In Vitro Matured Human Pluripotent Stem Cell-Derived Cardiomyocytes Form Grafts With Enhanced Structure and Function in Injured Hearts.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. METHODS: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. RESULTS: We demonstrated the economic generation of >1×108 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. CONCLUSIONS: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

Sensitivity and Validation of Porous Membrane Electrical Cell Substrate Impedance Spectroscopy (PM-ECIS) for Measuring Endothelial Barrier Properties.

Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here, we advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 µm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest being the most sensitive (p < 0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p < 0.001). Barrier disruption with both EGTA and thrombin was detectable by all electrode sizes. Resistances measured by PM-ECIS vs TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r > 0.9; p < 0.0001), but only with 750 µm electrodes for endothelial monolayers (r = 0.71; p = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.

Assessing engineered tissues and biomaterials using ultrasound imaging: In vitro and in vivo applications.

Quantitative assessment of the structural, functional, and mechanical properties of engineered tissues and biomaterials is fundamental to their development for regenerative medicine applications. Ultrasound (US) imaging is a non-invasive, non-destructive, and cost-effective technique capable of longitudinal and quantitative monitoring of tissue structure and function across centimeter to sub-micron length scales. Here we present the fundamentals of US to contextualize its application for the assessment of biomaterials and engineered tissues, both in vivo and in vitro. We review key studies that demonstrate the versatility and broad capabilities of US for clinical and pre-clinical biomaterials research. Finally, we highlight emerging techniques that further extend the applications of US, including for ultrafast imaging of biomaterials and engineered tissues in vivo and functional monitoring of stem cells, organoids, and organ-on-a-chip systems in vitro.

High-frequency quantitative ultrasound for the assessment of the acoustic properties of engineered tissues in vitro.

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the speed of sound, acoustic impedance, and acoustic attenuation of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were independent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contraction. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p < 0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accurate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity. STATEMENT OF SIGNIFICANCE: High-frequency ultrasound can measure the acoustic properties of engineered tissues non-invasively and non-destructively with µm-scale resolution. Acoustic properties, including acoustic attenuation, are related to intrinsic material properties, such as scatterer density. We developed an analytical approach to estimate the acoustic properties of cell-laden hydrogels that accounts for the frequency-dependent effects of attenuation in coupling media, the reflection/transmission of ultrasound waves at the coupling interfaces, and the dependency of measurements on hydrogel thickness. Despite up to 60% reduction in hydrogel thickness due to cell-mediated contraction, our approach enabled measurements of acoustic properties that were substantially independent of thickness. Acoustic attenuation increased significantly with increasing cell concentration (p < 0.001), demonstrating the ability of acoustic attenuation to reflect intrinsic physical properties of engineered tissues.

Design of a Mechanobioreactor to Apply Anisotropic, Biaxial Strain to Large Thin Biomaterials for Tissue Engineered Heart Valve Applications.

Repair and replacement solutions for congenitally diseased heart valves capable of post-surgery growth and adaptation have remained elusive. Tissue engineered heart valves (TEHVs) offer a potential biological solution that addresses the drawbacks of existing valve replacements. Typically, TEHVs are made from thin, fibrous biomaterials that either become cell populated in vitro or in situ. Often, TEHV designs poorly mimic the anisotropic mechanical properties of healthy native valves leading to inadequate biomechanical function. Mechanical conditioning of engineered tissues with anisotropic strain application can induce extracellular matrix remodelling to alter the anisotropic mechanical properties of a construct, but implementation has been limited to small-scale set-ups. To address this limitation for TEHV applications, we designed and built a mechanobioreactor capable of modulating biaxial strain anisotropy applied to large, thin, biomaterial sheets in vitro. The bioreactor can independently control two orthogonal stretch axes to modulate applied strain anisotropy on biomaterial sheets from 13 × 13 mm2 to 70 × 40 mm2. A design of experiments was performed using experimentally validated finite element (FE) models and demonstrated that biaxial strain was applied uniformly over a larger percentage of the cell seeded area for larger sheets (13 × 13 mm2: 58% of sheet area vs. 52 × 31 mm2: 86% of sheet area). Furthermore, bioreactor prototypes demonstrated that over 70% of the cell seeding area remained uniformly strained under different prescribed protocols: equibiaxial amplitudes between 5 to 40%, cyclic frequencies between 0.1 to 2.5 Hz and anisotropic strain ratios between 0:1 (constrained uniaxial) to 2:1. Lastly, proof-of-concept experiments were conducted where we applied equibiaxial (εx = εy = 8.75%) and anisotropic (εx = 12.5%, εy = 5%) strain protocols to cell-seeded, electrospun scaffolds. Cell nuclei and F-actin aligned to the vector-sum strain direction of each prescribed protocol (nuclei alignment: equibiaxial: 43.2° ± 1.8°, anisotropic: 17.5° ± 1.7°; p < 0.001). The abilities of this bioreactor to prescribe different strain amplitude, frequency and strain anisotropy protocols to cell-seeded scaffolds will enable future studies into the effects of anisotropic loading protocols on mechanically conditioned TEHVs and other engineered planar connective tissues.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e-8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

