Immune response profiles induced by silk-based biomaterials: a journey from 'immunogenicity' towards 'immuno-compatibility.

Silk is a widely accepted biomaterial for tissue regeneration owing to its tunable biomechanical properties and ease of chemical modification. However, a number of aspects associated with its clinical use are still debated. Indeed, to achieve clinical success, a biomaterial must favorably interact with host tissues without evoking local or systemic immuno-inflammatory responses. The analysis of immune responses associated with silk under in vitro and in vivo conditions provides useful insights, improving the understanding of the functional characteristics of silk biomaterials and further promoting their clinical application. Silk evokes moderate immune responses upon implantation in vivo, depending on the material structure, fabrication method, degradation time, and implantation in soft or hard tissue sites, which rapidly subside within a few days/weeks. In vitro studies indicate that its immune-stimulatory properties are largely due to inherent protein conformation and differential processing parameters. Strategically controlled levels of immune responses in vivo with marginal immunogenicity of silk-based biomaterials may contribute to matrix remodeling and replacement by native tissue matrix around the implanted site. Therefore, immunomodulatory strategies should be developed to promote the use of silk-based biomaterials as promising candidates for numerous clinical applications.

Enhancing Wound Healing With Sprayable Hydrogel Releasing Multi Metallic Ions: Inspired by the Body's Endogenous Healing Mechanism.

In the pursuit of new wound care products, researchers are exploring methods to improve wound healing through exogenous wound healing products. However, diverging from this conventional approach, this work has developed an endogenous support system for wound healing, drawing inspiration from the body's innate healing mechanisms governed by the sequential release of metal ions by body at wound site to promote different stages of wound healing. This work engineers a multi-ion-releasing sprayable hydrogel system, to mimic this intricate process, representing the next evolutionary step in wound care products. It comprises Alginate (Alg) and Fibrin (Fib) hydrogel infused with Polylactic acid (PLA) polymeric microcarriers encapsulating multi (calcium, copper, and zinc) nanoparticles (Alg-Fib-PLA-nCMB). Developed sprayable Alg-Fib-PLA-nCMB hydrogel show sustained release of beneficial multi metallic ions at wound site, offering a range of advantages including enhanced cellular function, antibacterial properties, and promotion of crucial wound healing processes like cell migration, ROS mitigation, macrophage polarization, collagen deposition, and vascular regeneration. In a comparative study with a commercial product (Midstress spray), developed Alg-Fib-PLA-nCMB hydrogel demonstrates superior wound healing outcomes in a rat model, indicating its potential for next generation wound care product, addressing critical challenges and offering a promising avenue for future advancements in the wound management.

Silk-gelatin hybrid hydrogel: a potential carrier for RNA therapeutics.

RNA-based therapeutics have exhibited remarkable potential in targeting genetic factors for disease intervention, exemplified by recent mRNA vaccines for COVID-19. Nevertheless, the intrinsic instability of RNA and challenges related to its translational efficiency remain significant obstacles to the development of RNA as therapeutics. This study introduces an innovative RNA delivery approach using a silk fibroin (SF) and positively charged gelatin (Gel) hydrogel matrix to enhance RNA stability for controlled release. As a proof of concept, whole-cell RNA was incorporated into the hydrogel to enhance interactions with RNA molecules. Additionally, molecular modeling studies were conducted to explore the interactions between SF, collagen, chitosan (Chi), and the various RNA species including ribosomal RNAs (28S, 18S, 8.5S, and 5S rRNAs), transfer RNAs (tRNA-ALA, tRNA-GLN, and tRNA-Leu), as well as messenger RNAs (mRNA-GAPDH, mRNA-ß actin, and mRNA-Nanog), shedding light on the RNA-polymer interaction and RNA stability; SF exhibits a more robust interaction with RNA compared to collagen/gel and chitosan. We confirmed the molecular interactions of SF and RNA by FTIR and Raman spectroscopy, which were further supported by AFM and contact angle measurement. This research introduces a novel RNA delivery platform and insights into biopolymer-RNA interactions, paving the way for tailored RNA delivery systems in therapeutics and biomedical applications.

3D bioprinting of stromal cells-laden artificial cornea based on visible light-crosslinkable bioinks forming multilength networks.

