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Cellular Proliferation, Self-Assembly, and Modulation of Signaling Pathways in Silk Fibroin Gelatin-Based 3D Bioprinted Constructs
Abstract
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 Bombyx mori silkworm as a promising biomaterial of choice for the formulation of bioink owing to its distinct characteristics involving rheology behavior, self-supporting filamentous extrusion, and a suitable biomaterial to achieve resolution printing. Further, we have elaborated on how SF gelatin bioink can in specific regulate the cellular differentiation pathway of progenitor cells, the mechanism of cellular self-assembly, cell migration, matrix remodeling, and self-orientation, leading to the desired tissue-specific construct. How features of bioink and fabrication design aspects can induce in vitro tissue patterning and anatomically relevant tissue organization have also been explored in this review. Importantly, we have tried to shift the understanding of bioprinted tissue regeneration from a cell-proliferation-centric and gene-expression-centric point of view to the complex role of the microenvironment present within the bioprinted constructs. We believe that shedding light on these factors would help in achieving the so-called "ideal 3D bioprinted construct" to meet the shortages of high-quality donor tissues for the regeneration of the damaged and diseased ones.
... The unique amino acid composition also allows SF to mimic the extracellular matrix of human tissues and organs, and makes it widely used in the biomedical materials and medical devices. [43][44][45][46][47] Cutting horizontally, the cross-sectional structure of SF is shown in Figure 2. The outer layer is sericin. Sericin occupies 30 % of the volume, which tightly connects the two insoluble silk protein fibroin that occupy the remaining 70 % of the . ...
... During the integration from the nanofibril to the fibroin, the interaction forces between the nanofibril result in a close parallel arrangement within the silk fibroin bundle. [43][44][45][46][47][48][49][50] In order to further explore the basic unitary structure of SF, we took the nanofibril as a starting point and zoomed in step by step. As shown in Figure 3, the H6L6P25 indicates that every six "heavy chain (391 kDa) -light chain ( Mainly engaged in scientific research on textile dyeing and finishing process and theory, surface modification and processing technology of textile materials, and application of nanotechnology on textile functional finishing. ...
... Junhong Qi, Anhui Fuyang, CPC member, Shanghai University of Engineering Science, the school of Textile and Fashion, Textile Chemistry and Dyeing and Finishing Engineering, Master's degree students, the main research direction for the surface modification of textile materials and textile functional finishing, is now published in the TiO2-based photocatalytic degradation of hazardous substances in the car research progress, Preparation of TiO2/graphene-based composites and investigation of catalytic properties under visible light and other papers. [43][44][45][46][47] mines that SF approximate human skin proteins and are able to be applied as structural substances. [51][52][53][54] It can be seen that the core amino acid sequence enriches the content of hydroxyl groups (À OH) on the heavy chain, and the large number of hydroxyl groups gives the heavy chain the ability to form very numerous and stable hydrogen bonds between and within molecules. ...
Due to excellent biocompatibility, sufficient raw material, robust mechanical properties and easy cross‐linking, Silk Fibroin (SF) is a promising protein for 3D printing inks and an ideal candidate for 3D scaffolds in fields like regenerative medicine, bioelectronics and bio‐optics. In order to meet the requirements of print accuracy, mechanical properties and form retention capabilities, the first step is to prepare SF 3D printing inks using physical, chemical or other strategies of cross‐linking. The basic chemical groups and physical structure of SF determines its ability to form 3D networks under different conditions using various cross‐linking strategies. In the preparation of SF‐based 3D printing inks, physical, chemical or other strategies of cross‐linking improve the qualities of printing, but each strategy has its advantages and disadvantages. This paper discusses different crosslinking strategies for SF to support the development of exciting potential for SF‐based 3D printing inks to meet more needs in the future.
... Although the precise level of resemblance needed for engineering a wholly functional tissue is still unknown [8], it has been observed in a few studies that regulating the architecture of the fabricated biological constructs while printing is a promising strategy to bridge the gap between the native and the bioprinted construct [8,9]. One mechanism in developing human embryonic tissue involves phenotypically identical cells experiencing self-assembly to produce a specific domain [10]. More research needs to be done on how bioink and fabrication design elements can generate in vitro tissue patterning and anatomically meaningful tissue organization. ...
... Even though a wide array of bioinks have been investigated for the engineering of articular cartilage tissue, there is still a feeble understanding of the role played by these biomaterials in regulating various molecular signaling pathways during in vitro chondrogenic differentiation [11]. In this regard, silk fibroin-gelatin (SF-G) has already been shown to intrinsically stimulate the Wnt pathway in primary chondrocytes and mesenchymal progenitor cells [10]. Another unique feature of the SF-G bioink is that it aids in abating hypertrophy during chondrogenesis [1,12,13]. ...
... Hence, gene expression analysis using hypertrophic markers such as Col 10A1, MMP 13, and protein expression was performed to ascertain which bioink helps abate hypertrophy during articular cartilage development. Although SF-G has been well established in regulating the Wnt/β-catenin and TGF-β signaling pathway [10,11], whether GelMA plays any role in regulating this and other signaling pathway remains inexplicable. Further, we analyzed which bioink produced a better fibrous collagen network of ECM when printed in this specific design. ...
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 Col2A1, SOX9, ACAN, and the genes upregulated on day 21 in RT-PCR, i.e., β-catenin, TGFβR1, Col1A1 in SF-G and PRG4, Col10A1, MMP13 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.

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... 71 Compared with collagen, GEL shows a lower antigenicity and better biocompatibility. [142][143][144] As a consequence, it has become an interesting material for biomedical purposes. 144 On the other hand, it is easily degradable and shows inadequate strength. ...
... On the other hand, those blends show decreased Young's modulus, tensile strength, and porosity when compared with SF. 144 As a consequence, the most common processing also includes crosslinking agents as stabilizers, the most common ones being genipin, 143 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) 79 and methylenebisacrylamide (MBA), 77 among others. 78,142 The use of these agents results in non-toxic structures with improved mechanical properties, 79 allowing their application as biomedical materials for building blocks for osteogenic tissue engineering, 79 cartilage repair, 143 and controllable drug delivery carriers, 142 among others. In addition, due to the GEL rheology and gelling ability, it has become a promising blend for SF-based structures for advanced manufacturing. ...
... Produced from Bombyx mori (B. mori), also known as the silkworm, this material has attracted attention in the 3D bioprinting segment, due to its excellent properties, such as biodegradability [152], biocompatibility [153], processing in different formats (hydrogels, films, membranes, etc.) and good mechanical properties [154]. Furthermore, when it comes to the manufacture of bioinks, another parameter that must be evaluated is the viability of cell growth [155]. ...
... biodegradability [152], biocompatibility [153], processing in different formats (hydrogels, films, membranes, etc.) and good mechanical properties [154]. Furthermore, when it comes to the manufacture of bioinks, another parameter that must be evaluated is the viability of cell growth [155]. ...
3D bioprinting has gained visibility in regenerative medicine and tissue engineering due to its applicability. Over time, this technology has been optimized and adapted to ensure a better printability of bioinks and biomaterial inks, contributing to developing structures that mimic human anatomy. Therefore, cross-linked polymeric materials, such as hydrogels, have been highly targeted for the elaboration of bioinks, as they guarantee cell proliferation and adhesion. Thus, this short review offers a brief evolution of the 3D bioprinting technology and elucidates the main hydrogels used in the process.
... The cell proliferation rate is a reliable assessment for in vitro study to evaluate the survivability of the biological cell after printing. The encapsulated cell in hydrogel has many growth limitations, including restricted cell connections due to the entrapment of the cells within the hydrogel [103]. Cell viability and proliferation rates have been identified as good selective indicators for cell sustainability. ...
... Besides, studies on gelatin-based bioinks only for encapsulated skin cells by using 3Dbioprinting have not been discovered yet. However, a study on the cellular proliferation rate for DFs and KCs against silk fibroin-gelatin bioinks gradually proven that the DFs and KCs indicate excellent cellular growth from day seven until day 21 [103]. The combination of silk fibroin-gelatin promotes cell adhesion site compared to silk fibroin only. ...
Skin substitutes can provide a temporary or permanent treatment option for chronic wounds. The selection of skin substitutes depends on several factors, including the type of wound and its severity. Full-thickness skin grafts (SGs) require a well-vascularised bed and sometimes will lead to contraction and scarring formation. Besides, donor sites for full-thickness skin grafts are very limited if the wound area is big, and it has been proven to have the lowest survival rate compared to thick- and thin-split thickness. Tissue engineering technology has introduced new advanced strategies since the last decades to fabricate the composite scaffold via the 3D-bioprinting approach as a tissue replacement strategy. Considering the current global donor shortage for autologous split-thickness skin graft (ASSG), skin 3D-bioprinting has emerged as a potential alternative to replace the ASSG treatment. The three-dimensional (3D)-bioprinting technique yields scaffold fabrication with the combination of biomaterials and cells to form bioinks. Thus, the essential key factor for success in 3D-bioprinting is selecting and developing suitable bioinks to maintain the mechanisms of cellular activity. This crucial stage is vital to mimic the native extracellular matrix (ECM) for the sustainability of cell viability before tissue regeneration. This comprehensive review outlined the application of the 3D-bioprinting technique to develop skin tissue regeneration. The cell viability of human skin cells, dermal fibroblasts (DFs), and keratinocytes (KCs) during in vitro testing has been further discussed prior to in vivo application. It is essential to ensure the printed tissue/organ constantly allows cellular activities, including cell proliferation rate and migration capacity. Therefore, 3D-bioprinting plays a vital role in developing a complex skin tissue structure for tissue replacement approach in future precision medicine.
