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Soft tissue defects are a common clinical challenge mostly caused by trauma, congenital anomalies and oncological surgery. Current soft tissue reconstruction options include synthetic materials (fillers and implants) and autologous adipose tissue transplantation through flap surgery and/or lipotransfer. Both reconstructive options hold important disadvantages to which vascularized adipose tissue engineering (VATE) strategies could offer solutions. In this review, we first summarized pivotal characteristics of functional adipose tissue such as the structure, function, cell types, development and extracellular matrix (ECM). Next, we discussed relevant cell sources and how they are applied in different state-of-the-art VATE techniques. Herein, biomaterial scaffolds and hydrogels, ECMs, spheroids, organoids, cell sheets, three dimensional printing and microfluidics are overviewed. Also, we included extracellular vesicles and emphasized their potential role in VATE. Lastly, current challenges and future perspectives in VATE are pointed out to help to pave the road towards clinical applications., (© 2023 IOP Publishing Ltd.)

Minimally invasive treatment via injectable delivery of cells has drawn extensive attention for tissue regeneration because it reduces the need for substantial open surgery and fits tissue defects with complex shapes, making it a suitable option for repairing articular cartilage defects. This work presents an alkaline treatment method to fabricate open-porous poly (lactic-co-glycolic acid) microspheres (OPMs) as bone marrow stromal cells (BMSCs) carriers for cartilage regeneration. OPMs have better biodegradation property and the extended pores can provide easier access for cells to the internal space. The BMSCs cultured with OPMs can display enhanced cell proliferation, up-regulated expression of cartilage-related mRNAs and proteins, and improved cartilage regeneration in vitro and in vivo. These results highlight the advantage and potential of using OPMs fabricated via simple alkaline treatment as injectable stem cell carriers for cartilage regeneration through minimally invasive procedures., (© 2021 Wiley Periodicals LLC.)

The development of 3D printing has significantly advanced the field of bone tissue engineering by enabling the fabrication of scaffolds that faithfully recapitulate desired mechanical properties and architectures. In addition, computer-based manufacturing relying on patient-derived medical images permits the fabrication of customized modules in a patient-specific manner. In addition to conventional 3D fabrication, progress in materials engineering has led to the development of 4D printing, allowing time-sensitive interventions such as programed therapeutics delivery and modulable mechanical features. Therapeutic interventions established via multi-dimensional engineering are expected to enhance the development of personalized treatment in various fields, including bone tissue regeneration. Here, recent studies utilizing 3D printed systems for bone tissue regeneration are summarized and advances in 4D printed systems are highlighted. Challenges and perspectives for the future development of multi-dimensional printed systems toward personalized bone regeneration are also discussed., (© 2021 Wiley-VCH GmbH.)

Vascularization is considered to be one of the key challenges in engineering functional 3D tissues. Engineering suturable vascular grafts containing pores with diameter of several tens of microns in tissue engineered constructs may provide an instantaneous blood perfusion through the grafts improving cell infiltration and thus, allowing rapid vascularization and vascular branching. The aim of this work was to develop suturable tubular scaffolds to be integrated in biofabricated constructs, enabling the direct connection of the biofabricated construct with the host blood stream, providing an immediate blood flow inside the construct. Here, tubular grafts with customizable shapes (tubes, Y-shape capillaries) and controlled diameter ranging from several hundreds of microns to few mm are fabricated based on poly(glycerol sebacate) (PGS)/poly(vinyl alcohol) (PVA) electrospun scaffolds. Furthermore, a network of pore channels of diameter in the order of 100 μ m was machined by laser femtosecond ablation in the tube wall. Both non-machined and laser machined tubular scaffolds elongated more than 100% of their original size have shown suture retention, being 5.85 and 3.96 N mm -2 respectively. To demonstrate the potential of application, the laser machined porous grafts were embedded in gelatin methacryloyl (GelMA) hydrogels, resulting in elastomeric porous tubular graft/GelMA 3D constructs. These constructs were then co-seeded with osteoblast-like cells (MG-63) at the external side of the graft and human umbilical vein endothelial cells inside, forming a bone osteon model. The laser machined pore network allowed an immediate endothelial cell flow towards the osteoblasts enabling the osteoblasts and endothelial cells to interact and form 3D structures. This rapid vascularization approach could be applied, not only for bone tissue regeneration, but also for a variety of tissues and organs., (© 2021 IOP Publishing Ltd.)

Engineering tissue-like scaffolds that can mimic the microstructure, architecture, topology, and mechanical properties of native tissues while offering an excellent environment for cellular growth has remained an unmet need. To address these challenges, multicompartment composite fibers are fabricated. These fibers can be assembled through textile processes to tailor tissue-level mechanical and electrical properties independent of cellular level components. Textile technologies also allow control of the distribution of different cell types and the microstructure of fabricated constructs and the direction of cellular growth within the 3D microenvironment. Here, we engineered composite fibers from biocompatible cores and biologically relevant hydrogel sheaths. The fibers are mechanically robust to being assembled using textile processes and could support adhesion, proliferation, and maturation of cell populations important for the engineering of skeletal muscles. We also demonstrated that the changes in the coating of the multicompartment fibers could potentially enhance myogenesis in vitro .

