Surface-Modified Diopside-Reinforced PCL Biopolymer Composites with Enhanced Interfacial Strength and Mechanical Properties for Orthopedic Applications.

The phase separation of ceramics in a biopolymer matrix makes it challenging to achieve satisfactory mechanical properties required for orthopedic applications. It has been found that silane coupling agents can modify the surface of the bioceramic phase by forming a molecular bridge between the polymer and the ceramic, resulting in improved interfacial strength and adhesion. Therefore, in the present study, silane-modified diopside (DI) ceramic and ε-polycaprolactone (PCL) biopolymer composites were fabricated by injection molding method. The silane modification of DI resulted in their uniform dispersion in the PCL matrix, whereas agglomeration was found in composites containing unmodified DI. The thermal stability of the silane-modified DI-containing composites also increased. The Young's modulus of the composite containing 50% w/w DI modified by 3% w/w silane increased by 103% compared to composites containing 50% w/w unmodified DI. The biodegradation of the unmodified composites was significantly high, indicating their weak interfacial strength with the PCL matrix (p ≤ 0.001). The osteoconductive behavior of the composites was also validated by in vitro cell-material studies. Overall, our findings supported that the silane-modified composites have improved surface roughness, mechanical, and osteoconductive properties compared to the unmodified composite and have the potential for orthopedic applications.

Hyaluronic Acid-Conjugated Thermoresponsive Polymer-Based Bioformulation Enhanced Wound Healing and Gut Barrier Repair of a TNBS-Induced Colitis Injury Ex Vivo Model in a Dynamic Perfusion Device.

Impairment of intestinal epithelium is a typical feature of inflammatory bowel disease (IBD) that causes leakage of bacteria and antigens from the intestinal lumen and thus results in persistent immune activation. Hence, healing and regeneration of the damaged gut mucosa is a promising therapeutic approach to achieve deep remission in IBD. Currently, available systemic therapies have moderate effects and are often associated with numerous side effects and malignancies. In this study, we aimed to develop a topical therapy by chemically conjugating a temperature-responsive polymer, i.e., poly(N-isopropylacrylamide), along with hyaluronic acid to obtain a sprayable therapeutic formulation that upon colon instillation adheres to the damaged gut mucosa due to its temperature-induced phase transition and mucoadhesive properties. An ex vivo adhesion experiment demonstrates that this therapeutic formulation forms a thin physical coating on the mucosal lining at a physiological temperature within 5 min. Physicochemical characterization of (P(NIPAM-co-NTBAM)-HA) established this formulation to be biocompatible, hemo-compatible, and non-immunogenic. Prednisolone was encapsulated within the polymer formulation to achieve maximum therapeutic efficacy in the case of IBD-like conditions as assessed in a custom-fabricated perfusion-based ex vivo model system. Histological analysis suggests that the prednisolone-encapsulated polymer formulation nearly restored the mucosal architecture after 2,4,6-trinitrobenzenesulfonic acid-induced damage. Furthermore, a significant (p ≤ 0.001) increase in mRNA levels of Muc-2 and ZO-1 in treated groups further confirmed the mucosal epithelial barrier restoration.

Advances in Fabrication Technologies for the Development of Next-Generation Cardiovascular Stents.

Coronary artery disease is the most prevalent cardiovascular disease, claiming millions of lives annually around the world. The current treatment includes surgically inserting a tubular construct, called a stent, inside arteries to restore blood flow. However, due to lack of patient-specific design, the commercial products cannot be used with different vessel anatomies. In this review, we have summarized the drawbacks in existing commercial metal stents which face problems of restenosis and inflammatory responses, owing to the development of neointimal hyperplasia. Further, we have highlighted the fabrication of stents using biodegradable polymers, which can circumvent most of the existing limitations. In this regard, we elaborated on the utilization of new fabrication methodologies based on additive manufacturing such as three-dimensional printing to design patient-specific stents. Finally, we have discussed the functionalization of these stent surfaces with suitable bioactive molecules which can prove to enhance their properties in preventing thrombosis and better healing of injured blood vessel lining.

Strontium-Substituted Nanohydroxyapatite-Incorporated Poly(lactic acid) Composites for Orthopedic Applications: Bioactive, Machinable, and High-Strength Properties.