... However, most of the current studies in this direction do not focus on assessing detailed molecular signaling pathways that would aid in developing phenotypically stable articular cartilage. Our earlier studies on 3D bioprinting for cartilage repair exquisitely highlighted modulation of HIF1 and canonical Wnt/β-catenin signaling pathways following the interaction of SF-G bioink with progenitor cells [7,8,36]; however, we could not provide proof of concept for the modulation of these signaling pathways at the protein level. In this light, we aimed to deepen this analysis by providing a detailed picture of the molecular signaling pathways modulating chondrogenic differentiation of hMSCs in bioprinted SF-G constructs to understand the intrinsic regenerative potential of the bioink. ...
... We have reported inherent regenerative potential of SF-G bioink as it induced activation of the Wnt/β-catenin signaling pathway in hMSCs [36]. However, there were indications for both activation and inhibition of Wnt/β-catenin signaling, probably leading to the observed expression of hypertrophic cartilage markers. ...
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.Graphic abstract
... 43 SF-G has been demonstrated to play a crucial role in intrinsically regulating the Wnt/b-catenin pathway in primary chondrocytes and mesenchymal progenitor cells. 43,44 Karahalilo glu et al. fabricated magnetic SF electrically mediated hydrogel (e-gel) constructs by an electrogelation process using 8% of concentrated Bombyx mori SF aqueous solution as a novel sort of bone-graft substitute. 45 The findings showed that introducing MNPs enhanced the mechanical characteristics of SF e-gel constructs, including increased hydrophilicity. ...
... The pro-chondrogenic role of 3D bioprinted SF-G bioink-based constructs has been exquisitely demonstrated by Chawla et al through an extensive proteomic analysis confirming the upregulation of Wnt signaling pathway, a key modulator of in vitro chondrogenesis [25]. Thus, these studies utilize the concept of auxiliary cell-type involvement in OA phenotype development and establishment of cell-ECM communication using a wide array of biomaterials to understand the disease severity and further devise therapeutic strategies to suppress the progression [21,26,27]. ...
The molecular niche of an osteoarthritic microenvironment comprises of 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 support chondrogenic differentiation due to upregulation in Wnt/β10 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 silk17 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 3D context along with the release of the soluble anti-inflammatory factors by the M2 cells significantly contributed to a better understanding regarding the molecular mechanisms responsible for immune cell-mediated OA healing.
... The sequence and structure of proteins determine the stiffness, swelling, and degradation rate of hydrogels. As a consequence, it can influence the behavior of cells and the regeneration of tissue within bioprinted constructs [40][41][42] . Furthermore, specific functional groups or peptides may be introduced into protein sequences to enhance cell adhesion, differentiation, or tissue regeneration [43] . ...
The fabrication of cell-laden protein-based hydrogels (PBHs) for bioprinting necessitates careful consideration of numerous factors to ensure optimal structure and functionality. Bioprinting techniques, such as single-cell, multi-cell, and cell aggregate bioprinting, are employed to encapsulate cells within PBHs bioink, enabling the creation of scaffolds for cartilage and bone regeneration. During the fabrication process, it is imperative to account for biophysical and biochemical factors that influence cell behavior and protein structure within the PBHs. Precise control of crosslinking methods, hydrogel rheological properties, and printing parameters is also crucial to achieve desired scaffold properties without compromising cell viability and protein integrity. This review primarily focuses on the influence of biophysical factors, including composition, microstructure, biodegradation, and crosslinking, as well as biochemical factors, including chemical structure, growth factors, and signaling molecules, on protein structure and cell behavior. Additionally, key considerations for bioprinting PBHs and their impact on the successful regeneration of tissues are discussed. Furthermore, the review highlights current advancements, existing challenges, and promising prospects in the development of cell-laden PBHs for bioprinting applications and the regeneration of bone and cartilage.
... It is thought that by changing the nature of the temporary scaffold or module used for assembly, the system can be optimized for different cell and tissue types, resulting in the construction of more complex and functional MTOs. 3D bioprinting should be used to create complex microenvironments, not just to build cell structures [100] . A scaffold-free strategy has been reported to successfully construct structures with curved surfaces and vascular-like structures [101,102] . ...
The biofabrication of multi-cellular tissues or organoids (MTOs) has been challenging in regenerative medicine for decades. Currently, two primary technological approaches are being explored: scaffold-based strategies utilizing three-dimensional (3D) bioprinting techniques and scaffold-free strategies employing bioassembly techniques. 3D bioprinting techniques include jetting-based, extrusion-based, and vat photopolymerization-based methods, and bioassembly techniques include Kenzan, fluid-based manipulation and microfluid, bioprinting-assisted tissue emergence, and aspiration-assisted technology methods. Scaffold-based strategies primarily concentrate on the construction of scaffold structures to provide an extracellular environment, while scaffold-free strategies primarily emphasize the assembly methods of building blocks. Different biofabrication technologies have their advantages and limitations. This review provides an overview of the mechanisms, advantages, and limitations of scaffold-based and scaffold-free strategies in tissue engineering. It also compares the strengths and weaknesses of these two strategies, along with their respective suitability under different conditions. Moreover, the significant challenges in the future development of convergence strategies, specifically the integration of scaffold-based and scaffold-free approaches, are examined in an objective manner. This review concludes that integrating scaffold-based and scaffold-free strategies could overcome the problems in the biofabrication of MTOs. A novel fabrication method, the BioMicroMesh method, is proposed based on the convergence strategy. Concurrently, the development of a desktop-scale integrated intelligent biofabrication device, the BioMicroMesh system, is underway. This system is tailored to the BioMicroMesh method and incorporates cell aggregate spheroids preparation, 3D bioprinting, bioassembly, and multi-organoid co-culture functions, providing an objective perspective on its capabilities.
... In the above study, the silk fibroin hydrogel was used for biomedical adhesive purpose and reported a wide range of viscosities (0-2000 Pa) for a different formulation of fibroin and poly(ethyleneglycol) (PEG) content. Several studies also claimed that the viscosity of hydrogel is highly regulated by temperature[8,96,[126][127][128][129][130].The methacrylated silk fibroin (5,10 and 15 %)-gelatin hydrogels prepared with 10-70Pa viscosity showed excellent biocompatibility, inflammatory infiltration, angiogenesis, nerve regeneration, ulcer and wound healing[104,131]. were 22.20, 25.90 and 31.90 ...
Hydrogel-based systems are widely used for conventional 3D-cell culture, where cells can be seeded on or embedded in 3D-matrix gels for cultivation. Several new approaches have emerged to develop innovative more performing polymeric biomaterials for tissue regeneration. Within this class of biomaterials, land and marine based-polymers (including among other collagens, silk, chitosan/chitin and alginates) have been explored to date. The best-known example of silk to date is the fiber produced by land-based animals like silkworms for the production of their cocoon. There are many successful studies already proving the empirical evidence of biomaterials from land-based silk in biomedical applications. Generally, silk-based hydrogels are mainly involved in the fabrication of different implants for skin, bone, cartilage and vascular-regeneration. The ideal silk fibroin hydrogels for skin, cosmetic and wound healing purposes should exhibit enhanced biological response which is mainly regulated by its tailored mechanical, rheological, viscoelastic properties, effective tissue regeneration ability, controllable swelling, hemostasis and biocompatibility. Accordingly, this review summarizes the rheological and viscoelastic properties of silk-fibroin based composite hydrogels obtained from various raw materials/composites, highlighting the relation of its rheological response to hydrogel biomaterial functions aiming biomedical applications.
... Toward this end, Chawla et al. [37•] successfully developed cartilage tissue equivalents by employing the mesenchymal stem cell MSC-encapsulated (SF-G) bioink, where a close resemblance in the signaling pathway was observed and hypertrophy was minimized in the 3D bioprinted construct concerning the signaling cascades encountered during embryonic cartilage development. In addition, it would be interesting to investigate the in vivo signaling crosstalk between Wnt and transforming growth factor β signaling, BMP signaling, and Hedgehog and Notch signaling, which occur in an in vitro network [38]. Furthermore, to precisely replicate the embryonic developmental program, the regulation of cell polarization, patterning, modulation of matrix arrangement, chemistry, and mechanical signals should be considered, promoting the structural reliability of 3D bioprinted constructs. ...