Recent Progress Toward Clinical Translation of Tissue-Engineered Heart Valves.

Surgical replacement remains the primary option to treat the rapidly growing number of patients with severe valvular heart disease. Although current valve replacements-mechanical, bioprosthetic, and cryopreserved homograft valves-enhance survival and quality of life for many patients, the ideal prosthetic heart valve that is abundantly available, immunocompatible, and capable of growth, self-repair, and life-long performance has yet to be developed. These features are essential for pediatric patients with congenital defects, children and young adult patients with rheumatic fever, and active adult patients with valve disease. Heart valve tissue engineering promises to address these needs by providing living valve replacements that function similarly to their native counterparts. This is best evidenced by the long-term clinical success of decellularised pulmonary and aortic homografts, but the supply of homografts cannot meet the demand for replacement valves. A more abundant and consistent source of replacement valves may come from cellularised valves grown in vitro or acellular off-the-shelf biomaterial/tissue constructs that recellularise in situ, but neither tissue engineering approach has yet achieved long-term success in preclinical testing. Beyond the technical challenges, heart valve tissue engineering faces logistical, economic, and regulatory challenges. In this review, we summarise recent progress in heart valve tissue engineering, highlight important outcomes from preclinical and clinical testing, and discuss challenges and future directions toward clinical translation.

Influence of substrate stiffness on the phenotype of heart cells.

Adult cardiomyocytes (CM) retain little capacity to regenerate, which motivates efforts to engineer heart tissues that can emulate the functional and mechanical properties of native myocardium. Although the effects of matrix stiffness on individual CM have been explored, less attention was devoted to studies at the monolayer and the tissue level. The purpose of this study was to characterize the influence of substrate mechanical stiffness on the heart cell phenotype and functional properties. Neonatal rat heart cells were seeded onto collagen-coated polyacrylamide (PA) substrates with Young's moduli of 3, 22, 50, and 144 kPa. Collagen-coated glass coverslips without PA represented surfaces with effectively "infinite" stiffness. The local elastic modulus of native neonatal rat heart tissue was measured to range from 4.0 to 11.4 kPa (mean value of 6.8 kPa) and for native adult rat heart tissue from 11.9 to 46.2 kPa (mean value of 25.6 kPa), motivating our choice of the above PA gel stiffness. Overall, by 120 h of cultivation, the lowest stiffness PA substrates (3 kPa) exhibited the lowest excitation threshold (ET; 3.5 +/- 0.3 V/cm), increased troponin I staining (52% positively stained area) but reduced cell density, force of contraction (0.18 +/- 0.1 mN/mm(2)), and cell elongation (aspect ratio = 1.3-1.4). Higher stiffness (144 kPa) PA substrates exhibited reduced troponin I staining (30% positively stained area), increased fibroblast density (70% positively stained area), and poor electrical excitability. Intermediate stiffness PA substrates of stiffness comparable to the native adult rat myocardium (22-50 kPa) were found to be optimal for heart cell morphology and function, with superior elongation (aspect ratio > 4.3), reasonable ET (ranging from 3.95 +/- 0.8 to 4.4 +/- 0.7 V/cm), high contractile force development (ranging from 0.52 +/- 0.2 to 1.60 +/- 0.6 mN/mm(2)), and well-developed striations, all consistent with a differentiated phenotype.

Molecular markers of early orthodontic tooth movement.