Traditional metal-alloy bone fixation devices provide structural support for bone repair but have limitations in actively promoting bone healing and often require additional surgeries for implant removal. In this study, we focused on addressing these challenges by fabricating biodegradable composites using poly(lactic acid) (PLA) and strontium-substituted nanohydroxyapatite (SrHAP) via melt compounding and injection molding. Various percentages of SrHAP (5, 10, 20, and 30% w/w) were incorporated into the PLA matrix. We systematically investigated the structural, morphological, thermal, mechanical, rheological, and dynamic mechanical properties of the prepared composites. Notably, the tensile modulus, a critical parameter for orthopedic implants, significantly improved from 2.77 GPa in pristine PLA to 3.73 GPa in the composite containing 10% w/w SrHAP. The incorporation of SrHAP (10% w/w) into the PLA matrix led to an increased storage modulus, indicating a uniform dispersion of SrHAP within the PLA and good compatibility between the polymer and nanoparticles. Moreover, we successfully fabricated screws using PLA composites with 10% (w/w) SrHAP, demonstrating their formability at room temperature and radiopacity when observed under X-ray microtomography (micro-CT). Furthermore, the water contact angle decreased from 93 ± 2° for pristine PLA to 75 ± 3° for the composite containing SrHAP, indicating better surface wettability. To assess the biological behavior of the composites, we conducted in vitro cell-material tests, which confirmed their osteoconductive and osteoinductive properties. These findings highlight the potential of our developed PLA/SrHAP10 (10% w/w) composites as machinable implant materials for orthopedic applications. In conclusion, our study presents the fabrication and comprehensive characterization of biodegradable composites comprising PLA and strontium-substituted nanohydroxyapatite (SrHAP). These composites exhibit improved mechanical properties, formability, and radiopacity while also demonstrating desirable biological behavior. Our results suggest that these PLA/SrHAP10 composites hold promise as machinable implant materials for orthopedic applications.

Recent advances in tailoring stimuli-responsive hybrid scaffolds for cardiac tissue engineering and allied applications.

To recapitulate bio-physical properties and functional behaviour of native heart tissues, recent tissue engineering-based approaches are focused on developing smart/stimuli-responsive materials for interfacing cardiac cells. Overcoming the drawbacks of the traditionally used biomaterials, these smart materials portray outstanding mechanical and conductive properties while promoting cell-cell interaction and cell-matrix transduction cues in such excitable tissues. To date, a large number of stimuli-responsive materials have been employed for interfacing cardiac tissues alone or in combination with natural/synthetic materials for cardiac tissue engineering. However, their comprehensive classification and a comparative analysis of the role played by these materials in regulating cardiac cell behaviour and in vivo metabolism are much less discussed. In an attempt to cover the recent advances in fabricating stimuli-responsive biomaterials for engineering cardiac tissues, this review details the role of these materials in modulating cardiomyocyte behaviour, functionality and surrounding matrix properties. Furthermore, concerns and challenges regarding the clinical translation of these materials and the possibility of using such materials for the fabrication of bio-actuators and bioelectronic devices are discussed.

Mimicking Native Heart Tissue Physiology and Pathology in Silk Fibroin Constructs through a Perfusion-Based Dynamic Mechanical Stimulation Microdevice.

In vitro cardiomyocyte (CM) maturation is an imperative step to replicate native heart tissue-like structures as cardiac tissue grafts or as drug screening platforms. CMs are known to interpret biophysical cues such as stiffness, topography, external mechanical stimulation or dynamic perfusion load through mechanotransduction and change their behavior, organization, and maturation. In this regard, a silk-based cardiac tissue (CT) coupled with a dynamic perfusion-based mechanical stimulation platform (DMM) for achieving maturation and functionality in vitro is tried to be delivered. Silk fibroin (SF) is used to fabricate lamellar scaffolds to provide native tissue-like anisotropic architecture and is found to be nonimmunogenic and biocompatible allowing cardiomyocyte attachment and growth in vitro. Further, the scaffolds display excellent mechanical properties by their ability to undergo cyclic compressions without any deformation when places in the DMM. Gradient compression strains (5% to 20%), mimicking the native physiological and pathological conditions, are applied to the cardiomyocyte culture seeded on lamellar silk scaffolds in the DMM. A strain-dependent difference in cardiomyocyte maturation, gene expression, sarcomere elongation, and extracellular matrix formation is observed. These silk-based CTs matured in the DMM can open up several avenues toward the development of host-specific grafts and in vitro models for drug screening.

Engineering Microsphere-Loaded Non-mulberry Silk-Based 3D Bioprinted Vascularized Cardiac Patches with Oxygen-Releasing and Immunomodulatory Potential.