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.
... Hydrogels have attracted great attention in the production of 3D bioprinting bioinks due to their good biocompatibility, high moisture content, and highly controllable 3D structure [8]. Currently, hydrogel materials that have been commonly used in 3D bioprinting include gelatin [9,10], sodium alginate [11,12], chitosan [13], agarose [14], fibrin [15], etc. ...
Vascular replacement is one of the most effective tools to solve cardiovascular diseases, but due to the limitations of autologous transplantation, size mismatch, etc., the blood vessels for replacement are often in short supply. The emergence of artificial blood vessels with 3D bioprinting has been expected to solve this problem. Blood vessel prosthesis plays an important role in the field of cardiovascular medical materials. However, a small-diameter blood vessel prosthesis (diameter < 6 mm) is still unable to achieve wide clinical application. In this paper, a response surface analysis was firstly utilized to obtain the relationship between the contact angle and the gelatin/sodium alginate mixed hydrogel solution at different temperatures and mass percentages. Then, the self-developed 3D bioprinter was used to obtain the optimal printing spacing under different conditions through row spacing, printing, and verifying the relationship between the contact angle and the printing thickness. Finally, the relationship between the blood vessel wall thickness and the contact angle was obtained by biofabrication with 3D bioprinting, which can also confirm the controllability of the vascular membrane thickness molding. It lays a foundation for the following study of the small caliber blood vessel printing molding experiment.
... In our earlier study, we have manifested the role of SF-gelatin bioink in the downregulation of hypertrophic markers and upregulated expression of the articular chondrogenic marker [22]. Furthermore, we have also demonstrated its role in the upregulation of the Wnt signaling pathway in MSCs and primary chondrocytes [22,61]. The probable reason for this expression obtained with SF solution and not observed with its SNP counterpart is that all the cells encapsulated within the bioprinted construct might not be able to get in contact with the SNPs due to their heterogeneous distribution. ...
Even though a substantial amount of research has been undertaken in the domains of bioprinting over the last few years, various challenges exist concerning printability. One of the grave challenges in developing a bioink with superior printability is the constraint of material availability, cross-linking, and other processing parameters that should warrant adequate functioning post bioprinting. This study demonstrates the development of a multicomponent shear-thinning bioink comprising Alginate and Gellan Gum with good mechanical, biological, and adequate printing properties. The addition of two types of silk nanoparticles (SNP), native and regenerated, acted as a reinforcement to the bioink, enhancing its printability. Additionally, silk fibroin solution was added to the bioink that served as a control apart from SNP. The addition of cationic SNP (native) to the anionic polymer mixture of Alginate-Gellan Gum exhibited a remarkable increase in the viscosity, storage modulus and mechanical property compared to the other experimental sets. Molecular characterization studies involving Real-Time PCR, gene expression, immunofluorescence, and histology analysis depicted enhanced chondrogenesis in the bioprinted construct containing the silk fibroin solution. The incorporated SNPs resulted in extracellular matrix secretion towards chondrogenesis of articular cartilage. Taken together, the method's broad applicability is likely to propel the field closer to the goal of precision bioprinting.
... The development of bioinks for such processes can often represent a bottleneck [12]. An ideal bioink should provide both biological and mechanical requirements of the printed tissues and maintain the appropriate structure of the printed construct [13]. Biological necessities include cytocompatibility, and modulation of signaling pathways, while mechanical requirements involve viscosity, degradability, and structural integrity post-printing [14]. ...
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.
... Silk proteins have also been shown to regulate Notch signalling by suppressing Hes-1 49 . Other studies with silk-gelatin bioinks have shown that there is a negative regulatory role on the IHH signalling pathway and Wnt/β-catenin signalling pathway to control hypertrophy in bone marrow stromal cells 50,51 . ...
Silk can be processed into a broad spectrum of material formats and is explored for a wide range of medical applications, including hydrogels for wound care. The current paradigm is that solution-stable silk fibroin in the hydrogels is responsible for their therapeutic response in wound healing. Here, we generated physically cross-linked silk fibroin hydrogels with tuned secondary structure and examined their ability to influence their biological response by leaching silk fibroin. Significantly more silk fibroin leached from hydrogels with an amorphous silk fibroin structure than with a beta sheet-rich silk fibroin structure, although all hydrogels leached silk fibroin. The leached silk was biologically active, as it induced vitro chemokinesis and faster scratch assay wound healing by activating receptor tyrosine kinases. Overall, these effects are desirable for wound management and show the promise of silk fibroin and hydrogel leaching in the wider healthcare setting.
... Like metallic devices, Bioprinted products currently lack regulatory guidelines as those processes are still in developmental stages. More fundamental data on Bioprinted products and a better understanding of the process-property relationship will help develop regulatory guidelines in the coming years [104][105][106]. For ceramics and glasses, reliability of 3D-printed products is needed, along with reproducible manufacturing practices. ...
3D printing, or additive manufacturing, is a transformative technology platform impacting various disciplines, including biomaterials and biomedical devices. We present scientists, engineers, and medical professionals' perspectives about 3D printing of biomaterials and biomedical devices in this special issue. This issue is geared towards understanding the structure–process–property relationships involving different materials in vitro, in vivo, and in silico environments. The focus issue covers polymer, ceramic, glass, metallic and composite biomaterials involving various 3D-printing processes such as fused deposition, powder bed fusion, Bioprinting, directed energy deposition, and binder jetting; addressing research topics including tissue engineering, drug delivery, porous metal implants, bioink development, cell–materials interactions, wear degradation, nano-bio materials, and challenges in point-of-care delivery. Such diversity of research topics is an accurate representation of what is happening in this field globally. This article presents some of the success stories, challenges, and future directions in the 3D printing of biomaterials and devices.Graphic abstract
... As a result, angiogenesis is promoted for an extended time period as compared to traditional scaffold-based approaches. Moreover, bioprinting offers the possibility of controlling the alignment of cells, as well as to strategically modulate cellular signaling pathways; thus recapitulating the specific microstructure of target tissue or organ compared to standard tissue fabrication approaches (Chakraborty and Ghosh 2020). ...
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.
... Thus, cell incorporation is an essential processing step that not only contemplates the choice of cell type(s) and maturity but also cell density. However, there are several works in which the term "bioink" is used to describe a protein-based hydrogel with good biological performance demonstrated through in vitro assays [38,145]. In fact, conventional approaches involve cell embedding or subsequent cell seeding on 3D material after printing has also been reported [40]. ...
In the last decade, three-dimensional (3D) extrusion bioprinting has been on the top trend for innovative technologies in the field of biomedical engineering. In particular, protein-based bioinks such as collagen, gelatin, silk fibroin, elastic, fibrin and protein complexes based on decellularized extracellular matrix (dECM) are receiving increasing attention. This current interest is the result of protein’s tunable properties, biocompatibility, environmentally friendly nature and possibility to provide cells with the adequate cues, mimicking the extracellular matrix’s function. In this review we describe the most relevant stages of the development of a protein-driven bioink. The most popular formulations, molecular weights and extraction methods are covered. The different crosslinking methods used in protein bioinks, the formulation with other polymeric systems or molecules of interest as well as the bioprinting settings are herein highlighted. The cell embedding procedures, the in vitro, in vivo, in situ studies and final applications are also discussed. Finally, we approach the development and optimization of bioinks from a sequential perspective, discussing the relevance of each parameter during the pre-processing, processing, and post-processing stages of technological development. Through this approach the present review expects to provide, in a sequential manner, helpful methodological guidelines for the development of novel bioinks.
... 18−22 For the first time, we reported that silk-gelatin (namely, SG) bioink can regulate several molecular signaling pathways like Wnt/βcatenin, Indian hedgehog, and bone morphogenetic protein signaling pathways. 23,24 These findings provide strong justification of using SF protein-based bioink for liver regeneration, as Wnt/β-catenin has been observed to play a significant role in liver tissue development and formation of a mature liver organ. 25,26 In this study, we hypothesized that (1) DCL ECM would serve as an excellent biomimetic bioink as it would provide liver-specific regenerative signals to the implanted cells, (2) combining silk with the DCL would help to improve the biophysical properties and printing fidelity of the DCL bioink, and (3) the 3D printed constructs developed using the SG− DCL ink would further help in simulating liver tissue-specific gene and protein expression during in vitro culture of hepatocytes. ...
... Silk fibroin need to combine with gelatin bioinks to produce putative cell attachments motifs. [49,65,69,78,79] Gelatin-Elastin Extrusion-based printing ...
