Hypothermic and cryogenic preservation of cardiac tissue-engineered constructs.

Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation. In this study, hypothermic preservation (4 °C) and two methods for cryopreservation (i.e., a slow and fast cooling approach to -196 °C and -150 °C, respectively) were investigated as potential solutions to extend the cTE construct implantation window. The cTE model used consisted of human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Cardiomyocyte viability, function (beat rate and calcium handling), and metabolic activity were investigated after rewarming. Our observations show that cardiomyocytes' age did not influence post-rewarming viability, however, it influenced construct function. Hypothermic preservation with HypoThermosol® ensured cardiomyocyte viability and function. Furthermore, fast freezing outperformed slow freezing, but both viability and function were severely reduced after rewarming. In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation with HypoThermosol® represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.

Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria.

Bacteria can be programmed to deliver natural materials with defined biological and mechanical properties for controlling cell growth and differentiation. Here, we present an elastic, resilient and bioactive polysaccharide derived from the extracellular matrix of Pantoea sp. BCCS 001. Specifically, it was methacrylated to generate a new photo crosslinkable hydrogel that we coined Pantoan Methacrylate or put simply PAMA. We have used it for the first time as a tissue engineering hydrogel to treat VML injuries in rats. The crosslinked PAMA hydrogel was super elastic with a recovery nearing 100 %, while mimicking the mechanical stiffness of native muscle. After inclusion of thiolated gelatin via a Michaelis reaction with acrylate groups on PAMA we could also guide muscle progenitor cells into fused and aligned tubes - something reminiscent of mature muscle cells. These results were complemented by sarcomeric alpha-actinin immunostaining studies. Importantly, the implanted hydrogels exhibited almost 2-fold more muscle formation and 50 % less fibrous tissue formation compared to untreated rat groups. In vivo inflammation and toxicity assays likewise gave rise to positive results confirming the biocompatibility of this new biomaterial system. Overall, our results demonstrate that programmable polysaccharides derived from bacteria can be used to further advance the field of tissue engineering. In greater detail, they could in the foreseeable future be used in practical therapies against VML.

Covalent Grafting of Functionalized MEW Fibers to Silk Fibroin Hydrogels to Obtain Reinforced Tissue Engineered Constructs.

Hydrogels are ideal materials to encapsulate cells, making them suitable for applications in tissue engineering and regenerative medicine. However, they generally do not possess adequate mechanical strength to functionally replace human tissues, and therefore they often need to be combined with reinforcing structures. While the interaction at the interface between the hydrogel and reinforcing structure is imperative for mechanical function and subsequent biological performance, this interaction is often overlooked. Melt electrowriting enables the production of reinforcing microscale fibers that can be effectively integrated with hydrogels. Yet, studies on the interaction between these micrometer scale fibers and hydrogels are limited. Here, we explored the influence of covalent interfacial interactions between reinforcing structures and silk fibroin methacryloyl hydrogels (silkMA) on the mechanical properties of the construct and cartilage-specific matrix production in vitro. For this, melt electrowritten fibers of a thermoplastic polymer blend (poly(hydroxymethylglycolide-co-ε-caprolactone):poly(ε-caprolactone) (pHMGCL:PCL)) were compared to those of the respective methacrylated polymer blend pMHMGCL:PCL as reinforcing structures. Photopolymerization of the methacrylate groups, present in both silkMA and pMHMGCL, was used to generate hybrid materials. Covalent bonding between the pMHMGCL:PCL blend and silkMA hydrogels resulted in an elastic response to the application of torque. In addition, an improved resistance was observed to compression (∼3-fold) and traction (∼40-55%) by the scaffolds with covalent links at the interface compared to those without these interactions. Biologically, both types of scaffolds (pHMGCL:PCL and pMHMGCL:PCL) showed similar levels of viability and metabolic activity, also compared to frequently used PCL. Moreover, articular cartilage progenitor cells embedded within the reinforced silkMA hydrogel were able to form a cartilage-like matrix after 28 days of in vitro culture. This study shows that hybrid cartilage constructs can be engineered with tunable mechanical properties by grafting silkMA hydrogels covalently to pMHMGCL:PCL blend microfibers at the interface.

Experimentally-guided in silico design of engineered heart tissues to improve cardiac electrical function after myocardial infarction.

Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.

Orthotopic equine study confirms the pivotal importance of structural reinforcement over the pre-culture of cartilage implants.

