Bio-Inspired Drug Delivery Noting that platelets naturally migrate to narrowed blood vessels characterized by high fluid shear stress, Korin et al. (p. 738 , published online 5 July; see the Perspective by Lavik and Ustin ) developed a nanoparticle-based therapeutic that uses a similar targeting mechanism to deliver a drug to vessels obstructed by blood clots. Aggregates of nanoparticles coated with the clot-dissolving drug tPA (tissue plasminogen activator) were designed to fall apart and release the drug only when encountering high fluid shear stress. In preclinical models, the bio-inspired therapeutic dissolved clots and restored normal blood flow at lower doses than free tPA, suggesting that this localized delivery system may help reduce the risk of side effects such as excessive bleeding.

Skin, the largest organ in the body, protects against toxins and microorganisms in the environment and serves to prevent dehydration of all non-aquatic animals. Immune surveillance, sensory detection, and self-healing are other critical functions of the skin. Loss of skin integrity because of injury or illness may result acutely in substantial physiologic imbalance and ultimately in significant disability or even death. It is estimated that, in 1992, there were 35.2 million cases of significant skin loss (US data) that required major therapeutic intervention. Of these, approximately 7 million wounds become chronic. Regardless of the specific advanced wound care product, the ideal goal would be to regenerate tissues such that both the structural and functional properties of the wounded tissue are restored to the levels before injury. The advent of tissue-engineered skin replacements revolutionized the therapeutic potential for recalcitrant wounds and for wounds that are not amenable to primary closure. This article will introduce the reader to the field of tissue engineering, briefly review tissue-engineered skin replacement from a historical perspective and then review current state-of-the-art concepts from our vantage point.

Abstract-Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response; however, the molecular basis remains unknown. Here, we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase-dependent activation and binding of additional ␤1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent "integrin-to-integrin" signaling mechanism mediated by force-induced activation of mechanically gated TRPV4 ion channels on the cell surface. Key Words: mechanical strain Ⅲ integrin Ⅲ TRPV4 Ⅲ endothelial cell Ⅲ reorientation Ⅲ cytoskeleton M echanical forces regulate vascular growth and development by influencing endothelial cell growth, survival, differentiation and migration. 1,2 Local mechanical cues conveyed by extracellular matrix (ECM) attributable to cyclic deformation of blood vessels, hemodynamic forces, or cellgenerated traction forces are also potent inducers of directional capillary blood vessel growth and vascular remodeling in vitro and in vivo. [3][4][5][6][7][8][9][10] For example, the initial step in neovascularization involves reorientation of a subset of capillary endothelial (CE) cells that spread and migrate perpendicular to the main axis of the preexisting vessel toward the angiogenic stimulus 11 ; however, the molecular mechanism responsible for this CE cell reorientation response is unknown. Many cell types, including large vessel endothelial cells, realign perpendicular to the direction of the applied force when they experience cyclic stretching (mechanical strain). [12][13][14][15] In the case of macrovascular endothelium, this reorientation response can be prevented by treatment with chemical inhibitors of stretch-activated (SA) ion channels. 15 But neither the identity of these channels nor the mechanism by which they elicit cell reorientation is known.Endothelial cells express most members of the transient receptor potential (TRP) family of ion channels 16 -18 and TRP vanilloid (TRPV)4 has been reported to mediate flowinduced vasodilation in large vessel endothelium. 19 -22 Here, we show that calcium influx through TRPV4 channels stimulated by mechanically stretching CE cells through their integrin-extracellular matrix (ECM) adhesions promotes cell reorientation by activating phosphatidylinositol 3-kinase (PI3K), thereby stimulating activ...

To successfully induce tissue repair or regeneration in vivo, bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli. In this report, we provide a fundamental understanding of how alterations in mechanical stimuli alone, produced by varying the viscoelastic properties of our bioengineered construct, modulate phenotypic behavior at the whole-cell level. Using a physiologically relevant ECM mimic composed of hyaluronan and fibronectin, we found that adult human dermal fibroblasts modify their mechanical response in order to match substrate stiffness. More specifically, the cells on stiffer substrates had higher modulus and a more stretched and organized actin cytoskeleton (and vice versa), which translated into larger traction forces exerted on the substrate. This modulation of cellular mechanics had contrasting effects on migration and proliferation, where cells migrated faster on softer substrates while proliferating preferentially on the stiffer ones. These findings implicate substrate rigidity as a critical design parameter in the development of bioengineered constructs aimed at eliciting maximal cell and tissue function.

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.

Tumor vessels are characterized by abnormal morphology and hyper-permeability that together cause inefficient delivery of chemotherapeutic agents. Although VEGF has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here, we show that the mechanosensitive ion channel TRPV4 regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TEC exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards ECM stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 KO mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anti-cancer drug Cisplatin, significantly reduced tumor growth in WT mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

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Tumor blood vessels exhibit abnormal structure and function that cause disturbed blood flow and high interstitial pressure, which impair delivery of anti-cancer agents. Past efforts to normalize the tumor vasculature have focused on inhibition of soluble angiogenic factors, such as VEGF; however, capillary endothelial (CE) cell growth and differentiation during angiogenesis are also influenced by mechanical forces conveyed by the extracellular matrix (ECM). Here, we explored the possibility that tumor CE cells form abnormal vessels because they lose their ability to sense and respond to these physical cues. These studies reveal that, in contrast to normal CE cells, tumor-derived CE cells fail to reorient their actin cytoskeleton when exposed to uniaxial cyclic strain, exhibit distinct shape sensitivity to variations in ECM elasticity, exert greater traction force, and display an enhanced ability to retract flexible ECM substrates and reorganize into tubular networks in vitro. These behaviors correlate with a constitutively high level of baseline activity of the small GTPase Rho and its downstream effector, Rho-associated kinase (ROCK). Moreover, decreasing Rho-mediated tension by using the ROCK inhibitor, Y27632, can reprogram the tumor CE cells so that they normalize their reorientation response to uniaxial cyclic strain and their ability to form tubular networks on ECM gels. Abnormal Rho-mediated sensing of mechanical cues in the tumor microenvironment may therefore contribute to the aberrant behaviors of tumor CE cells that result in the development of structural abnormalities in the cancer microvasculature.capillary ͉ contractility ͉ elasticity ͉ GTPase ͉ mechanotransduction