Co-culture of chondrocytes and stem cells: a review of head and neck cell lines for cartilage regeneration.

INTRODUCTION: Bioprinting, using "bio-inks" consisting of living cells, supporting structures and biological motifs to create customized constructs, is an emerging technique that aims to overcome the challenges of cartilaginous reconstruction of head and neck structures. Several living cell lines and culturing methods have been explored as bio-inks with varying efficacy. Co-culture of primary chondrocytes and stem cells (SCs) is one technique, well established for degenerative joint disease treatment, with potential for use in expanding chondrocyte populations for bio-inks. This study aims to evaluate the techniques for co-culture of primary chondrocytes and SCs for head and neck cartilage regeneration. METHODS: A literature review was performed through OVID/Web of Science/MEDLINE/BIOSIS Previews/Embase. Studies reporting on chondrocytes and SCs in conjunction with co-culture or cartilage regeneration were included. Studies not reporting on findings from chondrocytes/SCs of the head and neck were excluded. Extracted data included cell sources, co-culture ratios and histological, biochemical and clinical outcomes. RESULTS: 15 studies met inclusion criteria. Auricular cartilage was the most common chondrocyte source (n=10), then nasal septum (n=5), articular (n=1) and tracheal cartilage (n=1). Bone marrow was the most common SC source (n=9) then adipose tissue (n=7). Techniques varied, with co-culture ratios ranging from 1:1 to 1:10. All studies reported co-culture to be superior to SC mono-culture by all outcomes. Most studies reported superiority or equivalence of co-culture to chondrocyte mono-culture by all outcomes. When comparing clinical outcomes, co-culture constructs were equivalent to chondrocyte mono-culture in diameter, and equivalent or inferior in wet weight and height. CONCLUSION: Co-culture of primary chondrocytes and SCs is a promising technique for expanding chondrocyte populations, with at least equivalence to chondrocyte mono-culture and superior to SC mono-culture when seeded at the same chondrocyte densities. However, there remains a lack of consensus regarding the optimal cell sources and co-culture ratios.

Enhancing Cementitious Composites with Functionalized Graphene Oxide-Based Materials: Surface Chemistry and Mechanisms.

Graphene oxide-based materials (GOBMs) have been widely explored as nano-reinforcements in cementitious composites due to their unique properties. Oxygen-containing functional groups in GOBMs are crucial for enhancing the microstructure of cementitious composites. A better comprehension of their surface chemistry and mechanisms is required to advance the potential applications in cementitious composites of functionalized GOBMs. However, the mechanism by which the oxygen-containing functional groups enhance the response of cementitious composites is still unclear, and controlling the surface chemistry of GOBMs is currently constrained. This review aims to investigate the reactions and mechanisms for functionalized GOBMs as additives incorporated in cement composites. A variety of GOBMs, including graphene oxide (GO), hydroxylated graphene (HO-G), edge-carboxylated graphene (ECG), edge-oxidized graphene oxide (EOGO), reduced graphene oxide (rGO), and GO/silane composite, are discussed with regard to their oxygen functional groups and interactions with the cement microstructure. This review provides insight into the potential benefits of using GOBMs as nano-reinforcements in cementitious composites. A better understanding of the surface chemistry and mechanisms of GOBMs will enable the development of more effective functionalization strategies and open up new possibilities for the design of high-performance cementitious composites.

Collagen Alignment via Electro-Compaction for Biofabrication Applications: A Review.

As the most prevalent structural protein in the extracellular matrix, collagen has been extensively investigated for biofabrication-based applications. However, its utilisation has been impeded due to a lack of sufficient mechanical toughness and the inability of the scaffold to mimic complex natural tissues. The anisotropic alignment of collagen fibres has been proven to be an effective method to enhance its overall mechanical properties and produce biomimetic scaffolds. This review introduces the complicated scenario of collagen structure, fibril arrangement, type, function, and in addition, distribution within the body for the enhancement of collagen-based scaffolds. We describe and compare existing approaches for the alignment of collagen with a sharper focus on electro-compaction. Additionally, various effective processes to further enhance electro-compacted collagen, such as crosslinking, the addition of filler materials, and post-alignment fabrication techniques, are discussed. Finally, current challenges and future directions for the electro-compaction of collagen are presented, providing guidance for the further development of collagenous scaffolds for bioengineering and nanotechnology.

Controllable graphene oxide-based biocompatible hybrid interface as an anti-fibrotic coating for metallic implants.