Skin tissue engineering aimed to replace chronic tissue injury commonly occurred due to severe burn and chronic wound in diabetic ulcer patients. The normal skin is unable to be regenerated until the seriously injured tissue is disrupted and losing its function. 3D-bioprinting has been one of the effective methods for scaffold fabrication and is proven to replace the conventional method, which reported several drawbacks. In light of this, researchers have developed a new fabrication approach via 3D-bioprinting by combining biomaterials (bioinks) with cells and biomolecules followed by a suitable crosslinking approach. This advanced technology has been subcategorised into three different printing techniques including inject-based, laser-based, and extrusion-based printing. However, the printable quality of the currently available bioinks demonstrated shortcomings in the physicochemical and mechanical properties. This review aims to identify the limitations raised by using natural-based bioinks and the optimum temperature for various applied printing techniques. It is essential to ensure maintaining the acceptable printed scaffold property such as the optimum pore sizes and porosity that allow cell migration activity. In addition, the properties required for an ideal bioinks design for better scaffold printability were also summarised.
... The β-sheet nanocrystals of silk fibroin can be melted under heating without degradation, which is promising for thermal processing of solid silk fibroin materials, analogous to widely used thermoplastics. All of the above features make silk fibroin an intriguing ink component for three-dimensional (3D) printing/additive manufacturing [2,[22][23][24][25][26][27][28]. ...
Silk fibroin in material formats provides robust mechanical properties, and thus is a promising protein for 3D printing inks for a range of applications, including tissue engineering, bioelectronics, and bio-optics. Among the various crosslinking mechanisms, photo-crosslinking is particularly useful for 3D printing with silk fibroin inks due to the rapid kinetics, tunable crosslinking dynamics, light-assisted shape control, and the option to use visible light as a biocompatible processing condition. Multiple photo-crosslinking approaches have been applied to native or chemically modified silk fibroin, including photo-oxidation and free radical methacrylate polymerization. The molecular characteristics of silk fibroin, i.e., conformational polymorphism, provide a unique method for crosslinking and microfabrication via light. The molecular design features of silk fibroin inks and the exploitation of photo-crosslinking mechanisms suggest the exciting potential for meeting many biomedical needs in the future.
3D bioprinting has recently emerged as a successful biofabrication strategy for replicating the complex in vivo hepatic milieu. Significant research advances in this field have allowed for the fabrication of biomimetic hepatic tissues with potential applications in the healthcare (regeneration, transplantation, drug discovery) and diagnostic sectors (in vitro disease models). This article initially delves into describing the hepatic tissue architecture and function, followed by a rational exposition of how 3D bioprinting potentiates the better development of functional liver tissue compared to traditional tissue engineering approaches and 3D cell culture platforms. This review then highlights the recent breakthroughs and reliable strategies for replicating the liver structure and function through bioprinting approaches. In this context, we have systematically described the current landscape of hepatic bioprinting, initially focusing on the cell sources used, followed by the biomaterials and strategies implemented to prolong their in vitro viability. Proceeding forward, we have critically highlighted essential aspects of hepatic bioprinting, such as developing tissue-specific bioinks, strategies to induce vascularization within bioprinted liver constructs, and replication of native liver tissue heterogeneity through spatial distribution of multiple cell types in predetermined patterns. In our concluding remarks, we discuss the existing bottlenecks prevail in this field and provide our viewpoint regarding possible future directions to overcome them.
The self-healing of the articular cartilage lesions is difficult since it has a restricted self-healing capacity. However, surgical interventions have been implemented in osteoarthritis patients with limited success. Tissue-engineered cartilage can be used to address such issues. Over the years, researchers have employed three-dimensional bioprinting strategies for cartilage reconstruction. Although this technology potentially will enable researchers to fabricate the complex architecture of articular cartilage, there are quite a few hurdles yet to be addressed. A significant challenge is the selection of an ideal bioink formulation, which would encapsulate the cells without any cytotoxicity and demonstrate specific properties to be categorized as “bioprintable.” Most known biomaterials, either used alone or in combination, suffer from certain disadvantages; therefore the quest for an appropriate bioink for cartilage bioprinting is still on. In this chapter, we highlight the use of a combination of two protein-based biomaterials: (1) silk fibroin (SF), and (2) gelatin (G) for bioink development. Concerning the bioink formulation, we emphasize the various crosslinking strategies to prepare the SF–G bioink and the essential characteristics of the resultant ink before bioprinting. Lastly, we provide insight into the essential postbioprinting evaluation of the constructs and summarize the current challenges and future perspectives that could serve as a foundation for further research in this field.
Polylactic acid (PLA), a biodegradable polymer, has become a major raw material in the field of 3D printing. However, some defects such as high brittleness and average bioactivity limit its application. In this study, a novel 3D printing composite of animal bone powder/polyethylene glycol/polylactic acid was prepared and investigated. The structures and properties of bHA/PEG/PLA composites were compared with commercial micron‐sized hydroxyapatite (mHA) and nano hydroxyapatite (nHA) composites. The study results show that the bHA has the same chemical structure as mHA and nHA, but its average particle size is 390 nm, which is quite different from mHA and nHA. When the PEG content is 14%, the toughness of the PEG/PLA blends is good, and its elongation at break reaches 96.23%. The bending strength, tensile strength, and elongation at break of bHA/PEG/PLA composites reach 25.1 MPa, 16.3 MPa, and 11.32%, respectively, which are all higher than those of mHA/PEG/PLA and similar to those of nHA/PEG/PLA. The bHA/PEG/PLA biological scaffolds prepared by 3D printing show good biocompatibility in cytotoxicity experiments and have good application prospects in bone tissue engineering.
The significance of bioink suitability for the extrusion bioprinting of tissue-like constructs cannot be overemphasized. Gelatin, derived from the hydrolysis of collagen, not only can mimic the extracellular matrix to immensely support cell function, but also is suitable for extrusion under certain conditions. Thus, gelatin has been recognized as a promising bioink for extrusion bioprinting. However, the development of a gelatin-based bioink with satisfactory printability and bioactivity to fabricate complex tissue-like constructs with the desired physicochemical properties and biofunctions for a specific biomedical application is still in its infancy. Therefore, in this review, we aim to comprehensively summarize the state-of-the-art methods of gelatin-based bioink application for extrusion bioprinting. We firstly outline the properties and requirements of gelatin-based bioinks for extrusion bioprinting, highlighting the strategies to overcome their main limitations in terms of printability, structural stability and cell viability. Then, the challenges and prospects are further discussed regarding the development of ideal gelatin-based bioinks for extrusion bioprinting to create complex tissue-like constructs with preferable physicochemical properties and biofunctions.Graphic abstract
The three-dimensional (3D) printing and its extension four-dimensional (4D) printing technique show great promise for advanced additive manufacturing of functional and architected materials with engineered micro- and nanoscale features. The actual usage of additive manufacturing to produce 3D/4D structures is primarily dependent on the improvement of printable polymeric inks. Thermoresponsive polymers (TRPs) show broad application prospects in additive manufacturing since they enable changing the physicochemical properties of the system at a specific temperature. Continuing to progress in the understanding of TRP phenomena enable the design of new feedstocks and their compatibility with fabrication processes. Therefore, increasing the overlap between the fields of fundamental TRPs and application-based research efforts can be important to emerging the technology. In this review, we provide an overview of the functional design of the TRPs, the different classes of TRPs, and their application in 3D printing, ranging from biosensors via advanced self-assembly to a variety of biofabrication, biomedical, biotechnology, and food applications. Basic knowledge of imperative processes like gelation mechanisms of TRPs, bio-based adsorption on the surfaces and adhesion, and fabrication strategies using 3D printing is also provided. For diverse TRPs synthesis and the basic physical and chemical features are described and the mechanism of their TRPs behavior is highlighted. Fabrication methods of TRP surfaces have also been discussed describing several methods in detail.
The rheological characterization of any biopolymer solution is crucial for evaluating the overall printability or injectability of the hydrogel. However, the effect of cells in the cell-laden hydrogel's rheological profile is often ignored. As a result, there is a significant difference in the predicted and experimental outcome in the structural stability of the construct as well as on the cell viability, proliferation, and differentiation potential of the embedded cells. Our present study has addressed the effect of different cell densities (0.1 million cells/ml, 0.5 million cells/ml, 1 million cells/ml and 2 million cells/ml) of TVA-BMSCs on the flow property, modulus behaviour, gelation kinetics and printability of our proprietary silk fibroin-gelatin (5SF-6G) bioink. The cell-laden hydrogels demonstrated a characteristic shear thinning behaviour (low initial viscosity), low storage modulus and increased gelation time when compared to the acellular 5SF-6G hydrogel. The printability analysis also portrayed a square pore geometry with low spreading ratio in 1 million cells/ml encapsulated 5SF-6G hydrogel comparable to the acellular hydrogel. We postulated that incorporation of cells in the bioink interfered with the gelation mechanism of the mushroom tyrosinase in the 5SF-6G bioink by masking the active sites. Additionally, the mechanistic crosstalk between the cell-surface integrins with the cell-attachment motifs of the biomaterial alters the cellular biomechanics of the cell that in-turn profoundly impacts the rheological properties of the polymer blend. Therefore, cell density of 1 million cells/ml was considered the best fit for extrusion-based 3D bioprinting owing to its optimum rheological traits and printability index akin the acellular hydrogel.
