Rapid Volumetric Bioprinting of Pristine Protein-Based (Bio)inks

Abstract

Volumetric bioprinting (VBP) enables the rapid photopolymerization of three-dimensional constructs by modifying the illumination patterns within a build volume. However, only few unmodified, pristine protein-based bioinks can be used for VBP, making the resulting (bio)printed volumes incompatible for further modification steps required for extended applications, and thus limiting the wider adoption of VBP. We have recently developed new methods for VBP, where unmodified protein-based (bio)inks with tyrosine groups, including those based on silk, decellularized extracellular matrix (dECM), and gelatin, can be (bio)printed separately, in their pristine state, using the tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate/sodium persulfate (Ru/SPS) photoinitiator system to form sophisticated shapes and architectures. Here we provide the step-by-step instructions to complete the VBP process and include the characterization of these bioinks. Following post-treatment, the volumetrically printed silk sericin constructs show properties including reversible shrinkage and expansion, or shape-memory, whereas the volumetrically printed silk fibroin constructs exhibit broadly tunable mechanical performances ranging from a few hundred Pa to hundreds of MPa. Both types of silk-based (bio)inks as well as dECM (bio)inks are cytocompatible. We further cover several demonstrations which show the potential uses of volumetrically (bio)printed silk and dECM constructs in clinical and biomedical applications.

Introduction

Volumetric additive manufacturing achieves rapid photopolymerization by illuminating dynamic light patterns across the entire volume of a rotating vial containing the (bio)ink to achieve simultaneous crosslinking¹, while volumetric bioprinting (VBP) for fabrication of cell-embedded constructs was concurrently reported² (Fig. 1a). Its application spans a broad range of biomedical fields, including but not limited to medical devices^3,4, tissue engineering^5,6, and organoid engineering⁷. However, expanded applications of VBP are limited by the few selections of unmodified, pristine protein-based (bio)inks currently available^8,9, which reduce unnecessary modification steps possibly incompatible with certain applications.

Figure 1 |. Schematic showing the tyrosine-containing pristine protein-based (bio)ink preparation and VBP process.

a The customized VBP system, which mainly includes a projector, a vial-rotation motor, and a (bio)ink-containing vial. b Extraction and preparation of different types of tyrosine-containing pristine protein biomaterials. c Preparation of cell-laden bioink and the photocrosslinking mechanism of tyrosine-containing pristine protein-based (bio)ink. Ru: tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate; SPS: sodium persulfate.

Development of the protocol

Protein-based biomaterials such as those based on silk as well as decellularized extracellular matrix (dECM) contain abundant native tyrosine residues that could form di-tyrosine crosslinks after photooxidation in the presence of the tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate/sodium persulfate (Ru/SPS) system^10,11. Nonetheless, most protein-based materials used in photocuring biofabrication require chemical modifications, such as the introduction of methacryloyl or norbornene groups¹, to allow light-induced crosslinking. Meanwhile, the high-concentrations needed for these modified protein-based materials pose challenges in using them for further biomedical applications, owing to the resulting dense polymer networks that would impede nutrient-transport and sometimes mechanically inhibitive¹². Moreover, increased photoinitiator concentrations may be needed when using high concentrations of bioinks during photocuring processes, which could be harmful to cells as well¹⁰.

To overcome these challenges, we have developed unmodified protein-based (bio)inks (including silk-based (bio)inks, dECM (bio)inks, and gelatin (bio)inks, all formulated separately) that could be (bio)printed in their pristine form to build sophisticated constructs^10,11. We have identified silkworm silk derived from Bombyx mori (B. mori), a readily available natural protein composed primarily of silk sericin (SS) (the content of tyrosine groups at ~3.38 mol%) and silk fibroin (SF) (the content of tyrosine groups at ~5 mol%)^13,14 (Fig. 1b), as novel (bio)ink candidates. SF is traditionally utilized in high concentrations (>5%, w/v) for extrusion-based or digital light processing (DLP)-based (bio)printing¹⁵. We, instead, present the first instance of applying pure SS and SF for VBP. Moreover, compared with high-concentration silk-based (bio)inks used in DLP platforms, we have successfully (bio)printed varying concentrations of silk-based (bio)inks in our VBP platform, achieving a minimum concentration of 2.5% (w/v)¹⁰.

Additionally, our work includes the successful preparation of low-concentration pure dECM (bio)inks (Fig. 1b)¹¹, which are procured from decellularized tissues, retaining essential components that foster a cell-compatible microenvironment. We have volumetrically (bio)printed sophisticated structures with unmodified heart-derived dECM (h-dECM) (bio)inks (the content of tyrosine groups in h-dECM at ~0.15 mol%) and meniscus-derived dECM (Ms-dECM) (bio)inks (the content of tyrosine groups in Ms-dECM at ~1 mol%), although the inherent mechanical properties of dECM are typically poor for (bio)printing^16–18. Specifically, we have volumetrically bioprinted the heart-like constructs with rat cardiomyocyte (rCM)-laden h-dECM bioinks and human induced pluripotent stem cell (iPSC)-derived cardiomyocyte (hiPSC-CM)-laden h-dECM bioinks, separately; as well as meniscus-like constructs with human mesenchymal stem cell (hMSC)-laden Ms-dECM bioinks. The volumetrically bioprinted heart-like constructs achieved the spreading and contracting behaviors, and the volumetrically bioprinted meniscus-like constructs demonstrated appropriate chondrogenic differentiation and gene-expressions.

Further, unmodified porcine gelatin has also been successfully (bio)printed using VBP in this protocol (Fig. 1c), despite that its tyrosine content is less than 0.5 mol%¹⁹. The efficiency of this crosslinking process, coupled with the satisfactory (bio)printing performances, underscore the versatility and potential of utilizing a diverse type of unmodified protein-based biomaterials with VBP. Our pioneering efforts in developing a series of pristine, unmodified protein-based (bio)inks (silk, dECM, and gelatin as examples) not only expand the library of photocurable protein-based (bio)inks, but also provide a better understanding for the mechanism of rapid volumetric (bio)printing of these tyrosine-containing biomaterials. Additional possible (bio)ink options may include but are not limited to spider silk, Antheraea perenyi silk, and Antheraea mylitta silk (Supplementary Table S1). The methodologies for preparing these protein-based (bio)inks and their respective VBP processes are illustrated in the following sections, charting a route for future explorations and applications in VBP.

Overview of the procedures

The whole procedure includes the following four parts: (1) the VBP method; (2) the preparation procedures of silk (SS or SF) (bio)inks, dECM (h-dECM or Ms-dECM) (bio)inks, and gelatin (bio)inks; (3) the procedure of VBP of protein-based (bio)inks without embedding cells and their characterizations; and (4) the procedure of VBP of protein-based bioinks with embedded cells and their biomedical applications.

In Procedure 1, the preparation steps of protein-based (bio)inks are described in detail. We commence with the extraction process of SS and SF from B. mori cocoons (Procedure 1, steps 1–36), followed by the derivation of h-dECM and Ms-dECM solutions from porcine tissues (Procedure 1, steps 37–55). Then, the protocol describes the preparation processes of silk-based (bio)inks, dECM (bio)inks, and gelatin (bio)inks, all formulated separately, alongside the execution steps of the VBP process (Procedure 1, steps 56–69), which includes the optimizations of printing parameters and the evaluations of printing resolutions for all protein-based (bio)inks. Additionally, physicochemical properties of silk-based (bio)inks and dECM (bio)inks are assessed. Specifically, post-printing treatment and additional functional evaluations for the printed silk-based constructs are also presented.

In Procedure 2, the preparation steps of cell-laden silk-based bioinks and dECM bioinks as well as the following VBP process are described (Procedure 2, steps 1–24). In Procedure 2, NIH/3T3 fibroblasts are encapsulated in silk-based bioinks and dECM bioinks for cytocompatibility tests. Subsequently, the biological applications are described: (1) the volumetrically printed SF screw constructs with double-crosslinked networks induced by immersing in 70% (v/v) ethanol aqueous solution with post-seeded hMSCs; (2) the volumetrically bioprinted heart-like constructs with rCM-laden h-dECM; (3) the volumetrically bioprinted meniscus constructs with hMSC-laden Ms-dECM.

The versatility of tyrosine-containing protein-based bioinks allow for encapsulation of various cell types, enabling the fast creation of different tissue-like structures through VBP. Therefore, it is believed that a broad utility across diverse tissue engineering domains could be achieved. Here, we present guidelines for applying these pristine tyrosine-containing protein-based (bio)inks within the field of tissue engineering, with an expectation that they are adaptable to other biomedical applications as well.

Advantages and limitations of VBP of pristine protein-based (bio)inks

VBP shows outstanding advantages in fast (bio)printing speed, non-contact (bio)printing mode, and smooth surface quality compared to layer-by-layer (bio)printing strategies. However, the lack of (bio)inks is a key limitation for expanded biomedical applications of this technology.

The unmodified tyrosine-containing protein-based (bio)inks in this study are composed of photoactive polymers, photoinitiators/co-initiators, without or with cells, ensuring that the intrinsic properties of the proteins are preserved. Notably, in contrast to layer-by-layer (bio)printing methods, VBP of unmodified tyrosine-containing protein-based (bio)inks shows a fast fabrication speed (normally within 120 s); these protein-based (bio)inks could be volumetrically (bio)printed at low concentrations in most cases (down to 2.5% (w/v) for SS or SF, and 1% (w/v) for dECM, with an exception of 10% (w/v) for gelatin due to its extra-low tyrosine content), with high fidelity. Further, these protein-based bioinks could facilitate the creation of microenvironments with good cytocompatibility.