3D bioprinting has the potential for the rapid and precise engineering of hydrogel constructs that can mimic the structural and optical complexity of a healthy cornea. However, the use of existing light-activated bioinks for corneal printing is limited by their poor cytocompatibility, use of cytotoxic photoinitiators (PIs), low photo-crosslinking efficiency, and opaque/colored surface of the printed material. Herein, we report a fast-curable, non-cytotoxic, optically transparent bioprinting system using a new water-soluble benzoyl phosphinate-based PI and photocrosslinkable methacrylated hyaluronic acid (HAMA). Compared with commercially available PIs, the newly developed PI, lithium benzoyl (phenyl) phosphinate (BP), demonstrated increased photoinitiation efficiency under visible light and low cytotoxicity. Using a catalytic amount of BP, the HA-based bioinks quickly formed 3D hydrogel constructs under low-energy visible-light irradiation (405 nm, <1 J cm-2). The mechanical properties and printability of photocurable bioinks were further improved by blending low (10 kDa) and high (100 kDa) molecular weight (MW) HAMA by forming multilength networks. For potential applications as corneal scaffolds, stromal cell-laden dome-shaped constructs were fabricated using MW-blended HAMA/BP bioink and a digital light processing printer. The HA-based photocurable bioinks exhibited good cytocompatibility (80%-95%), fast curing kinetics (<5 s), and excellent optical transparency (>90% in the visible range), potentially making them suitable for corneal tissue engineering.

Covalent Conjugation of Small Molecule Inhibitors and Growth Factors to a Silk Fibroin-Derived Bioink to Develop Phenotypically Stable 3D Bioprinted Cartilage.

Implantation of a phenotypically stable cartilage graft could represent a viable approach for repairing osteoarthritic (OA) cartilage lesions. In the present study, we investigated the effects of modulating the bone morphogenetic protein (BMP), transforming growth factor beta (TGFß), and interleukin-1 (IL-1) signaling cascades in human bone marrow stromal cell (hBMSC)-encapsulated silk fibroin gelatin (SF-G) bioink. The selected small molecules LDN193189, TGFß3, and IL1 receptor antagonist (IL1Ra) are covalently conjugated to SF-G biomaterial to ensure sustained release, increased bioavailability, and printability, confirmed by ATR-FTIR, release kinetics, and rheological analyses. The 3D bioprinted constructs with chondrogenically differentiated hBMSCs were incubated in an OA-inducing medium for 14 days and assessed through a detailed qPCR, immunofluorescence, and biochemical analyses. Despite substantial heterogeneity in the observations among the donors, the IL1Ra molecule illustrated the maximum efficiency in enhancing the expression of articular cartilage components, reducing the expression of hypertrophic markers (re-validated by the GeneMANIA tool), as well as reducing the production of inflammatory molecules by the hBMSCs. Therefore, this study demonstrated a novel strategy to develop a chemically decorated, printable and biomimetic SF-G bioink to produce hyaline cartilage grafts resistant to acquiring OA traits that can be used for the treatment of degenerated cartilage lesions.

An Advanced Bioconjugation Strategy for Covalent Tethering of TGFß3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering.

The transforming growth factor-ß class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-ß3 (TGFß3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFß3 conjugated silk matrices than where TGFß3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFß3 and the role of the silk as a growth factor stabilizer. When cultured over TGFß3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.

Assessing the advantages of 3D bioprinting and 3D spheroids in deciphering the osteoarthritis healing mechanism using human chondrocytes and polarized macrophages.

The molecular niche of an osteoarthritic microenvironment comprises the native chondrocytes, the circulatory immune cells, and their respective inflammatory mediators. Although M2 macrophages infiltrate the joint tissue during osteoarthritis (OA) to initiate cartilage repair, the mechanistic crosstalk that dwells underneath is still unknown. Our study established a co-culture system of human OA chondrocytes and M2 macrophages in 3D spheroids and 3D bioprinted silk-gelatin constructs. It is already well established that Silk fibroin-gelatin bioink supports chondrogenic differentiation due to upregulation of the Wnt/ß-catenin pathway. Additionally, the presence of anti-inflammatory M2 macrophages significantly upregulated the expression of chondrogenic biomarkers (COL-II, ACAN) with an attenuated expression of the chondrocyte hypertrophy (COL-X), chondrocyte dedifferentiation (COL-I) and matrix catabolism (MMP-1 and MMP-13) genes even in the absence of the interleukins. Furthermore, the 3D bioprinted co-culture model displayed an upper hand in stimulating cartilage regeneration and OA inhibition than the spheroid model, underlining the role of silk fibroin-gelatin in encouraging chondrogenesis. Additionally, the 3D bioprinted silk-gelatin constructs further supported the maintenance of stable anti-inflammatory phenotype of M2 macrophage. Thus, the direct interaction between the primary OAC and M2 macrophages in the 3D context, along with the release of the soluble anti-inflammatory factors by the M2 cells, significantly contributed to a better understanding of the molecular mechanisms responsible for immune cell-mediated OA healing.

