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Innate immune cells play a pivotal role in controlling tissue repair and rejection after biomaterial implantation. Calcium supplementation regulates cellular responses and alter the pathophysiology of various diseases. A series of macrophage activations through differential plasticity has been observed after cell-to-material interactions. We investigated the role of calcium supplementation in controlling macrophage phenotypes in pro-inflammatory and pre-reparative states. Oxidative defence and mitochondria involvement in cellular plasticity and the sequential M0 to M1 and M1 to M2 transitions were observed after calcium supplementation. This study describes the molecular mechanism of reactive oxygen species and drives the interconnected cellular plasticity of macrophages in the presence of calcium. Gene expression, and immunostaining, revealed a relationship between MHC class II maturation and cellular plasticity. This study elucidated the role of controlled calcium supplementation under various conditions. These findings underscore the molecular mechanism of calcium-mediated immune induction and its favourable use in different calcium-containing biomaterials., essential for tissue regeneration.

The electronic National Agriculture Market (eNAM) is a pan-India online trading platform that links Agricultural Produce Market Committee markets to create a unified national market for agricultural produce. However, with the adoption of eNAM by the relevant stakeholders being relatively slow, Government of India has initiated a number of steps to address the issues related to post-harvest infrastructure, transportation of farm produce, etc. with particular focus on small and marginal farmers. This paper positions eNAM in the backdrop of marketing reforms and discusses the key enablers that would result in the successful implementation of this flagship scheme.

The development of blood vessels, referred to as angiogenesis, is an intricate process regulated spatially and temporally through a delicate balance between the qualitative and quantitative expression of pro and anti-angiogenic molecules. As angiogenesis is a prerequisite for solid tumors to grow and metastasize, a variety of tumor angiogenesis models have been formulated to better understand the underlying mechanisms and associated clinical applications. Studies have demonstrated independent mechanisms inducing angiogenesis in tumors such as (a) HIF-1/VEGF mediated paracrine interactions between a cancer cell and endothelial cells, (b) recruitment of progenitor endothelial cells, and (c) vasculogenic mimicry. Moreover, single-cell sequencing technologies have indicated endothelial cell heterogeneity among organ systems including tumor tissues. However, existing angiogenesis models often rely upon normal endothelial cells which significantly differ from tumor endothelial cells exhibiting distinct (epi)genetic and metabolic signatures. Besides, the existence of intra-individual variations necessitates the development of improved tumor vascular model systems for personalized medicine. In the present review, we summarize recent advancements of 3D tumor vascular model systems which include (a) tissue engineering-based tumor models; (b) vascular organoid models, and (c) organ-on-chips and their importance in replicating the tumor angiogenesis along with the associated challenges to design improved models.

We developed hybrid liver-specific three-dimensional (3D) printed scaffolds using a solubilized native decellularized liver (DCL) matrix and silk fibroin (SF) and investigated their ability to support functional cultures of hepatic cells. Rat livers were decellularized by perfusing detergents via the portal vein, solubilized using pepsin to form DCL, and characterized. SF blended with gelatin (8% w/v) was optimized with varying percentages of DCL to obtain silk gelatin-DCL bioink (SG-DCL). Different compositions of SG-DCL were studied by rheology for optimum versatility and print fidelity. 3D printed six-layered scaffolds were fabricated using a sophisticated direct-write 3D bioprinter. Huh7 cells were cultured on the 3D printed scaffolds for 3 weeks. 3D printed SG scaffolds without DCL along with 2D films (SG and SG-DCL) and 2D culture on tissue culture Petri dish control were used for comparative studies. The DCL matrix showed the absence of cells in histology and SEM. The combined SG-DCL ink at all of the studied DCL percentages (1-10%) revealed shear-thinning behavior in the printable range. The storage modulus value for the SG-DCL ink at all DCL percentages was higher than the loss modulus. In comparison to 2D controls, hepatic cells cultured on 3D SG-DCL revealed increased proliferation until 2 weeks and an upregulated expression of hepatocyte markers, including asialoglycoprotein receptor 1 (ASGR1). The Wnt pathway gene β-catenin was upregulated by more than 4-fold in 3D SG-DCL on day 3, while it showed a decline on day 7 as compared to 3D SG and also 2D controls. The expression of the epithelial cell adhesion molecule (EpCAM) was however lower in both 2D SG-DCL (2-fold) and 3D SG-DCL (2.5-fold) as compared to that in 2D controls. Immunofluorescence studies validated the protein expression of ASGR1 in 3D SG-DCL. Albumin (ALB) was not identified on SG scaffolds but prominently expressed in 3D SG-DCL constructs. In comparison to 2D SG, both ALB (1.8-fold) and urea (5-fold) were enhanced in cells cultured on 3D SG-DCL on day 7 of culture. Hence, the SG-DCL 3D printed scaffolds provide a conducive microenvironment for elevating differentiation and functions of hepatic cells possibly through an involvement of the Wnt/β-catenin signaling pathway.

