Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

peri-Adventitial delivery of smooth muscle cells in porous collagen scaffolds for treatment of experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

Assessment of mechanical and biocompatible performance of ultra-large nitinol endovascular devices fabricated via a low-energy laser joining process.

Nitinol is an excellent candidate material for developing various self-expanding endovascular devices due to its unique properties such as superelasticity, biocompatibility and shape memory effect. A low-energy laser joining technique suggests a high potential to create various large diameter Nitinol endovascular devices that contain complex geometries. The primary purpose of the study is to investigate the effects of laser joining process parameters with regard to the mechanical and biocompatible performance of Nitinol stents. Both the chemical composition and the microstructure of the laser-welded joints were evaluated using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). In vitro study results on cytotoxicity demonstrated that the joining condition of 8 Hz frequency and 1 kW laser power showed the highest degree of endothelial cell viability after thermal annealing in 500°C for 30 min. Also, in vitro study results showed the highest oxygen content at 0.9 kW laser power, 8 Hz frequency, and 0.3 mm spot size after the thermal annealing. Mechanical performance test results showed that the optimal condition for the highest disconnecting force was found at 1 Hz frequency and 1 kW power with 0.6 mm spot size. Two new endovascular devices have been fabricated using the optimized laser joining parameters, which have demonstrated successful device delivery and retrieval, as well as acute biocompatibility.

Pre-Clinical Cell Therapeutic Approaches for Repair of Volumetric Muscle Loss.

Extensive damage to skeletal muscle tissue due to volumetric muscle loss (VML) is beyond the inherent regenerative capacity of the body, and results in permanent functional debilitation. Current clinical treatments fail to fully restore native muscle function. Recently, cell-based therapies have emerged as a promising approach to promote skeletal muscle regeneration following injury and/or disease. Stem cell populations, such as muscle stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs), have shown a promising capacity for muscle differentiation. Support cells, such as endothelial cells, nerve cells or immune cells, play a pivotal role in providing paracrine signaling cues for myogenesis, along with modulating the processes of inflammation, angiogenesis and innervation. The efficacy of cell therapies relies on the provision of instructive microenvironmental cues and appropriate intercellular interactions. This review describes the recent developments of cell-based therapies for the treatment of VML, with a focus on preclinical testing and future trends in the field.

Use of Superelastic Nitinol and Highly-Stretchable Latex to Develop a Tongue Prosthetic Assist Device and Facilitate Swallowing for Dysphagia Patients.

We introduce a new tongue prosthetic assist device (TPAD), which shows the first prosthetic application for potential treatment of swallowing difficulty in dysphagia patients. The native tongue has a number of complex movements that are not feasible to mimic using a single mechanical prosthetic device. In order to overcome this challenge, our device has three key features, including (1) a superelastic nitinol structure that transfers the force produced by the jaws during chewing towards the palate, (2) angled composite tubes for guiding the nitinol strips smoothly during the motion, and (3) highly stretchable thin polymeric membrane as a covering sheet in order to secure the food and fluids on top of the TPAD for easy swallowing. A set of mechanical experiments has optimized the size and angle of the guiding tubes for the TPAD. The low-profile TPAD was successfully placed in a cadaver model and its mobility effectively provided a simplistic mimic of the native tongue elevation function by applying vertical chewing motions. This is the first demonstration of a new oral device powered by the jaw motions in order to create a bulge in the middle of the mouth mimicking native tongue behavior.

Engineering Biomimetic Materials for Skeletal Muscle Repair and Regeneration.

Although skeletal muscle is highly regenerative following injury or disease, endogenous self-regeneration is severely impaired in conditions of volume traumatic muscle loss. Consequently, tissue engineering approaches are a promising means to regenerate skeletal muscle. Biological scaffolds serve as not only structural support for the promotion of cellular ingrowth but also impart potent modulatory signaling cues that may be beneficial for tissue regeneration. In this work, the progress of tissue engineering approaches for skeletal muscle engineering and regeneration is overviewed, with a focus on the techniques to create biomimetic engineered tissue using extracellular cues. These factors include mechanical and electrical stimulation, geometric patterning, and delivery of growth factors or other bioactive molecules. The progress of evaluating the therapeutic efficacy of these approaches in preclinical models of muscle injury is further discussed.

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt57.5Cu14.7Ni5.3P22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

Nanopatterned bulk metallic glass-based biomaterials modulate macrophage polarization.