Deep Learning Augmented Osteoarthritis Grading Standardization.

Manual grading of cartilage histology images for investigating the extent and severity of osteoarthritis (OA) involves critical examination of the cell characteristics, which makes this task tiresome, tedious, and error prone. This results in wide interobserver variation, causing ambiguities in OA grade prediction. Such drawbacks of manual assessment can be overcome by implementing artificial intelligence-based automated image classification techniques such as deep learning (DL). Hence, we present the feasibility of training a deep neural network with cartilage histology images, which can grade the extent and severity of knee OA based on modified Mankin scoring system. The grading system used here for automating OA grading was simplified and modified based on the microscopic observations from the histology images, where three parameters (Safranin-O staining intensity, chondrocyte distribution and arrangement, and morphology) were considered for evaluating the OA progression. The histology images were tiled, labeled, and grouped together based on the developed grading system (Grade 0-3). Four different DL architectures were tried for image classification and the best performing model was selected by fivefold validation method. With a validation accuracy of ∼84%, 0.632 Cohen's kappa score, and an excellent receiver operating characteristic (ROC)-area under the ROC curve ranging between 0.89 and 0.99, DenseNet121 was selected among the four models as the best performing model, and was used for inferencing on new data. Final grades obtained from the models were in accordance with the grades provided by the medical experts. We hereby demonstrate that a DL architecture can be taught to interpret the degree of cartilage degradation, with excellent discriminatory ability across all four classes of OA severity. Unlike other works where radiographic images have been considered for grading of OA, we have considered histology images, which is a fundamental approach for grading OA extent and severity. This would bring a paradigm shift in histology-based assessment of OA, making this automated approach to be explored as an option for OA grading standardization. Ethical approval number-IAH-BMR-018/10-19.

Gelatin scaffold ameliorates proliferation & stem cell differentiation into the hepatic like cell and support liver regeneration in partial-hepatectomized mice model.

Stem cell-based tissue engineering is an emerging tool for developing functional tissues of choice. To understand pluripotency and hepatic differentiation of mouse embryonic stem cells (mESCs) on a three-dimensional (3D) scaffold, we established an efficient approach for generating hepatocyte-like cells (HLCs) from hepatoblast cells. We developed porous and biodegradable scaffold, which was stimulated with exogenous growth factors and investigated stemness and differentiation capacity of mESCs into HLCs on the scaffoldin-vitro. In animal studies, we had cultured mESCs-derived hepatoblast-like cells on the scaffold and then, transplanted them into the partially hepatectomized C57BL/6 male mice model to evaluate the effect of gelatin scaffold on hepatic regeneration. The 3D culture system allowed maintenance of stemness properties in mESCs. The step-wise induction of mESCs with differentiation factors leads to the formation of HLCs and expressed liver-specific genes, including albumin, hepatocyte nucleic factor 4 alpha, and cytokeratin 18. In addition, cells also expressed Ki67, indicating cells are proliferating. The secretome showed expression of albumin, urea, creatinine, alanine transaminase, and aspartate aminotransferase. However, the volume of the excised liver which aids regeneration has not been studied. Our results indicate that hepatoblast cells on the scaffold implanted in PH mouse indicates that these cells efficiently differentiate into HLCs and cholangiocytes, forming hepatic lobules with central and portal veins, and bile duct-like structures with neovascularization. The gelatin scaffold provides an efficient microenvironment for liver differentiation and regeneration bothin-vitroandin-vivo. These hepatoblasts cells would be a valuable source for 3D liver tissue engineering/transplantation in liver diseases.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

3D biofabrication and space: A 'far-fetched dream' or a 'forthcoming reality'?

The long duration space missions across the Low Earth Orbit (LEO) often expose the voyagers to an abrupt zero gravity influence. The severe extraterrestrial cosmic radiation directly causes a plethora of moderate to chronic healthcare crises. The only feasible solution to manage critical injuries on board is surgical interventions or immediate return to Earth. This led the group of space medicine practitioners to adopt principles from tissue engineering and develop human tissue equivalents as an immediate regenerative therapy on board. The current review explicitly demonstrates the constructive application of different tissue-engineered equivalents matured under the available ground-based microgravity simulation facilities. Further, it elucidates how augmenting the superiority of biomaterial-based 3D bioprinting technology can enhance their clinical applicability. Additionally, the regulatory role of weightlessness condition on the underlying cellular signaling pathways governing tissue morphogenesis has been critically discussed. This information will provide future directions on how 3D biofabrication can be used as a plausible tool for healing on-flight chronic health emergencies. Thus, in our review, we aimed to precisely debate whether 3D biofabrication is deployed to cater to on-flight healthcare anomalies or space-like conditions are being utilized for generating 3D bioprinted human tissue constructs for efficient drug screening and regenerative therapy.