In tissue engineering, foreign body reactions (FBRs) that may occur after the insertion of medical implants are a considerable challenge. Materials currently used in implants are mainly metals that are non-organic, and the lack of biocompatibility and absence of immune regulations may lead to fibrosis after long periods of implantation. Here, we introduce a highly biocompatible hybrid interface of graphene oxide (GO) and collagen type I (COL-I), where the topological nanostructure can effectively inhibit the differentiation of fibroblasts into myofibroblasts. The structure and roughness of this coating interface can be easily adjusted at the nanoscale level through changes in the GO concentration, thereby effectively inducing the polarization of macrophages to the M1 state without producing excessive amounts of pro-inflammatory factors. Compared to nanomaterials or the extracellular matrix as an anti-fibrotic interface, this hybrid bio-interface has superior mechanical strength, physical structures, and high inflammation. Evidenced by inorganic materials such as glass, titanium, and nitinol, GO-COL shows great potential for use in medical implants and cell-material interfaces.

An electroactive hybrid biointerface for enhancing neuronal differentiation and axonal outgrowth on bio-subretinal chip.

Retinal prostheses offer viable vision restoration therapy for patients with blindness. However, a critical requirement for maintaining the stable performance of electrical stimulation and signal transmission is the biocompatibility of the electrode interface. Here, we demonstrated a functionalized electrode-neuron biointerface composed of an annealed graphene oxide-collagen (aGO-COL) composite and neuronal cells. The aGO-COL exhibited an electroactive 3D crumpled surface structure and enhanced the differentiation efficiency of PC-12 â€‹cells. It is integrated into a photovoltaic self-powered retinal chip to develop a biohybrid retinal implant that facilitates biocompatibility and tissue regeneration. Moreover, aGO-COL micropatterns fabricated via 3D bioprinting can be used to create neuronal cell microarrays, which supports the possibility of retaining the high spatial resolution achieved through electrical stimulation of the retinal chip. This study paves the way for the next generation of biohybrid retinal implants based on biointerfaces.

Bioprinting of Chondrocyte Stem Cell Co-Cultures for Auricular Cartilage Regeneration.

Advances in 3D bioprinting allows not only controlled deposition of cells or cell-laden hydrogels but also flexibility in creating constructs that match the anatomical features of the patient. This is especially the case for reconstructing the pinna (ear), which is a large feature of the face and made from elastic cartilage that primarily relies on diffusion for nutrient transfer. The selection of cell lines for reconstructing this cartilage becomes a crucial step in clinical translation. Chondrocytes and mesenchymal stem cells are both studied extensively in the area of cartilage regeneration as they are capable of producing cartilage in vitro. However, such monoculture systems involve unfavorable processes and produce cartilage with suboptimal characteristics. Co-cultures of these cell types are known to alleviate these limitations to produce synergically active chondrocytes and cartilage. The current study utilized a 3D bioprinted scaffold made from combined gelatine methacryloyl and methacrylated hyaluronic acid (GelMA/HAMA) to interrogate monocultures and co-cultures of human septal chondrocytes (primary chondrocytes, PCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). This study is also the first to examine co-cultures of healthy human chondrocytes with human BM-hMSCs encapsulated in GelMA/HAMA bioprinted scaffolds. Findings revealed that the combination of MSCs and PCs not only yielded cell proliferation that mimicked MSCs but also produced chondrogenic expressions that mimicked PCs. These findings suggested that co-cultures of BM-hMSCs and healthy septal PCs can be employed to replace monocultures in chondrogenic studies for cartilage regeneration in this model. The opportunity for MSCs used to replace PCs alleviates the requirement of large cartilage biopsies that would otherwise be needed for sufficient cell numbers and therefore can be employed for clinical applications.

Effect of Graphene Addition on Polycaprolactone Scaffolds Fabricated Using Melt-Electrowriting.

Melt-electrowriting (MEW) is an emerging method that combines electrospinning and extrusion printing, allowing the fabrication of micron-scale structures suitable for tissue engineering. Compared to other additive fabrication methods, melt-electro written structures can offer more appropriate substrates for cell culture due to filament size and mechanical characteristics of the fabricated scaffolds. In this study, polycaprolactone (PCL)/graphene composites were investigated for fabrication of micron-size scaffolds through MEW. It was demonstrated that the addition of graphene can considerably improve the processability of PCL to fabricate micron-scale scaffolds with enhanced resolution. The tensile strength of the scaffold prepared from PCL/graphene composite (with only 0.5 wt.% graphene) was proved significantly (by more than 270%), better than that of the pristine PCL scaffold. Furthermore, graphene was demonstrated to be a suitable material for tailoring the degradation process to avoid undesirable bulk degradation, rapid mass loss and damage to the internal matrix of the polymer. The findings of this study offer a promising route for the fabrication of high-resolution scaffolds with micron-scale resolution for tissue engineering.

