Towards a Material-by-Design Approach to Electrospun Scaffolds for Tissue Engineering Based on Statistical Design of Experiments (DOE).

Electrospinning bears great potential for the manufacturing of scaffolds for tissue engineering, consisting of a porous mesh of ultrafine fibers that effectively mimic the extracellular matrix (ECM) and aid in directing stem cell fate. However, for engineering purposes, there is a need to develop material-by-design approaches based on predictive models. In this methodological study, a rational methodology based on statistical design of experiments (DOE) is discussed in detail, yielding heuristic models that capture the linkage between process parameters (Xs) of the electrospinning and scaffold properties (Ys). Five scaffolds made of polycaprolactone are produced according to a 22-factorial combinatorial scheme where two Xs, i.e., flow rate and applied voltage, are varied between two given levels plus a center point. The scaffolds were characterized to measure a set of properties (Ys), i.e., fiber diameter distribution, porosity, wettability, Young's modulus, and cell adhesion on murine myoblast C1C12 cells. Simple engineering DOE models were obtained for all Ys. Each Y, for example, the biological response, can be used as a driver for the design process, using the process-property model of interest for accurate interpolation within the design domain, enabling a material-by-design strategy and speeding up the product development cycle. The implications are also illustrated in the context of the design of multilayer scaffolds with microstructural gradients and controlled properties of each layer. The possibility of obtaining statistical models correlating between diverse output properties of the scaffolds is highlighted. Noteworthy, the featured DOE approach can be potentially merged with artificial intelligence tools to manage complexity and it is applicable to several fields including 3D printing.

Electrically conductive scaffolds mimicking the hierarchical structure of cardiac myofibers.

Electrically conductive scaffolds, mimicking the unique directional alignment of muscle fibers in the myocardium, are fabricated using the 3D printing micro-stereolithography technique. Polyethylene glycol diacrylate (photo-sensitive polymer), Irgacure 819 (photo-initiator), curcumin (dye) and polyaniline (conductive polymer) are blended to make the conductive ink that is crosslinked using free radical photo-polymerization reaction. Curcumin acts as a liquid filter and prevents light from penetrating deep into the photo-sensitive solution and plays a central role in the 3D printing process. The obtained scaffolds demonstrate well defined morphology with an average pore size of 300 ± 15 µm and semi-conducting properties with a conductivity of ~ 10-6 S/m. Cyclic voltammetry analyses detect the electroactivity and highlight how the electron transfer also involve an ionic diffusion between the polymer and the electrolyte solution. Scaffolds reach their maximum swelling extent 30 min after immersing in the PBS at 37 °C and after 4 weeks they demonstrate a slow hydrolytic degradation rate typical of polyethylene glycol network. Conductive scaffolds display tunable conductivity and provide an optimal environment to the cultured mouse cardiac progenitor cells.

Regenerative medicine technologies applied to transplant medicine. An update.

Regenerative medicine (RM) is changing how we think and practice transplant medicine. In regenerative medicine, the aim is to develop and employ methods to regenerate, restore or replace damaged/diseased tissues or organs. Regenerative medicine investigates using tools such as novel technologies or techniques, extracellular vesicles, cell-based therapies, and tissue-engineered constructs to design effective patient-specific treatments. This review illustrates current advancements in regenerative medicine that may pertain to transplant medicine. We highlight progress made and various tools designed and employed specifically for each tissue or organ, such as the kidney, heart, liver, lung, vasculature, gastrointestinal tract, and pancreas. By combing both fields of transplant and regenerative medicine, we can harbor a successful collaboration that would be beneficial and efficacious for the repair and design of de novo engineered whole organs for transplantations.

From Soft to Hard Biomimetic Materials: Tuning Micro/Nano-Architecture of Scaffolds for Tissue Regeneration.