Polarization of macrophages by chemical, topographical and mechanical cues presents a robust strategy for designing immunomodulatory biomaterials. Here, we studied the ability of nanopatterned bulk metallic glasses (BMGs), a new class of metallic biomaterials, to modulate murine macrophage polarization. Cytokine/chemokine analysis of IL-4 or IFNγ/LPS-stimulated macrophages showed that the secretion of TNF-α, IL-1α, IL-12, CCL-2 and CXCL1 was significantly reduced after 24-hour culture on BMGs with 55 nm nanorod arrays (BMG-55). Additionally, under these conditions, macrophages increased phagocytic potential and exhibited decreased cell area with multiple actin protrusions. These in vitro findings suggest that nanopatterning can modulate biochemical cues such as IFNγ/LPS. In vivo evaluation of the subcutaneous host response at 2 weeks demonstrated that the ratio of Arg-1 to iNOS increased in macrophages adjacent to BMG-55 implants, suggesting modulation of polarization. In addition, macrophage fusion and fibrous capsule thickness decreased and the number and size of blood vessels increased, which is consistent with changes in macrophage responses. Our study demonstrates that nanopatterning of BMG implants is a promising technique to selectively polarize macrophages to modulate the immune response, and also presents an effective tool to study mechanisms of macrophage polarization and function. STATEMENT OF SIGNIFICANCE: Implanted biomaterials elicit a complex series of tissue and cellular responses, termed the foreign body response (FBR), that can be influenced by the polarization state of macrophages. Surface topography can influence polarization, which is broadly characterized as either inflammatory or repair-like. The latter has been linked to improved outcomes of the FBR. However, the impact of topography on macrophage polarization is not fully understood, in part, due to a lack of high moduli biomaterials that can be reproducibly processed at the nanoscale. Here, we studied macrophage interactions with nanopatterned bulk metallic glasses (BMGs), a class of metallic alloys with amorphous microstructure and formability like polymers. We show that nanopatterned BMGs modulate macrophage polarization and transiently induce less fibrotic and more angiogenic responses. Overall, we demonstrate nanopatterning of BMG implants as a technique to polarize macrophages and modulate the FBR.

Wireless, intraoral hybrid electronics for real-time quantification of sodium intake toward hypertension management.

Recent wearable devices offer portable monitoring of biopotentials, heart rate, or physical activity, allowing for active management of human health and wellness. Such systems can be inserted in the oral cavity for measuring food intake in regard to controlling eating behavior, directly related to diseases such as hypertension, diabetes, and obesity. However, existing devices using plastic circuit boards and rigid sensors are not ideal for oral insertion. A user-comfortable system for the oral cavity requires an ultrathin, low-profile, and soft electronic platform along with miniaturized sensors. Here, we introduce a stretchable hybrid electronic system that has an exceptionally small form factor, enabling a long-range wireless monitoring of sodium intake. Computational study of flexible mechanics and soft materials provides fundamental aspects of key design factors for a tissue-friendly configuration, incorporating a stretchable circuit and sensor. Analytical calculation and experimental study enables reliable wireless circuitry that accommodates dynamic mechanical stress. Systematic in vitro modeling characterizes the functionality of a sodium sensor in the electronics. In vivo demonstration with human subjects captures the device feasibility for real-time quantification of sodium intake, which can be used to manage hypertension.

An in vivo pilot study of a microporous thin film nitinol-covered stent to assess the effect of porosity and pore geometry on device interaction with the vessel wall.

Sputter-deposited thin film nitinol constructs with various micropatterns were fabricated to evaluate their effect on the vessel wall in vivo when used as a covering for commercially available stents. Thin film nitinol constructs were used to cover stents and deployed in non-diseased swine arteries. Swine were sacrificed after approximately four weeks and the thin film nitinol-covered stents were removed for histopathologic evaluation. Histopathology revealed differences in neointimal thickness that correlated with the thin film nitinol micropattern. Devices covered with thin film nitinol with a lateral × vertical length = 20 × 40 µm diamond pattern had minimal neointimal growth with well-organized cell architecture and little evidence of ongoing inflammation. Devices covered with thin film nitinol with smaller fenestrations exhibited a relatively thick neointimal layer with inflammation and larger fenestrations showed migration of inflammatory and smooth muscle cells through the micro fenestrations. This "proof-of-concept" study suggests that there may be an ideal thin film nitinol porosity and pore geometry to encourage endothelialization and incorporation of the device into the vessel wall. Future work will be needed to determine the optimal pore size and geometry to minimize neointimal proliferation and in-stent stenosis.