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.

Skeletal muscle tissue engineering (SMTE) aims at repairing defective skeletal muscles. Until now, numerous developments are made in SMTE; however, it is still challenging to recapitulate the complexity of muscles with current methods of fabrication. Here, after a brief description of the anatomy of skeletal muscle and a short state-of-the-art on developments made in SMTE with "conventional methods," the use of 3D bioprinting as a new tool for SMTE is in focus. The current bioprinting methods are discussed, and an overview of the bioink formulations and properties used in 3D bioprinting is provided. Finally, different advances made in SMTE by 3D bioprinting are highlighted, and future needs and a short perspective are provided., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Due to the limited success rate of currently available vascular replacements, tissue engineering has received tremendous attention in recent years. A main challenge in the field of regenerative medicine is creating a mechanically functional tissue with a well-organized extracellular matrix, particularly of collagen and elastin. In this study, the native collagen scaffold derived from decellularized tendon sections, as a scaffold having the potential to be used for vascular tissue engineering applications, was studied. We showed that the elasticity of the scaffolds was improved when crosslinked with the bovine elastin. The effect of different concentrations of elastin on mechanical properties of the collagen scaffolds was evaluated of which 15% elastin concentration was selected for further analysis based on the results. Addition of 15% elastin to collagen scaffolds significantly decreased Young's modulus and the tensile stress at the maximum load and increased the tensile strain at the maximum load of the constructs as compared to those of the collagen scaffolds or control samples. Moreover, tubular elastin modified collagen scaffolds showed significantly higher burst pressure compared to the control samples. Smooth muscle cells and endothelial cells cultured on the elastin modified collagen scaffolds showed high viability (>80%) after 1, 3, and 7 days. Furthermore, the cells showed a high tendency to align with the collagen fibers within the scaffold and produced their own extracellular matrix over time. In conclusion, the results show that the decellularized tendon sections have a great potential to be used as scaffolds for vascular tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1225-1234, 2019., (© 2019 Wiley Periodicals, Inc.)

Impact Statement: In this Perspective, we discuss the impact of the past 25 years of tissue engineering on the development of clinical therapies. Based on their success and other significant research accomplishments, platforms of innovation were identified. Their discoveries will enable tissue engineering inspired therapies to meet the requirements necessary for large-scale manufacturing and Food and Drug Administration (FDA) approval for a diverse range of indications.

Fiber-based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro-environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell-laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM-MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP-12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell-laden aligned system can be used for engineering of scaffolds for tendon regeneration., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Here, electrospun fibers based on a blend of polycaprolactone (PCL), poly(ethylene glycol) (PEG), and gelatin methacryloyl (GelMA) were developed. The careful choice of this polymer combination allowed for the preparation of a biomaterial that preserved the mechanical strength of PCL, while at the same time improving the hydrophilicity of the blended material and human osteoblast maturation., Methods: The morphology, chemical structure, wettability, and mechanical properties before and after UV photocrosslinking were evaluated. Furthermore, human osteoblasts (hFOB) were cultivated for up to 21 days on the scaffolds, and their potential to upregulate cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition were investigated., Results: Contact angle measurement results showed that the developed scaffolds presented hydrophilic properties after PEG and GelMA incorporation before (25°) and after UV photocross-linking (69°) compared to pure PCL (149°). PCL:PEG:GelMA-UV displayed a slight increase in mechanical strength (elastic modulus ~37 MPa) over PCL alone (~33 MPa). Normally, an increase in strength of fibers leads to a decrease in elongation at break, due to the material becoming less deformable and stiffer, thus leading to breaks at low strain. This behavior was observed by comparing PCL (elongation at break ~106%) and PCL:PEG:GelMA-UV (~50%). Moreover, increases in ALP activity (10-fold at day 14) and calcium deposition (1.3-fold at day 21) by hFOBs were detected after PEG and GelMA incorporation after UV photocross-linking compared to pure PCL. Ultrathin and hydrophilic fibers were obtained after PEG and GelMA incorporation after UV photocrosslinking, but the strength of PCL was maintained. Interestingly, those ultrathin fiber characteristics improved hFOB functions., Conclusion: These findings appear promising for the use of these electrospun scaffolds, based on the combination of polymers used here for numerous orthopedic applications., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Bioprinting has emerged as a promising tool in tissue engineering and regenerative medicine. Various 3D printing strategies have been developed to enable bioprinting of various biopolymers and hydrogels. However, the incorporation of biological factors has not been well explored. As the importance of personalized medicine is becoming more clear, the need for the development of bioinks containing autologous/patient-specific biological factors for tissue engineering applications becomes more evident. Platelet-rich plasma (PRP) is used as a patient-specific source of autologous growth factors that can be easily incorporated to hydrogels and printed into 3D constructs. PRP contains a cocktail of growth factors enhancing angiogenesis, stem cell recruitment, and tissue regeneration. Here, the development of an alginate-based bioink that can be printed and crosslinked upon implantation through exposure to native calcium ions is reported. This platform can be used for the controlled release of PRP-associated growth factors which may ultimately enhance vascularization and stem cell migration., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue engineering (TE) is a highly interdisciplinary research field driven by the goal to restore, replace, or regenerate defective tissues. Throughout more than two decades of intense research, different technological approaches, which can be principally categorized into scaffold-based and scaffold-free strategies, have been developed. In this opinion article, we discuss the emergence of a third strategy in TE. This synergetic strategy integrates the advantages of both of these traditional approaches, while being clearly distinct from them. Its characteristic attributes, numerous practical benefits, and recent literature reports supporting our opinion, are discussed in detail., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bioinks with shear-thinning/rapid solidification properties and strong mechanics are usually needed for the bioprinting of three-dimensional (3D) cell-laden constructs. As such, it remains challenging to generate soft constructs from bioinks at low concentrations that are favorable for cellular activities. Herein, we report a strategy to fabricate cell-laden constructs with tunable 3D microenvironments achieved by bioprinting of gelatin methacryloyl (GelMA)/alginate core/sheath microfibers, where the alginate sheath serves as a template to support and confine the GelMA pre-hydrogel in the core during the extrusion process, allowing for subsequent UV crosslinking. This novel strategy minimizes the bioprinting requirements for the core bioink, and facilitates the fabrication of cell-laden GelMA constructs at low concentrations. We first showed the capability of generating various alginate hollow microfibrous constructs using a coaxial nozzle setup, and verified the diffusibility and perfusability of the bioprinted hollow structures that are important for the tissue engineering applications. More importantly, the hollow alginate microfibers were then used as templates for generating cell-laden GelMA constructs with soft microenvironments, by using GelMA pre-hydrogel as the bioink for the core phase during bioprinting. As such, GelMA constructs at extremely low concentrations (<2.0%) could be extruded to effectively support cellular activities including proliferation and spreading for various cell types. We believe that our strategy is likely to provide broad opportunities in bioprinting of 3D constructs with cell-favorable microenvironments for applications in tissue engineering and pharmaceutical screening.