Failure of tissues and organs resulting from degenerative diseases or trauma has caused huge economic and health concerns around the world. Tissue engineering represents the only possibility to revert this scenario owing to its potential to regenerate or replace damaged tissues and organs. In a regeneration strategy, biomaterials play a key role promoting new tissue formation by providing adequate space for cell accommodation and appropriate biochemical and biophysical cues to support cell proliferation and differentiation. Among other physical cues, the architectural features of the biomaterial as a kind of instructive stimuli can influence cellular behaviors and guide cells towards a specific tissue organization. Thus, the optimization of biomaterial micro/nano architecture, through different manufacturing techniques, is a crucial strategy for a successful regenerative therapy. Over the last decades, many micro/nanostructured biomaterials have been developed to mimic the defined structure of ECM of various soft and hard tissues. This review intends to provide an overview of the relevant studies on micro/nanostructured scaffolds created for soft and hard tissue regeneration and highlights their biological effects, with a particular focus on striated muscle, cartilage, and bone tissue engineering applications.

Intrinsically Conductive Polymers for Striated Cardiac Muscle Repair.

One of the most important features of striated cardiac muscle is the excitability that turns on the excitation-contraction coupling cycle, resulting in the heart blood pumping function. The function of the heart pump may be impaired by events such as myocardial infarction, the consequence of coronary artery thrombosis due to blood clots or plaques. This results in the death of billions of cardiomyocytes, the formation of scar tissue, and consequently impaired contractility. A whole heart transplant remains the gold standard so far and the current pharmacological approaches tend to stop further myocardium deterioration, but this is not a long-term solution. Electrically conductive, scaffold-based cardiac tissue engineering provides a promising solution to repair the injured myocardium. The non-conductive component of the scaffold provides a biocompatible microenvironment to the cultured cells while the conductive component improves intercellular coupling as well as electrical signal propagation through the scar tissue when implanted at the infarcted site. The in vivo electrical coupling of the cells leads to a better regeneration of the infarcted myocardium, reducing arrhythmias, QRS/QT intervals, and scar size and promoting cardiac cell maturation. This review presents the emerging applications of intrinsically conductive polymers in cardiac tissue engineering to repair post-ischemic myocardial insult.

Extrinsically Conductive Nanomaterials for Cardiac Tissue Engineering Applications.

Myocardial infarction (MI) is the consequence of coronary artery thrombosis resulting in ischemia and necrosis of the myocardium. As a result, billions of contractile cardiomyocytes are lost with poor innate regeneration capability. This degenerated tissue is replaced by collagen-rich fibrotic scar tissue as the usual body response to quickly repair the injury. The non-conductive nature of this tissue results in arrhythmias and asynchronous beating leading to total heart failure in the long run due to ventricular remodelling. Traditional pharmacological and assistive device approaches have failed to meet the utmost need for tissue regeneration to repair MI injuries. Engineered heart tissues (EHTs) seem promising alternatives, but their non-conductive nature could not resolve problems such as arrhythmias and asynchronous beating for long term in-vivo applications. The ability of nanotechnology to mimic the nano-bioarchitecture of the extracellular matrix and the potential of cardiac tissue engineering to engineer heart-like tissues makes it a unique combination to develop conductive constructs. Biomaterials blended with conductive nanomaterials could yield conductive constructs (referred to as extrinsically conductive). These cell-laden conductive constructs can alleviate cardiac functions when implanted in-vivo. A succinct review of the most promising applications of nanomaterials in cardiac tissue engineering to repair MI injuries is presented with a focus on extrinsically conductive nanomaterials.

3D Printing Decellularized Extracellular Matrix to Design Biomimetic Scaffolds for Skeletal Muscle Tissue Engineering.

Functional engineered muscles are still a critical clinical issue to be addressed, although different strategies have been considered so far for the treatment of severe muscular injuries. Indeed, the regenerative capacity of skeletal muscle (SM) results inadequate for large-scale defects, and currently, SM reconstruction remains a complex and unsolved task. For this aim, tissue engineered muscles should provide a proper biomimetic extracellular matrix (ECM) alternative, characterized by an aligned/microtopographical structure and a myogenic microenvironment, in order to promote muscle regeneration. As a consequence, both materials and fabrication techniques play a key role to plan an effective therapeutic approach. Tissue-specific decellularized ECM (dECM) seems to be one of the most promising material to support muscle regeneration and repair. 3D printing technologies, on the other side, enable the fabrication of scaffolds with a fine and detailed microarchitecture and patient-specific implants with high structural complexity. To identify innovative biomimetic solutions to develop engineered muscular constructs for the treatment of SM loss, the more recent (last 5 years) reports focused on SM dECM-based scaffolds and 3D printing technologies for SM regeneration are herein reviewed. Possible design inputs for 3D printed SM dECM-based scaffolds for muscular regeneration are also suggested.

