Direct Laser Writing: From Materials Synthesis and Conversion to Electronic Device Processing.

Direct Laser Writing (DLW) has been increasingly selected as a microfabrication route for efficient, cost-effective, high-resolution material synthesis and conversion. Concurrently, lasers participate in the patterning and assembly of functional geometries in several fields of application, of which electronics stand out. In this review, recent advances and strategies based on DLW for electronics microfabrication are surveyed and outlined, based on laser material growth strategies. First, the main DLW parameters influencing material synthesis and transformation mechanisms are summarized, aimed at selective, tailored writing of conductive and semiconducting materials. Additive and transformative DLW processing mechanisms are discussed, to open space to explore several categories of materials directly synthesized or transformed for electronics microfabrication. These include metallic conductors, metal oxides, transition metal chalcogenides and carbides, laser-induced graphene, and their mixtures. By accessing a wide range of material types, DLW-based electronic applications are explored, including processing components, energy harvesting and storage, sensing, and bioelectronics. The expanded capability of lasers to participate in multiple fabrication steps at different implementation levels, from material engineering to device processing, indicates their future applicability to next-generation electronics, where more accessible, green microfabrication approaches integrate lasers as comprehensive tools.

Unveiling Human Proteome Signatures of Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent but still poorly understood clinical entity. Its current pathophysiological understanding supports a critical role of comorbidities and their chronic effect on cardiac function and structure. Importantly, despite the replication of some HFpEF phenotypic features, to this day, experimental models have failed to bring new effective therapies to the clinical setting. Thus, the direct investigation of HFpEF human myocardial samples may unveil key, and possibly human-specific, pathophysiological mechanisms. This study employed quantitative proteomic analysis by advanced mass spectrometry (SWATH-MS) to investigate signaling pathways and pathophysiological mechanisms in HFpEF. Protein-expression profiles were analyzed in human left ventricular myocardial samples of HFpEF patients and compared with a mixed control group. Functional analysis revealed several proteins that correlate with HFpEF, including those associated with mitochondrial dysfunction, oxidative stress, and inflammation. Despite the known disease heterogeneity, proteomic profiles could indicate a reduced mitochondrial oxidative phosphorylation and fatty-acid oxidation capacity in HFpEF patients with diabetes. The proteomic characterization described in this work provides new insights. Furthermore, it fosters further questions related to HFpEF cellular pathophysiology, paving the way for additional studies focused on developing novel therapies and diagnosis strategies for HFpEF patients.

Evaluation of a co-culture of rapidly isolated chondrocytes and stem cells seeded on tri-layered collagen-based scaffolds in a caprine osteochondral defect model.

Cartilage has poor regenerative capacity and thus damage to the joint surfaces presents a major clinical challenge. Recent research has focussed on the development of tissue-engineered and cell-based approaches for the treatment of cartilage and osteochondral injuries, with current clinically available cell-based approaches including autologous chondrocyte implantation and matrix-assisted autologous chondrocyte implantation. However, these approaches have significant disadvantages due to the requirement for a two-stage surgical procedure and an in vitro chondrocyte expansion phase which increases logistical challenges, hospital times and costs. In this study, we hypothesized that seeding biomimetic tri-layered scaffolds, with proven regenerative potential, with chondrocyte/infrapatellar fat pad stromal cell co-cultures would improve their regenerative capacity compared to scaffolds implanted cell-free. Rapid cell isolation techniques, without the requirement for long term in vitro culture, were utilised to achieve co-cultures of chondrocytes and stromal cells and thus overcome the limitations of existing cell-based techniques. Cell-free and cell-seeded scaffolds were implanted in osteochondral defects, created within the femoral condyle and trochlear ridge, in a translational large animal goat model. While analysis showed trends towards delayed subchondral bone healing in the cell-seeded scaffold group, by the 12 month timepoint the cell-free and cell-seeded groups yield cartilage and bone tissue with comparable quality and quantity. The results of the study reinforce the potential of the biomimetic tri-layered scaffold to repair joint defects but failed to demonstrate a clear benefit from the addition of the CC/FPMSC co-culture to this scaffold. Taking into consideration the additional cost and complexity associated with the cell-seeded scaffold approach, this study demonstrates that the treatment of osteochondral defects using cell-free tri-layered scaffolds may represent a more prudent clinical approach.