OBJECTIVE: To understand the molecular basis of early orthodontic tooth movement by looking at the expression of KI-67, runt-related transcription factor 2 (Runx2), and tumor necrosis factor ligand superfamily member 11 (RANKL) proteins. MATERIALS AND METHODS: We employed a rat model of early orthodontic tooth movement using a split-mouth design (where contralateral side serves as a control) and performed immunohistochemical staining to map the spatial expression patterns of three proteins at 3 and 24 hours after appliance insertion. RESULTS: We observed increased expression of KI-67, a proliferation marker, and RANKL, a molecule associated with osteoclastic differentiation, in the compression sites of the periodontal ligament subjected to 3 hours of force. In contrast, there was increased expression of KI-67 and Runx2, a marker of osteoblast precursors, in tension areas after 24 hours of force. Decreased KI-67 expression in the mesial and distal regions of the periodontal ligament was observed at the midpoint of the tooth root. CONCLUSIONS: The early RANKL expression indicates that at this early stage cells are involved in osteoclast precursor signaling. Also, decreased KI-67 expression found near the midpoint of the tooth root is believed to represent the center of rotation, providing a molecular means of visualizing mechanical loading patterns.

Substrate architecture and fluid-induced shear stress during chondrocyte seeding: role of alpha5beta1 integrin.

Chondrocyte behaviour has been shown previously to be influenced by the architecture of the substrate on which the cells are grown. Chondrocytes cultured on fully porous titanium alloy substrates showed greater spreading and more matrix accumulation when compared to cells grown on porous-coated substrates with solid bases. We hypothesized that these features developed because of differences in fluid-induced shear stresses due to substrate architecture and that integrins mediate these responses. Computational fluid dynamics analyses predicted that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on porous-coated substrates with solid bases where media flow-through is restricted. To validate this model, the seeding protocol was modulated to affect fluid flow and this affected cell spreading and matrix accumulation as predicted. Integrin blocking experiments revealed that alpha5beta1 integrins regulated cell shape under these two conditions and when cell spreading was prevented the increased accumulation of collagen and proteoglycans by chondrocytes seeded on fully porous substrates did not occur. Identifying the substrate-induced mechanical and molecular mechanisms that influence chondrocyte behaviour and tissue formation may ultimately lead to the formation of a tissue that more closely resembles natural articular cartilage.

Microdevice arrays with strain sensors for 3D mechanical stimulation and monitoring of engineered tissues.

Native and engineered tissue development are regulated by the integrative effects of multiple microenvironmental stimuli. Microfabricated bioreactor array platforms can efficiently dissect cue-response networks, and have recently integrated critical 2D and 3D mechanical stimulation for greater physiological relevance. However, a limitation of these approaches is that assessment of tissue functional properties is typically limited to end-point analyses. Here we report a new deformable membrane platform with integrated strain sensors that enables mechanical stretching or compression of 3D cell-hydrogel arrays and simultaneous measurement of hydrogel construct stiffness in situ. We tested the ability of the integrated strain sensors to measure the evolution of the stiffness of cell-hydrogel constructs for two cases. First, we demonstrated in situ stiffness monitoring of degradable poly (ethylene glycol)-norbornene (PEG-NB) hydrogels embedded with mesenchymal stromal cells (MSCs) and cultured with or without cyclic tensile stimulation for up to 15 days. Whereas statically-cultured hydrogels degraded and softened throughout the culture period, mechanically-stimulated gels initially softened and then recovered their stiffness corresponding to extensive cell network and collagen production. Second, we demonstrated in situ measurement of compressive stiffening of MSC-seeded PEG-NB gels cultured statically under osteogenic conditions, corresponding to increased mineralization and cellularization. This measurement technique can be generalized to other relevant bioreactor and organ-on-a-chip platforms to facilitate online, non-invasive, and high-throughput functional analysis, and to provide insights into the dynamics of engineered tissue development that are otherwise not available.

Combinatorial screening of 3D biomaterial properties that promote myofibrogenesis for mesenchymal stromal cell-based heart valve tissue engineering.