A hostile myocardial microenvironment post ischemic injury (myocardial infarction) plays a decisive role in determining the fate of tissue-engineered approaches. Therefore, engineering hybrid 3D printed platforms that can modulate the MI microenvironment for improving implant acceptance has surfaced as a critical requirement for reconstructing an infarcted heart. Here, we have employed a non-mulberry silk-based conductive bioink comprising carbon nanotubes (CNTs) to bioprint functional 3D vascularized anisotropic cardiac constructs. Immunofluorescence staining, polymerase chain reaction-based gene expression studies, and electrophysiological studies showed that the inclusion of CNTs in the bioink played a significant role in upregulating matured cardiac biomarkers, sarcomere formation, and beating rate while promoting cardiomyocyte viability. These constructs were then microinjected with calcium peroxide and IL-10-loaded gelatin methacryloyl microspheres. Measurements of oxygen concentration revealed that these microspheres upheld the oxygen availability for maintaining cellular viability for at least 5 days in a hypoxic environment. Also, the ability of microinjected IL-10 microspheres to modulate the macrophages to anti-inflammatory M2 phenotype in vitro was uncovered using immunofluorescent staining and gene expression studies. Furthermore, in vivo subcutaneous implantation of microsphere-injected 3D constructs provided insights toward the extended time frame that was achieved for dealing with the hostile microenvironment for promoting host neovascularization and implant acceptance.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium-on-a-Chip Application.

Bioprinting holds great promise towards engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, we have developed a novel and mechanically robust biomaterial-ink based on non-mulberry silk fibroin protein. The silk-based ink demonstrated suitable mechanical properties required in terms of elasticity and stiffness (~40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allowed the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which were able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin-43, and maintain synchronously beating of cardiomyocytes. The printed constructs were found to be non-immunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue-on-a-chip, the silk-based ink was used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC-CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. We confirmed the ability of silk-based supporting hydrogel towards maturation and viability of hiPSC-CSs and endothelial cells, and for applications in evaluating drug toxicity.

Nanoparticle-Based Hybrid Scaffolds for Deciphering the Role of Multimodal Cues in Cardiac Tissue Engineering.

Myocardial microenvironment plays a decisive role in guiding the function and fate of cardiomyocytes, and engineering this extracellular niche holds great promise for cardiac tissue regeneration. Platforms utilizing hybrid hydrogels containing various types of conductive nanoparticles have been a critical tool for constructing engineered cardiac tissues with outstanding mechanical integrity and improved electrophysiological properties. However, there has been no attempt to directly compare the efficacy of these hybrid hydrogels and decipher the mechanisms behind how these platforms differentially regulate cardiomyocyte behavior. Here, we employed gelatin methacryloyl (GelMA) hydrogels containing three different types of carbon-based nanoparticles: carbon nanotubes (CNTs), graphene oxide (GO), and reduced GO (rGO), to investigate the influence of these hybrid scaffolds on the structural organization and functionality of cardiomyocytes. Using immunofluorescent staining for assessing cellular organization and proliferation, we showed that electrically conductive scaffolds (CNT- and rGO-GelMA compared to relatively nonconductive GO-GelMA) played a significant role in promoting desirable morphology of cardiomyocytes and elevated the expression of functional cardiac markers, while maintaining their viability. Electrophysiological analysis revealed that these engineered cardiac tissues showed distinct cardiomyocyte phenotypes and different levels of maturity based on the substrate (CNT-GelMA: ventricular-like, GO-GelMA: atrial-like, and rGO-GelMA: ventricular/atrial mixed phenotypes). Through analysis of gene-expression patterns, we uncovered that the engineered cardiac tissues matured on CNT-GelMA and native cardiac tissues showed comparable expression levels of maturation markers. Furthermore, we demonstrated that engineered cardiac tissues matured on CNT-GelMA have increased functionality through integrin-mediated mechanotransduction (via YAP/TAZ) in contrast to cardiomyocytes cultured on rGO-GelMA.

3D Printed Cartilage-Like Tissue Constructs with Spatially Controlled Mechanical Properties.

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.

3D Printing/Bioprinting Based Tailoring of in Vitro Tissue Models: Recent Advances and Challenges.

Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed.

Comprehensive Review on Silk at Nanoscale for Regenerative Medicine and Allied Applications.