However, tissues and organs are intricately assembled systems, comprising different cell types embedded within matrices with varying mechanical properties. Therefore, VBP of a single (bio)ink formulation may not satisfy the complex functional needs for tissues and organs, and methods for multi-material and multi-cellular VBP have been developed^20–23. Moreover, the concentrations of photoinitiator and the (bio)ink are important factors that impact (bio)printing resolution and cell viability. In this protocol, we have further evaluated the impact of different photoinitiator concentrations on printability and printing resolution within the range of protein-based (bio)inks that we have showcased.

Experimental design

Preparation of silk-based (bio)inks and dECM (bio)inks.

For printing the silk-based and dECM solutions, we use Ru/SPS as the photoinitiator complex, which engages with tyrosine residues present in the proteins, catalyzing crosslinking with light exposure^25,26.

For SS, B. mori cocoons are cut into pieces and boiled in Na₂CO₃ solution to remove SS from SF, and then the SS solution is dialyzed with deionized water (DW). For SF, B. mori cocoons are cut into pieces and boiled in aqueous Na₂CO₃ solution for varying durations to achieve different molecular weights. Then, the degummed SF is dissolved in lithium bromide (LiBr). After dialyzing the SF/LiBr solution with DW, the solution is centrifuged. Next, the precipitate is discarded, and the supernatant is filtered. The SS and SF solutions are loaded into dialysis bags and placed in the hood for concentrating. The final concentration is calculated by weighting the remaining mass after evaporating the water of the SS or SF solution (Fig. 2a).

Figure 2 |. Preparation of silk and dECM solutions.

a The routine for preparation of SS for silk fibers. b The routine for preparation of SF solutions from silk cocoons. c The routine for processing the porcine heart into h-dECM solution by decellularization and solubilization. d The routine for processing the porcine meniscus into Ms-dECM solution by decellularization and solubilization. B. mori: Bombyx mori; SS: silk sericin; SF: silk fibroin; DW: deionized water; SDS: sodium dodecyl sulfate; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix. c and d are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

For h-dECM, the fresh left ventricle is separated from the porcine heart (obtained from local butcher exempt of institutional review board approval). The tissue should be subjected to decellularization using a solution of sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS). After treating decellularized tissue with Triton X-100 in PBS, the tissue is stirred in isopropyl alcohol and rinsed with PBS to remove all the residual detergent (Fig. 2b). For Ms-dECM, the fresh porcine meniscus is harvested from fresh knee joints of pigs (obtained from local butcher exempt of institutional review board approval). The meniscus is cut into thin slices into 1-mm thickness, followed by freezing using liquid nitrogen and then grounding into rough powder. Then, the meniscus powder is decellularized in SDS solution in PBS. After treating decellularized tissue with trypsin-ethylenediaminetetraacetic acid (EDTA) solution in PBS, the sample is rinsed in PBS further to remove the residual chemicals. Both h-dECM and Ms-dECM powders can be dissolved completely in a pepsin acetic acid solution at room temperature (RT) (Fig. 2c).

The silk-based, dECM, and gelatin (bio)inks are prepared by mixing each separately with the Ru/SPS photoinitiator system in the dark at RT.

VBP system.

Silk, dECM, and gelatin constructs are volumetrically (bio)printed with a customized volumetric (bio)printer^10,11. The minimum printable volume with our VBP system is ~0.1 cm³ and maximum volume is ~1 cm³. The highest resolution achieved using these (bio)inks is ~50 μm in the XY-direction and ~25 μm in the Z-direction, depending on the bioink composition and photoinitiator concentration. The Radon transform algorithm, iterative algorithm, and filtered back-projection algorithm are used in VBP. All projection algorithms are controlled via custom MATLAB scripts and programmed to output patterned intensity-modulated images using the green-light (525 nm) channel.

Physical property characterizations of protein-based constructs.

To evaluate printability of the various protein-based (bio)inks, different formulations for each (bio)ink to print different constructions (screw for SF (bio)inks, temple for SS (bio)inks, cubes for both dECM (bio)inks and gelatin (bio)inks) are used (Fig. 3a–j). The photographs at different angles of the volumetrically printed constructs are taken using the digital camera to obtain the printability maps (Fig. 3k–o). To evaluate the penetration depth (D_p), we recommend using ultraviolet-visible (UV-vis) spectrophotometry at 525-nm of wavelength to assess the light-absorption profiles of (bio)inks with different formulations (Fig. 4a, b). We recommend using a computer-aided design (CAD) model of a solid rod with bars featuring a set of threads of different thicknesses (from 1 μm to 101 μm) and a CAD model of a radially arranged array of cubes to quantify the printing resolution; meanwhile, dosage assay and other commonly used test models are also recommended such as spikes, tips, and pillars for positive features, and channels, pores and voids for negative features^27,28 (Fig. 4c–e).

Figure 3 |. Printing performances of tyrosine-containing protein-based (bio)inks.

a-e Printability maps of SS (bio)ink, SF (bio)ink, h-dECM (bio)ink, Ms-dECM (bio)ink and gelatin (bio)ink with different formulations. √: printable; O: but the shape was not agreement with CAD; X: non-printable. f Quantified ratios of the width to height of the volumetrically printed SS temple construct with different formulations. The value close to 0.53 indicates matching with the designed CAD model. g Values of thread thickness of volumetrically printed SF screw construct with different formulations. The smaller values indicate the better resolutions. h-j Quantified ratios of the height to width of volumetrically printed cubic construct using h-dECM (bio)ink, Ms-dECM (bio)ink, and gelatin (bio)ink with different formulations. The value close to 1 indicates matching with the designed CAD model. k CAD models and photographs of volumetrically printed SS objects with printing parameters: 2.5% (w/v) SS with 0.5-mM Ru/5-mM SPS, light intensity measured at the printing volume: 3 mW cm⁻². The hollow triangle construct (60 s of printing time) and a brain-like construct (65 s of printing time). l CAD models and photographs of volumetrically printed SF objects under 3 mW cm⁻² of light intensity measured at the printing volume. Construct with dual spiral channel construct: 5% (w/v) SF with 0.25-mM Ru/2.5-mM SPS, 57 s of printing time; construct with H-shaped channel: 2.5% (w/v) SF with 0.25-mM Ru/2.5-mM SPS, 117 s of printing time. m CAD models and photographs of volumetrically printed objects using 1% (w/v) h-dECM with 0.5-mM Ru/5-mM SPS (light intensity measured at the printing volume: 3 mW cm⁻²). The ear construct was printed in 30 s, and the heart construct was printed in 30 s. n CAD models and photographs of volumetrically printed object using 1% (w/v) Ms-dECM with 0.25-mM Ru/2.5-mM SPS (light intensity measured at the printing volume: 3 mW cm⁻²). The bone construct was printed in 45 s, and the screw structure was printed in 30 s. o CAD models and photographs of volumetrically printed object using 10% (w/v) gelatin with 0.5-mM Ru/25-mM SPS (light intensity measured at the printing volume: 15 mW cm⁻²). The cylinder construct was printed in 150 s, and the cone structure was printed in 150 s. CAD: computer-aided design; SS: silk sericin; SF: silk fibroin; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix; Ru: tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate; SPS: sodium persulfate. a, b, f, g, k, and l are adapted with permission¹⁰, Copyright 2023, Springer Nature. c, d, h, i, m, and n are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

Figure 4 |. D_p and printing resolution of tyrosine-containing pristine protein-based (bio)inks.

a Schematic showing the effect of Ru/SPS concentration and protein-based (bio)inks concentration on printing resolution. b Dp of SS, SF, h-dECM, and Ms-dECM with different formulations. c(left to right) CAD image of the solid bar; thicknesses of the threads from 1 μm to 101 μm, microscopic images of the printed solid bar of 2.5% (w/v) SS, 2.5% (w/v) SF, 1% (w/v) h-dECM, and 1% (w/v) Ms-dECM. The printing parameters were 0.5-mM Ru/5-mM SPS for SS, h-dECM and Ms-dECM; 0.25-mM Ru/2.5-mM SPS for SF, light intensity measured at the printing volume: 3 mW cm⁻². d(left to right) CAD image of the radially arranged array of cubes, microscopic images of the printed arrays of cubes of 2.5% (w/v) SS, 2.5% (w/v) SF, 1% (w/v) h-dECM, and 1% (w/v) Ms-dECM. The printing parameters were 0.5-mM Ru/5-mM SPS for SS, h-dECM and Ms-dECM; 0.25-mM Ru/2.5-mM SPS for SF, light intensity measured at the printing volume: 3 mW cm⁻². e Quantified resolutions in X, Y and Z directions of the printed constructs of different pristine protein-based (bio)inks. CAD: computer-aided design; SS: silk sericin; SF: silk fibroin; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix; Ru: tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate; SPS: sodium persulfate. Statistical significances are expressed as **p<0.01, ***p<0.001, and ****p<0.0001 by two-way ANOVA with Tukey’s post hoc test; in figure b, n=3; in figure e, n=10. c(CAD, SS and SF) and d(CAD, SS and SF ) are adapted with permission¹⁰, Copyright 2023, Springer Nature. c(h-dECM and Ms-dECM) and d(h-dECM and Ms-dECM) are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

To test the physicochemical properties of the volumetrically printed protein-based constructs, we recommend characterizing the mechanical properties by universal testing machine (Fig. 5a–e) and checking the surface and internal morphologies by scanning electron microscopy (SEM) (Fig. 5f, g); the degradability of the volumetrically printed protein-based constructs is suggested to be evaluated as well (Fig. 5h–k). These procedures are based on previously described methods^10,11.