A novel low-profile thin-film nitinol/silk endograft for treating small vascular diseases.

Since the introduction of various endovascular graft materials such as expanded polytetrafluoroethylene (e-PTFE) and Dacron® polyester, they have been rapidly applied in endovascular devices for treating a variety of clinical situations. While present endovascular grafts have been successful in treating large blood vessels, there are still significant challenges and limitations for small and tortuous vessels to their use. Recently, our group has demonstrated the potential to use thin-film nitinol (TFN) as a novel material to develop endografts used in the treatment of a wide range of small vascular diseases because TFN is ultralow profile (that is, a few micrometers thick), relatively thromboresistant, and superelastic. While TFN has shown superior thromboresistance, its surface endothelialization is not rapid and sufficient. Therefore, our laboratory has been exploring the feasibility of using thin-film silk as a novel coating for facilitating rapid and confluent endothelial cell growth. The purpose of this study is to fabricate a low-profile composite endograft using thin layers of nitinol and silk, and to evaluate both thrombogenicity as well as endothelial cell and smooth muscle cell responses. This study also evaluates the functionality of the composite endograft using an in vitro blood circulation model. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 575-584, 2017.

Use of Micropatterned Thin Film Nitinol in Carotid Stents to Augment Embolic Protection.

Stenting is an alternative to endarterectomy for the treatment of carotid artery stenosis. However, stenting is associated with a higher risk of procedural stroke secondary to distal thromboembolism. Hybrid stents with a micromesh layer have been proposed to address this complication. We developed a micropatterned thin film nitinol (M-TFN) covered stent designed to prevent thromboembolism during carotid intervention. This innovation may obviate the need or work synergistically with embolic protection devices. The proposed double layered stent is low-profile, thromboresistant, and covered with a M-TFN that can be fabricated with fenestrations of varying geometries and sizes. The M-TFN was created in multiple geometries, dimensions, and porosities by sputter deposition. The efficiency of various M-TFN to capture embolic particles was evaluated in different atherosclerotic carotid stenotic conditions through in vitro tests. The covered stent prevented emboli dislodgement in the range of 70%-96% during 30 min duration tests. In vitro vascular cell growth study results showed that endothelial cell elongation, alignment and growth behaviour silhouettes significantly enhance, specifically on the diamond-shape M-TFN, with the dimensions of 145 µm × 20 µm and a porosity of 32%. Future studies will require in vivo testing. Our results demonstrate that M-TFN has a promising potential for carotid artery stenting.

In Vitro Study of a Superhydrophilic Thin Film Nitinol Endograft that is Electrostatically Endothelialized in the Catheter Prior to the Endovascular Procedure.

Electrostatic endothelial cell seeding has evolved as an exceptional technique to improve the efficiency of cell seeding in terms of frequency of attached cells and the amount of cell adhesion for the treatment of vascular diseases. In the recent times, both untreated and superhydrophilic thin film nitinol (TFN) have exhibited strong prospects as substrates for creation of small-diameter endovascular grafts due to their hallmark properties of superelasticity, ultra low-profile character, and grown hemocompatible oxide layer with the presence of a uniform endothelial layer on the surface. The purpose of the current study is to understand the effects of endothelial cell seeding parameters (i.e., applied voltage, incubation time, substrate chemistry, and cell suspension solution) to investigate the cell seeding phenomenon and to improve the cell adhesion and growth on the TFN surface under electrostatic transplantation. Both parallel plate and cylindrical capacitor models were used along with the Taguchi Design of Experiment (DOE) methods to design in vitro test parameters. A novel in vitro system for a cylindrical capacitor model was created using a micro flow pump, micro incubation system, and silicone tubings. The augmented endothelialization on thin film nitinol was developed to determine the effect of cell seeding and deployed in a 6 Fr intravascular catheter setup. Cell viability along with morphology and proliferation of adhered cells were evaluated using fluorescent and scanning electron microscopy. Our results demonstrated that the maximum number of cells attached on STFN in the catheter was observed in 5 V with the 2 h exposure of in the cell culture medium (CCM) solution. The condition showed 5 V voltage with 0.68 × 10-6 µC electrostatic charge and 5.11 V·mm-1 electric field. Our findings have first demonstrated that the electrostatic endothelialization on the superhydrophilic thin film nitinol endograft within the catheter prior to the endovascular procedure could enhance the biocompatibility for low-profile endovascular applications.