Fabricating 3D large-scale bone tissue constructs with functional vasculature has been a particular challenge in engineering tissues suitable for repairing large bone defects. To address this challenge, an extrusion-based direct-writing bioprinting strategy is utilized to fabricate microstructured bone-like tissue constructs containing a perfusable vascular lumen. The bioprinted constructs are used as biomimetic in vitro matrices to co-culture human umbilical vein endothelial cells and bone marrow derived human mesenchymal stem cells in a naturally derived hydrogel. To form the perfusable blood vessel inside the bioprinted construct, a central cylinder with 5% gelatin methacryloyl (GelMA) hydrogel at low methacryloyl substitution (GelMA LOW ) was printed. We also develop cell-laden cylinder elements made of GelMA hydrogel loaded with silicate nanoplatelets to induce osteogenesis, and synthesized hydrogel formulations with chemically conjugated vascular endothelial growth factor to promote vascular spreading. It was found that the engineered construct is able to support cell survival and proliferation during maturation in vitro. Additionally, the whole construct demonstrates high structural stability during the in vitro culture for 21 days. This method enables the local control of physical and chemical microniches and the establishment of gradients in the bioprinted constructs., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Engineering cardiac tissues and organ models remains a great challenge due to the hierarchical structure of the native myocardium. The need of integrating blood vessels brings additional complexity, limiting the available approaches that are suitable to produce integrated cardiovascular organoids. In this work we propose a novel hybrid strategy based on 3D bioprinting, to fabricate endothelialized myocardium. Enabled by the use of our composite bioink, endothelial cells directly bioprinted within microfibrous hydrogel scaffolds gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. Together with controlled anisotropy, this 3D endothelial bed was then seeded with cardiomyocytes to generate aligned myocardium capable of spontaneous and synchronous contraction. We further embedded the organoids into a specially designed microfluidic perfusion bioreactor to complete the endothelialized-myocardium-on-a-chip platform for cardiovascular toxicity evaluation. Finally, we demonstrated that such a technique could be translated to human cardiomyocytes derived from induced pluripotent stem cells to construct endothelialized human myocardium. We believe that our method for generation of endothelialized organoids fabricated through an innovative 3D bioprinting technology may find widespread applications in regenerative medicine, drug screening, and potentially disease modeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nanotechnology offers significant potential in regenerative medicine, specifically with the ability to mimic tissue architecture at the nanoscale. In this perspective, we highlight key achievements in the nanotechnology field for successfully mimicking the composition and structure of different tissues, and the development of bio-inspired nanotechnologies and functional nanomaterials to improve tissue regeneration. Numerous nanomaterials fabricated by electrospinning, nanolithography and self-assembly have been successfully applied to regenerate bone, cartilage, muscle, blood vessel, heart and bladder tissue. We also discuss nanotechnology-based regenerative medicine products in the clinic for tissue engineering applications, although so far most of them are focused on bone implants and fillers. We believe that recent advances in nanotechnologies will enable new applications for tissue regeneration in the near future.