The physical and chemical properties of a biomaterial integrate with soluble cues in the cell microenvironment to direct cell fate and function. Predictable biomaterial-based control of integrated cell responses has been investigated with two-dimensional (2D) screening platforms, but integrated responses in 3D have largely not been explored systematically. To address this need, we developed a screening platform using polyethylene glycol norbornene (PEG-NB) as a model biomaterial with which the polymer wt% (to control elastic modulus) and adhesion peptide types (RGD, DGEA, YIGSR) and densities could be controlled independently and combinatorially in arrays of 3D hydrogels. We applied this platform and regression modeling to identify combinations of biomaterial and soluble biochemical (TGF-ß1) factors that best promoted myofibrogenesis of human mesenchymal stromal cells (hMSCs) in order to inform our understanding of regenerative processes for heart valve tissue engineering. In contrast to 2D culture, our screens revealed that soft hydrogels (low PEG-NB wt%) best promoted spread myofibroblastic cells that expressed high levels of α-smooth muscle actin (α-SMA) and collagen type I. High concentrations of RGD enhanced α-SMA expression in the presence of TGF-ß1 and cell spreading regardless of whether TGF-ß1 was in the culture medium. Strikingly, combinations of peptides that maximized collagen expression depended on the presence or absence of TGF-ß1, indicating that biomaterial properties can modulate MSC response to soluble signals. This combination of a 3D biomaterial array screening platform with statistical modeling is broadly applicable to systematically identify combinations of biomaterial and microenvironmental conditions that optimally guide cell responses. STATEMENT OF SIGNIFICANCE: We present a novel screening platform and methodology to model and identify how combinations of biomaterial and microenvironmental conditions guide cell phenotypes in 3D. Our approach to systematically identify complex relationships between microenvironmental cues and cell responses enables greater predictive power over cell fate in conditions with interacting material design factors. We demonstrate that this approach not only predicts that mesenchymal stromal cell (MSC) myofibrogenesis is promoted by soft, porous 3D biomaterials, but also generated new insights which demonstrate how biomaterial properties can differentially modulate MSC response to soluble signals. An additional benefit of the process includes utilizing both parametric and non parametric analyses which can demonstrate dominant significant trends as well as subtle interactions between biochemical and biomaterial cues.

Deriving vascular smooth muscle cells from mesenchymal stromal cells: Evolving differentiation strategies and current understanding of their mechanisms.

Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function. Regeneration of functional vascular smooth muscle tissue to repair vascular diseases is an area of intense research in tissue engineering and regenerative medicine. For functional vascular smooth muscle tissue regeneration to become a practical therapy over the next decade, the field will need to have access to VSMC sources that are effective, robust and safe. While pluripotent stem cells hold good future promise to this end, more immediate translation is expected to come from approaches that generate functional VSMCs from adult sources of multipotent adipose-derived and bone marrow-derived mesenchymal stromal cells (ASCs and BMSCs). The research to this end is extensive and is dominated by studies relating to classical biochemical signalling molecules used to induce differentiation of ASCs and BMSCs. However, prolonged use of the biochemical induction factors is costly and can cause potential endotoxin contamination in the culture. Over recent years several non-traditional differentiation approaches have been devised to mimic defined aspects of the native micro-environment in which VSMCs reside to contribute to the differentiation of VSMC-like cells from ASCs and BMSCs. In this review, the promises and limitations of several non-traditional culture approaches (e.g., co-culture, biomechanical, and biomaterial stimuli) targeting VSMC differentiation are discussed. The extensive crosstalk between the underlying signalling cascades are delineated and put into a translational context. It is expected that this review will not only provide significant insight into VSMC differentiation strategies for vascular smooth muscle tissue engineering applications, but will also highlight the fundamental importance of engineering the cellular microenvironment on multiple scales (with consideration of different combinatorial pathways) in order to direct cell differentiation fate and obtain cells of a desired and stable phenotype. These strategies may ultimately be applied to different sources of stem cells in the future for a range of biomaterial and tissue engineering disciplines.

A microfabricated platform with hydrogel arrays for 3D mechanical stimulation of cells.

