet matrix cocultured with keratinocytes

We have developed a new method for the production of a dermal matrix equivalent. Human platelets were used to dilute human fibroblasts. The platelet mix was placed in a cell culture well. Addition of 200 microL of a thrombin solution caused gel formation. Gels were overlaid with standard Iscove's growth medium supplemented with 10% fetal bovine serum, insulin, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer. Medium was exchanged regularly. Keratinocytes were plated on top of selected gels and elevated to the air-liquid interface. The gels were harvested weekly, fixed, cut, and stained with hematoxylin and eosin stains and immunostains for collagens I, III, and IV and cytokeratins. Digital image analysis was used to quantitate collagen production. Growth factors, including transforming growth factor-beta (TGF-beta), platelet-derived growth factor, and vitamin C were added. Staining identified fibroblasts within the gels with a surrounding fibrous matrix. Immunostaining for cytokeratin identified keratinocytes on the gel surface. Immunostaining revealed the fibrous matrix to be composed of collagen I and III and some collagen IV. Digital image analysis demonstrated that greater TGF-beta concentration resulted in greater collagen production. These differences were statistically significant. With development of this construct, a viable dermal/epidermal replacement may be possible. TGF-beta enhances collagen production by fibroblasts in this matrix.     

Modification of type I collagenous gels by alveolar epithelial cells

Contraction of type I collagen gels is an in vitro model of tissue remodeling. In addition to fibroblasts, some epithelial cells can mediate this process. We therefore hypothesized that alveolar epithelial cells might contract extracellular matrices and have the potential to directly participate in the remodeling of the lung after alveolar injury. A549 cells were plated on top of collagen gels, and the gels were floated in culture medium. A549 cells contracted the gels in a time- and cell density-dependent manner. A549 cells, as well as human bronchial epithelial cells (HBEC) and rat alveolar epithelial cells (RalvEC) contracted collagen gels more when they were plated on top of the gel than when they were embedded inside, in contrast to human fetal lung fibroblast (HFL1), which contracted more when cast inside. The amount of hydroxyproline in the collagen gels remained unchanged throughout the contraction. Anti-beta(1) integrin antibody inhibited A549 cell-mediated contraction. Transforming growth factor beta augmented the contraction by A549 cells as well as that by HBEC and HFL1. Prostaglandin E(2) inhibited the contraction by HFL1 but did not affect the contraction by A549 cells, HBEC, or RalvEC. Cytomix (a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) inhibited the contraction by HFL1 but strongly enhanced the contraction by A549 cells. Cytomix also caused a morphologic change of A549 cells from a polygonal to a spindle shape. Immunocytochemistry showed that cytomix induced alpha-tubulin expression in A549 cells, whereas cytokeratin, vimentin, smooth muscle actin, beta(1) integrin, and paxillin expressions were not changed. This study thus demonstrates that alveolar epithelial cells can cause contraction of extracellular matrices and that this process is modulated by exogenous mediators, which also modify the microtubular system. Such an activity might contribute to alveolar remodeling after injury.     

Behavior and ultrastructure of in vitro ageing mouse embryo fibroblasts grown in collagen gels

We have compared the behaviour and the ultrastructure of embryonic mouse fibroblasts embedded into collagen gels at early, middle and late population doubling levels (PDL). Late mouse fibroblasts were able to induce gel contraction with a greater efficiency than young cells. We did not find any differences in the organization of these gels. Electron microscope observations on gels containing fibroblasts of different PDL showed that the collagen lattice induced new specific and distinct phenotypes. The well-known ultrastructural differences between young and late fibroblasts grown on plastic substrates were less prominent when these cells were embedded into collagen gels. The late fibroblasts grown into gels kept their large size and their lobulated nuclei and resembled fibroblasts grown on plastic surfaces. However, dramatic changes were observed in their pattern of microfilaments, in the dispersion of their chromatin and in their ergastoplasmic structure; these characteristics observed in late fibroblasts grown into gels were close to those of young cells. The new phenotypes of young, middle-aged and late fibroblasts in the collagen gels seemed to be stable and did not display the characteristics of an older phenotype on continued incubation. When the fibroblasts left the gel, they returned to their initial phenotype.     

Differences in tissue-remodeling potential of aortic and pulmonary heart valve interstitial cells

Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.     

Mechanisms and dynamics of mechanical strengthening in ligament-equivalent fibroblast-populated collagen matrices

We have measured the dynamics of extracellular matrix consolidation and strengthening by human dermal fibroblasts in hydrated collagen gels. Constraining matrix consolidation between two porous polyethylene posts held rigidly apart set up the mechanical stress which led to the formation of uniaxially oriented fibroblast-populated collagen matrices with a histology resembling a ligament. We measured the mechanical stiffness and tensile strength of these ligament equivalents (LEs) as a function of age at biweekly intervals up to 12 weeks in culture using a mechanical spectrometer customized for performing experiments under physiologic conditions. The LE load-strain curve changed as a function of LE age, increasing in stiffness and exhibiting less plastic-like behavior. At 12 weeks, LEs had acquired up to 30 times the breaking strength of 1-week-old LEs. Matrix strengthening occurred primarily through the formation of BAPN-sensitive, lysyl oxidase catalyzed crosslinks. Sulfated glycosaminoglycan (GAG) content increased monotonically with LE age, reaching levels that are characteristic of ligaments. Cells in the LEs actively incorporated [³H]proline and [³⁵S]sulfate into the extracellular matrix. Over the first three weeks, DNA content increased rapidly but thereafter remained constant. This data represent the first documentation of strengthening kinetics for cell-assembled biopolymer gels and the results suggest that this LE tissue may be a valuable model for studying the cellular processes responsible for tissue growth, repair, and remodeling.     