Figure 5 |. Physical properties of volumetrically printed tyrosine-containing pristine protein-based constructs.

a Schematic showing the routine of physical properties test with volumetrically printed constructs. b Compressive moduli of constructs volumetrically printed using 2.5% (w/v) and 5% (w/v) SS with 0.5-mM Ru/5-mM SPS. c Compressive moduli of constructs volumetrically printed using 2.5% (w/v) and 5% (w/v) SF with 0.5-mM Ru/5-mM SPS. d Compressive moduli of constructs volumetrically printed using 1% (w/v) h-dECM with 0.25-mM Ru/2.5-mM SPS and 0.5-mM Ru/5-mM SPS. e Compressive moduli of constructs volumetrically printed using 1% (w/v) Ms-dECM with 0.25-mM Ru/2.5-mM SPS and 0.5-mM Ru/5-mM SPS. f SEM images of cross-sections of the volumetrically printed constructs of f(top) SS, f(bottom) SF, g(top left and bottom left) h-dECM, and g(top right and bottom right) Ms-dECM with different formulations. h In vitro degradation profiles of the volumetrically printed SS constructs in PBS, 5 U mL⁻¹ of protease XIV PBS solution, and 40 U mL⁻¹ of α-chymotrypsin PBS solution. i In vitro degradation profiles of the volumetrically printed SF constructs in PBS, 5 U mL⁻¹ of protease XIV PBS solution, and 40 U mL⁻¹ of α-chymotrypsin PBS solution. j In vitro degradation profiles of the volumetrically printed h-dECM constructs in PBS, 5 U mL⁻¹ of collagenase IV PBS solution, and 40 U mL⁻¹ of α-chymotrypsin PBS solution k In vitro degradation profiles of the volumetrically printed Ms-dECM constructs in PBS, 5 U mL⁻¹ of collagenase IV PBS solution, and 40 U mL⁻¹ of α-chymotrypsin PBS solution. SS: silk sericin; SF: silk fibroin; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix; Ru: tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate; SPS: sodium persulfate. PBS: phosphate buffered saline. Statistical significances are expressed as *p<0.05, **p<0.01, and ***p<0.001, by two-way ANOVA with Tukey’s post hoc test. n=3. b, c, f, h, and i are adapted with permission¹⁰, Copyright 2023, Springer Nature. d, e, g, j, and k are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

Additionally, for SS and SF, which show major alterations in physical properties (such as volume, morphology, mechanical strength, resolution, etc.) after post-printing treatment (such as ethanol treatment), we recommend characterizing and comparing their physical property changes before and after post-printing treatment (Fig. 6).

Figure 6 |. Tunable mechanical strengths and secondary structure change of volumetrically printed silk objects before and after post-printing treatment.

a Shrinkage and expansion properties of volumetrically printed SS objects. Photographs of volumetrically printed hollow square (57 s of printing time) in the a(left) as-printed, a(center) shrunken (induced by immersion in 100% (v/v) ethanol for 2 h), and a(right) re-expanded (immersion in water for 2 h after shrunken) states. b SEM images of cross-sections of the volumetrically printed structures at different SS concentrations in the shrunken, and re-expanded states. c Compressive moduli of volumetrically printed 2.5% (w/v) and 5% (w/v) SS structures in the c(top) shrunken and c(bottom) re-expanded states. d(left to right) CAD image of the solid bar; thicknesses of the threads from 1 μm to 101 μm, microscopic images of the volumetrically printed solid bar of 5% (w/v) SS in as-printed, shrunken, and re-expanded states (0.5-mM Ru/5-mM SPS, 3 mW cm⁻² of light intensity measured at the printing volume, and 95 s of printing time). e-g Fiber diameter profiles of the volumetrically printed solid bar with different SS concentrations in the as-printed, shrunken, and re-expanded states. h(left) Illustration of the VBP process of a SF screw construct in a rotating vial. h(center) Photograph of the same screw after 70% (v/v) ethanol treatment for 24 h. Scale bar: 500 μm. h(right) Microscopic image of the same screw after evaporating the ethanol solution. Scale bar: 500 μm. i SEM images of cross-sections of the volumetrically printed 2.5% (w/v) SF screw, 5% (w/v) SF screw, 10% (w/v) SF screw with a double-crosslinked network (treated with 70% (v/v) ethanol for 24 h followed by 72 h of air-drying). j Compressive moduli of volumetrically printed 2.5% (w/v), 5% (w/v), and 10% (w/v) SF structures with a double-crosslinked network. k β-sheet contents of 2.5% (w/v), 5% (w/v), and 10% (w/v) SF structures with a double-crosslinked network. i Microscopic images showing volumetrically printed 2.5–10% (w/v) SF screws before (i.e., after 24 h-ethanol treatment but before air-drying) and after shrinking (i.e., induced by 72 h of air-drying after 24 h-ethanol treatment). The printing parameters were 0.25-mM Ru/2.5-mM SPS, and 3 mW cm-2 of light intensity measured at the printing volume. Scale bar: 500 μm. m Quantified thread thickness changes of the same SF screws before and after post-printing treatment. CAD: computer-aided design; SS: silk sericin; SF: silk fibroin; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix; Ru: tris (2,2-bipyridyl) dichlororuthenium (II) hexahydrate; SPS: sodium persulfate. Statistical significances are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by two-way ANOVA with Tukey’s post hoc test. In figures (e, f, g, and m), n=10; in figures (c, j, and k), n=3. a-m are adapted with permission¹⁰, Copyright 2023, Springer Nature.

Bioprinting and cytocompatibility.

Further, the optimal printing concentrations of these bioinks (2.5% and 5% SS, 2.5% and 5% SF, 1% h-dECM, and 1% Ms-dECM) are selected for biological evaluations (Fig. 7). For silk-based proteins, myoblasts (C2C12) are embedded within the SS and SF bioinks. Screw model (volume of the model: ~0.5 cm³; 45 s of printing time for SS+C2C12, 57 s of printing time for SF+C2C12), C60 model (volume of the model: ~0.5 cm³; SS+C2C12, 57 s of printing time), and channel-in-cube model (volume of the model: ~0.45 cm³; SS+C2C12, 45 s of printing time) are volumetrically bioprinted with the parameters of 0.5/5 mM Ru/SPS for SS and 0.25/2.5 mM Ru/SPS for SF, 5×10⁶ cells mL⁻¹, and 3 mW cm⁻² of light intensity measured at the printing volume. The metabolic activities of embedded cells are measured by the PrestoBlue or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent.

Figure 7 |. Cytocompatibility of cell-laden tyrosine containing pristine protein-based bioinks.

a Schematic showing the VBP process with cell-laden tyrosine containing pristine protein-based bioinks. b CAD models and microscopic images of the structures volumetrically bioprinted with C2C12-laden silk-based bioinks, and NIH/3T3 fibroblast-laden d-ECM-based bioinks. The C60 structure was bioprinted with 2.5% (w/v) SS and 0.25/2.5-mM Ru/SPS in 57 s. The screw structure was bioprinted with 5% (w/v) SF and 0.25/2.5-mM Ru/SPS in 45 s. The heart structure was bioprinted with 1% (w/v) h-dECM and 0.25/2.5-mM Ru/SPS in 45 s. The ear structure was bioprinted with 1% (w/v) Ms-dECM and 0.25/2.5-mM Ru/SPS in 30 s. Cells were all stained with CellTracker. c and d Live (green)/dead (red) images of C2C12 cultured within the volumetrically bioprinted SS and SF constructs at different concentrations with 0.25/2.5-mM Ru/SPS. e and f Live (green)/dead (red) images of NIH/3T3 fibroblasts cultured within 1% (w/v) h-dECM and 1% (w/v) Ms-dECM constructs at different concentrations of Ru/SPS. g-j Proliferation profiles of cells cultured within the volumetrically bioprinted SS, SF, h-dECM, and Ms-dECM constructs. k-n Viability profiles of cells cultured within the volumetrically bioprinted SS, SF, h-dECM and Ms-dECM constructs. CAD: computer-aided design; SS: silk sericin; SF: silk fibroin; h-dECM: heart-derived decellularized extracellular matrix; Ms-dECM: meniscus-derived decellularized extracellular matrix; Ru: tris (2,2-bipyridyl) dichlororuthenium (II) hexahydrate; SPS: sodium persulfate. Statistical significances are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by two-way ANOVA with Tukey’s post hoc test. n=3; ns= not significant. b (top 2 panels), c, d, g, h, k and l are adapted with permission¹⁰, Copyright 2023, Springer Nature. b (bottom 2 panels), e, f, i, j, m and n are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

For dECM proteins, NIH/3T3 fibroblasts are embedded within the dECM bioinks. Ear model (volume of the model: ~0.3 cm³; 30 s of printing time for 1% (w/v) NIH/3T3-laden Ms-dECM), heart model (volume of the model: ~0.5 cm³; 45 s of printing time for 1% (w/v) NIH/3T3-laden h-dECM), and screw model (volume of the model: ~0.5 cm³; 30 s of printing time for 1% (w/v) NIH/3T3-laden Ms-dECM) are volumetrically bioprinted with the parameters at 0.25/2.5 mM Ru/SPS and 3 mW cm⁻² of light intensity measured at the printing volume. Similarly, the metabolic activities of embedded cells are measured by the PrestoBlue and MTS reagents. Of note, the printing time does not necessarily scale with the volume of print in VBP²⁹.

Biomedical applications.

The volumetrically printed 10% (w/v) SF screw-like constructs, post-treated with 70% (w/v) ethanol followed by air drying, are seeded with hMSCs to evaluate cell differentiation. Additionally, the volumetrically printed 10% (w/v) SF screw-like constructs (without cells) are implanted in an ex vivo porcine femur model (Fig. 8a).