Major limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin–gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM. Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed in The Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3’UTR luciferase assays. The effect of miR-196a overexpression/inhibition was assessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies were performrd to study the effect of miR-196a on the GBM cellular transcriptome under hypoxia. We identified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196a yielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a in HIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs and in maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia. Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Three-dimensional (3D) bioprinting is a highly innovative and promising technology to render precise positioning of biologics together with living cells and extracellular matrix (ECM) constituents. In spite of such enthralling potential, the fabrication of a clinically relevant engineered tissue is quite challenging. A constellation of factors simulating the complex architecture of the native tissue, selection of the "ideal bioink", optimization of the biochemical, mechanical, and topographical functions of the cell-laden printed construct, cellular differentiation, their self-assembly, and remodeling into the desired lineage postprinting present major complications. Keeping this in view, we have attempted to highlight the use of silk fibroin (SF) protein from

In recent years, engineering biomimetic cellular microenvironments have been a top priority for regenerative medicine. Collagen II, which is arranged in arches, forms the predominant fiber network in articular cartilage. Due to the shortage of suitable microfabrication techniques capable of producing 3D fibrous structures, in vitro replication of the arch-like cartilaginous tissue constitutes one of the major challenges. Hence, in the present study, we report a 3D bioprinting approach for fabricating arch-like constructs using two types of bioinks, gelatin methacryloyl (GelMa) and silk fibroin-gelatin (SF-G). The bioprinted SF-G constructs displayed increased proliferation of the encapsulated human bone marrow-derived mesenchymal stem cells compared to the GelMA constructs. Biochemical assays, gene, and protein expression exhibited the superior role of SF-G in forming the fibrous collagen network and chondrogenesis. Protein-protein interaction study using Metascape evaluated the function of the proteins involved. Further GeneMANIA and STRING analysis using Col 2A1, SOX 9, ACAN, and the genes upregulated on day 21 in RT-PCR, i.e. β-catenin, TGFβR1, Col 1A1 in SF-G and PRG4, Col 10A1, MMP 13 in GelMA validated our in vitro results. These findings emphasized the role of SF-G in regulating the Wnt/β-catenin and TGF-β signaling pathways. Hence, the 3D bioprinted arch-like constructs possess a substantial potential for cartilage regeneration.