Bioprinting is an emerging tissue engineering technique that has attracted the attention of researchers around the world, for its ability to create tissue constructs that recapitulate physiological function. While the technique has been receiving hype, there are still limitations to the use of bioprinting in practical applications, much of which is due to inappropriate bioink design that is unable to recapitulate complex tissue architecture. Silk fibroin (SF) is an exciting and promising bioink candidate that has been increasingly popular in bioprinting applications because of its processability, biodegradability, and biocompatibility properties. However, due to its lack of optimum gelation properties, functionalization strategies need to be employed so that SF can be effectively used in bioprinting applications. These functionalization strategies are processing methods which allow SF to be compatible with specific bioprinting techniques. Previous literature reviews of SF as a bioink mainly focus on discussing different methods to functionalize SF as a bioink, while a comprehensive review on categorizing SF functional methods according to their potential applications is missing. This paper seeks to discuss and compartmentalize the different strategies used to functionalize SF for bioprinting and categorize the strategies for each bioprinting method (namely, inkjet, extrusion, and light-based bioprinting). By compartmentalizing the various strategies for each printing method, the paper illustrates how each strategy is better suited for a target tissue application. The paper will also discuss applications of SF bioinks in regenerating various tissue types and the challenges and future trends that SF can take in its role as a bioink material.
The present study scrutinized some of the crucial advancements in the synthesis and functionalisation of self-assembling biomaterials for application in biomedicine. The basic concept of self-organization was discussed along with the mechanisms and methods involved in its implementation with biomaterials. Further, several recent applications of this technology in the biological and medical domain, and the avenues for future research and development were presented. This study brought to focus the vast potential of basic and applied research involved, especially in the context of hybrids and composites, as well as the difference in pace of new developments for different types of biomolecular materials. As nanobiotechnology matures, the tools and techniques available for developing and controlling self-assembled biomaterials as well as studying their interaction with biological tissue, will grow exponentially. Presently, self-assembly remains a potent tool for the synthesis of functional biomaterials.
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.
The native cartilage extracellular matrix (ECM) is enriched in sulfated glycosaminoglycans with important roles in the signaling and phenotype of resident chondrocytes. Recapitulating the key ECM components within engineered tissues through biomimicking strategies has potential to improve the regenerative capacity of encapsulated cells and lead to better clinical outcome. Here, we developed a double-modified, biomimetic and tissue adhesive hydrogel for cartilage engineering. We demonstrated sequential modification of alginate with first sulfate moieties to mimic the high glycosaminoglycan content of native cartilage and then tyramine moieties to allow in situ enzymatic crosslinking with tyrosinase under physiological conditions. Tyrosinase-crosslinked alginate sulfate tyramine (ASTA) hydrogels showed strong adhesion to native cartilage tissue with higher bond strength compared to alginate tyramine (AlgTA). Both ASTA and AlgTA hydrogels supported the viability of encapsulated bovine chondrocytes and induced a strong increase in the expression of chondrogenic genes such as collagen 2, aggrecan and Sox9. Aggrecan and Sox9 gene expression of chondrocytes in ASTA hydrogels were significantly higher than those in AlgTA. Chondrocytes in both ASTA and AlgTA hydrogels showed potent deposition of cartilage matrix components collagen 2 and aggrecan after 3 weeks of culture whereas a decreased collagen 1 deposition was observed in the sulfated hydrogels. ASTA and AlgTA hydrogels with encapsulated human chondrocytes showed in vivo stability as well as cartilage matrix deposition upon subcutaneous implantation into mice for 4 weeks. Our data is the first demonstration of a double-modified alginate with sulfation and tyramination that allows in situ enzymatic crosslinking, strong adhesion to native cartilage and chondrogenic re-differentiation.
The Turing reaction-diffusion model and the French Flag Model are widely accepted in the field of development as the best models for explaining embryogenesis. Virtually all current attempts to understand cell differentiation in embryos begin and end with the assumption that some combination of these two models works. The result may become a bias in embryogenesis in assuming the problem has been solved by these two-chemical substance-based models. Neither model is applied consistently. We review the differences between the French Flag, Turing reaction-diffusion model, and a mechanochemical model called the differentiation wave/cell state splitter model. The cytoskeletal cell state splitter and the embryonic differentiation waves was first proposed in 1987 as a combined physics and chemistry model for cell differentiation in embryos, based on empirical observations on urodele amphibian embryos. We hope that the development of theory can be advanced and observations relevant to distinguishing the embryonic differentiation wave model from the French Flag model and reaction-diffusion equations will be taken up by experimentalists. Experimentalists rely on mathematical biologists for theory, and therefore depend on them for what parameters they choose to measure and ignore. Therefore, mathematical biologists need to fully understand the distinctions between these three models.
Silk spinning offers an evolution‐based manufacturing strategy for industrial polymer manufacturing, yet remains largely inaccessible as the manufacturing mechanisms in biological and synthetic systems, especially at the molecular level, are fundamentally different. The appealing characteristics of silk spinning include the sustainable sourcing of the protein material, the all‐aqueous processing into fibers, and the unique material properties of silks in various formats. Substantial progress has been made to mimic silk spinning in artificial manufacturing processes, despite the gap between natural and artificial systems. This report emphasizes the universal spinning conditions utilized by both spiders and silkworms to generate silk fibers in nature, as a scientific and technical framework for directing molecular assembly into high‐performance structures. The preparation of regenerated silk feedstocks and mimicking native spinning conditions in artificial manufacturing are discussed, as is progress and challenges in fiber spinning and 3D printing of silk‐composites. Silk spinning is a biomimetic model for advanced and sustainable artificial polymer manufacturing, offering benefits in biomedical applications for tissue scaffolds and implantable devices.
The field of bioprinting has made significant recent progress towards engineering tissues with increasing complexity and functionality. It remains challenging, however, to develop bioinks with optimal biocompatibility and good printing fidelity. Here, we demonstrate enhanced printability of a polymer-based bioink based on dynamic covalent linkages between nanoparticles and polymers, which retains good biocompatibility. Amine-presenting silica nanoparticles (NPs)(ca. 45 nm) were added to a polymeric ink containing oxidized alginate (OxA). The formation of reversible imine bonds between amines on the NPs and aldehydes of OxA lead to significantly improved rheological properties and high printing fidelity. In particular, the yield stress increased with increasing amounts of nanoparticles (14.5 Pa without nanoparticles, 79 Pa with 2 wt% nanoparticles). In addition, the presence of dynamic covalent linkages in the gel provided improved mechanical stability over 7 days compared to ionically crosslinked gels. The nanocomposite ink retained high printability and mechanical strength, resulting in generation of centimetre-scale porous constructs and an ear structure with overhangs and high structural fidelity. Furthermore, the nanocomposite ink supported both in vitro and in vivo maturation of bioprinted gels containing chondrocytes. This approach based on simple oxidation can be applied to any polysaccharide, thus the widely applicability of the method is expected to advance the field towards the goal of precision bioprinting.
Additive manufacturing in tissue engineering has significantly advanced in acceptance and use to address complex problems. However, there are still limitations to the technologies used and potential challenges that need to be addressed by the community. In this manuscript, we describe how the field can be advanced not only through the development of new materials and techniques but also through the standardization of characterization, which in turn may impact the translation potential of the field as it matures. Furthermore, we discuss how education and outreach could be modified to ensure end-users have a better grasp on the benefits and limitations of 3D printing to aid in their career development.
The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour ‘cell collision guidance’. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell–cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.
Three-dimensional (3D) bioprinting is an emerging manufacturing technology that layers living cells and biocompatible natural or synthetic materials to build complex, functional living tissue with the requisite 3D geometries. This technology holds tremendous promise across a plethora of applications as diverse as regenerative medicine, pathophysiological studies, and drug testing. Despite some success demonstrated in early attempts to recreate complex tissue structures, however, the field of bioprinting is very much in its infancy. There are a variety of challenges to building viable, functional, and lasting 3D structures, not the least of which is translation from a research to a clinical setting. In this review, the current translational status of 3D bioprinting is assessed for several major tissue types in the body (skin, bone/cartilage, cardiovascular, central/peripheral nervous systems, skeletal muscle, kidney, and liver), recent breakthroughs and current challenges are highlighted, and future prospects for this exciting research field are discussed. We begin with an overview of the technology itself, followed by a detailed discussion of the current approaches relevant for bioprinting different tissues for regenerative medicine.
Silk fibres are assembled via flow. While changes in the physiological environment of the gland as well as the shear rheology of silk are largely understood, the effect of extensional flow fields on native silk proteins is almost completely unknown. Here we demonstrate that filament stretching on a conventional tensile tester is a suitable technique to assess silk's extensional flow properties and its ability to form fibres under extensional conditions characteristic of natural spinning. We report that native Bombyx mori silk responds differently to extensional flow fields when compared to synthetic linear polymers, as evidenced by a higher Trouton ratio which we attribute to silk's increased interchain interactions. Finally, we show that native silk proteins can only be spun into stable fibres at low extension rates as a result of dehydration, suggesting that extensional fields alone are unable to induce natural fibre formation.