Figure 8 |. Proof-of-concept applications for volumetrically bioprinted tyrosine-containing pristine protein-based constructs.

a(left) hMSC immunostaining images showing RUNX2 and OCN expressions. a(center) Schematic, photographs, and micro-CT scanning image showing ex vivo implantation test in a porcine femur of the volumetrically printed 10% (w/v) SF screws with a double- crosslinked network induced by immersed in 70% (w/v) ethanol for 24 h followed by 72 h of air-drying. Scale bar: 1.5 mm. a(right) Expression levels of representative genes indicating osteoblast-formation during the 4-week culture period. Runx: runt-related transcription factor; ALP: alkaline phosphatase; COL1α1: collagen type I alpha 1 chain; OCN: osteocalcin; BMP2: bone morphogenetic protein 2; Osx: osterix; OPN: osteopontin. b(left) Schematic showing the VBP process of the heart-like structure and the process of rCM spreading. b(center) Brightfield and b(right) fluorescence micrographs showing immunostaining of hiPSC-CMs both on the surface and encapsulated in the heart-like constructs volumetrically bioprinted with 1% (w/v) h-dECM and 0.25/2.5-mM Ru/SPS reaching a stretching morphology and expressing sarcomeric-α-actinin after culturing for 10 days. c(left) Micrographs showing F-actin staining and immunostaining of hMSCs in the meniscus constructs volumetrically bioprinted with 1% (w/v) Ms-dECM and 0.25/2.5-mM Ru/SPS over 3 weeks of differentiation. c(center-left) Low-magnification micrograph showing F-actin staining of hMSC-ladened meniscus tissue volumetrically printed with 1% (w/v) Ms-dECM and 0.25/2.5-mM Ru at 3 weeks of differentiation. Inset shows confocal image of the high-density differentiated chondrocytes. c(center-right) Histology images of H&E and Masson’s trichrome for the meniscus constructs volumetrically bioprinted with 1% (w/v) Ms-dECM and 0.25/2.5-mM Ru/SPS containing hMSCs after 3 weeks of chondrogenic differentiation. c(right) Quantified expression levels of representative genes indicating chondrogenic differentiation status in 3 weeks. VBP: volumetric bioprinting; rCM: rat cardiac myocyte; H&E: hematoxylin and eosin; F-actin: filamentous actin; ACAN: aggrecan; COMP: cartilage oligomeric matrix protein; COL1α1: collagen type I alpha 1 chain; ELN: elastin; COL10A1: type X collagen. Statistical significances are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by two-way ANOVA with Tukey’s post hoc test. n=3. a is adapted with permission¹⁰, Copyright 2023, Springer Nature. b and c are adapted with permission¹¹, Copyright 2024, Wiley-VCH.

The feasibility of using dECM bioinks for VBP of tissue-specific constructs is demonstrated by two applications, a human heart-like construct and a meniscus-like construct. The h-dECM bioink, composed of rCM-laden h-dECM, is tested with two different concentrations of Ru/SPS. The meniscus constructs laden with hMSCs are volumetrically bioprinted using 1% (w/v) Ms-dECM with 0.25/2.5-mM Ru/SPS (Fig. 8b, c).

Materials

Reagents

! CAUTION When handling the chemicals used in this protocol, always wear suitable personal protective equipment (PPE), including a laboratory coat, nitrile gloves, safety goggles and, where indicated, face shield, respirator (or fume hood), and thermo-insulated gloves. Follow the relevant institutional and governmental safety guidelines and refer to the appropriate material data sheets. All synthesis steps should be performed within a chemical fume hood.

• Silkworm (B. mori) cocoons (Tajima Shoji; store at RT)

• Sodium carbonate (Na₂CO₃; Millipore Sigma, cat. no. 13418–1KG-R; store at RT)

▲CRITICAL Na₂CO₃ absorbs moisture from the air, it is important to keep the container tightly sealed after each use to maintain its integrity.

! CAUTION Na₂CO₃ can be harmful if inhaled in large quantities, potentially causing irritation or damage to the nasal passages.

• Porcine heart and meniscus (local slaughterhouses; store at 4 °C)

• Trypsin-EDTA (Thermo Fisher Scientific, cat. no. 15400–054)

• Triton X-100 (Sigma-Aldrich, cat. no. X100)

! CAUTION Triton X-100 can cause skin and eye irritation. Avoid inhalation and contact with skin or eyes.

• Isopropyl alcohol (Sigma-Aldrich, cat. no. W292912)

• SDS (Sigma-Aldrich, cat. no. L4509)

▲CRITICAL SDS is hygroscopic; ensure that the container cap is tightly closed after use.

! CAUTION SDS is toxic; inhalation, ingestion, or contact with skin can cause irritation or damage. Use appropriate protective equipment such as gloves and a mask when handling.

▲CRITICAL We recommend using fresh organs for dECM-extraction. The quality of the organs has a substantial impact on the printability of the obtained biomaterials.

• SS (from Procedure 1, steps 1–16; store at 4 °C)

• Ru photoinitiator (Advanced BioMatrix, cat. no. 5246, store at RT)

▲CRITICAL The dry powders of Ru and SPS are stable for over 1 year at RT. Once solubilized, use Ru and SPS within 2 weeks.

• SPS photoinitiator (Advanced BioMatrix, cat. no. 5247, store at RT)

▲CRITICAL Do not mix Ru and SPS together in advance. It will initiate a rapid redox reaction even in the absence of light³¹.

! CAUTION If inhaled, Ru/SPS may cause skin and eye irritation. When handling, PPE such as rubber gloves, airtight goggles, and a full-face shield mask are recommended.

• Degummed silk (from Procedure 1, steps 17–36; SF)

• LiBr (Sigma-Aldrich, cat. no. 213225)

! CAUTION LiBr is an anhydrous compound, which may cause skin irritation, severe eye damage, and acute toxicity if ingested. Always wear a face shield and respirator. Avoid any direct contact.

▲CRITICAL LiBr is extremely hygroscopic. Always minimize exposure to atmospheric air (seal the container with parafilm) to avoid contamination. Often best to utilize an unopened new container to avoid changes in water content.

• h-dECM powder and Ms-dECM powder (from Procedure 1, steps 37–55; store at −80 °C)

• Pepsin (Sigma-Aldrich, cat. no. P7000)

• Sodium hydroxide (Sigma-Aldrich, cat. no. 655104, store at RT)

• Acetic acid solution (Sigma-Aldrich, cat. no. 45754, store at RT)

▲CRITICAL Acetic acid and sodium hydroxide are corrosive; ensure that the container cap is tightly closed after use to prevent leakage and contamination.

! CAUTION Acetic acid and sodium hydroxide can cause severe skin burns and eye damage. Avoid inhalation of vapor, use appropriate protective equipment, and use it in chemical fume hood.

• Gelatin from porcine skin, type A (Sigma-Aldrich, cat. no. G2656, store at RT)

• SS and SF (bio)inks

• h-dECM and Ms-dECM (bio)inks

• Gelatin (bio)inks

▲CRITICAL Ru/SPS is easily activated when mixed in solution; always keep the solution away from light. The prepared (bio)inks with Ru/SPS should be used within the same day.

! CAUTION When observing the printing process, wear laser safety glasses to protect the eyes.

• Protease XIV (from Streptomyces griseus) (Pronase E; Merck, cat. no. 9036–06-0; store at −20 °C)

• α-chymotrypsin (Sigma-Aldrich, cat. no. C4129, store at −20 °C)

• Collagenase (Sigma-Aldrich, cat. no. C8051, store at −20 °C)

• Collagenase IV (Sigma-Aldrich, cat. no. C4–28, store at −20 °C)

• Dulbecco’s PBS (DPBS) powder (Thermo Fisher Scientific, cat. no. 21600010; store at RT

• 70% (v/v) ethanol (Sigma-Aldrich, cat. no. 493511)

• Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, cat. no. 11965–118)

• Fetal bovine serum (FBS) (Thermo Fisher Scientific, cat. no. 10437–028)

• Antibiotic-antimycotic (Anti-Anti) (Thermo Fisher Scientific, cat. no. 15240–062)

Cell lines

• Myoblasts (C2C12) (ATCC, cat. no. CRL-1772)

• NIH/3T3 fibroblasts (ATCC, cat. no. CRL-1730)

• MDA-MB-231 breast cancer cells (ATCC, cat. no. HTB-26)

• rCMs (ATCC, cat. no. CRL-1730)

• hMSCs (Lonza, cat. no. PT-2501)

• hiPSC-CMs (FUJIFILM, cat. no. R1092)

Cytocompatibility of silk-based and dECM constructs

• 10% (w/v) Formaldehyde (Sigma-Aldrich, cat. no. HT501128)

• Bovine serum albumin (BSA) (Sigma-Aldrich, cat. no. A4612)

• Alexa Fluor 488-phalloidin (Thermo Fisher Scientific, cat. no. A12379)

• 4’,6-diamidino-2-phenylindole (DAPI) (Millipore Sigma, cat. no. D9542–10MG)

• Live/Dead Viability/Cytotoxicity Kit (Thermo Fisher Scientific, cat. no. L3224; ethidium homodimer and calcein-AM are included in this kit)

• MTS Assay Kit (Thermo Fisher Scientific, cat. no. 50–221-5358)

• PrestoBlue (Thermo Fisher Scientific, cat. no. A13261)

Cell differentiation and immunostaining

• hMSC growth medium (Lonza, cat. no. PT-3001)

• hMSC osteogenic differentiation medium (Lonza, cat. no. PT-3002)

• hMSC chondrogenic differentiation medium (Lonza, cat. no. PT-3003)

• Mouse anti-RUNX2 antibody (Invitrogen, cat. no. NP0326BOX)

• Mouse anti-osteocalcin antibody (Abcam, cat. no. ab198228)

• iCell cardiomyocytes media kit (FUJIFILM, cat. no. R1151; cardiomyocyte medium is included in this kit)

• Goat anti-mouse IgG H&L (Alexa Fluor 488) (Abcam, cat. no. ab150113)

• Anti-sarcomeric-α-actinin antibody (Abcam, cat. no. ab137346)

• Mouse anti-aggrecan (Abcam, cat. no. ab3778)

• Rabbit anti-collagen II antibody (Abcam, cat. no. ab34712)

RNA-isolation and real-time reverse-transcription qPCR

• QuantiTect reverse transcription kit (Qiagen, cat. no. 205311)

• SYBR Green qPCR Master Mix (Thermo Fisher Scientific, cat. no. A25742; store at −20 °C)

• RNeasy mini kit (Qiagen, cat. no.74104)

• hMSC chondrogenic differentiation medium BulletKit (Lonza, cat. no. PT-3003)

Ex vivo implantation of SF screw-like constructs

• Volumetrically printed SF screw-like constructs (Procedure 2, steps 1–6)

• Fresh porcine femur (local supermarket)

Micro computed tomography (Micro-CT) analyses

• The SF screw-implanted porcine femur from Procedure 2, step 58.