3D porous hydroxyapatite (HAP) scaffolds produced by conventional foaming processes have limited control over the scaffold’s pore size, geometry, and pore interconnectivity. In addition, random internal pore architecture often results in limited clinical success. Imitating the intricate 3D architecture and the functional dynamics of skeletal deformations is a difficult task, highlighting the necessity for a custom-made, on-demand tissue replacement, for which 3D printing is a potential solution. To combat these problems, here we report the ability of 3D printed HAP scaffolds for in vivo bone regeneration in a rat tibial defect model. Rapid prototyping using the direct-write technique to fabricate 25 mm2 HAP scaffolds were employed for precise control over geometry (both external and internal) and scaffold chemistry. Bone ingrowth was determined using histomorphometry and a novel micro-computed tomography (micro-CT) image analysis. Substantial bone ingrowth was observed in implants that filled the defect site. Further validating this quantitatively by micro-CT, the Bone mineral density (BMD) of the implant at the defect site was 1024 mgHA ccm−1, which was approximately 61.5% more than the BMD found with the sham control at the defect site. In addition, no evident immunoinflammatory response was observed in the hematoxylin and eosin micrographs. Interestingly, the present study showed a positive correlation with the outcomes obtained in our previous in vitro study. Overall, the results suggest that 3D printed HAP scaffolds developed in this study offer a suitable matrix for rendering patient-specific and defect-specific bone formation and warrant further testing for clinical application.

Very few tissue-engineered constructs could achieve the desired results in human clinical trials. The main reason is their inability to recapitulate the cellular conformation, biological, and mechanical functions of the native tissue. Here, we highlight the future avenues of tissue regeneration combining developmental biology, organoids, and 3D bioprinting. A deep mechanistic insight into the embryonic level and recapitulating them would be the most promising strategy in next-generation tissue engineering. Rather than focusing on the adult tissue features, the latest developmental re-engineering strategies replicate the developmental phases of tissue development. Integrating developmental re-engineering with 3D bioprinting can regulate several signaling pathways. This would further help to fabricate mini-organ constructs for transplantation or in vitro screening of drugs using an organ-on-a-chip platform.

The substantially growing gap between suitable donors and patients waiting for new organ transplantation has compelled tissue engineers to look for suitable patient-specific alternatives. Lately, a decellularized extracellular matrix (dECM), obtained primarily from either discarded human tissues/organs or other species, has shown great promise in the constrained availability of high-quality donor tissues. In this review, we have addressed critical gaps and often-ignored aspects of understanding the innate and adaptive immune response to the dECM. Firstly, although most of the studies claim preservation of the ECM ultrastructure, almost all methods employed for decellularization would inevitably cause a certain degree of disruption to the ECM ultrastructure and modulation in secondary conformations, which may elicit a distinct immunogenic response. Secondly, it is still a major challenge to find ways to conserve the native biochemical, structural and biomechanical cues by making a judicious decision regarding the choice of decellularization agents/techniques. We have critically analyzed various decellularization protocols and tried to find answers on various aspects such as whether the secondary structural conformation of dECM proteins would be preserved after decellularization. Thirdly, to keep the dECM ultrastructure as close to the native ECM we have raised the question "How good is good enough?" Even residual cellular antigens or nucleic acid fragments may elicit antigenicity leading to a low-grade immune response. A combinative knowledge of macrophage plasticity in the decellularized tissue and limits of decellularization will help achieve the native ultrastructure. Lastly, we have shifted our focus on the scientific basis of the presently accepted criteria for decellularization, and the effect on immune response concerning the interaction between the decellularized extracellular matrix and macrophages with the subsequent influence of T-cell activation. Amalgamating suitable decellularization approaches, sufficient knowledge of macrophage plasticity and elucidation of molecular pathways together will help fabricate functional immune informed decellularized tissues in vitro that will have substantial implications for efficient clinical translation and prediction for in vivo reprogramming and tissue regeneration.