Tissue-level alveolar epithelium model for recapitulating SARS-CoV-2 infection and cellular plasticity.

Pulmonary sequelae following COVID-19 pneumonia have been emerging as a challenge; however, suitable cell sources for studying COVID-19 mechanisms and therapeutics are currently lacking. In this paper, we present a standardized primary alveolar cell culture method for establishing a human alveolar epithelium model that can recapitulate viral infection and cellular plasticity. The alveolar model is infected with a SARS-CoV-2 pseudovirus, and the clinically relevant features of the viral entry into the alveolar type-I/II cells, cytokine production activation, and pulmonary surfactant destruction are reproduced. For this damaged alveolar model, we find that the inhibition of Wnt signaling via XAV939 substantially improves alveolar repair function and prevents subsequent pulmonary fibrosis. Thus, the proposed alveolar cell culture strategy exhibits potential for the identification of pathogenesis and therapeutics in basic and translational research.

Investigation of the role of the autophagic protein LC3B in the regulation of human airway epithelium cell differentiation in COPD using a biomimetic model.

Chronic obstructive pulmonary disease (COPD) is one of the most lethal chronic disease worldwide; however, the establishment of reliable in vitro models for exploring the biological mechanisms of COPD remains challenging. Here, we determined the differences in the expression and characteristics of the autophagic protein LC3B in normal and COPD human small airway epithelial cells and found that the nucleus of COPD cells obviously accumulated LC3B. We next established 3D human small airway tissues with distinct disease characteristics by regulating the biological microenvironment, extracellular matrix, and air-liquid interface culture methods. Using this biomimetic model, we found that LC3B affects the differentiation of COPD cells into basal, secretory, mucous, and ciliated cells. Moreover, although chloroquine and ivermectin effectively inhibited the expression of LC3B in the nucleus, chloroquine specifically maintained the performance of LC3B in cytoplasm, thereby contributing to the differentiation of ciliated cells and subsequent improvement in the beating functions of the cilia, whereas ivermectin only facilitated differentiation of goblet cells. We demonstrated that the autophagic mechanism of LC3B in the nucleus is one factor regulating the ciliary differentiation and function of COPD cells. Our innovative model can be used to further analyze the physiological mechanisms in the in vitro airway environment.

Graphene Oxide-Based Nanomaterials: An Insight into Retinal Prosthesis.

Retinal prosthesis has recently emerged as a treatment strategy for retinopathies, providing excellent assistance in the treatment of age-related macular degeneration (AMD) and retinitis pigmentosa. The potential application of graphene oxide (GO), a highly biocompatible nanomaterial with superior physicochemical properties, in the fabrication of electrodes for retinal prosthesis, is reviewed in this article. This review integrates insights from biological medicine and nanotechnology, with electronic and electrical engineering technological breakthroughs, and aims to highlight innovative objectives in developing biomedical applications of retinal prosthesis.

Synthesis and 3D Printing of Conducting Alginate-Polypyrrole Ionomers.

Hydrogels composed of calcium cross-linked alginate are under investigation as bioinks for tissue engineering scaffolds due to their variable viscoelasticity, biocompatibility, and erodibility. Here, pyrrole was oxidatively polymerized in the presence of sodium alginate solutions to form ionomeric composites of various compositions. The IR spectroscopy shows that mild base is required to prevent the oxidant from attacking the alginate during the polymerization reaction. The resulting composites were isolated as dried thin films or cross-linked hydrogels and aerogels. The products were characterized by elemental analysis to determine polypyrrole incorporation, electrical conductivity measurements, and by SEM to determine changes in morphology or large-scale phase separation. Polypyrrole incorporation of up to twice the alginate (monomer versus monomer) provided materials amenable to 3D extrusion printing. The PC12 neuronal cells adhered and proliferated on the composites, demonstrating their biocompatibility and potential for tissue engineering applications.

3D hybrid printing platform for auricular cartilage reconstruction.

