α5ß1-Integrin promotes tension-dependent mammary epithelial cell invasion by engaging the fibronectin synergy site.

Tumors are fibrotic and characterized by abundant, remodeled, and cross-linked collagen that stiffens the extracellular matrix stroma. The stiffened collagenous stroma fosters malignant transformation of the tissue by increasing tumor cell tension to promote focal adhesion formation and potentiate growth factor receptor signaling through kinase. Importantly, collagen cross-linking requires fibronectin (FN). Fibrotic tumors contain abundant FN, and tumor cells frequently up-regulate the FN receptor α5ß1 integrin. Using transgenic and xenograft models and tunable two- and three-dimensional substrates, we show that FN-bound α5ß1 integrin promotes tension-dependent malignant transformation through engagement of the synergy site that enhances integrin adhesion force. We determined that ligation of the synergy site of FN permits tumor cells to engage a zyxin-stabilized, vinculin-linked scaffold that facilitates nucleation of phosphatidylinositol (3,4,5)-triphosphate at the plasma membrane to enhance phosphoinositide 3-kinase (PI3K)-dependent tumor cell invasion. The data explain why rigid collagen fibrils potentiate PI3K activation to promote malignancy and offer a perspective regarding the consistent up-regulation of α5ß1 integrin and FN in many tumors and their correlation with cancer aggression.

A self-assembling peptide matrix used to control stiffness and binding site density supports the formation of microvascular networks in three dimensions.

A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ∼60 to 6000Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ∼2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.

Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties.

The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.

Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis.

Appropriate choice of biomaterial supports is critical for the study of capillary morphogenesis in vitro as well as to support vascularization of engineered tissues in vivo. Self-assembling peptides are a class of synthetic, ionic, oligopeptides that spontaneously assemble into gels with an ECM-like microarchitecture when exposed to salt. In this paper, the ability of four different self-assembling peptide gels to promote endothelial cell adhesion and capillary morphogenesis is explored. Human umbilical vein endothelial cells (HUVECs) were cultured within ionic self-assembling peptide family members, RAD16-I ((RADA)(4)), RAD16-II ((RARADADA)(2)), KFE-8 ((FKFE)(2)), or KLD-12 ((KLDL)(3)). HUVECs suspended in RAD16-I or RAD16-II gels elongated and formed interconnected capillary-like networks resembling in vivo capillaries, while they remained round and formed clusters within KFE-8 or KLD-12 gels. As HUVECs attach to RAD16-I and RAD16-II significantly better than the other peptides, these differences appear to be explained by differences in cell adhesion. Although adhesion likely occurs via bound adhesion proteins, there appears to be no difference in protein binding to the peptides investigated. Results indicate that, although these oligopeptides have similar mechanisms of self- assembly, their primary sequence can greatly affect cell adhesion. Additionally, a subset of these biomimetic ECM-like materials support capillary morphogenesis and thus may be useful for supporting vascularization.

The stiffness of three-dimensional ionic self-assembling peptide gels affects the extent of capillary-like network formation.

Improving our ability to control capillary morphogenesis has implications for not only better understanding of basic biology, but also for applications in tissue engineering and in vitro testing. Numerous biomaterials have been investigated as cellular supports for these applications and the biophysical environment biomaterials provide to cells has been increasingly recognized as an important factor in directing cell function. Here, the ability of ionic self-assembling peptide gels to support capillary morphogenesis and the effect of their mechanical properties is investigated. When placed in a physiological salt solution, these oligopeptides spontaneously self-assemble into gels with an extracellular matrix (ECM)-like microarchitecture. To evaluate the ability of three-dimensional (3D) self-assembled peptide gels to support capillary-like network formation, human umbilical vein endothelial cells (HUVECs) were embedded within RAD16-I ((RADA)4) or RAD16-II ((RARADADA)2) peptide gels with various stiffness values. As peptide stiffness is decreased cells show increased elongation and are increasingly able to contract gels. The observation that capillary morphogenesis is favored in more malleable substrates is consistent with previous reports using natural biomaterials. The structural properties of peptide gels and their ability to support capillary morphogenesis in vitro make them promising biomaterials to investigate for numerous biomedical applications.