Tissue-engineered valves with commissural alignment

We present a novel approach to producing bioartificial valves using the tissue-equivalent method of entrapping cells within a biopolymer gel and using a mold design that presents appropriate mechanical constraints to the cell-induced gel compaction to yield both the fibril alignment and the geometry of a native valve. Bileaflet valves were fabricated from bovine collagen and neonatal human dermal fibroblasts as proof of principle. The resultant valves possessed both commissure-tocommissure alignment of collagen fibers in the leaflets and circumferential alignment in the root. While this alignment was manifested in planar biaxial tensile mechanical properties, histology of the leaflets revealed an aligned collagen matrix but lacking other extracellular matrix (ECM) components present in the native valve. The apparent lack of ECM production by the fibroblasts after contracting and aligning the collagen fibrils is consistent with peak loads during biaxial testing being only approximately 10% of native leaflet values and a 0:1 coupling index that was only approximately 50% of native leaflet values despite exhibiting comparable values for the anisotropy index.     

Effects of substratum surface topography on the organization of cells and collagen fibers in collagen gel cultures

This study investigated the orientation of fibroblasts and collagen cultured on microfabricated grooved or smooth titanium surfaces, as well as on tissue culture polystyrene, in the presence or absence of collagen gels. The gels were first added either to the confluent fibroblast culture on the surface (cell-gel condition) or to the fibroblasts were suspended within the collagen gel and then placed onto the surface (gel condition). Cells and collagen were observed with differential interference, polarization, and confocal laser scanning microscopy. Although the smooth surfaces had no effect on cell orientation in the gel for the first 2 weeks of culture, cells did orient with grooves regardless of the culture conditions. There was evidence for orthogonal multilayering of cells under the cell-gel condition at 4 weeks, and collagen alignment reflected cell alignment. The interaction of the collagen gel with the surface depended on whether the cell-gel or the gel condition was employed. In the former condition, the gel contracted toward the substratum, whereas the gel condition resulted in the formation of a ring of collagen loosely attached to the substratum. These results suggest that the order in which fibroblasts encounter substratum and extracellular matrix can influence the eventual matrix-cell interactions, and that substratum topography can influence matrix and cell orientation in zones not immediately in contact with the surface.     

Prostacyclin analogs inhibit fibroblast contraction of collagen gels through the cAMP-PKA pathway

Prostacyclin is an arachidonic acid metabolite that modulates vascular tone within the lung. The current study evaluated the hypothesis that prostacyclin can also modulate tissue remodeling by affecting fibroblast-mediated contraction of extracellular matrix. To accomplish this, fibroblasts were cultured in three-dimensional native type I collagen gels in the presence of prostacyclin analogs: carbaprostacyclin, iloprost, and beraprost. All three analogs significantly inhibited contraction of the three-dimensional collagen gels mediated by three different fibroblasts. All three analogs significantly inhibited fibronectin release and reduced fibroblast fibronectin mRNA expression. Addition of exogenous fibronectin restored the contractile activity to fibroblasts incubated in the presence of all three analogs. Iloprost and beraprost significantly activated cAMP-dependent protein kinase-A (PKA), and an action through this pathway was confirmed by blockade of the inhibitory effect on contraction and fibronectin release with the PKA inhibitor KT-5720. In contrast, carbaprostacyclin, which is not as selective for the prostacyclin (IP) receptor, did not activate PKA, and its effects on contraction and fibronectin release were not fully blocked by KT-5720. Finally, the cAMP analogs N(6)-Benzoyl- (6-Bnz-) cAMP and dibutyryl-cAMP inhibited contraction, and this contrasted with the activity of an Epac selective agonist 8-pCPT-2'-O-Me-cAMP, which had no effect. Taken together, these results indicate that prostacyclin, acting through the IP receptor and by activating PKA, can lead to inhibition of fibronectin release and can subsequently inhibit fibroblast-mediated collagen gel contraction. The ability of prostacyclin to modulate fibroblast function suggests that prostacyclin can contribute to tissue remodeling.     

Prediction of equibiaxial loading stress in collagen-based extracellular matrix using a three-dimensional unit cell model

Mechanical signals are important factors in determining cell fate. Therefore, insights as to how mechanical signals are transferred between the cell and its surrounding three-dimensional collagen fibril network will provide a basis for designing the optimum extracellular matrix (ECM) microenvironment for tissue regeneration. Previously we described a cellular solid model to predict fibril microstructure–mechanical relationships of reconstituted collagen matrices due to unidirectional loads (Acta Biomater 2010;6:1471–86). The model consisted of representative volume elements made up of an interconnected network of flexible struts. The present study extends this work by adapting the model to account for microstructural anisotropy of the collagen fibrils and a biaxial loading environment. The model was calibrated based on uniaxial tensile data and used to predict the equibiaxial tensile stress–stretch relationship. Modifications to the model significantly improved its predictive capacity for equibiaxial loading data. With a comparable fibril length (model 5.9–8 μm, measured 7.5 μm) and appropriate fibril anisotropy the anisotropic model provides a better representation of the collagen fibril microstructure. Such models are important tools for tissue engineering because they facilitate prediction of microstructure–mechanical relationships for collagen matrices over a wide range of microstructures and provide a framework for predicting cell–ECM interactions.