Chitosan-collagen-hydroxyapatite membranes for tissue engineering.

Tissue engineering is growing in developing new technologies focused on providing effective solutions to degenerative pathologies that affect different types of connective tissues. The search for biocompatible, bioactive, biodegradable, and multifunctional materials has grown significantly in recent years. Chitosan, calcium phosphates collagen, and their combination as composite materials fulfill the required properties and could result in biostimulation for tissue regeneration. In the present work, the chitosan/collagen/hydroxyapatite membranes were prepared with different concentrations of collagen and hydroxyapatite. Cell adhesion was evaluated by MTS assay for two in vitro models. Additionally, cytotoxicity of the different membranes employing hemolysis of erythrocytes isolated from human blood was carried out. The structure of the membranes was analyzed by X-rays diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal stability properties by thermogravimetric methods (TGA). The highest cell adhesion after 48 h was obtained for chitosan membranes with the highest hydroxyapatite and collagen content. All composite membranes showed good cell adhesion and low cytotoxicity, suggesting that these materials have a significant potential to be used as biomaterials for tissue engineering. Graphical abstract.

Loading of erythropoietin on biphasic calcium phosphate bioceramics promotes osteogenesis and angiogenesis by regulating EphB4/EphrinB2 molecules.

Improving osteogenesis and angiogenesis using different cells and drugs is critical in the field of bone tissue engineering. Recent research has found that erythropoietin (EPO) plays an important role in both osteogenesis and angiogenesis. In this study, we grafted polydopamine and EPO onto the surface of biphasic calcium phosphate. The characterization and release property of the modified bioceramics were assessed. Cell proliferation, expression of osteoblastic and endothelial markers, and EphB4/EphrinB2 molecules were investigated while employing co-cultures of two different cells [rat vein endothelial cells (VECs) and rat bone marrow mesenchymal stromal cells (BMSCs)]. The modified bioceramics were finally implanted into the SD rats' femurs and followed by investigating the bone defect repair efficacy and the expression of EphB4/EphrinB2 molecules in vivo. The results indicated that the modified bioceramics could control the release of EPO continuously. The osteogenesis and angiogenesis were improved along with the increased expression of EphB4/EphrinB2 molecules. The expression of EphB4/EphrinB2 molecules was also significantly increased in vivo and the bone defect was repaired effectively. Overall, our findings demonstrated that EPO loading on biphasic calcium phosphate bioceramics could promote both osteogenesis and angiogenesis. The results suggest that EphB4/EphrinB2 may be crucial in the process. Graphical abstract.

Phenotypic change of mesenchymal stem cells into smooth muscle cells regulated by dynamic cell-surface interactions on patterned arrays of ultrathin graphene oxide substrates.

The topographical interface of the extracellular environment has been appreciated as a principal biophysical regulator for modulating cell functions, such as adhesion, migration, proliferation, and differentiation. Despite the existed approaches that use two-dimensional nanomaterials to provide beneficial effects, opportunities evaluating their impact on stem cells remain open to elicit unprecedented cellular responses. Herein, we report an ultrathin cell-culture platform with potential-responsive nanoscale biointerfaces for monitoring mesenchymal stem cells (MSCs). We designed an intriguing nanostructured array through self-assembly of graphene oxide sheets and subsequent lithographical patterning method to produce chemophysically defined regions. MSCs cultured on anisotropic micro/nanoscale patterned substrate were spontaneously organized in a highly ordered configuration mainly due to the cell-repellent interactions. Moreover, the spatially aligned MSCs were spontaneously differentiated into smooth muscle cells upon the specific crosstalk between cells. This work provides a robust strategy for directing stem cells and differentiation, which can be utilized as a potential cell culture platform to understand cell-substrate or cell-cell interactions, further developing tissue repair and stem cell-based therapies.

Clinical Applications of Additive Manufacturing Models in Neurosurgery: a Systematic Review