Histological evaluations

• Formalin (Sigma-Aldrich, cat. no. R03379)

Equipment

• Sterile 5-mL, 10-mL, and 25-mL serological pipettes (SPL Life Science, cat. no. 91005, 91010, and 91025, respectively)

• Sterile 15-mL and 50-mL conical tubes (SPL Life Sciences, cat. no. 50015 and 50250, respectively)

• Pipette aid (Drummond Scientific, cat. no. 4–000-201)

• 50-mL, 2-L, and 5-L glass beakers (DWK Life Science, cat. no. 14000–50, 211066301, and 211067306, respectively)

• Sterile 3-mL and 10-mL syringes (Kovax-Syringe Korea Vaccine, cat. no. 22G 11/4”)

• Microplate reader (Biotek, EON microplate reader)

• 10-μL, 100-μL, 200-μL, and 1000-μL micropipettes and appropriate sterile tips (Eppendorf)

• 0.22-μm syringe filter (Sigma, cat. no. Z741969)

• Volumetric bioprinter (see ‘VBP setup’)

• Printing vial (DWK Life Science, cat. no. W225122, 12-mm diameter)

• Centrifuge (Eppendorf, model. no. 5810R)

• Refrigerators set to 4 °C and −20 °C (Thermo Fisher Scientific, model. no. TSX1205GA and TSX2320FD, respectively)

• Deep freezer set to −80 °C (Thermo Fisher Scientific, model. no. 902GP-ULTS)

• Dialysis bag (Thermo Fisher Scientific, cat. no. Spectrum S432706, 12–14-kDa MWCO)

• Freeze-dryer (Operon, model. no. FDCF-12003)

• Balance (AS ONE, model. no. AXA5003)

• Aluminum foil (AS ONE, cat. no. CP-1850–01)

• 1.7-mL, 2.0-mL, and 5.0-mL conical microcentrifuge tubes (Axygen, cat. no. MCT-175-C and MCT-200-C; and Eppendorf, cat. no. 0030119401)

• 6-well plate, 48-well plate, and 96-well plate (Thermo Fisher Scientific, cat. no. 07–200-83, 07–200-86, and 14–245-71, respectively)

• Incubator (AS ONE, model. no. CC-2559–01)

• Spatula (AS ONE, cat. no. 6–524-01)

• UV-vis spectrophotometry (Molecular Devices, model. no. SpectraMax M3)

• Digital camera (Canon, model. no. 70D)

• Inverted fluorescence microscope (Nikon, model. no. Ti-E)

• Mechanical tester (Instron, model. no. 6800 SERIES)

• Confocal Raman spectrometer (Horiba Scientific, model. no. XploRA plus)

• Fourier-transform infrared (FTIR) spectrometer (JASCO, model. no. 6200)

• SEM (Carl-Zeiss, model. no. Ultra 55 field-emission SEM)

• Screwdriver (SATA, model. no. 08008)

• Micro-CT X-ray imaging system (version X-Tek HMXST 225)

• UV-vis spectrophotometry (Molecular Devices, model. no. SpectraMax M3)

Software

• For CAD: SolidWorks, 3ds Max, AutoCAD, Unigraphics NX

• For VBP control: MATLAB, Python

• For data analyses: Origin, GraphPad, SPSS

• For image analyses: ImageJ

VBP setup

• Volumetric bioprinter. The volumetric bioprinter is built and customized within our laboratory^10,11. The specifications of our customized bioprinter are as follows: i) an LED light engine with resolution of 50-μm pixel size (912×1140 pixels; PRO4500, Wintech, USA); ii) the LED light engine has a maximum output power of 1,100 mW; iii) a lens module (focal length 40 mm, working distance 230 mm, and field of view 20 × 12.5 mm²); iv) a transparent glass vial with a diameter of 12 mm, with a rotation mount at a 6.3° s⁻¹ of rotation rate. The building area of the transparent glass vial is 51.5 × 350 (R × H) mm³. The detailed information on VBP system designs including algorithms, software, scripts, hardware, and related specifications are recommended to follow this open resource: https://github.com/computed-axial-lithography/CAL-software-Matlab, which is readily accessible. The STL files of the main parts used for the VBP setup are provided as Supplementary Data 1–6. The workflow of VBP process is shown in Supplementary Fig. S1.

  ▲CRITICAL In this protocol and previous studies on VBP published by our laboratory^10,11, we built the VBP system instead of using any commercial ones. While this protocol may be adaptable to commercial bioprinters, the parameters likely need to be adjusted.

Procedure 1

Preparation of SS ● Timing 3 days

! CAUTION Wear appropriate PPE such as laboratory coat, gloves, and safety goggles.

! CAUTION Perform all functionalization steps in a chemical fume hood.

▲CRITICAL Ensure that all laboratory equipment and glassware are clean by rinsing with DW and drying them properly to avoid chemical contamination during degumming, dialysis, and lyophilization.

• 1Cut dry cocoons with scissors into dime-sized pieces and remove any silkworms in the cocoons. Weigh out 20 g of cocoon pieces into a large weighing dish.
• 2Prepare a 500-mL glass beaker filled with 200 mL of DW, cover it with a lid, and heat it up until boiling.

  ! CAUTION Do not leave the beaker unattended during heating.

• 3Weigh out 4.24 g of Na₂CO₃ (to prepare 0.04-M Na₂CO₃) into the boiling water and let it completely dissolve.

  ! CAUTION Ensure wearing PPE correctly and keep adding Na₂CO₃ slowly to avoid boiling over which would sputter from the boiling beaker.

• 4Add the cocoon pieces into the beaker and continue boiling for 30 min. Poke with two glass rods every 10 min to promote dispersion of the silk (Fig. 9a).

  ▲CRITICAL STEP For reproducibility, boil for exactly 30 min every time.

  ▲CRITICAL STEP Add more DW to make sure that the silk can be immersed entirely during the boiling process.

  ? TROUBLESHOOTING (Table 1)

• 5Pick up and move the silk with stainless-steel tongs. Squeeze excess DW out of the silk with hands while wearing Teflon gloves. Cool the SS and Na₂CO₃ solution.

  ! CAUTION The SF fibers and SS solution would be hot, hand-protectors such as Teflon gloves should be used.

• 6Purify the cold SS solution, centrifuge at 3000 g for 10 min at 4 °C to remove impurities.
• 7Hydrate four 200-mm-long 12-to-14-kDa dialysis bags in DW for 5 min.
• 8Double-knot one end of the membrane and introduce 50 mL of DW through the other end of the membrane.
• 9While holding the tied end of the dialysis bag with one hand, use the index and middle fingers of the other hand to close the open end of the tube. Invert the dialysis bag containing water 5 times to rinse the inside of the membrane before discarding the water.

  ▲CRITICAL STEP Check for any leakage in the membrane by gentle squeezing before proceeding to the next step. A fine spray of liquid indicates a hole in the membrane; discard and use a new membrane if any leaks are identified.

• 10Insert 50 mL of the solution from step 6 into each dialysis bag via a plastic funnel.
• 11Expunge all air from the dialysis bag and clip the open end of the membrane at more than 110 mm above the solution.
• 12Dialyze with at least 4 L of DW to remove Na₂CO₃ in a 5-L glass beaker at RT. Change water three times a day (for example, 10 am, 4 pm, and 10 pm or 9 am, 4 pm, and 9 pm daily) for 3 days.

  ▲CRITICAL STEP The aliquot volume in the membrane would increase during the dialysis process, ensure firm clipping to avoid spillage of the aliquot into the dialysis water.

• 13Separate the dialyzed solution from step 12, centrifuge at 3000 g for 20 min at 4 °C to discard the precipitate, and the supernatant is filtered with 0.22-μm filters.
• 14Insert 100 mL of the solution from step 13 into the dialysis bag via a plastic funnel and place it in the hood for concentrating.
• 15
  The final concentration (Z%) of SS solution is calculated by weighting the remaining mass (M g) after evaporating the water (N g) as follow:

  $$\frac{\mathrm{M}}{\mathrm{M}+\mathrm{N}}\mathrm{*}100\mathrm{\%}=\mathrm{Z}\mathrm{\%}\mathrm{o}\mathrm{f}\mathrm{S}\mathrm{S}$$

  ▲CRITICAL STEP It is recommended to dilute the final concentration based on the desired hydrogel concentration, e.g., 2.5% (w/v) or 5% (w/v).

• 16Store the solution from step 15 at 4 °C.

  ■ PAUSE POINT The SS solution can be stored at 4 °C for up to a week.

  ▲CRITICAL STEP SS will be denatured if it stays over 1 week, which potentially influences the printability. The method for evaluating the storability and stability of SS is provided in Supplementary Fig. S3, whereas the method for detecting the denatured SS is illustrated in Supplementary Fig. S4.