Biomaterials currently used in cardiac tissue engineering have certain limitations, such as lack of electrical conductivity and appropriate mechanical properties, which are two parameters playing a key role in regulating cardiac cell behavior. Here, the myocardial tissue constructs are engineered based on reduced graphene oxide (rGO)-incorporated gelatin methacryloyl (GelMA) hybrid hydrogels. The incorporation of rGO into the GelMA matrix significantly enhances the electrical conductivity and mechanical properties of the material. Moreover, cells cultured on composite rGO-GelMA scaffolds exhibit better biological activities such as cell viability, proliferation, and maturation compared to ones cultured on GelMA hydrogels. Cardiomyocytes show stronger contractility and faster spontaneous beating rate on rGO-GelMA hydrogel sheets compared to those on pristine GelMA hydrogels, as well as GO-GelMA hydrogel sheets with similar mechanical property and particle concentration. Our strategy of integrating rGO within a biocompatible hydrogel is expected to be broadly applicable for future biomaterial designs to improve tissue engineering outcomes. The engineered cardiac tissue constructs using rGO incorporated hybrid hydrogels can potentially provide high-fidelity tissue models for drug studies and the investigations of cardiac tissue development and/or disease processes in vitro., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three-dimensional neural tissue engineering has made great strides in developing neural disease models and replacement tissues for patients. However, the need for biomimetic tissue models and effective patient therapies remains unmet. The recent push to expand 2D neural tissue engineering into the third dimension shows great potential to advance the field. Another area which has much to offer to neural tissue engineering is stem cell research. Stem cells are well known for their self-renewal and differentiation potential and have been shown to give rise to tissues with structural and functional properties mimicking natural organs. Application of these capabilities to 3D neural tissue engineering may be highly useful for basic research on neural tissue structure and function, engineering disease models, designing tissues for drug development, and generating replacement tissues with a patient's genetic makeup. Here, we discuss the vast potential, as well as the current challenges, unique to integration of 3D fabrication strategies and stem cells into neural tissue engineering. We also present some of the most significant recent achievements, including nerve guidance conduits to facilitate better healing of nerve injuries, functional 3D biomimetic neural tissue models, physiologically relevant disease models for research purposes, and rapid and effective screening of potential drugs.

There is an immense need for tissue engineered blood vessels. However, current tissue engineering approaches still lack the ability to build native blood vessel-like perfusable structures with multi-layered vascular walls. This paper demonstrated a new method to fabricate tri-layer biomimetic blood vessel-like structures on a microfluidic platform using photocrosslinkable gelatin hydrogel. The presented method enables fabrication of physiological blood vessel-like structures with mono-, bi- or tri-layer vascular walls. The diameter of the vessels, the total thickness of the vessel wall and the thickness of each individual layer of the wall were independently controlled. The developed fabrication process is a simple and rapid method, allowing the physical fabrication of the vascular structure in minutes, and the formation of a vascular endothelial cell layer inside the vessels in 3-5 days. The fabricated vascular constructs can potentially be used in numerous applications including drug screening, development of in vitro models for cardiovascular diseases and/or cancer metastasis, and study of vascular biology and mechanobiology.

Carbon nanotube (CNT)-based nanocomposites often possess properties such as high stiffness, electrical conductivity, and thermal stability and have been studied for various biomedical and biotechnological applications. However, the current design approaches utilize CNTs as physical fillers, and thus, the true potential of CNT-based nanocomposites has not been realized. Here, we introduce a general approach to fabricating stiff, elastomeric nanocomposites from poly(glycerol sebacate) (PGS) and CNTs. The covalent crosslinking between the nanotubes and polymer chains resulted in novel property combinations that are not observed in conventional nanocomposites. The addition of 1% CNTs resulted in a five-fold increase in the tensile modulus and a six-fold increase in compression modulus compared with PGS alone, which is far superior to the previously reported studies for CNT-based nanocomposites. Despite a significant increase in mechanical stiffness, the elasticity of the network was not compromised and the resulting nanocomposites showed more than 94% recovery. This study demonstrates that the chemical conjugation of CNTs to a PGS backbone results in stiff and elastomeric nanocomposites. Additionally, in vitro studies using human mesenchymal stem cells (hMSCs) indicated that the incorporation of CNTs into the PGS network significantly enhanced the differentiation potential of the seeded hMSCs, rendering them potentially suitable for applications ranging from scaffolding in musculoskeletal tissue engineering to biosensors in biomedical devices.