As scaffolds approach dimensions that are of clinical relevance, mechanical integrity and distribution becomes an important factor to the overall success of the implant. Hydrogels often lack the structural integrity and mechanical properties for use in vivo or handling. The inclusion of a structural support during the printing process, referred to as hybrid printing, allows the implant to retain structure and protect cells during maturation without needing to compromise its biological performance. In this study, scaffolds for the purpose of auricular cartilage reconstruction were evaluated via a hybrid printing approach using methacrylated Gelatin (GelMA) and Hyaluronic acid (HAMA) as the cell-laden hydrogel, Polycaprolactone (PCL) as structural support and Lutrol F-127 as sacrificial material. Furthermore, printing parameters such as nozzle diameter, strand spacing and filament orientation scaffolds were investigated. Compression and bending tests showed that increasing nozzle sizes decrease the compressive modulus of printed scaffolds, with up to 82% decrease in modulus when comparing between a 400 µm and 200 µm sized nozzle tip at the same strand spacing. On the contrary, strand spacing and orientation influences mainly the bending modulus due to the greater porosity and changes in pore size area. Using a 400 µm sized nozzle, scaffolds fabricated have a measured compression and bending modulus in the range similar to the native cartilage. The viability and proliferation of human mesenchymal stem cells delivered within the bioink was not affected by the printing process. Using results obtained from mechanical testing, a scaffold with matching mechanical properties across six distinct regions mimicking the human auricular cartilage can be completed in one single print process. The use of PCL and GelMA-HAMA as structural support and cell-laden hydrogel respectively are an excellent combination to provide tailored mechanical integrity, while maintaining porosity and protection to cells during differentiation.

Poly(3,4-ethylenedioxythiophene):dextran sulfate (PEDOT:DS) - a highly processable conductive organic biopolymer.

A novel water-dispersible conducting polymer analogous to poly(3,4-dioxythiophene):polystyrene sulfonate (PEDOT:PSS) has been chemically synthesized in a single reaction in high yield. PEDOT:DS, a new member of the polythiophene family, is composed of a complex between PEDOT and the sulfonated polysaccharide polyanion dextran sulfate. Drop-cast films of aqueous suspensions of the material display a native conductivity of up to 7 ± 1 S cm(-1), increasing to 20 ± 2 S cm(-1) after treatment with ethylene glycol and thermal annealing. Mass ratios of the precursors NaDS and EDOT were varied from 5:1 to 2:1 and a decrease in the NaDS:EDOT ratio produces tougher, less hygroscopic films of higher conductivity. Ultraviolet-visible spectroelectrochemistry yields spectra typical of PEDOT complexes. Cyclic voltammetry reveals that PEDOT:DS is electrochemically active from -1.0 to 0.8 V vs. Ag/Ag(+) in acetonitrile, with similar characteristics to PEDOT:PSS. Water dispersions of PEDOT:DS are successfully processed by drop casting, spray coating, inkjet printing and extrusion printing. Furthermore, laser etching of dried films allows the creation of patterns with excellent definition. To assess the cytotoxicity of PEDOT:DS, L-929 cells were cultured with a polymer complex concentration range of 0.002 to 0.2 g l(-1) in cell culture medium. No significant difference is found between the proliferation rates of L-929 cells exposed to PEDOT:DS and those in plain medium after 96h. However, PEDOT:PSS shows around 25% less cell growth after 4 days, even at the lowest concentration. Taken together, these results suggest PEDOT:DS has exceptional potential as an electromaterial for the biointerface.

Bio-ink properties and printability for extrusion printing living cells.

Additive biofabrication (3D bioprinting) makes it possible to create scaffolds with precise geometries, control over pore interconnectivity and architectures that are not possible with conventional techniques. Inclusion of cells within the ink to form a "bio-ink" presents the potential to print 3D structures that can be implanted into damaged/diseased tissue to promote highly controlled cell-based regeneration and repair. The properties of an 'ink' are defined by its formulation and critically influence the delivery and integrity of structure formed. Importantly, the ink properties need to conform to biological requirements necessary for the cell system that they are intended to support and it is often challenging to find conditions for printing that facilitate this critical aspect of tissue bioengineering. In this study, alginate (Alg) was selected as the major component of the 'bio-ink' formulations for extrusion printing of cells. The rheological properties of alginate-gelatin (Alg-Gel) blends were compared with pre-crosslinked alginate and alginate solution to establish their printability whilst maintaining their ability to support optimal cell growth. Pre-crosslinked alginate on its own was liquid-like during printing. However, by controlling the temperature, Alg-Gel formulations had higher viscosity, storage modulus and consistency which facilitated higher print resolution/precision. Compression and indentation testing were used to examine the mechanical properties of alginate compared to Alg-Gel. Both types of gels yielded similar results with modulus increasing with alginate concentration. Decay in mechanical properties over time suggests that Alg-Gel slowly degrades in cell culture media with more than 60% decrease in initial modulus over 7 days. The viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not affected by the printing process, indicating that the Alg-Gel matrix provides a potential means to print 3D constructs that may find application in myoregenerative applications.