Recent advancement in understanding cancer etiology has highlighted epigenetic deregulation as an important phenomenon leading to poor prognosis in glioblastoma (GBM). Polycomb repressive complex 2 (PRC2) is one such important epigenetic modifier reportedly altered in GBM. However, its defined mechanism in tumorigenesis still remains elusive. In present study, we analyzed our in-house ChIPseq data for H3k27me3 modified miRNAs and identified miR-490-3p to be the most common target in GBM with significantly downregulated expression in glioma patients in both TCGA and GBM patient cohort. Our functional analysis delineates for the first time, a central role of PRC2 catalytic unit EZH2 in directly regulating expression of this miRNA and its host gene CHRM2 in GBM. In accordance, cell line treatment with EZH2 siRNA and 5-azacytidine also confirmed its coregulation by CpG and histone methylation based epigenetic mechanisms. Furthermore, induced overexpression of miR-490-3p in GBM cell lines significantly inhibited key hallmarks including cellular proliferation, colony formation and spheroid formation, as well as epithelial-to-mesenchymal transition (EMT), with downregulation of multiple EMT transcription factors and promigratory genes (MMP9, CCL5, PIK3R1, ICAM1, ADAM17 and NOTCH1). We also for the first time report TGFBR1 and TGIF2 as two direct downstream effector targets of miR-490-3p that are also deregulated in GBM. TGIF2, a novel target, was shown to promote migration and EMT that could partially be rescued by miR-490-3p overexpression. Overall, this stands as a first study that provides a direct link between epigenetic modulator EZH2 and oncogenic TGF-β signaling involving novel miR-490-3p/TGIF2/TGFBR1 axis, that being targetable might be promising in developing new therapeutic intervention strategies for GBM.

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

Keratoconus (KC) is a bilateral corneal dystrophy and a multifactorial, multigenic disorder with an etiology involving a strong environmental component and complex inheritance patterns. The underlying pathophysiology of KC is poorly understood because of potential crosstalk between genetic-epigenetic variants possibly triggered by the environmental factors. Here, we decode the etiopathological basis of KC using genomic, transcriptomic, proteomic and metabolic approaches. The lack of relevant models that accurately imitate this condition has been particularly limiting in terms of the effective management of KC. Tissue-engineered in vitro models of KC could address this need and generate valuable insights into its etiopathology for the establishment of disease models to accelerate drug discovery.

3D printing has experienced swift growth for biological applications in the field of regenerative medicine and tissue engineering. Essential features of bioprinting include determining the appropriate bioink, printing speed mechanics, and print resolution while also maintaining cytocompatibility. However, the scarcity of bioinks that provide printing and print properties and cell support remains a limitation. Silk Fibroin (SF) displays exceptional features and versatility for inks and shows the potential to print complex structures with tunable mechanical properties, degradation rates, and cytocompatibility. Here we summarize recent advances and needs with the use of SF protein from Bombyx mori silkworm as a bioink, including crosslinking methods for extrusion bioprinting using SF and the maintenance of cell viability during and post bioprinting. Additionally, we discuss how encapsulated cells within these SF-based 3D bioprinted constructs are differentiated into various lineages such as skin, cartilage, and bone to expedite tissue regeneration. We then shift the focus towards SF-based 3D printing applications, including magnetically decorated hydrogels, in situ bioprinting, and a next-generation 4D bioprinting approach. Future perspectives on improvements in printing strategies and the use of multicomponent bioinks to improve print fidelity are also discussed.

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin–gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.

To date, the development of phenotypically stable, functionally equivalent engineered cartilage tissue constructs remains elusive. This study explored chondrogenic differentiation and suppression of hypertrophic differentiation in tyrosinase cross-linked silk-gelatin bioink using different cell modalities (dispersed, aggregates) for chondrocytes and mesenchymal progenitor cells (hMSCs) compared against the "gold standard" hMSC spheroids. Chondrogenic differentiation of hMSC spheroids (without silk-gelatin) showed a constant increase in hypertrophy over 21 days (gradual upregulated expression of COL10A1, MMP13). On the contrary, hMSC-laden constructs (both dispersed and aggregates) in bioink showed upregulated hypoxia (HIF1A) which positively regulated the expression of chondrogenic markers (aggrecan, COMP1) over chondrocyte-laden constructs. The gelatin component in the bioink induced MMP2 activity, which degraded the synthesized matrix, creating a pericellular zone for the accumulation of growth factors and newly synthesized matrices. We believe that the combinatorial effect of these accumulated factors as well as the hypoxia-regulated HDAC4 pathway played a pivotal role in stabilizing the chondrogenic phenotype of differentiated hMSCs along with suppressed hypertrophy. Therefore, the results suggest that tyrosinase cross-linked silk-gelatin bioink offers a suitable material composition for 3D bioprinting of cartilage constructs. Further standardization is warranted to investigate the biological mechanisms minimizing hypertrophic differentiation of hMSC/chondrocytes toward development of improved cartilage constructs.