3D bioprinting field is making remarkable progress; however, the development of critical sized engineered tissue construct is still a farfetched goal. Silk fibroin offers a promising choice for bioink material. Nature has imparted several unique structural features in silk protein to ensure spinnability by silkworms or spider. Researchers have modified the structure–property relationship by reverse engineering to further improve shear thinning behavior, high printability, cytocompatible gelation, and high structural fidelity. In this review, it is attempted to summarize the recent advancements made in the field of 3D bioprinting in context of two major sources of silk fibroin: silkworm silk and spider silk (native and recombinant). The challenges faced by current approaches in processing silk bioinks, cellular signaling pathways modulated by silk chemistry and secondary conformations, gaps in knowledge, and future directions acquired for pushing the field further toward clinic are further elaborated.
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 the 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 fibres, by conducting detailed gene expression profiling using 26 different osteogenic markers, followed by further validation by immunohistochemistry. Our study provides novel insights into direct regulatory role of nonmulberry silk fibroin braids on Indian hedgehog and Parathyroid signaling pathways in controlling osteogenic differentiation of cultured hFOBs, a phenomenon not very evident in the mulberry silk textile braids. While both silk braids enabled adequate cellular attachment, proliferation and extracellular collagen matrix, 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 tissue to be a prospective tissue equivalent with potential of providing the essential instructive elements for activating pathways of organogenesis. This engineered constructs has potential to use as an in vitro model for drug testing and designing for bone regeneration strategies.
Cell migration is dependent on the dynamic formation and disassembly of actin filament–based structures, including lamellipodia, filopodia, invadopodia, and membrane blebs, as well as on cell–cell and cell–extracellular matrix adhesions. These processes all involve Rho family small guanosine triphosphatases (GTPases), which are regulated by the opposing actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho GTPase activity needs to be precisely tuned at distinct cellular locations to enable cells to move in response to different environments and stimuli. In this review, we focus on the ability of RhoGEFs and RhoGAPs to form complexes with diverse binding partners, and describe how this influences their ability to control localized GTPase activity in the context of migration and invasion.
Alginate, a natural acidic polysaccharide extracted from marine brown seaweeds, is composed of different blocks of β-(1, 4)-D-mannuronate (M) and its C-5 epimer α-(1, 4)-L-guluronate (G). Alginate-derived guluronate oligosaccharide (GOS) readily activates macrophages. However, to understand its role in immune responses, further studies are needed to characterize GOS transport and signalling. Our results show that GOS is recognized by and upregulates Toll-like receptor 4 (TLR4) on RAW264.7 macrophages, followed by its endocytosis via TLR4. Increased expression of TLR4 and myeloid differentiation protein 2 (MD2) results in Akt phosphorylation and subsequent activation of both nuclear factor-κB (NF-κB) and mechanistic target of rapamycin (mTOR). Moreover, GOS stimulates mitogen-activated protein kinases (MAPKs); notably, c-Jun N-terminal kinase (JNK) phosphorylation depends on TLR4 initiation. All these events contribute to the production of inflammatory mediators, either together or separately. Our findings also reveal that GOS induces cytoskeleton remodelling in RAW264.7 cells and promotes macrophage proliferation in mice ascites, both of which improve innate immunity. Conclusively, our investigation demonstrates that GOS, which is dependent on TLR4, is taken up by macrophages and stimulates TLR4/Akt/NF-κB, TLR4/Akt/mTOR and MAPK signalling pathways and exerts impressive immuno-stimulatory activity.
Three-dimensional (3D) bioprinting is on the cusp of permitting the direct fabrication of artificial living tissue. Multicellular building blocks (bioinks) are dispensed layer by layer and scaled for the target construct. However, only a few materials are able to fulfill the considerable requirements for suitable bioink formulation, a critical component of efficient 3D bioprinting. Alginate, a naturally occurring polysaccharide, is clearly the most commonly employed material in current bioinks. Here, we discuss the benefits and disadvantages of the use of alginate in 3D bioprinting by summarizing the most recent studies that used alginate for printing vascular tissue, bone and cartilage. In addition, other breakthroughs in the use of alginate in bioprinting are discussed, including strategies to improve its structural and degradation characteristics. In this review, we organize the available literature in order to inspire and accelerate novel alginate-based bioink formulations with enhanced properties for future applications in basic research, drug screening and regenerative medicine.
The mechanism by which native silk feedstocks are converted to solid fibres in nature has attracted much interest. To address this question, the present work used rheology to investigate the gelation of Bombyx mori native silk feedstock. Exceeding a critical shear stress appeared to be more important than shear rate, during flow-induced initiation. Compositional changes (salts, pH etc.,) were not required, although their possible role in vivo is not excluded. Moreover, after successful initiation, gel strength continued to increase over a considerable time under effectively quiescent conditions, without requiring further application of the initial stimulus. Gelation by elevated temperature or freezing was also observed. Prior to gelation, literature suggests that silk protein adopts a random coil configuration, which argued against the conventional explanation of gelation, based on hydrophilic and hydrophobic interactions. Instead, a new hypothesis is presented, based on entropically-driven loss of hydration, which appears to explain the apparently diverse methods by which silk feedstocks can be gelled.
Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.
3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.
Silk fibroin (SF) is a typical fibrous protein that is secreted by silkworms and spiders. It has been used in a variety of areas, and especially for tissue-engineering scaffolds, due to its sound processability, mechanical properties, biodegradability, and biocompatibility. With respect to gelation, the SF gelation time is long in aqueous solutions, so a novel approach is needed to shorten this time. The solubility of regenerated SF is sound in formic acid (FA), which is a carboxylic acid of the simplest structure. In this study, SF was dissolved in formic acid, and the addition of salts then induced a rapid gelation that accompanied a solution-color change. Based on the gelation behaviors of the SF solution according to different SF and salt concentrations, the gelation mechanism was investigated.
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has an extremely poor prognosis. Surgical resection is always inapplicable to HCC patients diagnosed at an advanced tumor stage. The mechanisms underlying HCC cell proliferation remain obscure. In the present study, SWItch/sucrose nonfermentable catalytic subunit SNF2 (SNF2H) expression was tested in HCC tissues and Wnt/β-catenin pathway activation upon overexpression of SNF2H or knockdown of SNF2H expression was investigated in cultured HCC cells. It was demonstrated that SNF2H is a vital factor for HCC growth. The SNF2H expression level is increased in HCC tissues compared with paratumoral liver tissues. SNF2H promotes HCC cell proliferation and colony formation ability in vitro. SNF2H may increase the protein level of β-catenin and enhance its nuclear accumulation in HCC cells, thereby leading to the activation of the Wnt/β-catenin signaling pathway. In conclusion, the present results indicate that SNF2H plays a vital role in HCC cell growth, suggesting that SNF2H may be a promising therapeutic target for HCC treatment.
Whilst much is known about the properties of silks, the means by which native silk feedstocks are spun still represent a gap in our knowledge. Rheology of the native silk feedstocks is germane to an understanding of the natural spinning process. Yet, an overview of the literature reveals subtle limitations and inconsistencies between studies, which has been largely attributed to sample-to-sample variation when testing these exquisitely flow-sensitive materials. This ambiguity has prevented reliable, consistent inferences from standard polymer rheology and constitutes an obstacle to further development.
To address this challenge, we present the largest study to date into the rheological properties of native silk feedstocks from Bombyx mori larvae. A combination of shear and oscillatory measurements were used to examine in detail the relationships between concentration, low shear viscosity, relaxation times, complex modulus and estimates of the molecular weights between entanglements. The results from this highly detailed survey will provide a sound basis for further experimental or theoretical work and lay the foundations for future bio-inspired processing of proteins.
A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.
Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.
Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.
Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.
Statement of significance:
Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.
Despite significant investment in tissue engineering over the past 20 years, few tissue engineered products have made it to market. One of the reasons is the poor control over the 3D arrangement of the scaffold’s components. Biofabrication is a new field of research that exploits 3D printing technologies with high spatial resolution for the simultaneous processing of cells and biomaterials into 3D constructs suitable for tissue engineering. Cell-encapsulating biomaterials used in 3D bioprinting are referred to as bioinks. This review consists of: (1) an introduction of biofabrication, (2) an introduction of 3D bioprinting, (3) the requirements of bioinks, (4) existing bioinks, and (5) a specific example of a recombinant spider silk bioink. The recombinant spider silk bioink will be used as an example because its unmodified hydrogel format fits the basic requirements of bioinks: to be printable and at the same time cytocompatible. The bioink exhibited both cytocompatible (self-assembly, high cell viability) and printable (injectable, shear-thinning, high shape fidelity) qualities. Although improvements can be made, it is clear from this system that, with the appropriate bioink, many of the existing faults in tissue-like structures produced by 3D bioprinting can be minimized.
Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Macromolecular crowding is known to have a significant impact on the aggregation and folding of proteins. In this study, the role of different macromolecular crowders such as polyethylene glycol (PEG 200 and PEG 6000) and dextran (dextran 6000 and 70000) on the self‐assembly of silk fibroin protein is explored. The data show that crowders induce a clear transition from random coil to β‐sheet secondary conformation in the fibroin as demonstrated by ThT fluorescence spectroscopy, circular dichroism, and ATR‐FTIR findings. However, this transition is not observed in case of control sample (fibroin without crowders). Excluded volume effects accelerate the kinetics of self‐association of the fibroin protein. Increasing the molecular weight of crowders increases the required time for maximum aggregation. This study suggests that treating the fibroin with different macromolecular crowders has modulated the propensity of fibroin going into β‐sheet conformation. Macromolecular crowders are known to have a significant influence on various biochemical process, viz. protein folding, protein aggregation, and conformational transitions by mimicking the in vivo molecular confinement conditions in vitro. Based on this, different macromolecular crowders are used in the current study for modulating the self‐assembly process of silk fibroin in order to fabricate the biomaterials and scaffolds.
This review addresses critical gaps and often-ignored aspects of immune response to a decellularized ECM.
Several synthetic and natural materials are used in soft tissue engineering and regenerative medicine with varying degrees of success. Among them, silkworm silk protein fibroin, a naturally occurring protein-based biomaterial, exhibits many promising characteristics such as biocompatibility, controllable biodegradability, tunable mechanical properties, aqueous preparation, minimal inflammation in host tissue, low cost and ease of use. Silk fibroin is often used alone or in combination with other materials in various formats and is also a promising delivery system for bioactive compounds as part of such repair scenarios. These properties make silk fibroin an excellent biomaterial for skin tissue engineering and repair applications. This review focuses on the promising characteristics and recent advances in the use of silk fibroin for skin wound healing and/or soft-tissue repair applications. The benefits and limitations of silk fibroin as a scaffolding biomaterial in this context are also discussed. STATEMENT OF SIGNIFICANCE: Silk protein fibroin is a natural biomaterial with important biological and mechanical properties for soft tissue engineering applications. Silk fibroin is obtained from silkworms and can be purified using alkali or enzyme based degumming (removal of glue protein sericin) procedures. Fibroin is used alone or in combination with other materials in different scaffold forms, such as nanofibrous mats, hydrogels, sponges or films tailored for specific applications. The investigations carried out using silk fibroin or its blends in skin tissue engineering have increased dramatically in recent years due to the advantages of this unique biomaterial. This review focuses on the promising characteristics of silk fibroin for skin wound healing and/or soft-tissue repair applications.
Currently available tissue engineered skin-equivalents fail to replicate anatomically relevant features, e.g. undulated morphology of the dermal-epidermal junction, resulting in inadequate recapitulation of biologically meaningful cellular signaling pathways. This study reports fabrication of 3D bioprinted, human cell-based full thickness skin model possessing anatomically relevant structural, mechanical and biochemical features akin to human skin. The unique undulated feature of the epidermis-dermis junction could be recapitulated in the 3D bioprinted skin-equivalents. Extensive migration of the keratinocytes within the construct along with differentiation events reminded reepithelialisation. Cell-cell interaction along with diffusible factors promoted expression of differentiation and cornification markers in region-specific manner. Architectural features and silk bioink microenvironment triggered deposition of basement membrane specific-proteins at the interface. Most interestingly, extensive transcriptomics and proteomics analysis accentuated striking similarity of the 3D bioprinted full thickness skin model to the native human skin, with involvement of a number of pathways related to skin development and physiology, such as skin development, extracellular matrix organization, keratinization/cornification and collagen fibril organization. Such bioprinted in vitro human skin models would offer tremendous potential for screening cosmetic products and drugs, as well as to understand the complex physiological processes relevant to human skin thereby bridging the gap between conventional monolayer or 3D cultures and animal models.
Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shapespecific, 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 which 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 mesenchymal stem 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 signalling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signalling pathway, The forkhead box O (FOXO) pathway and Hippo pathways in bone marrow-derived mesenchymal stem cell-laden bioprinted constructs.
Silk is a natural polymer sourced mainly from spiders and silkworms. Due to its biocompatibility, biodegradability, and mechanical properties, it has been heavily investigated for biomedical applications. It can be processed into a number of formats, such as scaffolds, films, and nanoparticles. Common methods of production create constructs with limited complexity. 3D printing allows silk to be printed into more intricate designs, increasing its potential applications. Extrusion and inkjet printing are the primary ways silk has been 3D printed, though other methods are beginning to be investigated. Silk has been integrated into bioink with other polymers, both natural and synthetic. The addition of silk is primarily done to offer more desirable viscosity characteristics and mechanical properties for printing. Silk-based bioinks have been used to fabricate medical devices and tissues. This article discusses recent research and printing parameters important for 3D printing with silk.
A major challenge in bone tissue engineering is to develop clinically conformant load bearing bone constructs in patient-specific manner. A paradigm shift would be combination of developmental engineering and 3D bioprinting to optimize strategies focusing on close simulation of in vivo developmental processes using in vitro tissue engineering approaches. This study demonstrates that silk-gelatin bioink could activate the canonical Wnt/β-catenin and Indian Hedgehog (IHH) pathways during osteogenic differentiation of mesenchymal stem cells (TVA-BMSC), laden in 3D bioprinted constructs. Temporal gene expression related to early and terminal osteogenic differentiation of the TVA-BMSC in 3D bioprinted constructs closely followed the in vivo processes. As evidenced by early differentiation markers (RUNX2 and COL I), mid and mid-to-late stage markers (ALP, ON, OPN and OCN) as well as terminal osteocytic genes (PDPN, DMP1 and SOST). Furthermore, combinatorial effect of addition of T3 and simulation of endochondral ossification route could activate the Parathyroid hormone (PTH), IHH and Wnt/β-catenin pathways thus improving the osteogenic differentiation potential of stem cells and improved mineralization. The endochondral ossification observed in vitro in our study shows stark similarities to in vivo endochondral ossification based limb skeletal development, specifically 1) chondrogenic condensation and hypertrophic cartilaginous template development, 2) involvement of IHH signaling indicative of the development of bony collar by perichondral ossification, 3) involvement of Wnt/β-catenin signaling, 4) involvement of PTH signaling, 5) synthesis and deposition of bone specific mineral. Thus, induction of differentiation of progenitor cells to osteoblasts in 3D bioprinted constructs, while recapitulating the developmental biology-inspired endochondral ossification route, may offer an important therapeutic proposition to develop clinically conformant bone construct.
Silk fibroin (SF) hydrogel is a promising candidate in biomaterial field; however its application is quite limited by long-gelation time. In the present study, we developed a novel strategy named soft freezing to accelerate the process and control the sol-gel transition of SF protein. SF protein was induced to self-assembly by soft freezing process for achieving the reconstructed SF solution with metastable structure. It was found that the soft freezing process triggers the structural transition of mainly SF protein from enriching with random structure initially to enriching ordered structure. Gelation kinetics showed that the reconstructed SF solution allowed enhanced sol-gel transition within several hours, even at extremely low concentration, and gelation time could be regulated in several hours to several days, depending on freezing time and initial concentration. The attractive features of the method described here include the accelerated gelation, free of chemical agents, and reducing processing complexity. The SF solution with short gelation time will be applicable as cell encapsulation and drug delivery for tissue engineering, which greatly expand the applications of SF hydrogels.
Bioprinting is an emerging technology with various applications in making functional tissue constructs to replace injured or diseased tissues. It is a relatively new approach that provides high reproducibility and precise control over the fabricated constructs in an automated manner, potentially enabling high-throughput production. During the bioprinting process, a solution of a biomaterial or a mixture of several biomaterials in the hydrogel form, usually encapsulating the desired cell types, termed the bioink, is used for creating tissue constructs. This bioink can be cross-linked or stabilized during or immediately after bioprinting to generate the final shape, structure, and architecture of the designed construct. Bioinks may be made from natural or synthetic biomaterials alone, or a combination of the two as hybrid materials. In certain cases, cell aggregates without any additional biomaterials can also be adopted for use as a bioink for bioprinting processes. An ideal bioink should possess proper mechanical, rheological, and biological properties of the target tissues, which are essential to ensure correct functionality of the bioprinted tissues and organs. In this review, we provide an in-depth discussion of the different bioinks currently employed for bioprinting, and outline some future perspectives in their further development.
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-à-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.
Silk-based natural polymers can regulate osteogenesis by mimicking the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotype is not yet demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (non-regenerated) spun non-mulberry silk A.mylitta, possessing mechanical stiff-ness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human pre-osteoblasts onto these braids and cultured them under varied temperatures (33.5oC and 39oC), soluble factors (Dexamethasone, Ascorbic acid and β-glycerophosphate) and cytokines (TGF-β1). In particular, cell dendrites were conspic-uously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1 and sclerostin). A.mylitta silk braids alone were sufficient to induce this differentiation, albeit on-ly transiently. Therefore we believe that the combinatorial effect of A.mylitta silk (surface chemistry, braid rigidity, topography), osteogenic differentiation factors and TGF-β1 were critical in stabilizing mature osteocytic phenotype. Interestingly, Wnt signaling promoted os-teocytic differentiation as evidenced by upregulated expression of β-catenin in the presence of growth factor cocktail. This study highlights the role of non-mulberry silk braids in regu-lating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D in vitro bone model systems.