  ? TROUBLESHOOTING (Table 1)

Figure 9 |. Representative images corresponding to the troubleshooting Table 1.

a Complete and incomplete degumming of cocoons (Table 1, Procedure 1, step 4). b Degummed cocoons not thoroughly interacting with the LiBr solution (Table 1, Procedure 1, step 26). c Low-quality and high-quality proteins with the quick photocrosslinking test (Table 1, Procedure 1, step 35). d Incomplete and complete decellularization of h-dECM (Table 1, Procedure 1, step 40). e Printing results with low-quality SS (Table 1, Procedure 1, step 58). f Not entirely dissolved and entirely dissolved Ms-dECM (Table 1, Procedure 1, step 61). g Uneven and even cell distributions after bioprinting with cell-laden bioink (Table 1, Procedure 2, steps 9, 24). h Cell seeded evenly or unevenly on the printed screw structure (Table 1, Procedure 2, step 41). i Low cell density of hiPSC-CMs (Table 1, Procedure 2, step 89). c, f and i are adapted with permission¹¹, Copyright 2024, Wiley-VCH. h is adapted with permission¹⁰, Copyright 2023, Springer Nature.

Table 1.

Troubleshooting for Procedure 1
Step	Problem	Possible reason	Solution
4	Degumming of cocoons is not good. Cocoons are still hard after degumming and only little SS is taken off from cocoons.	The cocoons are not entirely immersed in water during the degumming process.	Cut the cocoons into smaller pieces and press them into the bottom of the beaker. During the degumming, poke with two glass rods every 10 min to promote good dispersion of cocoons and also to make sure all the cocoons are immersed in the boiling water.
16, 35, 46, 55	Protein quality between batches is not consistent.	The original cocoons’ quality is not same.	For quantifying the nitrogen content of protein, we recommend using the Kjeldahl method32. For quantifying tyrosine content in extracted proteins, we recommend using UV-vis spectroscopy33 and high-performance liquid chromatography34.
26	SF does not entirely dissolve in LiBr solution after 4 h.	SF are not thoroughly interacting with the LiBr solution.	Before adding the LiBr solution, press SF into the bottom of a 100-mL beaker to remove as much air as possible. Use a glass rod to stir the SF fibers. Replace the LiBr solution with a new container in case of water-uptake by the salt.
40	Good amount of tissue still looks red in the 1% (v/v) SDS/PBS buffer after 72 h.	The decellularization of heart tissue is not complete.	Make sure that the heart tissue slides are not too thick. Increase the tissue-to-solution ratio to 1:24 if necessary.
58	SS (bio)ink cannot be photocrosslinked.	The dialysis process of SS solution is not completed.	Separate dialysis bags into several 5-L dialysis beakers to make sure they are well-dialyzed.
61	Ms-dECM powders do not entirely dissolve in the pepsin/0.5-M acetic acid solution after 72 h.	The sizes of Ms-dECM powders are too large.	Remove the large pieces of Ms-dECM after serval grounding rounds.
64	Failure to form thermal dECM gel.	Incorrect pH-neutralization (proper pH: 7.2–7.4).	Slowly add 1-M sodium hydroxide solution drop by drop. Once the pH value exceeds 5, switch to using a 2-μL pipette for finer control.
74–87	There are some issues that might happen during the VBP process, such as: i) Uneven crosslinking of printed construct. ii) Crosslinking occurs on the surface of the printing vial. iii) Misalignment or distortion of printed construct.	i) The rotation speed of printing vial is not properly synchronized with the image projection rate, leading to misaligned light exposure during reconstruction. ii) The concentration of photoinitiator such as Ru/SPS is too high. iii) The concentration of cell embedded in the bioink is too high.	i) Ensure that the vial rotation speed matches the image-update frequency. ii) Test a proper concentration of photoinitiator before VBP. iii) Test a proper concentration of cell before VBP.
Troubleshooting for Procedure 2
9, 24	Encapsulated cells are not distributed evenly.	Insufficient mixing of the bioink and cells or the bioink is left for too long time before bioprinting.	Mix cell-laden bioink very well by gently and repetitively pipetting, and then transfer into the printing vial immediately.
9, 24, 64, 98	Resolution of bioprinting with cells is low.	Printing resolution is reduced with strong light scattering caused by the increasing cell concentration.	Reduce cell concentration, or when high cell densities are needed, introduce cytocompatible refractive index-matching compounds such as iodixanol7.
41	hMSCs are not seeded on the screw surface evenly.	In the second round of seeding incubation, the side of SF screws has not been changed.	Make sure the side of SF screws is changed and observes using a microscope, and to move the samples carefully and slowly.
89	There are no spontaneous beating movements after 5 days.	The culturing time is not sufficient. The cell density might be too low.	Culture the samples up to 2 weeks and observe them daily. Increase the cell density.

Preparation of SF ● Timing 3 days

! CAUTION Wear appropriate PPE such as laboratory coat, nitrile gloves, and safety goggles.

! CAUTION Perform all functionalization experiments in a chemical fume hood.

▲CRITICAL Ensure that all laboratory equipment and glassware are clean by rinsing with DW and drying them properly to avoid chemical contamination during degumming, dialysis, and lyophilization.

• 17Perform Procedure 1, steps 1–5.
• 18Place SF in a 5-L beaker filled with 5 L of DW.
• 19Place SF in DW for 15 min, squeeze it by hand and discard the DW.
• 20Repeat Procedure 1, steps 18 and 19.

  ! CAUTION Squeeze until there are no bubbles to ensure complete removal of Na₂CO₃ from SF.

• 21After the third-time wash, pick up the SF fibers, squeeze them well and then spread them out on clean pieces of large aluminum foil.
• 22Allow the SF fibers to dry overnight in chemical fume hood.

  ■ PAUSE POINT Dry SF fibers could be stored indefinitely at RT. For long-term storage, wrap them in a clean aluminum foil. Be sure to indicate the date and weight on the label.

• 23
  Calculate the amount (X g) of LiBr needed to prepare a 9.3-M LiBr stock as follows:

  $$\left(86.85\frac{\mathrm{g}}{\mathrm{m}\mathrm{o}\mathrm{l}}\right)\left(9.3\frac{\mathrm{m}\mathrm{o}\mathrm{l}}{\mathrm{L}}\right)\left(\frac{\mathrm{L}\mathrm{i}\mathrm{B}\mathrm{r}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}}{\mathrm{1,000}\mathrm{m}\mathrm{L}}\right)=\mathrm{X}\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{L}\mathrm{i}\mathrm{B}\mathrm{r}$$
• 24To prepare a 28-mL LiBr solution, dissolve 27.3 g of LiBr in a beaker containing 18 mL of DW. Stir gently with a stainless-steel spatula until completely dissolved, and then fill the volume to 28 mL with DW to produce the 9.3-M LiBr solution.

  ! CAUTION The dissolution of LiBr in DW is exothermic; be mindful of the heat generated. We recommend adding DW slowly and wearing protectors such as Teflon gloves.

  ▲CRITICAL STEP LiBr has a low density, and its volume should be considered while preparing the solution. Therefore, ensure that LiBr is dissolved completely before making the solution up to the final required volume.

• 25Soak 7 g of SF fibers in the beaker containing 28 mL of the LiBr solution to prepare a 25% (w/v) SF solution. Cover the beaker first with cling film then aluminum foil and transfer the beaker into an oven for 4 h of incubation at 60 °C.

  ▲CRITICAL STEP Ensure that SF is immersed completely in the LiBr solution. Use a 50-mL beaker that can hold both SF and LiBr solution without spillage so that LiBr solution will eventually cover the SF fibers. If a liquid overflow occurs or SF fibers cannot be completely covered, tear the SF fibers into multiple small pieces, and add them successively.

  ▲CRITICAL STEP Add a magnetic rotor to the LiBr solution or use glass rods to mix the solution every 10 min to accelerate the dissolution of SF fibers.

• 26Allow SF fibers to dissolve completely before proceeding to the next step. The obtained SF solution should be viscous and transparent amber in color, without a trace of any intact fiber (Fig. 9b).

  ? TROUBLESHOOTING (Table 1)

• 27Hydrate a 200-mm-long 12-to-14-kDa dialysis bag in DW for 5 min.
• 28Double-knot one end of the bag and introduce 50 mL of DW through the other end of the bag.
• 29While holding the tied end of the dialysis bag with one hand, use the index and middle fingers of the other hand to close the open end of the tube. Invert the dialysis bag containing water 5 times to rinse the inside of the bag before discarding the water.

  ▲CRITICAL STEP Check for any leakage in the bag by gentle squeezing before proceeding to the next step. A fine spray of liquid indicates a hole in the bag; discard and use a new bag if any leaks are identified.

• 30Insert the SF solution into the dialysis bag.
• 31Expunge all air from the dialysis bag and clip the open end of the bag at more than 110 mm above the solution.
• 32Dialyze with at least 4 L of DW to remove LiBr in a 5 L of glass beaker at 4 °C. Change water three times a day (for example, 10 am, 4 pm and 10 pm or 9 am, 4 pm and 9 pm daily) for at least 3 days.

  ▲CRITICAL STEP The aliquot volume in the bag would increase during the dialysis process, ensure firm clipping to avoid spillage of the aliquot into the dialysis water.

• 33Separate the dialyzed solution, centrifuge at 9000 g for 20 min at 4 °C to discard precipitate, and the supernatant is filtered with 0.22-μm filters.
• 34Insert 50 mL of the solution into the dialysis bag via a plastic funnel and place it in the fume hood for concentrating.
• 35
  The final concentration (Y%) of SF solution is calculated by weighing the remaining mass (M g) after evaporating water (N g) as follows (Fig. 9c):

  $$\frac{\mathrm{M}}{\mathrm{M}+\mathrm{N}}\mathrm{*}100\mathrm{\%}=\mathrm{Y}\mathrm{\%}\mathrm{o}\mathrm{f}\mathrm{S}\mathrm{F}$$

  ▲CRITICAL STEP It is recommended to dilute the final concentration based on the desired hydrogel concentration, e.g., 2.5% (w/v) or 5% (w/v).

