Production-scale fibronectin nanofibers promote wound closure and tissue repair in a dermal mouse model.

Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (∼1.2 ×105 s-1) and shear (∼3 ×105 s-1) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (∼10 mL/min), thus producing nanofiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.

A tissue-engineered scale model of the heart ventricle.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.

Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering.

Biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. In this context several synthetic polymers have been investigated, however a concise systematic comparative analyses is missing. Therefore, the present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymers structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGA-poly-4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly-caprolactone (PCL) patches. These patches were analyzed after 21days of culture qualitative by histology and quantitative by determining the amount of DNA, glycosaminoglycan and hydroxyproline. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds (p<0.05). Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro.

JetValve: Rapid manufacturing of biohybrid scaffolds for biomimetic heart valve replacement.

Tissue engineered scaffolds have emerged as a promising solution for heart valve replacement because of their potential for regeneration. However, traditional heart valve tissue engineering has relied on resource-intensive, cell-based manufacturing, which increases cost and hinders clinical translation. To overcome these limitations, in situ tissue engineering approaches aim to develop scaffold materials and manufacturing processes that elicit endogenous tissue remodeling and repair. Yet despite recent advances in synthetic materials manufacturing, there remains a lack of cell-free, automated approaches for rapidly producing biomimetic heart valve scaffolds. Here, we designed a jet spinning process for the rapid and automated fabrication of fibrous heart valve scaffolds. The composition, multiscale architecture, and mechanical properties of the scaffolds were tailored to mimic that of the native leaflet fibrosa and assembled into three dimensional, semilunar valve structures. We demonstrated controlled modulation of these scaffold parameters and show initial biocompatibility and functionality in vitro. Valves were minimally-invasively deployed via transapical access to the pulmonary valve position in an ovine model and shown to be functional for 15 h.

Phototactic guidance of a tissue-engineered soft-robotic ray.

Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal--a tissue-engineered ray--to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 1/10 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.

Acute pergolide exposure stiffens engineered valve interstitial cell tissues and reduces contractility in vitro.

Medications based on ergoline-derived dopamine and serotonin agonists are associated with off-target toxicities that include valvular heart disease (VHD). Reports of drug-induced VHD resulted in the withdrawal of appetite suppressants containing fenfluramine and phentermine from the US market in 1997 and pergolide, a Parkinson's disease medication, in 2007. Recent evidence suggests that serotonin receptor activity affected by these medications modulates cardiac valve interstitial cell activation and subsequent valvular remodeling, which can lead to cardiac valve fibrosis and dysfunction similar to that seen in carcinoid heart disease. Failure to identify these risks prior to market and continued use of similar drugs reaffirm the need to improve preclinical evaluation of drug-induced VHD. Here, we present two complimentary assays to measure stiffness and contractile stresses generated by engineered valvular tissues in vitro. As a case study, we measured the effects of acute (24 h) pergolide exposure to engineered porcine aortic valve interstitial cell (AVIC) tissues. Pergolide exposure led to increased tissue stiffness, but it decreased both basal and active contractile tone stresses generated by AVIC tissues. Pergolide exposure also disrupted AVIC tissue organization (i.e., tissue anisotropy), suggesting that the mechanical properties and contractile functionality of these tissues are governed by their ability to maintain their structure. We expect further use of these assays to identify off-target drug effects that alter the phenotypic balance of AVICs, disrupt their ability to maintain mechanical homeostasis, and lead to VHD.

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning.

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning.

Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro.

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G' = 0.3 kPa) to stiff (G' = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1 Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.