This paper discusses "bioink", bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader.
In the field of soft tissue reconstruction, custom implants could address the need for materials that can fill complex geometries. Our aim was to develop a material system with optimal rheology for material extrusion, that can be processed in physiological and non-toxic conditions and provide structural support for soft tissue reconstruction. To meet this need we developed silk based bioinks using gelatin as a bulking agent and glycerol as a non-toxic additive to induce physical crosslinking. We developed these inks optimizing printing efficacy and resolution for patient-specific geometries that can be used for soft tissue reconstruction. We demonstrated in vitro that the material was stable under physiological conditions and could be tuned to match soft tissue mechanical properties. We demonstrated in vivo that the material was biocompatible and could be tuned to maintain shape and volume up to three months while promoting cellular infiltration and tissue integration.
The development of functional biomaterials for tissue engineering and medical applications has received increasing attention. While it has been known for decades that di-tyrosine bonds are a key component to many biopolymer materials in native tissues, only recently have these motifs been exploited in the development of new biomaterials. Here we first review the importance of tyrosine-tyrosine chemical bonds in the assembly and mechanical properties of natural materials. Next we discuss the chemistries available for crosslinking via tyrosine bonds and how these interactions have been applied to biomaterials. The goal of this review is to highlight di-tyrosine bonding in biomaterial development, the reactions used to form them and their utility in crosslinking native and chemically substituted phenolic side chains, as an underutilized tool in the de novo development of biomaterials.
Till date development of phenotypically stable, functionally equivalent engineered cartilage tissue constructs remains elusive. This study explored chondrogenic differentiation and hypertrophic suppression in tyrosinase crosslinked 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 constant increase in hypertrophy over 21 days (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 (ACAN, COMP1) over chondrocyte-laden constructs. The gelatin component in the bioink induced MMP2 activity which degraded the synthesized matrix creating a pericellular zone for accumulation of growth factors and newly synthesized matrices. We believe that the combinatorial effect of these accumulated factors as well as hypoxia-regulated HDAC4 pathway played a pivotal role in stabilizing chondrogenic phenotype of differentiated hMSCs along with suppressed hypertrophy. Therefore the results suggest that tyrosinase crosslinked silk-gelatin bioink offers a suitable material composition for cartilage tissue engineering. Further standardization is warranted to investigate the biological mechanisms minimizing hypertrophic differentiation of hMSC/chondrocytes towards development of improved cartilage constructs.
Silk fibroin (SF) scaffolds have been widely used in tissue engineering. However, a critical challenge for 3D SF scaffolds remains to provide a more appropriate microenvironment with a nanofibrous network to enhance cell viability and guide cell migration, thus further promoting tissue regeneration. In this study, a novel SF scaffold containing micro/nano fibers was prepared by a facile two-step freeze-drying technology. Carbodiimide-activated SF solution was diluted to 0.2 wt%, and then poured into pre-fabricated porous SF scaffolds. Consequently, well-dispersed fibrous networks with the fiber size of 511±217 nm were produced within the pores of SF scaffolds after liquid nitrogen immersion, followed by lyophilization. The results of in vitro culture of dermal fibroblast cells and umbilical vein endothelial cells on fibrous SF scaffolds demonstrated that the introduction of the micro/nano fibers significantly enhanced cell attachment, proliferation and migration by providing 3D topographic cues. In vivo, the SF scaffolds were implanted into dorsal full-thickness wounds of Sprague-Dawley rats as dermal equivalents to evaluate the effect of the fibrous microstructure on dermal tissue reconstruction. The results demonstrated that the fibrous SF scaffolds promoted the tissue neogenesis and collagen matrix formation by providing a fibrous ECM-like topography. This new fibrous SF scaffold offers potential for dermal tissue regeneration.
3D printing is an additive manufacturing (AM) technique that has quickly disrupted traditional design and manufacturing strategies. New structures can be manufactured that could not be fabricated using other methods. These new capabilities are considered by many to hallmark a historic shift representative of a new industrial revolution. Exciting utilities of this evolving technology are the fields of biomedical engineering and translational medicine, particularly in applying three-dimensional (3D) printing toward enabling on-demand fabrication of customized tissue scaffolds and medical device geometries. AM techniques are promising a future where on-demand production of patient-specific living tissues is a reality. In this review, we cover the rapid evolution and widespread concepts of a bio-"ink" and bioprinted devices and tissues from the past two decades as well as review the various additive manufacturing methods that have been used toward 3D bioprinting of cells and scaffolds with a special look at the benefits and practical considerations for each method. Despite being a young technology, the evolution and impact of AM in the fields of tissue engineering and regenerative medicine has progressed rapidly. We finish the review by looking toward the future of bioprinting and identify some of the current bottlenecks facing the blossoming industry.
Controlling the mechanism of self assembly in proteins has emerged as a potent tool for various biomedical applications. Silk fibroin self assembly consists of gradual conformational transition from random coil to β-sheet structure. In this work we elucidated the intermediate secondary conformation in the presence of Ca2+ ions during fibroin self assembly. The interaction of fibroin and calcium ions resulted in a predominantly α-helical intermediate conformation, which was maintained to certain extent even in the final conformation illustrated by circular dichroism and attenuated total reflectance-fourier transform infrared spectroscopy. Further, to elucidate the mechanism behind this interaction molecular modelling of the N-terminal region of fibroin with Ca2+ ions was performed. Negatively charged glutamate and aspartate amino acids play a key role in the electrostatic interaction with positively charged calcium ions. Therefore, insights about modulation of self assembly mechanism of fibroin could potentially be utilized to develop silk-based biomaterials consists of the desired secondary conformation.
Cells in developing organs undergo a series of changes in their transcriptional state until a complete repertoire of cell types is specified. These changes in cell identity, together with the control of tissue growth, determine the pattern of gene expression in the tissue. Recent studies explore the dynamics of pattern formation during development and provide new insights into the control mechanisms. Changes in morphogen signalling and transcriptional networks control the specification of cell types. This is often followed by a distinct second phase, where pattern is elaborated by tissue growth. Here, we discuss the transitions between distinct phases in pattern formation. We consider the implications of the underlying mechanisms for understanding how reproducible patterns form during development.
Silk-based bio-inks were developed for 2-D and 3-D printing. By incorporating non-toxic polyols into silk solutions, two-part formulations with self-curing features at room temperature were generated. By varying the formulations the crystallinity of the silk polymer matrix could be controlled to support printing in 2-D and 3-D formats interfaced with CAD geometry and with good feature resolution. The self-curing phenomenon was tuned and exploited in order to demonstrate the formation of both structural and support materials. Biocompatible aqueous protein inks for printing that avoid the need for chemical or photo initiators and that form aqueous-stable structures with good resolution at ambient temperatures provide useful options for biofunctionalization and a broad range of applications.
Coordinated movement of large groups of cells is required for many biological processes, such as gastrulation and wound healing. During collective cell migration, cell-cell and cell-extracellular matrix (ECM) adhesions must be integrated so that cells maintain strong interactions with neighboring cells and the underlying substratum. Initiation and maintenance of cadherin adhesions at cell-cell junctions and integrin-based cell-ECM adhesions require integration of mechanical cues, dynamic regulation of the actin cytoskeleton, and input from specific signaling cascades, including Rho family GTPases. Here, we summarize recent advances made in understanding the interplay between these pathways at cadherin-based and integrin-based adhesions during collective cell migration and highlight outstanding questions that remain in the field.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Cells "spin" a mineralized spiderweb: BMP-2-loaded silk material is inkjet-printed into a spiderweb pattern on a Petri dish and seeded with mesenchymal stem cells (MSCs) by F. G. Omenetto and co-workers, in work on page 4273. Upon culture in osteogenic media, the osteo-inductive print topographically controls the differentiation of MSCs, resulting in the localized formation of mineralized tissue-like material (stained with Alizarin red in the front cover picture).
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Bioprinting is an emerging technology to fabricate artificial tissues and organs through additive manufacturing of living cells in a tissues-specific pattern by stacking them layer by layer. Two major approaches have been proposed in the literature: bioprinting cells in a scaffold matrix to support cell proliferation and growth, and bioprinting cells without using a scaffold structure. Despite great progress, particularly in scaffold-based approaches along with recent significant attempts, printing large-scale tissues and organs is still elusive. This paper demonstrates recent significant attempts in scaffold-based and scaffold-free tissue printing approaches, discusses the advantages and limitations of both approaches, and presents a conceptual framework for bioprinting of scale-up tissue by complementing the benefits of these approaches.