An overview of thin film nitinol endovascular devices.

Thin film nitinol has unique mechanical properties (e.g., superelasticity), excellent biocompatibility, and ultra-smooth surface, as well as shape memory behavior. All these features along with its low-profile physical dimension (i.e., a few micrometers thick) make this material an ideal candidate in developing low-profile medical devices (e.g., endovascular devices). Thin film nitinol-based devices can be collapsed and inserted in remarkably smaller diameter catheters for a wide range of catheter-based procedures; therefore, it can be easily delivered through highly tortuous or narrow vascular system. A high-quality thin film nitinol can be fabricated by vacuum sputter deposition technique. Micromachining techniques were used to create micro patterns on the thin film nitinol to provide fenestrations for nutrition and oxygen transport and to increase the device's flexibility for the devices used as thin film nitinol covered stent. In addition, a new surface treatment method has been developed for improving the hemocompatibility of thin film nitinol when it is used as a graft material in endovascular devices. Both in vitro and in vivo test data demonstrated a superior hemocompatibility of the thin film nitinol when compared with commercially available endovascular graft materials such as ePTFE or Dacron polyester. Promising features like these have motivated the development of thin film nitinol as a novel biomaterial for creating endovascular devices such as stent grafts, neurovascular flow diverters, and heart valves. This review focuses on thin film nitinol fabrication processes, mechanical and biological properties of the material, as well as current and potential thin film nitinol medical applications.

In vitro assessment of the trackability of neurovascular intermediate catheters: a comparative analysis.

The advent of new flexible intermediate catheters facilitated manual aspiration thrombectomy (MAT) for treating neurovascular ischaemic diseases. While these catheters are somewhat flexible, most catheters were not designed specifically for aspiration. Trackability is an important property of catheters facilitating catheter advancement in highly tortuous cerebrovasculature. In this study, a novel in vitro trackability test system has been developed using micro pressure transducers and silicone tubes. The exerted force from the catheter tips were quantitatively evaluated while the catheter passed the curved regions. The trackability of three different types of catheters were compared, i.e. Penumbra 054, Concentric DAC 057 and Reverse Reflex. The exerted forces obtained from the first sensor (Sensor 1) during passing the second curve showed the maximum values through the entire transcatheter procedure. When compared, the exerted forces were least for the Penumbra 054 (0.272 ± 0.012 N), representing highly trackable systems in highly tortuous vessel navigation.

Improved osteoblast response to UV-irradiated PMMA/TiO2 nanocomposites with controllable wettability.

Osteoblast response was evaluated with polymethylmethacrylate (PMMA)/titanium dioxide (TiO2) nanocomposite thin films that exhibit the controllable wettability with ultraviolet (UV) treatment. In this study, three samples of PMMA/TiO2 were fabricated with three different compositional volume ratios (i.e., 25/75, 50/50, and 75/25) followed by UV treatment for 0, 4, and 8 h. All samples showed the increased hydrophilicity after UV irradiation. The films fabricated with the greater amount of TiO2 and treated with the longer UV irradiation time increased the hydrophilicity more. The partial elimination of PMMA on the surface after UV irradiation created a durable hydrophilic surface by (1) exposing higher amount of TiO2 on the surface, (2) increasing the hydroxyl groups on the TiO2 surface, and (3) producing a mesoporous structure that helps to hold the water molecules on the surface longer. The partial elimination of PMMA on the surface was confirmed by Fourier transform infrared spectroscopy. Surface profiler and atomic force microscopy demonstrated the increased surface roughness after UV irradiation. Both scanning electron microscopy and energy-dispersive X-ray spectroscopy demonstrated that particles containing calcium and phosphate elements appeared on the 8 h UV-treated surface of PMMA/TiO2 25/75 samples after 4 days soaking in Dulbecco's Modified Eagle Medium. UV treatment showed the osteoblast adhesion improved on all the surfaces. While all UV-treated hydrophilic samples demonstrated the improvement of osteoblast cell adhesion, the PMMA/TiO2 25/75 sample after 8 h UV irradiation (n = 5, P value = 0.000) represented the best cellular response as compared to other samples. UV-treated PMMA/TiO2 nanocomposite thin films with controllable surface properties represent a high potential for the biomaterials used in both orthopedic and dental applications.