Materials at the nanoscale offer numerous avenues to be explored and exploited in diverse realms. Among others, proteinaceous biomaterials such as silk hold immense prospects in the domain of nanoengineering. Silk offers a unique combination of desirable facets like biocompatibility; extraordinary mechanical properties, such as elongation, elasticity, toughness, and modulus; and tunable biodegradability which are far better than most naturally occurring and engineered materials. Much of these properties are due to the molecular structure of the silk protein and it is self-assembly into hierarchical structures. Taking advantage of the hierarchical assembly, a large number of fabrication strategies have now emerged that allow the tailoring of silk structure of at the nanoscale. Harnessing the favorable properties of silk, such methods offer a promising direction toward producing structurally and functionally optimized silk nanomaterials. This review discusses the critical structure-property relationship in silk that occurs at the nanoscale and also aims to bring out the recent status in the approaches for fabrication, characterization, and the gamut of applications of various silk-based nanomaterials (nanoparticles, nanofibers, and nanocomposites) in the niche of translational research. Harnessing the favorable nanostructure of silk, the review also takes into account the impetus of silk in avant-garde applications such as chemo-biosensing, energy harvesting, microfluidics, and environmental applications.

Magnetic Actuator Device Assisted Modulation of Cellular Behavior and Tuning of Drug Release on Silk Platform.

Externally applied physical forces and mechanical stimulations have been found to be instructive to cells which lead to their signaling or differentiation. Further, bioreactors and functional biomaterials have been designed based on this principle to modulate cellular behavior under in vitro conditions. Herein, we have designed a magnetic actuator device (MAD) to understand the fundamental responses of two different phenomena: the effect of actuation on cardiac muscle cells and drug delivery under the influence of pulsed magnetic field. Silk fibroin (SF)-based magnetically responsive matrix, developed by incorporating magnetic iron oxide nanoparticles (IONP) within silk nanofibers was actuated with MAD. The silk matrix was seeded with cells and drugs independently to study effect of physical actuation by MAD on cellular behavior and drug release properties. Neonatal rat cardiomyocytes and H9c2 cells were used for studying the former while model drug was used to observe the latter. Pulsed magnetic stimulation promoted proliferation of cells at a significantly higher rate in comparison to those under static conditions, p ≤ 0.01. For instance, a significantly higher expression of Connexin 43 gene was observed in both H9c2 and primary rat cardiomyocytes under magnetic stimulation compared to nonstimulation conditions after day 14, p ≤ 0.01. A differential drug release profile corresponding to respective actuation frequency was observed while studying drug release properties. Overall, the device can be applied as a non-invasive technique to stimulate cardiac cells grown under laboratory conditions for developing functional artificial construct coupled with additional regulated drug release properties. The study thus demonstrates versatile applications of MAD in biomedical and tissue engineering.

Stacked silk-cell monolayers as a biomimetic three dimensional construct for cardiac tissue reconstruction.

Heart failure, due to necrosis of heart tissue, interminably poses a significant burden on world-wide health care systems. In this context, a facile tissue engineering approach using mulberry (Bombyx mori) and non-mulberry (Antheraea assama) silk fibroin (SF) has been delved into. Amalgamating the efficacious attributes of cell sheet tissue engineering and robusticity of silk biomaterial, we developed a 3-D construct using silk films to promote cardiac tissue regeneration. Herein, the fabricated patterned silk films were physico-chemically characterized and analysed for their compatibility with cardiomyocytes. The presence of nanogrooves on silk films provided contact guidance to the growing cardiomyocytes allowing them to form unidirectionally aligned cell monolayers. Non-mulberry silk films exhibiting significantly greater mechanical strength and low immunogenicity in vitro and in vivo supported better growth, proliferation and maturation of both primary rat cardiomyocytes (PCMs) and H9c2 cells. The directional cue and presence of cell binding motifs such as RGD in A. assama films favoured the growth and maturation of cardiomyocytes to their functional phenotype. Cardiomyocyte maturation was attested by significant (p≤ 0.05) upregulation of myosin heavy chain-α (∼1.25 fold), connexin 43 (∼2 fold), and troponin I (∼1.25 fold) genes in PCMs grown on non-mulberry silk films. The patterned silk-cardiomyocyte monolayers were then stacked onto each other while maintaining their alignment to form a 3-D construct which exhibited structural integrity and uniform cellular distribution. Taking together, this work attests the suitability of non-mulberry A. assama silk fibroin as a potential biomaterial and prospects of exploring silk-cardiomyocyte monolayers for cardiac tissue engineering applications.