Heart valve-related disorders are among the major causes of death worldwide. Although prosthetic valves are widely used to treat this pathology, current prosthetic grafts cannot grow with the patient while maintaining normal valve mechanical and hemodynamic properties. Tissue engineering may provide a possible solution to this issue through using biodegradable scaffolds and patients' own cells. Despite their similarity to heart valve tissue, most hydrogel scaffolds are not mechanically suitable for the dynamic stresses of the heart valve microenvironment. In this study, we integrated electrospun poly(glycerol sebacate) (PGS)-poly(ɛ-caprolactone) (PCL) microfiber scaffolds, which possess enhanced mechanical properties for heart valve engineering, within a hybrid hydrogel made from methacrylated hyaluronic acid and methacrylated gelatin. Sheep mitral valvular interstitial cells were encapsulated in the hydrogel and evaluated in hydrogel-only, PGS-PCL scaffold-only, and composite scaffold conditions. Although the cellular viability and metabolic activity were similar among all scaffold types, the presence of the hydrogel improved the three-dimensional distribution of mitral valvular interstitial cells. As seen by similar values in both the Young's modulus and the ultimate tensile strength between the PGS-PCL scaffolds and the composites, microfibrous scaffolds preserved their mechanical properties in the presence of the hydrogels. Compared to electrospun or hydrogel scaffolds alone, this combined system may provide a more suitable three-dimensional structure for generating scaffolds for heart valve tissue engineering., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the past few years, a considerable amount of effort has been devoted toward the development of biomimetic scaffolds for cardiac tissue engineering. However, most of the previous scaffolds have been electrically insulating or lacked the structural and mechanical robustness to engineer cardiac tissue constructs with suitable electrophysiological functions. Here, we developed tough and flexible hybrid scaffolds with enhanced electrical properties composed of carbon nanotubes (CNTs) embedded aligned poly(glycerol sebacate):gelatin (PG) electrospun nanofibers. Incorporation of varying concentrations of CNTs from 0 to 1.5% within the PG nanofibrous scaffolds (CNT-PG scaffolds) notably enhanced fiber alignment and improved the electrical conductivity and toughness of the scaffolds while maintaining the viability, retention, alignment, and contractile activities of cardiomyocytes (CMs) seeded on the scaffolds. The resulting CNT-PG scaffolds resulted in stronger spontaneous and synchronous beating behavior (3.5-fold lower excitation threshold and 2.8-fold higher maximum capture rate) compared to those cultured on PG scaffold. Overall, our findings demonstrated that aligned CNT-PG scaffold exhibited superior mechanical properties with enhanced CM beating properties. It is envisioned that the proposed hybrid scaffolds can be useful for generating cardiac tissue constructs with improved organization and maturation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(ɛ-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclay-enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering.

Like a carpet for cells, micropatterned polymeric nanosheets are developed toward local cell delivery. The nanosheets direct morphogenesis of retinal pigment epithelial (RPE) cells and allow for the injection of an engineered RPE monolayer through syringe needles without the loss of cell viability. Such an ultrathin carrier has the promise of a minimally invasive delivery of cells into narrow tissue spaces., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Kappa carrageenan (κ-CA) is a natural-origin polymer that closely mimics the glycosaminoglycan structure, one of the most important constituents of native tissues extracellular matrix. Previously, it has been shown that κ-CA can crosslink via ionic interactions rendering strong, but brittle hydrogels. In this study, we introduce photocrosslinkable methacrylate moieties on the κ-CA backbone to create physically and chemically crosslinked hydrogels highlighting their use in the context of tissue engineering. By varying the degree of methacrylation, the effect on hydrogel crosslinking was investigated in terms of hydration degree, dissolution profiles, morphological, mechanical, and rheological properties. Furthermore, the viability of fibroblast cells cultured inside the photocrosslinked hydrogels was investigated. The combination of chemical and physical crosslinking procedures enables the formation of hydrogels with highly versatile physical and chemical properties, while maintaining the viability of encapsulated cells. To our best knowledge, this is the first study reporting the synthesis of photocrosslinkable κ-CA with controllable compressive moduli, swelling ratios and pore size distributions. Moreover, by micromolding approaches, spatially controlled geometries and cell distribution patterns could be obtained, thus enabling the development of cell-material platforms that can be applied and tailored to a broad range of tissue engineering strategies., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We engineered functional cardiac patches by seeding neonatal rat cardiomyocytes onto carbon nanotube (CNT)-incorporated photo-cross-linkable gelatin methacrylate (GelMA) hydrogels. The resulting cardiac constructs showed excellent mechanical integrity and advanced electrophysiological functions. Specifically, myocardial tissues cultured on 50 μm thick CNT-GelMA showed 3 times higher spontaneous synchronous beating rates and 85% lower excitation threshold, compared to those cultured on pristine GelMA hydrogels. Our results indicate that the electrically conductive and nanofibrous networks formed by CNTs within a porous gelatin framework are the key characteristics of CNT-GelMA leading to improved cardiac cell adhesion, organization, and cell-cell coupling. Centimeter-scale patches were released from glass substrates to form 3D biohybrid actuators, which showed controllable linear cyclic contraction/extension, pumping, and swimming actuations. In addition, we demonstrate for the first time that cardiac tissues cultured on CNT-GelMA resist damage by a model cardiac inhibitor as well as a cytotoxic compound. Therefore, incorporation of CNTs into gelatin, and potentially other biomaterials, could be useful in creating multifunctional cardiac scaffolds for both therapeutic purposes and in vitro studies. These hybrid materials could also be used for neuron and other muscle cells to create tissue constructs with improved organization, electroactivity, and mechanical integrity.