Layer-specific stem cell differentiation in tri-layered tissue engineering biomaterials: Towards development of a single-stage cell-based approach for osteochondral defect repair.

Successful repair of osteochondral defects is challenging, due in part to their complex gradient nature. Tissue engineering approaches have shown promise with the development of layered scaffolds that aim to promote cartilage and bone regeneration within the defect. The clinical potential of implanting these scaffolds cell-free has been demonstrated, whereby cells from the host bone marrow MSCs infiltrate the scaffolds and promote cartilage and bone regeneration within the required regions of the defect. However, seeding the cartilage layer of the scaffold with a chondrogenic cell population prior to implantation may enhance cartilage tissue regeneration, thus enabling the treatment of larger defects. Here the development of a cell seeding approach capable of enhancing articular cartilage repair without the requirement for in vitro expansion of the cell population is explored. The intrinsic ability of a tri-layered scaffold previously developed in our group to direct stem cell differentiation in each layer of the scaffold was first demonstrated. Following this, the optimal chondrogenic cell seeding approach capable of enhancing the regenerative capacity of the tri-layered scaffold was demonstrated with the highest levels of chondrogenesis achieved with a co-culture of rapidly isolated infrapatellar fat pad MSCs (FPMSCs) and chondrocytes (CCs). The addition of FPMSCs to a relatively small number of CCs led to a 7.8-fold increase in the sGAG production over chondrocytes in mono-culture. This cell seeding approach has the potential to be delivered within a single-stage approach, without the requirement for costly in vitro expansion of harvested cells, to achieve rapid repair of osteochondral defects.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization.

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold's intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Human Extracellular-Matrix Functionalization of 3D hiPSC-Based Cardiac Tissues Improves Cardiomyocyte Maturation.

Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Recent discoveries have demonstrated the potential of the extracellular matrix (ECM) as a critical regulator in development, homeostasis, and injury of the cardiac microenvironment. Within this context, this work aimed to assess the impact of human cardiac ECM in the phenotype and maturation features of hiPSC-CM. Human ECM was isolated from myocardium tissue through a physical decellularization approach. The cardiac tissue decellularization process reduced DNA content significantly while maintaining ECM composition in terms of sulfated glycosaminoglycans (s-GAG) and collagen content. These ECM particles were successfully incorporated in three-dimensional (3D) hiPSC-CM aggregates (CM+ECM) with no impact on viability and metabolic activity throughout 20 days in 3D culture conditions. Also, CM+ECM aggregates displayed organized and longer sarcomeres, with improved calcium handling when compared to hiPSC-CM aggregates. This study shows that human cardiac ECM functionalization of hiPSC-based cardiac tissues improves cardiomyocyte maturation. The knowledge generated herein provides essential insights to streamline the application of ECM in the development of hiPSC-based therapies targeting cardiac diseases.

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features.

The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC-CM and single cells of hiPSC-derived EC and MC (hiPSC-EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC-EC+MC survival was promoted by the co-culture with hiPSC-CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC-EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC-CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC-CM, hiPSC-EC+MC, and extracellular matrix induced the maturation of hiPSC-CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.

Tissue-specific extracellular matrix scaffolds for the regeneration of spatially complex musculoskeletal tissues.

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

Stem cells display a donor dependent response to escalating levels of growth factor release from extracellular matrix-derived scaffolds.

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular-matrix (ECM)-derived scaffolds loaded with escalating levels of transforming growth factor (TGF)-ß3. It was also sought to determine if stem cells display a donor-dependent response to different doses of TGF-ß3, from low (5 ng) to high (200 ng), released from such scaffolds. It was found that ECM-derived scaffolds possess the capacity to bind and release increasing amounts of TGF-ß3, with between 60% and 75% of this growth factor released into the media over the first 12 days of culture. After seeding these scaffolds with human infrapatellar fat pad-derived stem cells (FPSCs), it was found that cartilage-specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak-loading cartilage ECM-derived scaffolds with high levels of TGF-ß3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM-derived scaffold coupled with appropriate levels of TGF-ß3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 John Wiley & Sons, Ltd.