  ? TROUBLESHOOTING (Table 1)

• 36Store the SF solution at 4 °C.

  ■ PAUSE POINT SF solution can be stored at 4 °C for up to a week.

  ▲CRITICAL STEP SF will be self-crosslinked if it stays over 1 week, which potentially influence the printability. The method for evaluating the storability and stability of SF is provided in Supplementary Fig. S3.

  ▲CRITICAL STEP Use SF and SS together would cause immunogenic reaction, and should be cautioned especially for in vivo applications.

Preparation of h-dECM ● Timing 2 weeks

• 37Separate the left ventricle from the whole porcine heart.

  ▲CRITICAL STEP Select the freshly sourced tissues carefully, adhering to a standardized protocol we refined specifically for the VBP purpose¹¹.

• 38The separated left ventricles are sliced into 1-mm thickness and stirred in 5 L of DW for 1 h to remove the blood.
• 39Weigh out 50 g of SDS into 5 mL 1× PBS solution (to prepare 1% (w/v) SDS/PBS) and let it completely dissolve.

  ! CAUTION SDS can cause respiratory tract irritation, including coughing, shortness of breath, and sore throat. Repeated exposure can lead to allergic respiratory reactions or decreased pulmonary function. Keep SDS away from skin, eyes, and clothing. Appropriate PPE should be worn when weighing it.

• 40Add the tissues into 1% (w/v) SDS/PBS in a tissue-to-solution ratio of 1:16 (w/v) for 72 h (Fig. 9d).

  ▲CRITICAL STEP The SDS solution should be refreshed every 24 h.

  ? TROUBLESHOOTING (Table 1)

• 41The tissues are removed and treated with 1% (w/v) Triton X-100 in 1× PBS for 1 h.

  ▲CRITICAL STEP The well-decellularized tissues would turn to white color. Remove the pieces that are still red or brown in color.

• 42Stir the tissue in isopropyl alcohol for 2 h.
• 43The decellularized tissues need to be immediately rinsed with 1× PBS for 72 h to remove all the residual detergent.

  ▲CRITICAL STEP PBS solution should be refreshed every 24 h.

• 44Transfer the decellularized tissues to 50 mL of tubes and freeze at −80 °C for overnight.
• 45Lyophilize the frozen tissue until entirely dried (~3 days).
• 46Submerge the material in liquid nitrogen for 2 min and ground the meniscal tissues into fine powders and store at −80 °C.

  ▲CRITICAL STEP It will take multiple rounds of grinding to obtain fine powders, which will be helpful for the following digestion process.

  ! CAUTION Wear PPE such as laboratory coat, thermal gloves, and goggles when using liquid nitrogen.

  ▲CRITICAL STEP To minimize variability across experiments, we recommend preparing h-dECM in large quantities following the optimized preparation method at one time.

  ■ PAUSE POINT The prepared h-dECM powder can be stored at −80 °C for at least 2 months.

  ? TROUBLESHOOTING (Table 1)

Preparation of Ms-dECM ● Timing 2 weeks

• 47Harvest the porcine meniscus from fresh knee joints of pigs.

  ▲CRITICAL STEP Select the freshly sourced tissues carefully, adhering to a standardized protocol we refined specifically for the VBP purpose¹¹.

• 48Cut the meniscus into thin slices at 1-mm thickness and clean in 5 L of DW for 1 h.
• 49Freeze the meniscus pieces at −80 °C for 2 h and ground into rough powders.
• 50Add the meniscus powders in 1% (w/v) SDS in PBS solution for 72 h.

  ! CAUTION SDS can cause respiratory tract irritation, including coughing, shortness of breath, and sore throat. Repeated exposure can lead to allergic respiratory reactions or decreased pulmonary function. Keep SDS away from skin, eyes, and clothing. Appropriate PPE should be worn when weighing it.

  ▲CRITICAL STEP The SDS solution should be refreshed every 24 h.

• 51The meniscus powder is treated with 0.1% (w/v) EDTA solution in PBS for 24 h.

  ▲CRITICAL STEP The well-decellularized tissues would turn to white color. Remove the pieces that are still red or brown in color.

• 52The treated tissue is rinsed in 1× PBS for 72 h to remove the residual chemicals.

  ▲CRITICAL STEP PBS solution should be refreshed every 24 h.

• 53Transfer the decellularized tissues to 50-mL tubes and freeze at −80 °C overnight.
• 54Lyophilize the frozen tissue until entirely dried (~3 days).
• 55Submerge the material in liquid nitrogen for 2 min and ground the meniscal tissues into fine powders and store at −80 °C.

  ▲CRITICAL STEP It will take multiple rounds of grinding to obtain fine powders, which will be helpful for the following digestion process.

  ! CAUTION Wear PPE such as laboratory coat, thermal gloves, and goggles when using liquid nitrogen.

  ▲CRITICAL STEP To minimize variability across experiments, we recommend preparing Ms-dECM in large quantities following the optimized preparation method at one time.

  ■ PAUSE POINT The prepared Ms-dECM powder can be stored at −80 °C for at least 2 months.

  ? TROUBLESHOOTING (Table 1)

Preparation of silk-based (bio)inks ● Timing 30 min to 1 h

• 56Dissolve 7.396 mg of Ru in 500 μL of PBS to prepare a 20-mM Ru stock solution; and dissolve 23.875 mg of SPS in 500 μL of PBS to prepare a 200-mM SPS stock solution.

  ▲CRITICAL STEP Ru/SPS can be activated by light, leading to a premature gelation of the (bio)ink. Hence, always block light when preparing photocurable (bio)ink (e.g., cover the container with aluminum foil or work in a dark room).

  ▲CRITICAL STEP Ensure that Ru/SPS dissolves completely; Ru and SPS should be added to (bio)inks at a fixed concentration ratio of 1:10 without premix. Insufficient dissolution will lead to poor VBP quality.

  ▲CRITICAL STEP Use PBS to dissolve Ru/SPS for maintaining a biologically compatible environment for cell-laden bioinks and following biomedical applications. It is also worth noting that the concentrations of Ru/SPS used here are way below the solubility limits.

• 57Dilute the high concentration of the above SS/SF solution with PBS to make 2.5–10% (w/v) SS or 2.5–5% (w/v) SF (bio)ink.

  ▲CRITICAL STEP The oxygen content within the SS or SF solution without adding photoinitiators should be equilibrated prior to printing by storing in a container for up to several days at 4 °C.

• 58By adding different volume of Ru/SPS stock solution into each SS or SF solution, different (bio)inks with gradient Ru/SPS concentrations from 0.125/1.25 mM to 2/20 mM are obtained (Fig. 9e).

  ? TROUBLESHOOTING (Table 1)

• 59Remove air bubble in the SS or SF (bio)ink by vertex and centrifuge for 30 s at 2000 g.

  ■PAUSE POINT The prepared (bio)ink can be stored for 2 to 4 h in the dark at RT or 4 °C.

Preparation of dECM (bio)ink ● Timing 3 days

• 60Dissolve h-dECM powder in pepsin/0.5-M acetic acid solution at 15 mg mL⁻¹ at RT for 72 h. The ratio of pepsin/h-dECM is 1:10 (w/w).
• 61Dissolve Ms-dECM powder in pepsin/0.5-M acetic acid solution at 15 mg mL⁻¹ at RT for 72 h. The ratio of pepsin/Ms-dECM is 1:10 (w/w) (Fig. 9f).

  ▲CRITICAL STEP The above solutions need to be placed on a shaker to ensure full dissolution of the material.

  ? TROUBLESHOOTING (Table 1)

• 62Centrifuge the solution at a speed of 3000 g for 20 min to remove the undigested parts of the material or other impurities.
• 63Collect the transparent dECM supernatant from the centrifuged solution and store at 4 °C.

  ▲CRITICAL STEP The oxygen content within the dECM solution without adding photoinitiators should be equilibrated prior to printing by storing in a container for several days at 4 °C.

• 64The dECM solution is neutralized (pH 7.4) using 10-N sodium hydroxide solution on the ice box.

  ▲CRITICAL STEP The neutralized dECM materials are thermosensitive, which may self-crosslink at RT, and therefore the materials should be kept in the ice box before the VBP process.

  ? TROUBLESHOOTING (Table 1)

• 65Add Ru/SPS stock solution into different concentrations of dECM solutions according to the predetermined formulations.

  ▲CRITICAL STEP Ru/SPS can be activated by light, leading to a premature gelation of the (bio)ink. Hence, always block light when preparing the (bio)ink (e.g., cover the container with aluminum foil or work in a dark room).

  ▲CRITICAL STEP Ensure that Ru/SPS dissolves completely; Ru and SPS should be added to the (bio)ink at a fixed concentration ratio of 1:10 without premix. Insufficient dissolution will lead to poor VBP quality.

• 66Remove air bubble in the dECM (bio)ink by vertex and centrifuge for 30 s at 2000 g.

  ■PAUSE POINT The prepared dECM (bio)ink can be stored for 2 to 4 h in the dark at RT or 4 °C.

Preparation of gelatin (bio)inks ● Timing 30 min

• 67Dissolve porcine gelatin powder (type A) in PBS.

  ▲CRITICAL STEP After adding PBS to the porcine gelatin powder, it should be kept in the 50 °C water bath for over 10 min to ensure complete dissolution.

  ▲CRITICAL STEP The porcine gelatin solution should be kept in 37 °C water bath before printing.

  ▲CRITICAL STEP The oxygen content within the gelatin solution without adding photoinitiators should be equilibrated prior to printing by storing in a container for several days at 4 °C.

• 68Add Ru/SPS stock solution at a fixed concentration ratio of 1:50 into different concentrations of gelatin solutions according to the predetermined formulations.
• 69Remove air bubble in the gelatin (bio)ink by vertex and centrifuge for 30 s at 2000 g.

