Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging.

Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogs were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm-specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

Strain-induced bands of Büngner formation promotes axon growth in 3D tissue-engineered constructs.

Treatment of peripheral nerve lesions remains a major challenge due to poor functional recovery; hence, ongoing research efforts strive to enhance peripheral nerve repair. In this study, we aimed to establish three-dimensional tissue-engineered bands of Büngner constructs by subjecting Schwann cells (SCs) embedded in fibrin hydrogels to mechanical stimulation. We show for the first time that the application of strain induces (i) longitudinal alignment of SCs resembling bands of Büngner, and (ii) the expression of a pronounced repair SC phenotype as evidenced by upregulation of BDNF, NGF, and p75NTR. Furthermore, we show that mechanically aligned SCs provide physical guidance for migrating axons over several millimeters in vitro in a co-culture model with rat dorsal root ganglion explants. Consequently, these constructs hold great therapeutic potential for transplantation into patients and might also provide a physiologically relevant in vitro peripheral nerve model for drug screening or investigation of pathologic or regenerative processes.

Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Cyclic Tensile Stress Induces Skeletal Muscle Hypertrophy and Myonuclear Accretion in a 3D Model.

Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and ß-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle. Impact statement Sustained adaption to mechanical stress presents a key feature for skeletal muscle functionality and growth. Knowledge of these processes, however, is mostly based on in vivo or 2D cell culture models, both of which entail significant shortcomings. Herein, we generated highly hypertrophic tissue-engineered skeletal muscle-like constructs that are comparable to the results of successful in vivo models of adaption to mechanical stimuli, achieving an outcome that only few in vitro approaches have reached. Second, we aimed at studying the underlying molecular mechanisms, which is of interest since there is little knowledge of the intracellular events during hypertrophy upon mechanical stimulation.

Effects of Endochondral and Intramembranous Ossification Pathways on Bone Tissue Formation and Vascularization in Human Tissue-Engineered Grafts.

Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways' constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body's repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture.

Optimizing the Surface Structural and Morphological Properties of Silk Thin Films via Ultra-Short Laser Texturing for Creation of Muscle Cell Matrix Model.

Temporary scaffolds that mimic the extracellular matrix's structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity-the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix.

Changes in Elastic Moduli of Fibrin Hydrogels Within the Myogenic Range Alter Behavior of Murine C2C12 and Human C25 Myoblasts Differently.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates ("2D") or embedded in fibrin hydrogels ("3D") with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels' fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7-30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.

Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering.

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.