To engineer tissue-like structures, cells must organize themselves into three-dimensional (3D) networks that mimic the native tissue microarchitecture. Microfabricated hydrogel substrates provide a potentially useful platform for directing cells into biomimetic tissue architecture in vitro. Here, we present microgrooved methacrylated gelatin hydrogels as a suitable platform to build muscle-like fibrous structures in a facile and highly reproducible fashion. Microgrooved hydrogel substrates with two different ridge sizes (50 and 100 μm) were fabricated to assess the effect of the distance between engineered myofibers on the orientation of the bridging C2C12 myoblasts and the formation of the resulting multinucleated myotubes. It was shown that although the ridge size did not significantly affect the C2C12 myoblast alignment, the wider-ridged micropatterned hydrogels generated more myotubes that were not aligned to the groove direction as compared to those on the smaller-ridge micropatterns. We also demonstrated that electrical stimulation improved the myoblast alignment and increased the diameter of the resulting myotubes. By using the microstructured methacrylated gelatin substrates, we built free-standing 3D muscle sheets, which contracted when electrically stimulated. Given their robust contractility and biomimetic microarchitecture, engineered tissues may find use in tissue engineering, biological studies, high-throughput drug screening, and biorobotics.

Substantial progress has been made in the field of cardiovascular tissue engineering with an ever increasing number of clinically viable implants being reported. However, poor cellular integration of constructs remains a major problem. Limitations in our knowledge of cell/substrate interactions and their impact upon cell proliferation, survival and phenotype are proving to be a major hindrance. Advances in nanotechnology have allowed researchers to fabricate scaffolds which mimic the natural cell environment to a greater extent; allowing the elucidation of appropriate physical cues which influence cell behaviour. The ability to manipulate cell/substrate interactions at the micro/nano scale may help to create a viable cellular environment which can integrate effectively with the host tissue. This review summarises the influence of nanotopographical features on cell behaviour and provides details of some popular fabricating techniques to manufacture 3D scaffolds for tissue engineering. Recent examples of the translation of this research into fabricating clinically viable implants for the regeneration of cardiovascular tissues are also provided.

The success of tissue engineering will rely on the ability to generate complex, cell seeded three-dimensional (3D) structures. Therefore, methods that can be used to precisely engineer the architecture and topography of scaffolding materials will represent a critical aspect of functional tissue engineering. Previous approaches for 3D scaffold fabrication based on top-down and process driven methods are often not adequate to produce complex structures due to the lack of control on scaffold architecture, porosity, and cellular interactions. The proposed projection stereolithography (PSL) platform can be used to design intricate 3D tissue scaffolds that can be engineered to mimic the microarchitecture of tissues, based on computer aided design (CAD). The PSL system was developed, programmed and optimized to fabricate 3D scaffolds using gelatin methacrylate (GelMA). Variation of the structure and prepolymer concentration enabled tailoring the mechanical properties of the scaffolds. A dynamic cell seeding method was utilized to improve the coverage of the scaffold throughout its thickness. The results demonstrated that the interconnectivity of pores allowed for uniform human umbilical vein endothelial cells (HUVECs) distribution and proliferation in the scaffolds, leading to high cell density and confluency at the end of the culture period. Moreover, immunohistochemistry results showed that cells seeded on the scaffold maintained their endothelial phenotype, demonstrating the biological functionality of the microfabricated GelMA scaffolds., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hydrogels that mimic biological extracellular matrix (ECM) can provide cells with mechanical support and signaling cues to regulate their behavior. However, despite the ability of hydrogels to generate artificial ECM that can modulate cellular behavior, they often lack the mechanical strength needed for many tissue constructs. Here, we present reinforced CNT-gelatin methacrylate (GelMA) hybrid as a biocompatible, cell-responsive hydrogel platform for creating cell-laden three-dimensional (3D) constructs. The addition of carbon nanotubes (CNTs) successfully reinforced GelMA hydrogels without decreasing their porosity or inhibiting cell growth. The CNT-GelMA hybrids were also photopatternable allowing for easy fabrication of microscale structures without harsh processes. NIH-3T3 cells and human mesenchymal stem cells (hMSCs) readily spread and proliferated after encapsulation in CNT-GelMA hybrid microgels. By controlling the amount of CNTs incorporated into the GelMA hydrogel system, we demonstrated that the mechanical properties of the hybrid material can be tuned making it suitable for various tissue engineering applications. Furthermore, due to the high pattern fidelity and resolution of CNT incorporated GelMA, it can be used for in vitro cell studies or fabricating complex 3D biomimetic tissue-like structures., (© 2011 American Chemical Society)

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue-engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue-engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation. By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue-engineered heart valve may one day be realized. A viable tissue-engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

The aim of this study was to investigate the feasibility of fabricating porous crosslinked chitosan hydrogels in an aqueous phase using dense gas CO(2) as a foaming agent. Highly porous chitosan hydrogels were formed by using glutaraldehyde and genipin as crosslinkers. The method developed here eliminates the formation of a skin layer, and does not require the use of surfactants or other toxic reagents to generate porosity. The chitosan hydrogel scaffolds had an average pore diameter of 30-40 μm. The operating pressure had a negligible effect on the pore characteristics of chitosan hydrogels. Temperature, reaction period, type of biopolymer and crosslinker had a significant impact on the pore size and characteristics of the hydrogel produced by dense gas CO(2). Scanning electron microscopy and histological analysis confirmed that the resulting porous structures allowed fibroblasts seeded on these scaffolds to proliferate into the three-dimensional (3-D) structure of these chitosan hydrogels. Live/dead staining and MTS analysis demonstrated that fibroblast cells proliferated over 7 days. The fabricated hydrogels exhibited comparable mechanical strength and swelling ratio and are potentially useful for soft tissue engineering applications such as skin and cartilage regeneration., (Copyright © 2010. Published by Elsevier Ltd.)