Introduction Three-dimensional (3D) printing technologies provide a practical and anatomical way to reproduce precise tailored-made models of the patients and of the diseases. Those models can allow surgical planning, besides training and surgical simulation in the treatment of neurosurgical diseases. Objective The aim of the present article is to review the scenario of the development of different types of available 3D printing technologies, the processes involved in the creation of biomodels, and the application of those advances in the neurosurgical field. Methods We searched for papers that addressed the clinical application of 3D printing in neurosurgery on the PubMed, Ebsco, Web of Science, Scopus, and Science Direct databases. All papers related to the use of any additivemanufacturing technique were included in the present study. Results Studies involving 3D printing in neurosurgery are concentrated on threemain areas: (1) creation of anatomical tailored-made models for planning and training; (2) development of devices and materials for the treatment of neurosurgical diseases, and (3) biological implants for tissues engineering. Biomodels are extremely useful in several branches of neurosurgery, and their use in spinal, cerebrovascular, endovascular, neuro-oncological, neuropediatric, and functional surgeries can be highlighted. Conclusions Three-dimensional printing technologies are an exclusive way for direct replication of specific pathologies of the patient. It can identify the anatomical variation and provide a way for rapid construction of training models, allowing the medical resident and the experienced neurosurgeon to practice the surgical steps before the operation.

Engineered 3D vessel-on-chip using hiPSC-derived endothelial- and vascular smooth muscle cells.

Crosstalk between endothelial cells (ECs) and pericytes or vascular smooth muscle cells (VSMCs) is essential for the proper functioning of blood vessels. This balance is disrupted in several vascular diseases but there are few experimental models which recapitulate this vascular cell dialogue in humans. Here, we developed a robust multi-cell type 3D vessel-on-chip (VoC) model based entirely on human induced pluripotent stem cells (hiPSCs). Within a fibrin hydrogel microenvironment, the hiPSC-derived vascular cells self-organized to form stable microvascular networks reproducibly, in which the vessels were lumenized and functional, responding as expected to vasoactive stimulation. Vascular organization and intracellular Ca2+ release kinetics in VSMCs could be quantified using automated image analysis based on open-source software CellProfiler and ImageJ on widefield or confocal images, setting the stage for use of the platform to study vascular (patho)physiology and therapy.

A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction.

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

Mechanical Studies of the Third Dimension in Cancer: From 2D to 3D Model.

From the development of self-aggregating, scaffold-free multicellular spheroids to the inclusion of scaffold systems, 3D models have progressively increased in complexity to better mimic native tissues. The inclusion of a third dimension in cancer models allows researchers to zoom out from a significant but limited cancer cell research approach to a wider investigation of the tumor microenvironment. This model can include multiple cell types and many elements from the extracellular matrix (ECM), which provides mechanical support for the tissue, mediates cell-microenvironment interactions, and plays a key role in cancer cell invasion. Both biochemical and biophysical signals from the extracellular space strongly influence cell fate, the epigenetic landscape, and gene expression. Specifically, a detailed mechanistic understanding of tumor cell-ECM interactions, especially during cancer invasion, is lacking. In this review, we focus on the latest achievements in the study of ECM biomechanics and mechanosensing in cancer on 3D scaffold-based and scaffold-free models, focusing on each platform's level of complexity, up-to-date mechanical tests performed, limitations, and potential for further improvements.

Magnetic levitation assisted biofabrication, culture, and manipulation of 3D cellular structures using a ring magnet based setup.

Diamagnetic levitation is an emerging technology for remote manipulation of cells in cell and tissue level applications. Low-cost magnetic levitation configurations using permanent magnets are commonly composed of a culture chamber physically sandwiched between two block magnets that limit working volume and applicability. This work describes a single ring magnet-based magnetic levitation system to eliminate physical limitations for biofabrication. Developed configuration utilizes sample culture volume for construct size manipulation and long-term maintenance. Furthermore, our configuration enables convenient transfer of liquid or solid phases during the levitation. Before biofabrication, we first calibrated/ the platform for levitation with polymeric beads, considering the single cell density range of viable cells. By taking advantage of magnetic focusing and cellular self-assembly, millimeter-sized 3D structures were formed and maintained in the system allowing easy and on-site intervention in cell culture with an open operational space. We demonstrated that the levitation protocol could be adapted for levitation of various cell types (i.e., stem cell, adipocyte and cancer cell) representing cells of different densities by modifying the paramagnetic ion concentration that could be also reduced by manipulating the density of the medium. This technique allowed the manipulation and merging of separately formed 3D biological units, as well as the hybrid biofabrication with biopolymers. In conclusion, we believe that this platform will serve as an important tool in broad fields such as bottom-up tissue engineering, drug discovery and developmental biology.

Evaluation of natural gum-based cryogels for soft tissue engineering.