Even after several decades of research, the most optimal source of silk for promoting osteogenesis in situ is still a subject of debate. A major gap in existing knowledge is role of underlying signaling mechanisms in both the mulberry and nonmulberry silk species that leads to the development of differential levels of osteogenesis. In our previous study, we elucidated the role of Wnt/β-catenin signaling for promoting superior osteogenic differentiation in nonmulberry silk braids in the presence of TGF-β and pro-osteogenic supplements. Here, we provide a comparative osteogenic analysis of the two most popular silk species (mulberry and nonmulberry silk), in the form of silk braids prepared from natively spun fibers, by conducting detailed gene expression profiling using 25 different osteogenic markers, followed by further validation by immunohistochemistry. Our study provides novel insights into the direct regulatory role of nonmulberry silk fibroin braids on hedgehog and parathyroid signaling pathways in controlling osteogenic differentiation of cultured human fetal osteoblasts (hFOBs), a phenomenon not very evident in the mulberry silk textile braids. Although both silk braids enabled adequate cellular attachment, proliferation, and extracellular collagen matrix formation, superior expression of osteogenic markers (ALP, VDR, Runx2), matrix proteins (Col1A2, OPN), and signaling molecules (GLI1, GLI2, Shh) with characteristic terminal osteocytic phenotype could only be observed in nonmulberry silk. Therefore, our study provided detailed insights into the development of engineered bone to be a prospective tissue equivalent with potential to provide the essential instructive elements for activating physiological pathways of bone differentiation. Such engineered constructs have potential for use as an in vitro model for drug testing and as scaffolds for bone regeneration strategies.

Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high-quality donor tissues. However, the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this study we tested the hypothesis that cross-linking the decellularized corneas with chondroitin sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Cross-linking of the decellularized cornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis. An in vitro immune response study involving healthy monocytes and differentiated macrophages with their surface marker analysis by pHrodo red, Lysotracker red, ER tracker, and CD63, LAMP-2 antibodies confirmed that the cross-linked decellularized matrices elicited the least immune response compared to the decellularized ones. We implanted three sets of corneal scaffolds obtained from goat, i.e., native, decellularized, and decellularized corneas conjugated with chondroitin sulfate into the rabbit stroma. Histology analysis, three months after implantation into the rabbit corneal stromal region, confirmed the restoration of the collagen fibril conformation and the migration of cells to the implanted constructs, affirming proper graft integration. Hence we conclude that the chondroitin sulfate cross-linked decellularized corneal matrix may serve as an efficient alternative to the allograft and human cadaveric corneas.

The direct write printing method has gained popularity in synthesizing scaffolds for tissue engineering. To achieve an excellent printability of scaffolds, a thorough evaluation of rheological properties is required. We report the synthesis, characterization, rheology, and direct-write printing of chitosan - graphene oxide (CH - GO) nanocomposite hydrogels at a varying concentration of GO in 3 and 4 wt% CH polymeric gels. Rheological characterization of CH - GO hydrogels shows that an addition of only 0.5 wt% of GO leads to a substantial increase in storage modulus (G'), viscosity, and yield stress of 3 and 4 wt% of CH hydrogels. A three-interval thixotropy test (3ITT) shows that 3 wt% CH with 0.5 wt% GO hydrogel has 94% recovery of G' after 7 sequential stress cycles and is the best candidate for direct-write printing. Neuronal cell culture on 3 wt% CH with 0.5 wt% hydrogels reveals that GO promotes the differentiation of SH-SY5Y cells.