Improved microvascular network in vitro by human blood outgrowth endothelial cells relative to vessel-derived endothelial cells.

Evidence suggests that bone marrow-derived cells circulating in adult blood, sometimes called endothelial progenitor cells, contribute to neovascularization in vivo and give rise to cells expressing endothelial markers in culture. To explore the utility of blood-derived cells expressing an endothelial phenotype for creating tissue-engineered microvascular networks, we employed a three-dimensional in vitro angiogenesis model to compare microvascular network formation by human blood outgrowth endothelial cells (HBOECs) with three human vessel-derived endothelial cell (EC) types: human umbilical vein ECs (HUVECs), and adult and neonatal human microvascular ECs. Under every condition investigated, HBOECs within collagen gels elongated significantly more than any other cell type. Under all conditions investigated, gel contraction and cell elongation were correlated, with HBOECs demonstrating the largest generation of force. HBOECs did not exhibit a survival advantage, nor did they enhance elongation of HUVECs when the two cell types were cocultured. Network formation of both HBOECs and HUVECs was inhibited by blocking antibodies to alpha2beta1, but not alpha(v)beta3, integrins. Taken together, these data suggest that superior network exhibited by HBOECs relative to vessel-derived endothelial cells is not due to a survival advantage, use of different integrins, or secretion of an autocrine/paracrine factor, but may be related to increased force generation.

The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro.

When suspended in collagen gels, endothelial cells elongate and form capillary-like networks containing lumens. Human blood outgrowth endothelial cells (HBOEC) suspended in relatively rigid 3 mg/ml floating collagen gels, formed in vivo-like, thin, branched multi-cellular structures with small, thick-walled lumens, while human umbilical vein endothelial cells (HUVEC) formed fewer multi-cellular structures, had a spread appearance, and had larger lumens. HBOEC exert more traction on collagen gels than HUVEC as evidenced by greater contraction of floating gels. When the stiffness of floating gels was decreased by decreasing the collagen concentration from 3 to 1.5 mg/ml, HUVEC contracted gels more and formed thin, multi-cellular structures with small lumens, similar in appearance to HBOEC in floating 3 mg/ml gels. In contrast to floating gels, traction forces exerted by cells in mechanically constrained gels encounter considerable resistance. In constrained collagen gels (3 mg/ml), both cell types appeared spread, formed structures with fewer cells, had larger, thinner-walled lumens than in floating gels, and showed prominent actin stress fibers, not seen in floating gels. These results suggest that the relative magnitudes of cellular force generation and apparent matrix stiffness modulate capillary morphogenesis in vitro and that this balance may play a role in regulating angiogenesis in vivo.

Bone morphogenetic protein 9: a potent modulator of cartilage development in vitro.

Few publications describe the activity of bone morphogenetic protein-9 (BMP-9), but the consensus of these largely in vivo studies is that while BMP-9 can induce ectopic bone formation at relatively large concentrations, it is primarily active in non-skeletal locations--including the liver, nervous system and marrow. To study the effects of BMP-9 on chondrogenesis in a well-defined environment, calf articular chondrocytes were seeded onto biodegradable PGA scaffolds. The resulting cell-polymer constructs were cultured in either control medium or medium supplemented with 1, 10, 50 or 100 ng/ml of BMP-9. After 4 weeks of in vitro culture, all concentrations of BMP-9 increased the total mass of the constructs, and the amounts of collagen, glycosaminoglycans (GAG) and cells per construct. On a mass percentage basis, BMP-9 tended to increase GAG, to decrease the relative amount of collagen and had little effect on the relative amount of cells. BMP-9 elicited qualitatively similar responses as BMP-2, -12 and -13. However, in contrast to BMP-12 and -13, BMP-9 (at concentrations > or = 10 ng/ml) induced hypertrophic chondrocyte formation and was the only BMP tested to induce mineralization. Taken together, these data suggest that BMP-9 is a potent modulator of cartilage development in vitro.

Bone morphogenetic proteins-2, -12, and -13 modulate in vitro development of engineered cartilage.

Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in either control medium or medium supplemented with 1, 10, or 100 ng/mL of bone morphogenetic proteins (BMPs) BMP-2, BMP-12, or BMP-13. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks in vitro macroscopically and histologically resembled native cartilage. Addition of 100 ng/mL of BMP-2, BMP-12, or BMP-13 increased the total mass of the constructs relative to the controls by 121%, 80%, and 62%, respectively, which was accompanied by increases in the absolute amounts of collagen, glycosaminoglycans (GAG), and cells. The addition of 100 ng/mL of BMP-2, BMP-12, or BMP-13 increased the weight percentage of GAG in the constructs by 27%, 18%, and 15%, and decreased the weight percent of total collagen to 63%, 89%, and 83% of controls, respectively. BMP-2, but not BMP-12 or BMP-13 promoted chondrocyte hypertrophy. Taken together, these data suggest that BMP-2, BMP-12, and BMP-13 increase growth rate and modulate the composition of engineered cartilage and that 100 ng/mL of BMP-2 has the greatest effect. In addition, in vitro engineered cartilage provides a system for studying the effects of BMPs on chondrogenesis in a well-defined environment.

Systemic delivery of human growth hormone using genetically modified tissue-engineered microvascular networks: prolonged delivery and endothelial survival with inclusion of nonendothelial cells.

Endothelial cells have the potential to provide efficient long-term delivery of therapeutic proteins to the circulation if a sufficient number of genetically modified endothelial cells can be incorporated into the host vasculature and if these cells persist for an adequate period of time. Here we describe the ability of nonendothelial cells to modulate the survival of implanted endothelial cells and their incorporation into host vasculature. Bovine aortic endothelial cells (BAECs) suspended in Matrigel and cultured in vitro remained spherical and decreased in number over time. Subcutaneous implantation of gels containing BAECs secreting human growth hormone (hGH) in mice initially resulted in detectable plasma hGH levels, which were undetectable after 2 weeks. When mixed with fibroblasts and suspended in Matrigel, hGH-secreting BAECs formed microvascular networks in vitro. Implantation of these gels resulted in plasma hGH levels that decreased slightly over 2 weeks and then remained stable for at least 6 weeks. BAECs incorporated into blood vessels within both the implant and fibrous capsule that surrounded and invaded implants. Within implants containing BAECs and fibroblasts, viable BAECs were present for at least 6 weeks at a higher density than in implants containing BAECs alone at 3 weeks. These results indicate that implanted BAECs can incorporate into host blood vessels and that inclusion of fibroblasts in this system prolongs BAEC survival and hGH delivery.

IGF-I and mechanical environment interact to modulate engineered cartilage development.

Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured for four weeks using in vitro systems providing different mechanical environments (static and mixed Petri dishes, static and mixed flasks, and rotating vessels) and different biochemical environments (medium with and without supplemental insulin-like growth factor I, IGF-I). Under all conditions, the resulting engineered tissue histologically resembled cartilage and contained its major constituents: glycosaminoglycans, collagen, and cells. The mechanical environment and supplemental IGF-I (a) independently modulated tissue morphology, growth, biochemical composition, and mechanical properties (equilibrium modulus) of engineered cartilage as previously reported; (b) interacted additively or in some cases nonadditively producing results not suggested by the independent responses, and (c) in combination produced tissue superior to that obtained by modifying these factors individually.

Biomaterial-microvasculature interactions.

The utility of implanted sensors, drug-delivery systems, immunoisolation devices, engineered cells, and engineered tissues can be limited by inadequate transport to and from the circulation. As the primary function of the microvasculature is to facilitate transport between the circulation and the surrounding tissue, interactions between biomaterials and the microvasculature have been explored to understand the mechanisms controlling transport to implanted objects and ultimately improve it. This review surveys work on biomaterial-microvasculature interactions with a focus on the use of biomaterials to regulate the structure and function of the microvasculature. Several applications in which biomaterial-microvasculature interactions play a crucial role are briefly presented. These applications provide motivation and framework for a more in-depth discussion of general principles that appear to govern biomaterial-microvasculature interactions (i.e., the microarchitecture and physio-chemical properties of a biomaterial as well as the local biochemical environment).