Porous Scaffolds Derived from Devitalized Tissue Engineered Cartilaginous Matrix Support Chondrogenesis of Adult Stem Cells.

ECM-derived scaffolds have previously been developed from devitalized native cartilage and successfully used in tissue engineering. Such ECM-based biomaterials are commonly derived from animal tissue, which may not represent the ideal source for applications in human. Native human ECM can be used as an alternative to xenogeneic tissue; however, its supply may be limited, leading to the need for a more readily available source of such biomaterials. The objective of this study was to compare devitalized native and tissue engineered cartilaginous ECM as chondro-permissive scaffolds for tissue engineering. To this end, porous scaffolds were produced using ECM derived from porcine articular cartilage and cartilaginous sheets engineered using human bone marrow stem cells. An identical process was used to produce scaffolds from three different types of devitalized ECMs, namely that derived from porcine cartilage (Native), human engineered cartilaginous sheets (Eng), and human engineered cartilaginous sheets generated in the presence of growth factor releasing microspheres (Eng-MS). Scaffolds produced using both devitalized engineered and native ECM possessed similar mechanical properties, pore size and GAG content, although were compositionally distinct. After being seeded with human infrapatellar fat pad stem cells, the engineered ECM-derived scaffolds (no Microspheres) supported less robust cartilage matrix deposition than native ECM scaffolds. However, more chondro-permissive scaffolds could be generated using cartilaginous ECM engineered in the presence of TGF-ß1 releasing microspheres. Eng-MS scaffolds supported comparable levels of GAG synthesis to native ECM scaffolds. These results demonstrate that engineered ECM can be used to produce scaffolds for cartilage tissue engineering, overcoming stock limitations and other barriers associated with native autogeneic, allogeneic, and xenogeneic tissues. Such engineered ECM holds significant promise as an off-the-shelf chondro-permissive scaffold for articular cartilage repair.

Scaffolds Derived from ECM Produced by Chondrogenically Induced Human MSC Condensates Support Human MSC Chondrogenesis.

Osteoarthritis is a leading cause of disability affecting an increasing number of individuals. However, cartilage replacement therapies are inadequate, and better cartilage regeneration products must be developed. In this work, we describe a human mesenchymal stem cell (hMSC)-based approach for fabricating extracellular matrix (ECM) scaffolds from tissue-engineered cartilage sheets and then for inducing chondrogenesis of reseeded hMSCs within the ECM scaffolds. Two types of ECM scaffolds were fabricated: one from high-density hMSC sheets cultured with media-supplemented transforming growth factor beta-1 (TGF-ß1; -MS) and the other from high-density hMSC sheets incorporated with TGF-ß1-laden gelatin microspheres (+MS), which significantly enhance chondrogenesis within the sheet system. Interestingly, when scaffolds were reseeded with hMSCs, -MS scaffolds lead to significantly more glycosaminoglycan (GAG) accumulation than +MS scaffolds. Importantly, ECM scaffolds could be soak loaded with TGF-ß1 to produce cartilage of similar quality as that of constructs cultured with TGF-ß1 in the media, thereby removing the need for supplementing the media with the growth factor. Lastly, tissues formed with the scaffolds were larger with more uniform cartilage matrix elaboration compared to scaffold-free groups making this strategy a clinically promising auto- or allogeneic therapy.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

Infrapatellar Fat Pad Stem Cells: From Developmental Biology to Cell Therapy.

The ideal cell type to be used for cartilage therapy should possess a proven chondrogenic capacity, not cause donor-site morbidity, and should be readily expandable in culture without losing their phenotype. There are several cell sources being investigated to promote cartilage regeneration: mature articular chondrocytes, chondrocyte progenitors, and various stem cells. Most recently, stem cells isolated from joint tissue, such as chondrogenic stem/progenitors from cartilage itself, synovial fluid, synovial membrane, and infrapatellar fat pad (IFP) have gained great attention due to their increased chondrogenic capacity over the bone marrow and subcutaneous adipose-derived stem cells. In this review, we first describe the IFP anatomy and compare and contrast it with other adipose tissues, with a particular focus on the embryological and developmental aspects of the tissue. We then discuss the recent advances in IFP stem cells for regenerative medicine. We compare their properties with other stem cell types and discuss an ontogeny relationship with other joint cells and their role on in vivo cartilage repair. We conclude with a perspective for future clinical trials using IFP stem cells.