Harnessing Inorganic Nanoparticles to Direct Macrophage Polarization for Skeletal Muscle Regeneration.

Modulation of macrophage plasticity is emerging as a successful strategy in tissue engineering (TE) to control the immune response elicited by the implanted material. Indeed, one major determinant of success in regenerating tissues and organs is to achieve the correct balance between immune pro-inflammatory and pro-resolution players. In recent years, nanoparticle-mediated macrophage polarization towards the pro- or anti-inflammatory subtypes is gaining increasing interest in the biomedical field. In TE, despite significant progress in the use of nanomaterials, the full potential of nanoparticles as effective immunomodulators has not yet been completely realized. This work discusses the contribution that nanotechnology gives to TE applications, helping native or synthetic scaffolds to direct macrophage polarization; here, three bioactive metallic and ceramic nanoparticles (gold, titanium oxide, and cerium oxide nanoparticles) are proposed as potential valuable tools to trigger skeletal muscle regeneration.

Information-Driven Design as a Potential Approach for 3D Printing of Skeletal Muscle Biomimetic Scaffolds.

Severe muscle injuries are a real clinical issue that still needs to be successfully addressed. Tissue engineering can represent a potential approach for this aim, but effective healing solutions have not been developed yet. In this regard, novel experimental protocols tailored to a biomimetic approach can thus be defined by properly systematizing the findings acquired so far in the biomaterials and scaffold manufacturing fields. In order to plan a more comprehensive strategy, the extracellular matrix (ECM), with its properties stimulating neomyogenesis and vascularization, should be considered as a valuable biomaterial to be used to fabricate the tissue-specific three-dimensional structure of interest. The skeletal muscle decellularized ECM can be processed and printed, e.g., by means of stereolithography, to prepare bioactive and biomimetic 3D scaffolds, including both biochemical and topographical features specifically oriented to skeletal muscle regenerative applications. This paper aims to focus on the skeletal muscle tissue engineering sector, suggesting a possible approach to develop instructive scaffolds for a guided healing process.

Smart ECM-Based Electrospun Biomaterials for Skeletal Muscle Regeneration.

The development of smart and intelligent regenerative biomaterials for skeletal muscle tissue engineering is an ongoing challenge, owing to the requirement of achieving biomimetic systems able to communicate biological signals and thus promote optimal tissue regeneration. Electrospinning is a well-known technique to produce fibers that mimic the three dimensional microstructural arrangements, down to nanoscale and the properties of the extracellular matrix fibers. Natural and synthetic polymers are used in the electrospinning process; moreover, a blend of them provides composite materials that have demonstrated the potential advantage of supporting cell function and adhesion. Recently, the decellularized extracellular matrix (dECM), which is the noncellular component of tissue that retains relevant biological cues for cells, has been evaluated as a starting biomaterial to realize composite electrospun constructs. The properties of the electrospun systems can be further improved with innovative procedures of functionalization with biomolecules. Among the various approaches, great attention is devoted to the "click" concept in constructing a bioactive system, due to the modularity, orthogonality, and simplicity features of the "click" reactions. In this paper, we first provide an overview of current approaches that can be used to obtain biofunctional composite electrospun biomaterials. Finally, we propose a design of composite electrospun biomaterials suitable for skeletal muscle tissue regeneration.

Nanoporous Microsponge Particles (NMP) of Polysaccharides as Universal Carriers for Biomolecules Delivery.