Cellular microenvironments present cells with multiple stimuli, including not only soluble biochemical and insoluble matrix cues but also mechanical factors. Biomaterial array platforms have been used to combinatorially and efficiently probe and define two-dimensional (2D) and 3D microenvironmental cues to guide cell functions for tissue engineering applications. However, there are few examples of array platforms that include dynamic mechanical forces, particularly to enable stretching of 3D cell-seeded biomaterials, which is relevant to engineering connective and cardiovascular tissues. Here we present a deformable membrane platform that enables 3D dynamic mechanical stretch of arrayed biomaterial constructs. Cell-seeded polyethylene glycol norbornene (PEG-NB) hydrogels were bound to miniaturized deformable membranes via a thiol-ene reaction with off-stoichiometry thiol-ene based polydimethylsiloxane (OSTE-PDMS) as the membrane material. Bonding to OSTE-PDMS enabled the 3D hydrogel microconstructs to be cyclically deformed and stretched by the membrane. As a first demonstration, human mesenchymal stromal cells (MSCs) embedded in PEG-NB were stretched for several days. They were found to be viable, spread in the 3D hydrogels, and exhibited a contractile myofibroblast phenotype when exposed to dynamic 3D mechanical deformation. This platform, which is readily scalable to larger arrays, enables systematic interrogation of the relationships between combinations of 3D mechanobiological cues and cellular responses, and thus has the potential to identify strategies to predictably control the construction of functional engineered tissues. STATEMENT OF SIGNIFICANCE: Current high-throughput biomaterial screening approaches fail to consider the effects of dynamic mechanical stimulation, despite its importance in a wide variety of regenerative medicine applications. To meet this need, we developed a deformable membrane platform that enables 3D dynamic stretch of arrayed biomaterial constructs. Our approach combines microtechnologies fabricated with off-stoichiometry thiol-ene based polydimethylsiloxane membranes that can covalently bond cell-seeded polyethylene glycol norbornene 3D hydrogels, a model biomaterial with tunable adhesive, elastic and degradation characteristics. As a first demonstration, we show that human mesenchymal stromal cells embedded in hydrogels and subjected to dynamic mechanical stimulation undergo myofibroblast differentiation. This system is readily scaled up to larger arrays, and will enable systematic and efficient screening of combinations of 3D mechanobiological and biomaterial cues on cell fate and function.

Effects of a bone-like mineral film on phenotype of adult human mesenchymal stem cells in vitro.

Multipotent cell types are rapidly becoming key components in a variety of tissue engineering schemes, and mesenchymal stem cells (MSCs) are emerging as an important tool in bone tissue regeneration. Although several soluble signals influencing osteogenic differentiation of MSCs in vitro are well-characterized, relatively little is known about the influence of substrate signals. This study was aimed at elucidating the effects of a bone-like mineral (BLM), which is vital in the process of bone bonding to orthopedic implant materials, on the osteogenic differentiation of human MSCs in vitro. Growth of a BLM film (carbonate apatite, Ca/P = 1.55) on poly(lactide-co-glycolide) (PLG) substrates was achieved via surface hydrolysis and subsequent incubation in a modified simulated body fluid. The BLM film demonstrated significantly increased adsorption of fibronectin, and supported enhanced proliferation of human mesenchymal stem cells (hMSCs) relative to PLG substrates. In the absence of osteogenic supplements hMSCs did not display a high expression of osteogenic markers on BLM or PLG. In the presence of osteogenic supplements hMSCs exhibited greater expression of osteogenic markers on PLG substrates than on BLM substrates, as measured by alkaline phosphatase activity and osteocalcin production. Taken together, these data support the concept that substrate signals significantly influence MSC growth and differentiation, highlighting the importance of carrier material composition in stem cell-based tissue engineering schemes.

Miniaturized platform with on-chip strain sensors for compression testing of arrayed materials.

We report a microfabricated mechanical testing platform with on-chip strain sensors for in situ mechanical characterization of arrayed materials. The device is based on deformable elastomeric membranes that are actuated by pressure that is delivered through an underlying channel network. The bulging membranes compress material samples that are confined between the membranes and a rigid top-plate. Carbon nanotube-based strain sensors that exhibit strain-dependent electrical resistivity were integrated within the membranes to provide continuous read-out of membrane deflection amplitude. We used this platform to study the cyclic compression of several different silicone samples and thereby measured their elastic moduli. The results obtained using our miniaturized platform were in excellent agreement with those obtained using a commercially available mechanical testing platform and clearly demonstrated the utility of our platform for the mechanical testing of small samples in parallel. The miniaturized platform can significantly increase mechanical testing efficiency, particularly when testing of iterative sample formulations is required.

Hydrogel substrate stiffness and topography interact to induce contact guidance in cardiac fibroblasts.