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.

For tissue engineering applications, scaffolds should be porous to enable rapid nutrient and oxygen transfer while providing a three-dimensional (3D) microenvironment for the encapsulated cells. This dual characteristic can be achieved by fabrication of porous hydrogels that contain encapsulated cells. In this work, we developed a simple method that allows cell encapsulation and pore generation inside alginate hydrogels simultaneously. Gelatin beads of 150-300 microm diameter were used as a sacrificial porogen for generating pores within cell-laden hydrogels. Gelation of gelatin at low temperature (4 degrees C) was used to form beads without chemical crosslinking and their subsequent dissolution after cell encapsulation led to generation of pores within cell-laden hydrogels. The pore size and porosity of the scaffolds were controlled by the gelatin bead size and their volume ratio, respectively. Fabricated hydrogels were characterized for their internal microarchitecture, mechanical properties and permeability. Hydrogels exhibited a high degree of porosity with increasing gelatin bead content in contrast to nonporous alginate hydrogel. Furthermore, permeability increased by two to three orders while compressive modulus decreased with increasing porosity of the scaffolds. Application of these scaffolds for tissue engineering was tested by encapsulation of hepatocarcinoma cell line (HepG2). All the scaffolds showed similar cell viability; however, cell proliferation was enhanced under porous conditions. Furthermore, porous alginate hydrogels resulted in formation of larger spheroids and higher albumin secretion compared to nonporous conditions. These data suggest that porous alginate hydrogels may have provided a better environment for cell proliferation and albumin production. This may be due to the enhanced mass transfer of nutrients, oxygen and waste removal, which is potentially beneficial for tissue engineering and regenerative medicine applications.

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm x 5 mm x 81 microm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 microm thick per layer) with controlled spatial resolution (proximal axis: 18.0 +/- 7.0 microm and distal axis: 0.5 +/- 4.9 microm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 +/- 2 cells/mm(2) at 1 million cells/mL, 122 +/- 20 cells/mm(2) at 5 million cells/mL, and 216 +/- 38 cells/mm(2) at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.

Development of biodegradable tough elastomeric scaffolds are important for engineering tissues such as myocardium and heart valves that experience dynamic environments in vivo. Biomaterial scaffolds should ideally provide appropriate physical, chemical and mechanical cues to the seeded cells to closely mimic the native ECM. Collagen fibers form an important component of native myocardium as well as heart valve leaflets and provide necessary tensile properties to these tissues. Amongst various polymers, collagen mimicking biodegradable elastomer, Poly-(glycerol-sebacate) (PGS) has shown great promise in microfabricated scaffolds for cardiac tissue engineering. However, its use is limited by its solubility and the ability to cast nano-/microfibrous structures. For its superior mechanical properties, thermal or UV crosslinking of the pre-polymer is required under high temperatures and vacuum limiting fabrication of fibers. In this work, we fabricated electrospun PGS fibers were fabricated by simply blending it with biodegradable polycaprolactone (PCL) polymer without any post-processing. It was hypothesized that microfibrous PGS-PCL scaffolds would provide appropriate physical (fibrous structure) and chemical (balanced hydrophilicity and hydrophobicity) to the cells in addition to the mechanical properties.

Synthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

Interconnected pathways in 3D bioartificial organs are essential to retaining cell activity in thick functional 3D tissues. 3D bioprinting methods have been widely explored in biofabrication of functionally patterned tissues; however, these methods are costly and confined to thin tissue layers due to poor control of low-viscosity bioinks. Here, cell-laden hydrogels that could be precisely patterned via water-soluble gelatin templates are constructed by economical extrusion 3D printed plastic templates. Tortuous co-continuous plastic networks, designed based on triply periodic minimal surfaces (TPMS), serve as a sacrificial pattern to shape the secondary sacrificial gelatin templates. These templates are eventually used to form cell-encapsulated gelatin methacryloyl (GelMA) hydrogel scaffolds patterned with the complex interconnected pathways. The proposed fabrication process is compatible with photo-crosslinkable hydrogels wherein prepolymer casting enables incorporation of high cell populations with high viability. The cell-laden hydrogel constructs are characterized by robust mechanical behavior. In vivo studies demonstrate a superior cell ingrowth into the highly permeable constructs compared to the bulk hydrogels. Perfusable complex interconnected networks within cell-encapsulated hydrogels may assist in engineering thick and functional tissue constructs through the permeable internal channels for efficient cellular activities in vivo.