Intelligent control of integrated on-board charger with improved power quality and reduced charging transients.

This paper proposes an intelligent control scheme for a two-stage integrated onboard electric vehicle (EV) battery charger connected to a single-phase household outlet which offers a close to ideal battery charging profile with power factor correction feature. Generally, the front-end AC-DC​ conversion stage is controlled by dual loop proportional-integral (PI) controllers, and tuning their gain constants is a difficult task. Furthermore, to achieve a close to ideal charging profile for an EV battery, the DC-DC conversion stage switches from constant current (CC) and constant voltage (CV) mode after a certain state of charge (SOC) which may lead to discontinuity in the charging current and voltage. This paper attempts to solve these issues by proposing an intelligent control scheme that includes the dynamic estimation of PI controller gain constants as well as provides a seamless mode transfer feature for battery charging. It is achieved by using fuzzy-PI-based control in the AC-DC conversion stage and Bayesian Regularization (BR) algorithm trained artificial neural network (ANN)-based control in the DC-DC conversion stage. The performance of the proposed control scheme is assessed both in steady-state and transient conditions in MATLAB® Simulink environment by comparing it against similar control schemes. The proposed intelligent control approach improves the dynamic response of DC link voltage, offers unity power factor operation and maintains the line current harmonics within IEEE 519 standards even during the switchover from CC to CV charging mode. Also, there is a decrease of 85% in the third harmonic component of the source current, 23.2% improvement in DC link voltage undershoot and 6.5% reduction in DC link voltage overshoot with reduced settling times using the proposed unified control scheme.

Development of a biomimetic arch-like 3D bioprinted construct for cartilage regeneration using gelatin methacryloyl and silk fibroin-gelatin bioinks.

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 vitroreplication 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.ß-catenin, TGFßR1, Col 1A1 in SF-G and PRG4, Col 10A1, MMP 13 in GelMA validated ourin vitroresults. These findings emphasized the role of SF-G in regulating the Wnt/ß-catenin and TGF-ßsignaling pathways. Hence, the 3D bioprinted arch-like constructs possess a substantial potential for cartilage regeneration.

Mechanistic crosstalk of extracellular calcium-mediated regulation of maturation and plasticity in human monocytes.

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.

Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies.

Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state (with the expression of collagen type X, matrix metalloproteinases-13, and alkaline phosphatase) analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause-effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies.

Developmental biology-inspired tissue engineering by combining organoids and 3D bioprinting.

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.

Recent advances in bioprinting using silk protein-based bioinks.

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.

3D printed hydroxyapatite promotes congruent bone ingrowth in rat load bearing defects.

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 forin vivobone regeneration in a rat tibial defect model. Rapid prototyping using the direct-write technique to fabricate 25 mm2HAP 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 previousin vitrostudy. 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.

Macrophage Polarization Profiling on Native and Regenerated Silk Biomaterials.

We investigated the plasticity and polarization of THP-1 cells on native and regenerated silk-based biomaterials to address the basic paradigm of immune response. Here, we report redox kinetics, adhesion morphology, and nitric oxide release patterns to identify specific subtypes of macrophages at different time points. Water-annealed silk film and native fibrous braids from Bombyx mori silkworms showed higher anti-inflammatory cytokine profiles or M2 subtypes (as evidenced by the enhanced expression of interleukin-10, interleukin-13, and interleukin-4). Ethanol-treated Bombyx mori silk films and Antheraea mylitta braids exhibited higher levels of pro-inflammatory cytokines or the M1 subtype (as evidenced by enhanced expression of interleukin-1, interleukin-6, interleukin-8, interferon-γ, TNF-α, and GM-CSF) in contact with healthy THP monocytes for 14 days; such a long study is unprecedented. Cytokine microarray analysis revealed the transition (M0-M1, M1-M2), plasticity, and stable phenotype of THP-1 cells in a variable stage in contact with different physicochemical properties of silk-based biomaterials. The detailed immunogenicity in the context of the physicochemical properties of native and regenerative silk-based biomaterials will enable us to accurately predict the possibility of a pro-/anti-inflammatory response. It will helps to predict the in vivo reprogramming and avoid fibrosis formation to enhance their clinical translational potential.

3D tumor angiogenesis models: recent advances and challenges.

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.