Previous studies demonstrated the importance of substrate stiffness and topography on the phenotype of many different cell types including fibroblasts. Yet the interaction of these two physical parameters remains insufficiently characterized, in particular for cardiac fibroblasts. Most studies focusing on contact guidance use rigid patterned substrates. It is not known how the ability of cardiac fibroblasts to follow grooves and ridges changes as the substrate stiffness is decreased to match the range of stiffness found in native heart tissues. This report demonstrates a significant interactive effect of substrate stiffness and topography on cardiac fibroblast elongation and orientation using polyacrylamide substrates of different stiffness and topography.

Lessons from (patho)physiological tissue stiffness and their implications for drug screening, drug delivery and regenerative medicine.

Diseased tissues are noted for their compromised mechanical properties, which contribute to organ failure; regeneration entails restoration of tissue structure and thereby functions. Thus, the physical signature of a tissue is closely associated with its biological function. In this review, we consider a mechanics-centric view of disease and regeneration by drawing parallels between in vivo tissue-level observations and corroborative cellular evidence in vitro to demonstrate the importance of the mechanical stiffness of the extracellular matrix in these processes. This is not intended to devalue the importance of biochemical signaling; in fact, as we discuss, many mechanical stiffness-driven processes not only require cooperation with biochemical cues, but they ultimately converge at common signaling cascades to influence cell and tissue function in an integrative manner. The study of how physical and biochemical signals collectively modulate cell function not only brings forth a more holistic understanding of cell (patho)biology, but it also creates opportunities to control material properties to improve culture platforms for research and drug screening and aid in the rationale design of biomaterials for molecular therapy and tissue engineering applications.

Measurement of layer-specific mechanical properties in multilayered biomaterials by micropipette aspiration.

Many biomaterials and tissues are complex multilayered structures in which the individual layers have distinct mechanical properties that influence the mechanical behavior and define the local cellular microenvironment. Characterization of the mechanical properties of individual layers in intact tissues is technically challenging. Micropipette aspiration (MA) is a proven method for the analysis of local mechanical properties of soft single-layer biomaterials, but its applicability for multilayer structures has not been demonstrated. We sought to determine and validate MA experimental parameters that would permit measurement of the mechanical properties of only the top layer of an intact multilayer biomaterial or tissue. To do so, we performed parametric nonlinear finite-element (FE) analyses and validation experiments using a multilayer gelatin system. The parametric FE analyses demonstrated that measurement of the properties of only the top layer of a multilayer structure is sensitive to the ratio of the pipette inner diameter (D) to top layer thickness (ttop), and that accurate measurement of the top layer modulus requires D/ttop<1. These predictions were confirmed experimentally by MA of the gelatin system. Using this approach and an inverse FE method, the mean effective modulus of the fibrosa layer of intact porcine aortic valve leaflets was determined to be greater than that of the ventricularis layer (P<0.01), consistent with data obtained by tensile testing of dissected layers. This study provides practical guidelines for the use of MA to measure the mechanical properties of single layers in intact multilayer biomaterials and tissues.

Functional characterization of human coronary artery smooth muscle cells under cyclic mechanical strain in a degradable polyurethane scaffold.

There are few synthetic elastomeric biomaterials that simultaneously provide the required biological conditioning and the ability to translate biomechanical stimuli to vascular smooth muscle cells (VSMCs). Biomechanical stresses are important physiological elements that regulate VSMC function, and polyurethane elastomers are a class of materials capable of facilitating the translation of stress induced biomechanics. In this study, human coronary artery smooth muscle cells (hCASMCs), which were seeded into a porous degradable polar/hydrophobic/ionic (D-PHI) polyurethane scaffold, were subjected to uniaxial cyclic mechanical strain (CMS) over a span of four weeks using a customized bioreactor. The distribution, proliferation and contractile protein expression of hCASMCs in the scaffold were then analyzed and compared to those grown under static conditions. Four weeks of CMS, applied to the elastomeric scaffold, resulted in statistically greater DNA mass, more cell area coverage and a better distribution of cells deeper within the scaffold construct. Furthermore, CMS samples demonstrated improved tensile mechanical properties following four weeks of culture, suggesting the generation of more extracellular matrix within the polyurethane constructs. The expression of smooth muscle α-actin, calponin and smooth muscle myosin heavy chain and the absence of Ki-67+ cells in both static and CMS cultures, throughout the 4 weeks, suggest that hCASMCs retained their contractile character on these biomaterials. The study highlights the importance of implementing physiologically-relevant biomechanical stimuli in the development of synthetic elastomeric tissue engineering scaffolds.