We read with interest the recent study by Yang et al , who reported the construction of three-dimensional bioprinted hepatorganoids (3DP-HOs) using HepaRG cells and bioinks.1 We congratulate the authors for this pioneering study that demonstrates the fabrication and in vivo application of functional 3DP-HOs in mice models of liver failure. The study would pave the way for the use of three-dimensional (3D) bioprinted primary hepatocytes and/or hepatic progenitor cells as possible regenerative therapies in liver diseases. However, there are some caveats in the study, which need to be highlighted. First, the study showed that after 7 days of differentiation in vitro, HepaRG cells in 3DP-HOs expressed hepatic marker genes and exhibited albumin secretion (figure 2). However, it is to be noted that most of …

Bone morphogenetic protein (BMP) signalling plays a significant role during embryonic cartilage development and has been associated with osteoarthritis (OA) pathogenesis, being in both cases involved in triggering hypertrophy. Inspired by recent findings that BMP inhibition counteracts hypertrophic differentiation of human mesenchymal progenitors, we hypothesized that selective inhibition of BMP signalling would mitigate hypertrophic features in OA cartilage. First, a 3D in vitro OA micro-cartilage model was established using minimally expanded OA chondrocytes that was reproducibly able to capture OA-like hypertrophic features. BMP signalling was then restricted by means of two BMP receptor type I inhibitors, resulting in reduction of OA hypertrophic traits while maintaining synthesis of cartilage extracellular matrix. Our findings open potential pharmacological strategies for counteracting cartilage hypertrophy in OA and support the broader perspective that key signalling pathways known from developmental processes can guide the understanding, and possibly the mitigation, of adult pathological features.

The slings and arrows of two-dimensional (2D) graphene and one-dimensional (1D) carbon nanotubes embody a high risk-to-benefit ratio, which is challenging for their biocompatibility. Herein, we report for the first time the synthesis of hydrogel nanotubes (HNTs) with ice helices as exotic nanostructures via free radical-mediated aqueous copolymerization. Extensive embryonic 2D polymer nanosheets were produced after 10 h of reaction which buckled out-of-plane into HNTs, inspired by the ancient art of origami. Water drawn into the hydrophobic hollow conduit freezes spontaneously, acting as an epicenter for the genesis of an ice helix trapped in a kinetically stable arrangement with regular periodicity, extending rational synthesis into the nanoscale regime. In this seminal work, the mechanism for the formation of ice helices inside the HNTs was delineated with the aid of transmission electron microscopy (TEM), X-ray diffraction patterns (XRD) and Raman spectroscopy. Endowed with unique biocompatibility, these HNTs aided the rapid establishment of wound barrier properties with concomitant cell proliferation guided by a provisional matrix mimicking the extracellular matrix niche. The HNTs support a permissive milieu for vascular sufficiency with well-proliferated fibroblasts at the wound bed.

Culturing osteocytes on planer surfaces on Petri dish in vitro fails to recapitulate their natural orientation as well as biological functionality in human bone. Ability to recapitulate spatial arr...