In articular cartilage (AC), the collagen arcades provide the tissue with its extraordinary mechanical properties. As these structures cannot be restored once damaged, functional restoration of AC defects remains a major challenge. We report that the use of a converged bioprinted, osteochondral implant, based on a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electrowritten polycaprolactone micrometer-scale fibers in a zonal fashion, inspired by native collagen architecture, can provide long-term mechanically stable neo-tissue in an orthotopic large animal model. The design of this novel implant was achieved via state-of-the-art converging of extrusion-based ceramic printing, melt electrowriting, and extrusion-based bioprinting. Interestingly, the cell-free implants, used as a control in this study, showed abundant cell ingrowth and similar favorable results as the cell-containing implants. Our findings underscore the hypothesis that mechanical stability is more determining for the successful survival of the implant than the presence of cells and pre-cultured extracellular matrix. This observation is of great translational importance and highlights the aptness of advanced 3D (bio)fabrication technologies for functional tissue restoration in the harsh articular joint mechanical environment.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

GelMA/TCP nanocomposite scaffold for vital pulp therapy.

Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) fibrous scaffold incorporating beta-tricalcium phosphate (TCP) particles for pulp capping. A comprehensive morphological, physical-chemical, and mechanical characterization of the engineered fibrous scaffolds was performed. In vitro bioactivity, cell compatibility, and odontogenic differentiation potential of the scaffolds in dental pulp stem cells (DPSCs) were also evaluated. A pre-clinical in vivo model was used to determine the therapeutic role of the GelMA/TCP scaffolds in promoting hard tissue formation. Morphological, chemical, and thermal analyses confirmed effective TCP incorporation in the GelMA nanofibers. The GelMA+20%TCP nanofibrous scaffold exhibited bead-free morphology and suitable mechanical and degradation properties. In vitro, GelMA+20%TCP scaffolds supported apatite-like formation, improved cell spreading, and increased deposition of mineralization nodules. Gene expression analysis revealed upregulation of ALPL, RUNX2, COL1A1, and DMP1 in the presence of TCP-laden scaffolds. In vivo, analyses showed mild inflammatory reaction upon scaffolds' contact while supporting mineralized tissue formation. Although the levels of Nestin and DMP1 proteins did not exceed those associated with the clinical reference treatment (i.e., mineral trioxide aggregate), the GelMA+20%TCP scaffold exhibited comparable levels, thus suggesting the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion. Our innovative GelMA/TCP scaffold represents a simplified and efficient alternative to conventional pulp-capping biomaterials. STATEMENT OF SIGNIFICANCE: Vital pulp therapy (VPT) aims to preserve dental pulp vitality and avoid root canal treatment. Biomaterials that bolster mineralized tissue regeneration with ease of use are still lacking. We successfully engineered gelatin methacryloyl (GelMA) electrospun scaffolds incorporated with beta-tricalcium phosphate (TCP) for VPT. Notably, electrospun GelMA-based scaffolds containing 20% (w/v) of TCP exhibited favorable mechanical properties and degradation, cytocompatibility, and mineralization potential indicated by apatite-like structures in vitro and mineralized tissue deposition in vivo, although not surpassing those associated with the standard of care. Collectively, our innovative GelMA/TCP scaffold represents a simplified alternative to conventional pulp capping materials such as MTA and Biodentine™ since it is a ready-to-use biomaterial, requires no setting time, and is therapeutically effective.

3D Printed Magneto-Active Microfiber Scaffolds for Remote Stimulation and Guided Organization of 3D In Vitro Skeletal Muscle Models.

This work reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a magnetized polycaprolactone-based composite. In situ iron oxide nanoparticle deposition on oxidized graphene yields homogeneously dispersed magnetic particles with sizes above 0.5 µm and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with high magnetic content (up to 20 weight %) are obtained and processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the creation of skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material, with elastic tensile deformability. External magnetic fields below 300 mT are sufficient to trigger out-of-plane reversible deformation. In vitro culture of C2C12 myoblasts on three-dimensional (3D) Matrigel/collagen/MEW scaffolds showed that microfibers guided the formation of 3D myotube architectures, and the presence of magnetic particles does not significantly affect viability or differentiation rates after 8 days. Centimeter-sized skeletal muscle constructs allowed for reversible, continued, and dynamic magneto-mechanical stimulation. Overall, these innovative microfiber scaffolds provide magnetically deformable platforms suitable for dynamic culture of skeletal muscle, offering potential for in vitro disease modeling.

Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration.

Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.

Microstructural differences in the osteochondral unit of terrestrial and aquatic mammals.