Coupling Freshly Isolated CD44(+) Infrapatellar Fat Pad-Derived Stromal Cells with a TGF-ß3 Eluting Cartilage ECM-Derived Scaffold as a Single-Stage Strategy for Promoting Chondrogenesis.

An alternative strategy to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold is used to provide an environment conducive to proliferation and tissue-specific differentiation in vivo. The objective of this study is to develop a cartilage extracellular matrix (ECM)-derived scaffold that could facilitate the rapid proliferation and chondrogenic differentiation of freshly isolated stromal cells. By freeze-drying cryomilled cartilage ECM of differing concentrations, it is possible to produce scaffolds with a range of pore sizes. The migration, proliferation, and chondrogenic differentiation of infrapatellar fat pad-derived stem cells (FPSCs) depend on the concentration/porosity of these scaffolds, with greater sulphated glycosaminoglycan (sGAG) accumulation observed in scaffolds with larger-sized pores. It is then sought to determine if freshly isolated fat pad-derived stromal cells, seeded onto a transforming growth factor (TGF)-ß3 eluting ECM-derived scaffold, could promote chondrogenesis in vivo. While a more cartilage-like tissue could be generated using culture expanded FPSCs compared to nonenriched freshly isolated cells, fresh CD44(+) stromal cells are capable of producing a tissue in vivo that stained strongly for sGAGs and type II collagen. These findings open up new possibilities for in-theatre cell-based therapies for joint regeneration.

Infrapatellar fat pad-derived stem cells maintain their chondrogenic capacity in disease and can be used to engineer cartilaginous grafts of clinically relevant dimensions.

A therapy for regenerating large cartilaginous lesions within the articular surface of osteoarthritic joints remains elusive. While tissue engineering strategies such as matrix-assisted autologous chondrocyte implantation can be used in the repair of focal cartilage defects, extending such approaches to the treatment of osteoarthritis will require a number of scientific and technical challenges to be overcome. These include the identification of an abundant source of chondroprogenitor cells that maintain their chondrogenic capacity in disease, as well as the development of novel approaches to engineer scalable cartilaginous grafts that could be used to resurface large areas of damaged joints. In this study, it is first demonstrated that infrapatellar fat pad-derived stem cells (FPSCs) isolated from osteoarthritic (OA) donors possess a comparable chondrogenic capacity to FPSCs isolated from patients undergoing ligament reconstruction. In a further validation of their functionality, we also demonstrate that FPSCs from OA donors respond to the application of physiological levels of cyclic hydrostatic pressure by increasing aggrecan gene expression and the production of sulfated glycosaminoglycans. We next explored whether cartilaginous grafts could be engineered with diseased human FPSCs using a self-assembly or scaffold-free approach. After examining a range of culture conditions, it was found that continuous supplementation with both transforming growth factor-ß3 (TGF-ß3) and bone morphogenic protein-6 (BMP-6) promoted the development of tissues rich in proteoglycans and type II collagen. The final phase of the study sought to scale-up this approach to engineer cartilaginous grafts of clinically relevant dimensions (≥2 cm in diameter) by assembling FPSCs onto electrospun PLLA fiber membranes. Over 6 weeks in culture, it was possible to generate robust, flexible cartilage-like grafts of scale, opening up the possibility that tissues engineered using FPSCs derived from OA patients could potentially be used to resurface large areas of joint surfaces damaged by trauma or disease.

Controlled release of transforming growth factor-ß3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells.

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-ß3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen-hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen-HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-ß3, the final phase of the study explored whether this scaffold could be used as a TGF-ß3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-ß3 that was loaded onto the scaffold was released in a controlled manner over the first 10days of culture, with comparable long-term chondrogenesis observed in these TGF-ß3-loaded constructs compared to scaffolds where the TGF-ß3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.