The development of transplantable cell sheets of functional keratocytes embedded within an aligned collagen type I matrix is a viable approach for constructing a bioequivalent of corneal stroma. Thermoresponsive materials based on poly(N-isopropylacrylamide) (PolyNIPA) have been utilized to recover carrier-free corneal cell sheets by inducing temperature changes. In this study, we employed direct-write assembly (DWA) to develop microperiodic parallel patterns of silk–PolyNIPA and gelatin–PolyNIPA. Semi-interpenetrating networks of PolyNIPA hybrids (with silk/gelatin) exhibited temperature-responsive nature and thereby have potential use in cell sheet engineering. Silk–PolyNIPA and gelatin–PolyNIPA hybrids demonstrated a hydrophobic surface at 37 °C (i.e. above their lower critical solution temperature) with a contact angle of 59° ± 0.3° and 55° ± 3°, respectively, whereas the surface roughness of silk–PolyNIPA was double that of gelatin–PolyNIPA. The reduction of temperature to 20 °C resulted in a decrease in the value of surface roughness and water contact angle for both hybrids. All four parallel patterned substrates guided corneal cell alignment along the direction of the patterns. Collagen type-I and aggrecan gene expression was higher when the cells were grown over the gelatin–PolyNIPA matrix after 3 weeks of culture when compared to silk–PolyNIPA. In addition, a significantly higher metabolic activity as well as enhanced vinculin expression of keratocytes on the gelatin–PolyNIPA matrix indicated the improved cytocompability compared to the silk, gelatin and silk–PolyNIPA matrices. Interestingly, the detachment of keratocytes cell sheet was achieved from the silk–PolyNIPA and gelatin–PolyNIPA planar films only within 10 min and 30 min, respectively, but the patterns could not yield intact sheet recovery. Hence, we conclude that while gelatin–PolyNIPA hybrids with parallel patterns fabricated using DWA will benefit from the application of cellular alignment, some optimization in the pattern parameters may be required for rapid sheet recovery from such substrates. Understanding the keratocytes responses to such hybrid biomaterials suggests viable options to develop a corneal stromal bioequivalent.

Current therapeutic strategies to reduce scarring in full thickness skin defect offer limited success due to poor understanding of scar tissue formation and the underlying signaling pathways. There is an urgent need to develop human cell based in vitro scar tissue models as animal testing is associated with ethical and logistic complications and inter-species variations. Pro-inflammatory cytokines play critical role in regulating scar development through complex interplay and interaction with the ECM and corresponding signaling pathways. In this context, we assessed the responses of cultured fibroblasts with respect to their differentiation into myofibroblasts using optimised cytokines (TGF-β1, IL-6 and IL-8) for scar formation in 2D (tissue culture plate, collagen type I coated plate) vs 3D collagen type I gel based constructs. We attempted to deduce the role of dimensionality of cell culture matrix in modulating differentiation, function and phenotype of cultured fibroblasts. Validation of the developed model showed similarity to etiology and pathophysiology of in vivo hypertrophic scar with respect to several features: 1) transition of fibroblasts to myofibroblasts with convincing expression of α-SMA stress fibers; 2) contraction; 3) excessive collagen and fibronectin secretion; 4) expression of fibrotic ECM proteins (SPARC and Tenascin); 5) low MMP secretion. Most importantly, we elucidated the involvement of TGF-β/SMAD and Wnt/β-catenin pathways in developing in vitro dermal scar. Hence, this relatively simple in vitro human scar tissue equivalent may serve as an alternative for testing and designing of novel therapeutics and help in extending our understanding of the complex interplay of cytokines and related dermal scar specific signaling. Statement of Significance Scarring of the skin affects almost millions of people per year in the developed world alone, nevertheless the complex pathophysiology and the precise signaling mechanisms responsible for this phenomenon of skin scarring are still unknown. A number of anti-scar drugs are being developed and being tested on animals and monolayer models. However, testing the efficacy of these drugs on lab based 3D in vitro models may prove extremely useful in recapitulating the 3D microenvironment of the native scar tissue. In that context in this study we have demonstrated the development of 3D in vitro dermal scar model, by optimizing a constellation of factors, such as combination of cytokines (TGF-β1,IL-6,IL-8) and cellular dimensionality in inducing the differentiation of dermal fibroblasts to myofibroblasts. This in vitro scar model was successful in replicating hallmark features of hypertrophic scar such as excessive synthesis of fibrotic extracellular matrix, perturbed matrix homeostasis, contraction, diminished MMP synthesis. The study also highlighted significant involvement of TGF-β/SMAD and Wnt/β-catenin signaling pathways in in vitro scar formation.