During evolution, animals have returned from land to water, adapting with morphological modifications to life in an aquatic environment. We compared the osteochondral units of the humeral head of marine and terrestrial mammals across species spanning a wide range of body weights, focusing on microstructural organization and biomechanical performance. Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of the microarchitecture-function relationship is pivotal for understanding articular biology and, hence, for the development of durable functional regenerative approaches for treatment of joint damage, which are thus far lacking.

Injectable hydrogels for cartilage and bone tissue regeneration: A review.

Annually, millions of patients suffer from irreversible injury owing to the loss or failure of an organ or tissue caused by accident, aging, or disease. The combination of injectable hydrogels and the science of stem cells have emerged to address this persistent issue in society by generating minimally invasive treatments to augment tissue function. Hydrogels are composed of a cross-linked network of polymers that exhibit a high-water retention capacity, thereby mimicking the wet environment of native cells. Due to their inherent mechanical softness, hydrogels can be used as needle-injectable stem cell carrier materials to mend tissue defects. Hydrogels are made of different natural or synthetic polymers, displaying a broad portfolio of eligible properties, which include biocompatibility, low cytotoxicity, shear-thinning properties as well as tunable biological and physicochemical properties. Presently, novel ongoing developments and native-like hydrogels are increasingly being used broadly to improve the quality of life of those with disabling tissue-related diseases. The present review outlines various future and in-vitro applications of injectable hydrogel-based biomaterials, focusing on the newest ongoing developments of in-situ forming injectable hydrogels for bone and cartilage tissue engineering purposes.

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct.

To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml-1collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml-1collagen and 1 mg·ml-1fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct.

Load-induced fluid pressurisation in hydrogel systems before and after reinforcement by melt-electrowritten fibrous meshes.

Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) technique enables three-dimensional printing of structured fibrous mesh with small fibre diameter (20 µm). The MEW mesh with 20 µm fibre diameter can synergistically increase the instantaneous mechanical stiffness of soft hydrogels. However, the reinforcing mechanism of the MEW meshes is not well understood, and may involve load-induced fluid pressurisation. Here, we examined the reinforcing effect of MEW meshes in three hydrogels: gelatin methacryloyl (GelMA), agarose and alginate, and the role of load-induced fluid pressurisation in the MEW reinforcement. We tested the hydrogels with and without MEW mesh (i.e., hydrogel alone, and MEW-hydrogel composite) using micro-indentation and unconfined compression, and analysed the mechanical data using biphasic Hertz and mixture models. We found that the MEW mesh altered the tension-to-compression modulus ratio differently for hydrogels that are cross-linked differently, which led to a variable change to their load-induced fluid pressurisation. MEW meshes only enhanced the fluid pressurisation for GelMA, but not for agarose or alginate. We speculate that only covalently cross-linked hydrogels (GelMA) can effectively tense the MEW meshes, thereby enhancing the fluid pressure developed during compressive loading. In conclusion, load-induced fluid pressurisation in selected hydrogels was enhanced by MEW fibrous mesh, and may be controlled by MEW mesh of different designs in the future, thereby making fluid pressure a tunable cell growth stimulus for tissue engineering involving mechanical stimulation.

Multi-leveled Nanosilicate Implants Can Facilitate Near-Perfect Bone Healing.

Several studies have shown that nanosilicate-reinforced scaffolds are suitable for bone regeneration. However, hydrogels are inherently too soft for load-bearing bone defects of critical sizes, and hard scaffolds typically do not provide a suitable three-dimensional (3D) microenvironment for cells to thrive, grow, and differentiate naturally. In this study, we bypass these long-standing challenges by fabricating a cell-free multi-level implant consisting of a porous and hard bone-like framework capable of providing load-bearing support and a softer native-like phase that has been reinforced with nanosilicates. The system was tested with rat bone marrow mesenchymal stem cells in vitro and as a cell-free system in a critical-sized rat bone defect. Overall, our combinatorial and multi-level implant design displayed remarkable osteoconductivity in vitro without differentiation factors, expressing significant levels of osteogenic markers compared to unmodified groups. Moreover, after 8 weeks of implantation, histological and immunohistochemical assays indicated that the cell-free scaffolds enhanced bone repair up to approximately 84% following a near-complete defect healing. Overall, our results suggest that the proposed nanosilicate bioceramic implant could herald a new age in the field of orthopedics.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Unveiling the potential of melt electrowriting in regenerative dental medicine.