Different polysaccharides-namely dextran, carboxymethyl dextran, alginate, and hyaluronic acid-were compared for the synthesis of nanoporous microsponges particles (NMPs) obtained from a one-pot self-precipitation/cross-linking process. The morphologies and sizes of the NMPs were evaluated comparatively with respect to polymer-to-polymer and cross-linker solvents (water-based vs. DMSO). We found that the radial distribution of the polymer in the near-spherical NMPs was found to peak either at the core or in the corona of the particle, depending both on the specific polymer or the solvent used for the formation of NMPs. The NMP porosity and the swelling capability were evaluated via scanning electron microscopy (SEM). The degradation study indicated that after 10 h incubation with a reducing agent, approximately 80% of the NMPs were disassembled into soluble polysaccharide chains. The adsorption and release capacity of each type of NMP were evaluated using fluorescently labeled bovine serum albumin and lysozyme as model proteins, highlighting a release time typically much longer than the corresponding adsorption time. The dependence of the adsorption-release performance on pH was demonstrated as well. Confocal microscopy images allowed us to probe the different distribution of labeled proteins inside the NMP. The safety and non-cytotoxicity of NMPs were evaluated after incubation with fibroblast 3T3 cells and showed that all types of NMPs did not adversely affect the cell viability for concentrations up to 2.25 µg/mL and an exposure time up to 120 h. Confocal microscopy imaging revealed also the effective interaction between NMPs and fibroblast 3T3 cells. Overall, this study describes a rapid, versatile, and facile approach for preparing a universal non-toxic, nanoporous carrier for protein delivery under physiological conditions.

Turning regenerative technologies into treatment to repair myocardial injuries.

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.

Extracellular matrix-based hydrogels obtained from human tissues: a work still in progress.

PURPOSE OF REVIEW: The current review summarizes contemporary decellularization and hydrogel manufacturing strategies in the field of tissue engineering and regenerative medicine. RECENT FINDINGS: Decellularized extracellular matrix (ECM) bioscaffolds are a valuable biomaterial that can be purposed into various forms of synthetic tissues such as hydrogels. ECM-based hydrogels can be of animal or human origin. The use of human tissues as a source for ECM hydrogels in the clinical setting is still in its infancy and current literature is scant and anecdotal, resulting in inconclusive results. SUMMARY: Thus far the methods used to obtain hydrogels from human tissues remains a work in progress. Gelation, the most complex technique in obtaining hydrogels, is challenging due to remarkable heterogeneity of the tissues secondary to interindividual variability. Age, sex, ethnicity, and preexisting conditions are factors that dramatically undermine the technical feasibility of the gelation process. This is contrasted with animals whose well defined anatomical and histological characteristics have been selectively bred for the goal of manufacturing hydrogels.

Scaffold-in-Scaffold Potential to Induce Growth and Differentiation of Cardiac Progenitor Cells.

The design of reliable biocompatible and biodegradable scaffolds remains one of the most important challenges for tissue engineering. In fact, properly designed scaffolds must display an adequate and interconnected porosity to facilitate cell spreading and colonization of the inner layers, and must release physical signals concurring to modulate cell function to ultimately drive cell fate. In this study, a combination of optimal mechanical and biochemical properties has been considered to design a one-component three-dimensional (3D) multitextured hydrogel scaffold to favor cell-scaffold interactions. A polyethylene glycol diacrylate woodpile (PEGDa-Wp) structure of the order of 100 µm has been manufactured using a microstereolithography process. Subsequently, the PEGDa-Wp has been embedded in a PEGDa hydrogel to obtain a 3D scaffold-in-scaffold (3D-SS) system. Finally, the 3D-SS capability to address cell fate has been assessed using human Lin- Sca-1+ cardiac progenitor cells (hCPCs). Results have shown that a multitextured 3D scaffold represents a favorable microenvironment to promote hCPC differentiation and orientation. In fact, while cultured on 3D-SS, hCPCs adopt an ordered 3D spatial orientation and activate the expression of structural proteins, such as the α-sarcomeric actinin, a specific marker of the cardiomyocyte phenotype, and connexin 43, the principal gap junction protein of the heart. Although preliminary, this study demonstrates that complex multitextured scaffolds closely mimicking the extracellular matrix structure and function are efficient in driving progenitor cell fate. A leap forward will be determined by the use of advanced 3D printing technologies that will improve multitextured scaffold manufacturing and their biological efficiency.