In this study, three natural biomaterials, Locust bean gum (LBG), Xanthan gum (XG), and Mastic gum (MG), were combined to form cryogel scaffolds. Thermal and chemical characterizations revealed the successful blend formation from LBG-XG (LX) and LBG-XG-MG (LXM) polymers. All blends resulted in macro-porous scaffolds with interconnected pore structures under the size of 400 µm. The swollen cryogels had similar mechanical properties compared with other polysaccharide-based cryogels. The mean tensile and compressive modulus values of the wet cryogels were in the range of 3.5-11.6 kPa and 82-398 kPa, respectively. The sustained release of the small molecule Kartogenin from varying concentrations and ratios of cryogels was in between 32 and 66% through 21 days of incubation. Physical, mechanical, and chemical properties make LX and LXM polysaccharide-based cryogels promising candidates for cartilage and other soft tissue engineering, and drug delivery applications.

Surface acoustic wave (SAW) techniques in tissue engineering.

Recently, the introduction of surface acoustic wave (SAW) technique for microfluidics has drawn a lot of attention. The pattern and mutual communication in cell layers, tissues, and organs play a critical role in tissue homeostasis and regeneration and may contribute to disease occurrence and progression. Tissue engineering aims to repair and regenerate damaged organs, depending on biomimetic scaffolds and advanced fabrication technology. However, traditional bioengineering synthesis approaches are time-consuming, heterogeneous, and unmanageable. It is hard to pattern cells in scaffolds effectively with no impact on cell viability and function. Here, we summarize a biocompatible, easily available, label-free, and non-invasive tool, surface acoustic wave (SAW) technique, which is getting a lot of attention in tissue engineering. SAW technique can realize accurate sorting, manipulation, and cells' pattern and rapid formation of spheroids. By integrating several SAW devices onto lab-on-a-chip platforms, tissue engineering lab-on-a-chip system was proposed. To the best of our knowledge, this is the first report to summarize the application of this novel technique in the field of tissue engineering.

A Self-Powered Piezo-Bioelectric Device Regulates Tendon Repair-Associated Signaling Pathways through Modulation of Mechanosensitive Ion Channels.

Tendon disease constitutes an unmet clinical need and remains a critical challenge in the field of orthopaedic surgery. Innovative solutions are required to overcome the limitations of current tendon grafting approaches, and bioelectronic therapies show promise in treating musculoskeletal diseases, accelerating functional recovery through the activation of tissue regeneration-specific signaling pathways. Self-powered bioelectronic devices, particularly piezoelectric materials, represent a paradigm shift in biomedicine, negating the need for battery or external powering and complementing existing mechanotherapy to accelerate the repair processes. Here, the dynamic response of tendon cells to a piezoelectric collagen-analogue scaffold comprised of aligned nanoscale fibers made of the ferroelectric material poly(vinylidene fluoride-co-trifluoroethylene) is shown. It is demonstrated that motion-powered electromechanical stimulation of tendon tissue through piezo-bioelectric device results in ion channel modulation in vitro and regulates specific tissue regeneration signaling pathways. Finally, the potential of the piezo-bioelectronic device in modulating the progression of tendinopathy-associated processes in vivo, using a rat Achilles acute injury model is shown. This study indicates that electromechanical stimulation regulates mechanosensitive ion channel sensitivity and promotes tendon-specific over non-tenogenic tissue repair processes.

Electrospun nano-fibrous bilayer scaffold prepared from polycaprolactone/gelatin and bioactive glass for bone tissue engineering.

This work is focused on integrating nanotechnology with bone tissue engineering (BTE) to fabricate a bilayer scaffold with enhanced biological, physical and mechanical properties, using polycaprolactone (PCL) and gelatin (Gt) as the base nanofibrous layer, followed by the deposition of a bioactive glass (BG) nanofibrous layer via the electrospinning technique. Electrospun scaffolds were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Surface area and porosity were evaluated using the nitrogen adsorption method and mercury intrusion porosimetry. Moreover, scaffold swelling rate, degradation rate and in vitro bioactivity were examined in simulated body fluid (SBF) for up to 14 days. Mechanical properties of the prepared scaffolds were evaluated. Cell cytotoxicity was assessed using MRC-5 cells. Analyses showed successful formation of bead-free uniform fibers and the incorporation of BG nanoparticles within fibers. The bilayer scaffold showed enhanced surface area and total pore volume in comparison to the composite single layer scaffold. Moreover, a hydroxyapatite-like layer with a Ca/P molar ratio of 1.4 was formed after 14 days of immersion in SBF. Furthermore, its swelling and degradation rates were significantly higher than those of pure PCL scaffold. The bilayer's tensile strength was four times higher than that of PCL/Gt scaffold with greatly enhanced elongation. Cytotoxicity test revealed the bilayer's biocompatibility. Overall analyses showed that the incorporation of BG within a bilayer scaffold enhances the scaffold's properties in comparison to those of a composite single layer scaffold, and offers potential avenues for development in the field of BTE.