Tissue engineered cartilage has never been evaluated with an aim to distinguish between transient and articular cartilage. A major drawback of existing state-of-the art engineered cartilage is cellular hypertrophy, leading to development of transient cartilage which ultimately undergoes endochondral ossification to form bone trabeculae. As a paradigm shift, using 3D bioprinting, we have evaluated six different conditions for best outcome vis-a-vis articular cartilage differentiation as assessed by expression of a constellation of markers (like Autotaxin, lubricin etc). Our study strongly suggests that BMSCs undergo hypertrophic differentiation in the presence of TGF-β1, while in the absence of TGF-β1 BMSCs encapsulated in 3D bioprinted silk-gelatin bioink matrix undergo articular cartilage differentiation. Our study provides novel insights into direct regulatory role of silk-gelatin bioink on IHH and Wnt signaling pathways in controlling hypertrophy during chondrogenic differentiation of BMSCs. 3D bioprinted silk-gelatin constructs enabled adequate cellular attachment, proliferation and most importantly, articular cartilage differentiation. Interestingly, we observed close similarities between the signaling pathways associated with the 3D bioprinted constructs with respect to the signaling pathways with embryonic cartilage development suggesting our engineered cartilage tissue to be a prospective tissue equivalent with potential of providing the essential instructive elements for activating pathways of organogenesis in patient-specific manner.

Corneal scarring is the major source of permanent blindness worldwide. The complex pathophysiology of corneal scarring is not comprehensibly understood as it involves the interaction of a constellation of pro-fibrotic cytokines influencing several signaling pathways involved in corneal scar development. In the present study, an attempt has been made to generate a relatively simple in vitro corneal scar model using primary corneal keratocytes by exogenously providing an optimized dose of combination of cytokines (TGF-β1, IL-6, and IL-8) involved in scar formation in situ. Data obtained from gene and protein expression analysis depicted enhanced ECM production with discrete expression of myofibroblast specific markers. The protein-protein interactions associated these proteins to various pathways involved in wound healing, cellular migration, and cytoskeletal remodeling justifying high relevance to in vivo scar formation. Hence the developed model can be used to acquire understanding about corneal scar pathophysiology and thus might be useful for designing the treatment modalities and efficacies for controlling scar formation.

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk A. mylitta, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and β-glycerophosphate), and cytokine (TGF-β1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, an...

A photocurable silk fibroin hydrogel is prepared, for the first time, using natural silk protein fibroin and biophotosensitizer riboflavin. Riboflavin is excited by ultraviolet light to generate a triplet state which is transferred to produce active oxygen radicals with singlet oxygen as the main component. Active oxygen radicals can induce chemical cross-linking of amino-, phenol- and other groups in the silk fibroin macromolecules to form a photocurable hydrogel. The different biophysical characterizations of the gelation of this modified fibroin protein solution were studied by using Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, microplate reader and texture analyzer. The aggregate structures, surface morphologies, mechanical properties, light transmission and degradation properties of the gel were studied. The investigations showed that the silk fibroin/riboflavin hydrogels predominantly have random coils or alpha helix structures. These gels show resilience up to 90% after 80% compression and a light transmission of up to 97%. The cell culture experiment exhibits that the hydrogel has a satisfactory cytocompatibility., The work is supported by National Natural Science Foundation of China (Grant No. 51373114), Six Talent Peaks Project in Jiangsu Province (Grant No. SWYY-038), PAPD and Natural Science Foundation of Jiangsu Province, China (Grant No. BK20171239). SCK presently holds a European Research Area Chair position at the 3B's Research Group, University of Minho, Portugal, supported by the European Union Framework Programme for Research and Innovation HORIZON 2020 under grant agreement n° 668983 — FoReCaST.