Cardiac Progenitor Cell Extraction from Human Auricles.

For many years, myocardial tissue has been considered terminally differentiated and, thus, incapable of regenerating. Recent studies have shown, instead, that cardiomyocytes, at least in part, are slowly substituted by new cells originating by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient. The protocol here reported describes how from auricles a population of multipotent, cardiogenic cells can be isolated, cultured, and differentiated. Further studies are needed to fully exploit this cell population, but, sampling auricles, it could be possible to treat cardiac patients using their own cells circumventing rejection or organ shortage limitations.

Dietary Flaxseed Mitigates Impaired Skeletal Muscle Regeneration: in Vivo, in Vitro and in Silico Studies.

BACKGROUND: Diets enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to exert a positive impact on muscle diseases. Flaxseed is one of the richest sources of n-3 PUFA acid α-linolenic acid (ALA). The aim of this study was to assess the effects of flaxseed and ALA in models of skeletal muscle degeneration characterized by high levels of Tumor Necrosis Factor-α (TNF). METHODS: The in vivo studies were carried out on dystrophic hamsters affected by muscle damage associated with high TNF plasma levels and fed with a long-term 30% flaxseed-supplemented diet. Differentiating C2C12 myoblasts treated with TNF and challenged with ALA represented the in vitro model. Skeletal muscle morphology was scrutinized by applying the Principal Component Analysis statistical method. Apoptosis, inflammation and myogenesis were analyzed by immunofluorescence. Finally, an in silico analysis was carried out to predict the possible pathways underlying the effects of n-3 PUFAs. RESULTS: The flaxseed-enriched diet protected the dystrophic muscle from apoptosis and preserved muscle myogenesis by increasing the myogenin and alpha myosin heavy chain. Moreover, it restored the normal expression pattern of caveolin-3 thereby allowing protein retention at the sarcolemma. ALA reduced TNF-induced apoptosis in differentiating myoblasts and prevented the TNF-induced inhibition of myogenesis, as demonstrated by the increased expression of myogenin, myosin heavy chain and caveolin-3, while promoting myotube fusion. The in silico investigation revealed that FAK pathways may play a central role in the protective effects of ALA on myogenesis. CONCLUSIONS: These findings indicate that flaxseed may exert potent beneficial effects by preserving skeletal muscle regeneration and homeostasis partly through an ALA-mediated action. Thus, dietary flaxseed and ALA may serve as a useful strategy for treating patients with muscle dystrophies.

α-Linolenic Acid Reduces TNF-Induced Apoptosis in C2C12 Myoblasts by Regulating Expression of Apoptotic Proteins.

Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions such as adjuvant strategy for preventing these conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested as having a positive impact on muscle diseases. We recently demonstrated that a diet enriched with plant derived n3-fatty acid, α-linolenic acid (ALA), exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we here investigate on the expression level of the anti- and pro-apoptotic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of tumor necrosis factor-α (TNF). The results demonstrated that ALA protective effect on C2C12 myoblasts was associated with a decrease in caspase-3 activity and an increase of the Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescuing Bcl-2 expression and to revert Bax translocation to mitochondria both affected in an opposite way by TNF, a major pro-inflammatory cytokine expressed in damaged skeletal muscle. Therefore, ALA counteracts inflammatory signals in the muscle microenvironment and may represent a valuable strategy for ameliorating skeletal muscle pathologies.

Hippo pathway effectors control cardiac progenitor cell fate by acting as dynamic sensors of substrate mechanics and nanostructure.

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

Histatins: salivary peptides with copper(II)- and zinc(II)-binding motifs: perspectives for biomedical applications.

Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

Stable phenotype and function of immortalized Lin-Sca-1+ cardiac progenitor cells in long-term culture: a step closer to standardization.

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin(-)Sca-1(+) CPCs (iCPC(Sca-1)) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC(Sca-1) were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC(Sca-1) differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin(-)Sca-1(+) CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC(Sca-1) significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC(Sca-1) are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC(Sca-1) as an in vitro model system for Lin(-)Sca-1(+) progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC(Sca-1) can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.