Ice Templating Soft Matter: Fundamental Principles and Fabrication Approaches to Tailor Pore Structure and Morphology and Their Biomedical Applications.

Porous scaffolds are widely used in biomedical applications where pore size and morphology influence a range of biological processes, including mass transfer of solutes, cellular interactions and organization, immune responses, and tissue vascularization, as well as drug delivery from biomaterials. Ice templating, one of the most widely utilized techniques for the fabrication of porous materials, allows control over pore morphology by controlling ice formation in a suspension of solutes. By fine-tuning freezing and solute parameters, ice templating can be used to incorporate pores with tunable morphological features into a wide range of materials using a simple, accessible, and scalable process. While soft matter is widely ice templated for biomedical applications and includes commercial and clinical products, the principles underpinning its ice templating are not reviewed as well as their inorganic counterparts. This review describes and critically evaluates fundamental principles, fabrication and characterization approaches, and biomedical applications of ice templating in polymer-based biomaterials. It describes the utility of porous scaffolds in biomedical applications, highlighting biological mechanisms impacted by pore features, outlines the physical and thermodynamic mechanisms underpinning ice templating, describes common fabrication setups, critically evaluates complexities of ice templating specific to polymers, and discusses future directions in this field.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Synthetic extracellular matrices with tailored adhesiveness and degradability support lumen formation during angiogenic sprouting.

A major deficit in tissue engineering strategies is the lack of materials that promote angiogenesis, wherein endothelial cells from the host vasculature invade the implanted matrix to form new blood vessels. To determine the material properties that regulate angiogenesis, we have developed a microfluidic in vitro model in which chemokine-guided endothelial cell sprouting into a tunable hydrogel is followed by the formation of perfusable lumens. We show that long, perfusable tubes only develop if hydrogel adhesiveness and degradability are fine-tuned to support the initial collective invasion of endothelial cells and, at the same time, allow for matrix remodeling to permit the opening of lumens. These studies provide a better understanding of how cell-matrix interactions regulate angiogenesis and, therefore, constitute an important step towards optimal design criteria for tissue-engineered materials that require vascularization.

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering.

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.

Nanotechnology Facilitated Cultured Neuronal Network and Its Applications.

The development of a biomimetic neuronal network from neural cells is a big challenge for researchers. Recent advances in nanotechnology, on the other hand, have enabled unprecedented tools and techniques for guiding and directing neural stem cell proliferation and differentiation in vitro to construct an in vivo-like neuronal network. Nanotechnology allows control over neural stem cells by means of scaffolds that guide neurons to reform synaptic networks in suitable directions in 3D architecture, surface modification/nanopatterning to decide cell fate and stimulate/record signals from neurons to find out the relationships between neuronal circuit connectivity and their pathophysiological functions. Overall, nanotechnology-mediated methods facilitate precise physiochemical controls essential to develop tools appropriate for applications in neuroscience. This review emphasizes the newest applications of nanotechnology for examining central nervous system (CNS) roles and, therefore, provides an insight into how these technologies can be tested in vitro before being used in preclinical and clinical research and their potential role in regenerative medicine and tissue engineering.

Chitosan scaffolds with enhanced mechanical strength and elastic response by combination of freeze gelation, photo-crosslinking and freeze-drying.

In this study, a new scaffold fabrication method based on the combination of a series of stabilization processes was set up to obtain chitosan scaffolds with improved mechanical properties for regeneration of load-bearing tissues. Specifically, thermally induced phase separation (TIPS) of chitosan solutions was used to obtain an open structure which was then stabilized by freeze-gelation and photo cross-linking. Freeze-gelation combined with freeze-drying permitted to obtain a porous structure with a 95 µm-mean pore size suitable for osteoblast cells' housing. Photo-crosslinking improved by ca. three times the scaffold compressive modulus, passing from 0,8 MPa of the uncrosslinked scaffolds to 2,2 MPa of the crosslinked one. Hydrated crosslinked scaffolds showed a good elastic response, with an 80% elastic recovery for at least 5 consecutive compressive cycles. The herein reported method has the advantage to not require the use of potentially toxic cross-linking agents and may be extended to other soft materials.

Compliant 3D frameworks instrumented with strain sensors for characterization of millimeter-scale engineered muscle tissues.

Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.