For nearly three decades, tissue engineering strategies have been leveraged to devise effective therapeutics for dental, oral, and craniofacial (DOC) regenerative medicine and treat permanent deformities caused by many debilitating health conditions. In this regard, additive manufacturing (AM) allows the fabrication of personalized scaffolds that have the potential to recapitulate native tissue morphology and biomechanics through the utilization of several 3D printing techniques. Among these, melt electrowriting (MEW) is a versatile direct electrowriting process that permits the development of well-organized fibrous constructs with fiber resolutions ranging from micron to nanoscale. Indeed, MEW offers great prospects for the fabrication of scaffolds mimicking tissue specificity, healthy and pathophysiological microenvironments, personalized multi-scale transitions, and functional interfaces for tissue regeneration in medicine and dentistry. Excitingly, recent work has demonstrated the potential of converging MEW with other AM technologies and/or cell-laden scaffold fabrication (bioprinting) as a favorable route to overcome some of the limitations of MEW for DOC tissue regeneration. In particular, such convergency fabrication strategy has opened great promise in terms of supporting multi-tissue compartmentalization and predetermined cell commitment. In this review, we offer a critical appraisal on the latest advances in MEW and its convergence with other biofabrication technologies for DOC tissue regeneration. We first present the engineering principles of MEW and the most relevant design aspects for transition from flat to more anatomically relevant 3D structures while printing highly-ordered constructs. Secondly, we provide a thorough assessment of contemporary achievements using MEW scaffolds to study and guide soft and hard tissue regeneration, and draw a parallel on how to extrapolate proven concepts for applications in DOC tissue regeneration. Finally, we offer a combined engineering/clinical perspective on the fabrication of hierarchically organized MEW scaffold architectures and the future translational potential of site-specific, single-step scaffold fabrication to address tissue and tissue interfaces in dental, oral, and craniofacial regenerative medicine. STATEMENT OF SIGNIFICANCE: Melt electrowriting (MEW) techniques can further replicate the complexity of native tissues and could be the foundation for novel personalized (defect-specific) and tissue-specific clinical approaches in regenerative dental medicine. This work presents a unique perspective on how MEW has been translated towards the application of highly-ordered personalized multi-scale and functional interfaces for tissue regeneration, targeting the transition from flat to anatomically-relevant three-dimensional structures. Furthermore, we address the value of convergence of biofabrication technologies to overcome the traditional manufacturing limitations provided by multi-tissue complexity. Taken together, this work offers abundant engineering and clinical perspectives on the fabrication of hierarchically MEW architectures aiming towards site-specific implants to address complex tissue damage in regenerative dental medicine.

Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues.

Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 µm and large 500 µm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 µm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 µm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.

Self-assembling peptide-laden electrospun scaffolds for guided mineralized tissue regeneration.

OBJECTIVES: Electrospun scaffolds are a versatile biomaterial platform to mimic fibrillar structure of native tissues extracellular matrix, and facilitate the incorporation of biomolecules for regenerative therapies. Self-assembling peptide P11-4 has emerged as a promising strategy to induce mineralization; however, P11-4 application has been mostly addressed for early caries lesions repair on dental enamel. Here, to investigate P11-4's efficacy on bone regeneration, polymeric electrospun scaffolds were developed, and then distinct concentrations of P11-4 were physically adsorbed on the scaffolds. METHODS: P11-4-laden and pristine (P11-4-free) electrospun scaffolds were immersed in simulated body fluid and mineral precipitation identified by SEM. Functional groups and crystalline phases were analyzed by FTIR and XRD, respectively. Cytocompatibility, mineralization, and gene expression assays were conducted using stem cells from human exfoliated deciduous teeth. To investigate P11-4-laden scaffolds potential to induce in vivo mineralization, an established rat calvaria critical-size defect model was used. RESULTS: We successfully synthesized nanofibrous (∼ 500 nm fiber diameter) scaffolds and observed that functionalization with P11-4 did not affect the fibers' diameter. SEM images indicated mineral precipitation, while FTIR and XRD confirmed apatite-like formation and crystallization for P11-4-laden scaffolds. In addition, P11-4-laden scaffolds were cytocompatible, highly stimulated cell-mediated mineral deposition, and upregulated the expression of mineralization-related genes compared to pristine scaffolds. P11-4-laden scaffolds led to enhanced in vivo bone regeneration after 8 weeks compared to pristine PCL. SIGNIFICANCE: Electrospun scaffolds functionalized with P11-4 are a promising strategy for inducing mineralized tissues regeneration in the craniomaxillofacial complex.