The design of new hydrogel-based biomaterials with tunable physical and biological properties is essential for the advancement of applications related to tissue engineering and regenerative medicine. For instance, interpenetrating polymer network (IPN) and semi-IPN hydrogels have been widely explored to engineer functional tissues due to their characteristic microstructural and mechanical properties. Here, we engineered IPN and semi-IPN hydrogels comprised of a tough pectin grafted polycaprolactone (pectin-g-PCL) component to provide mechanical stability, and a highly cytocompatible gelatin methacryloyl (GelMA) component to support cellular growth and proliferation. IPN hydrogels were formed by calcium ion (Ca2+)-crosslinking of pectin-g-PCL chains, followed by photocrosslinking of the GelMA precursor. Conversely, semi-IPN networks were formed by photocrosslinking of the pectin-g-PCL and GelMA mixture, in the absence of Ca2+ crosslinking. IPN and semi-IPN hydrogels synthesized with varying ratios of pectin-g-PCL to GelMA, with and without Ca2+-crosslinking, exhibited a broad range of mechanical properties. For semi-IPN hydrogels, the aggregation of microcrystalline cores led to formation of hydrogels with compressive moduli ranging from 3.1 to 10.4 kPa. For IPN hydrogels, the mechanistic optimization of pectin-g-PCL, GelMA, and Ca2+ concentrations resulted in hydrogels with comparatively higher compressive modulus, in the range of 39 kPa-5029 kPa. Our results also showed that IPN hydrogels were cytocompatible in vitro and could support the growth of three-dimensionally (3D) encapsulated MC3T3-E1 preosteoblasts in vitro. The simplicity, technical feasibility, low cost, tunable mechanical properties, and cytocompatibility of the engineered semi-IPN and IPN hydrogels highlight their potential for different tissue engineering and biomedical applications.

Nanoparticles have been used for engineering composite materials to improve the intrinsic properties and/or add functionalities to pristine polymers. The majority of the studies have focused on the incorporation of spherical nanoparticles within the composite fibers. Herein, we incorporate anisotropic branched-shaped zinc oxide (ZnO) nanoparticles into fibrous scaffolds fabricated by electrospinning. The addition of the branched particles resulted in their protrusion from fibers, mimicking the architecture of a rose stem. We demonstrated that the encapsulation of different-shape particles significantly influences the physicochemical and biological activities of the resultant composite scaffolds. In particular, the branched nanoparticles induced heterogeneous crystallization of the polymeric matrix and enhance the ultimate mechanical strain and strength. Moreover, the three-dimensional (3D) nature of the branched ZnO nanoparticles enhanced adhesion properties of the composite scaffolds to the tissues. In addition, the rose stem-like constructs offered excellent antibacterial activity, while supporting the growth of eukaryote cells.

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation.

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4 weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p

Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models.

Gelatin methacryloyl (GelMA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics. GelMA hydrogels closely resemble some essential properties of native extracellular matrix (ECM) due to the presence of cell-attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in GelMA-based scaffolds. GelMA is also versatile from a processing perspective. It crosslinks when exposed to light irradiation to form hydrogels with tunable mechanical properties. It can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, self-assembly, and microfluidic techniques to generate constructs with controlled architectures. Hybrid hydrogel systems can also be formed by mixing GelMA with nanoparticles such as carbon nanotubes and graphene oxide, and other polymers to form networks with desired combined properties and characteristics for specific biological applications. Recent research has demonstrated the proficiency of GelMA-based hydrogels in a wide range of tissue engineering applications including engineering of bone, cartilage, cardiac, and vascular tissues, among others. Other applications of GelMA hydrogels, besides tissue engineering, include fundamental cell research, cell signaling, drug and gene delivery, and bio-sensing.

Tissue engineered heart valves (TEHV) can be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, a directional electrospinning technique is used to fabricate fibrous poly(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resemble native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds are tuned by changing the ratio of PGS:PCL to mimic the native heart valve's mechanical properties. Primary human valvular interstitial cells (VICs) attach and align along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells are also biochemically active in producing heart-valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimick the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.

Biological scaffolds with tunable electrical and mechanical properties are of great interest in many different fields, such as regenerative medicine, biorobotics, and biosensing. In this study, dielectrophoresis (DEP) was used to vertically align carbon nanotubes (CNTs) within methacrylated gelatin (GelMA) hydrogels in a robust, simple, and rapid manner. GelMA-aligned CNT hydrogels showed anisotropic electrical conductivity and superior mechanical properties compared with pristine GelMA hydrogels and GelMA hydrogels containing randomly distributed CNTs. Skeletal muscle cells grown on vertically aligned CNTs in GelMA hydrogels yielded a higher number of functional myofibers than cells that were cultured on hydrogels with randomly distributed CNTs and horizontally aligned CNTs, as confirmed by the expression of myogenic genes and proteins. In addition, the myogenic gene and protein expression increased more profoundly after applying electrical stimulation along the direction of the aligned CNTs due to the anisotropic conductivity of the hybrid GelMA-vertically aligned CNT hydrogels. We believe that platform could attract great attention in other biomedical applications, such as biosensing, bioelectronics, and creating functional biomedical devices.