  ■PAUSE POINT The prepared gelatin (bio)ink can be stored for 2 to 4 h in the dark at RT or 4 °C.

D_p assessment ● Timing 1.5 h

• 70Prepare 500 μL of (bio)inks with different formulations by adding gradient Ru/SPS from 0/0 to 1/10 mM.
• 71Add 100 μL of each formulation of the (bio)ink to a well of a transparent 96-well plate with three replicates.

  ▲CRITICAL STEP The volume height of the tested (bio)ink in the well is critical for calculating ${\mathrm{D}}_{\mathrm{p}}$.

• 72Measuring absorbance of (bio)inks using UV-vis spectrophotometry at a wavelength from 400 nm to 700 nm with a step size of 5 nm.
• 73
  ${\mathrm{D}}_{\mathrm{p}}$of 525-nm projected light into silk-based or dECM (bio)ink with or without Ru/SPS is calculated as follow:

  $$D_p=\frac{1}{\alpha }$$

  $$\alpha =\mathit{ln}\left(10\right)\times \left(\mathrm{A}/\mathrm{t}\right)$$

  ▲CRITICAL STEP ${\mathrm{D}}_{\mathrm{p}}$ is defined as the depth at which the intensity of the radiation inside the material falls to 1/e (roughly 37%) of its original value at (or more properly, just beneath) the surface.

  ▲CRITICAL STEP A is the absorbance value of materials at the 525-nm wavelength, and t is the height of the tested material.

VBP of silk-based (bio)inks ● Timing 30–120 s

▲CRITICAL It should be noted that for VBP, the printing time does not scale with the size of the print²⁹.

• 74Launch the printer and open the software.
• 75Import the standard triangle language (STL) file of the CAD model into the software, adjust the image, slice the STL file, and save it as a .mat file.

  ▲CRITICAL STEP Ensure that the printer is connected before clicking on the print icon.

• 76Set the printing parameters (such as printing time, LED power, see Procedure 1, VBP setup).

  ▲CRITICAL STEP The wavelength of printer is set at 525-nm wavelength, which can be replaced with other wavelengths depending on the photoinitiator used.

• 77Gently transfer the silk-based (bio)ink into the printing vial.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Use a 1-mL pipette to gently remove any bubble at the surface of the (bio)ink or inside the (bio)ink in the printing vial.

• 78Place the printing vial onto the printing stage of the VBP system.
• 79After printing, remove the printing vial from the printing stage and gently rinse the printed constructs twice with PBS to remove the un-crosslinked (bio)ink.

VBP of dECM (bio)inks ● Timing 30–120 s

• 80Perform Procedure 1, steps 74–76.
• 81Gently transfer the dECM (bio)ink into the printing vial.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Keep the dECM (bio)ink in icebox in dark. Remove any bubble in the vial.

• 82Place the printing vial onto the printing stage of the VBP system.
• 83After printing, remove the printing vial from the printing stage and gently rinse the printed constructs twice with PBS to remove the un-crosslinked (bio)ink.

VBP of gelatin (bio)inks ● Timing 30–120 s

• 84Perform Procedure 1, steps 74–76.
• 85Gently transfer the gelatin (bio)ink into the printing vial.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Use a 1-mL pipette to gently remove any bubble at the surface of the (bio)ink or inside the (bio)ink in the printing vial.

• 86Place the printing vial onto the printing stage of the VBP system.
• 87After printing, remove the printing vial from the printing stage and gently rinse the printed constructs twice with PBS to remove the un-crosslinked (bio)ink.

  ▲CRITICAL STEP The troubleshooting for the VBP process can be found in Table 1.

  ? TROUBLESHOOTING (Table 1)

Printability assessment ● Timing 3 h

• 88Print samples with different shapes under optimal conditions using a desired concentration of silk-based, dECM, or gelatin (bio)ink prepared in advance.
• 89Use the inverted fluorescence microscope at the suitable focal plane to take images and use a customized Python program to calculate the Jaccard similarity index to measure the similarity of printed construct compares to the CAD model³⁰.

  ▲CRITICAL STEP Jaccard similarity index ranges from 0 to 100%, and the higher percentage means higher similarity of printed construct compares to the CAD model, indicating better printability (Supplementary Fig. S2). The scripts (run by MATLAB) for printability assessment are provided in Supplementary Script. The CAD models (STL files) used for VBP in this protocol are provided in Supplementary Data 7–24.

Printing resolution assessment ● Timing 3 h

• 90Design a 3D model of a solid bar featuring a set of separated parallel threads of different thicknesses (from 1 μm to 101 μm) for Z-axis resolution test; and design a 3D model of a radially arranged array of cubes with different diameters for measuring the X-Y resolution (Fig. 4).
• 91Print the above constructs and take images using an inverted fluorescence microscope at the suitable focal plane to measure the smallest feature using ImageJ.

Mechanical property measurement ● Timing 2–3 h

• 92Load the compression force at a displacement rate of 5 mm min⁻¹ until the specimen (e.g., hydrogel disk with a diameter of 10 mm and a height of 5 mm) breaks, to acquire the stress (Pa) and strain (%) values from the compressive stress-strain curve at the point of failure.

  ▲CRITICAL STEP Ensure that the hydrogel surface is flat and parallel to the bottom of the movable platform.

• 93Examine the tensile strength on the UTM QMESYS with tensile jigs at a stretch velocity of 5 mm min⁻¹.
• 94Use a dumbbell-shaped column hydrogel (16 × 7 × 2 (L × W × H) mm³) to obtain the readings.

  ▲CRITICAL STEP To prevent drying out, all mechanical tests should be conducted at RT and a humidity of 80% when possible.

Raman spectroscopy ● Timing 2–3 h

• 95Freeze the volumetrically printed hydrogels at −80 °C for at least 12 h.
• 96Lyophilize the frozen hydrogel until entirely dried (~3 days).

  ! CAUTION Contact of liquid nitrogen with the skin or eyes may cause serious frostbite injury. Avoid any direct contact and wear appropriate PPEs.

• 97Raman spectra are obtained using a confocal Raman spectrometer.
• 98The Raman spectra are calibrated using a silicon wafer with a characteristic peak at 520 cm⁻¹ and are post-processed via the LabSpec 6 software.
• 99Place the black cap (a standard Raman spectrometer sample-holder cap with a central aperture designed for focusing the laser beam onto the specimen) with the small hole in the middle on the Raman probe head and the slice of dried hydrogel on the top of it.
• 100Initiate the Raman spectrometer and perform the spectral acquisition.

  ▲CRITICAL STEP For materials that exhibit weak Raman signals, a longer integration time is needed.

  ! CAUTION Too long integration time could lead to complete saturation of the detector.

  ▲CRITICAL STEP Collect multiple spectra from each sample.

FTIR spectroscopy ● Timing 3 h

• 101Freeze the volumetrically printed hydrogels at −80 °C for at least 12 h.
• 102Lyophilize the frozen hydrogel until entirely dried (~3 days).
• 103Break the sample into small pieces, approximately 3 mm in diameter.
• 104FTIR analyses are carried out on an FTIR spectrometer.

  ▲CRITICAL STEP Set 32 scans and a resolution of 4 cm⁻¹ to record the spectrum for each test.

  ▲CRITICAL STEP Collect multiple spectra from each sample.

• 105Test and measure samples in every different experimental condition.
• 106The secondary structure contents of SF are determined by performing peak deconvolution over the amide I region (1720 cm⁻¹ to 1580 cm⁻¹) using the Origin software.

SEM imaging ● Timing 1.5–3 h

• 107Freeze the volumetrically printed hydrogels at −80 °C for at least 12 h.
• 108Lyophilize the frozen hydrogel until entirely dried (~3 days).

  ▲CRITICAL STEP Critical point drying is used very often to better-preserve the structures since freeze-drying sometimes leads to artifacts in pore sizes.

• 109Break the sample to obtain a complete cross-section.

  ▲CRITICAL STEP Due to the difficulty in controlling the cross-section of the material, we recommend preparing multiple replicates and break the samples at different positions to obtain multiple cross-sections.

• 110Fix a carbon tape on a clean sample holder.
• 111Paste the hydrogel samples on the carbon tape-fixed sample holder.

  ▲CRITICAL STEP Lyophilization may result in a shrinkage of surface of the hydrogels. Therefore, expose their inner structures for SEM observation is needed.

• 112Coat the samples with a thin 5-nm layer of gold/palladium for 15 s at 15-mA discharge current with an ion-sputter.
• 113Take micrographs from each sample at an accelerating voltage of 1.2–1.3 kV under field-emission SEM.

Post-printing processing for inducing the double-crosslinked network of printed SF constructs ●Timing 3–4 days

• 114The volumetrically printed SF constructs (without cells) with single-photocrosslinked networks are immersed in 70% (v/v) ethanol aqueous solution to induce the formation of β-sheet conformation within the same SF constructs for 2 h (only for taking photographs after printing) or 24 h (for all other experiments related to induction of β-sheet conformation) at RT.
• 115The SF constructs possessing a double-crosslinked network are left in a chemical fume hood to allow evaporation of the remaining ethanol for 72 h.
• 116Harvest the dried SF constructs and store at RT.

Post-printing processing for shrinkage and expansion property evaluations of SS constructs ● Timing 2 days

• 117The volumetrically printed SS constructs (without cells) with a single photocrosslinked network are immersed in 100% (v/v) ethanol aqueous solution to induce the formation of β-sheet conformation within the same SS constructs for up to 24 h.
• 118Measure the dimensional changes in diameter, width, and height of the printed SS constructs by a vernier caliper at different time points.
• 119Treat the shrunken SS constructs with DW for up to 24 h.
• 120Measure the dimensional changes in diameter, width, and height of the re-expanded SS constructs by a vernier caliper at different time points.

In vitro water-uptake tests ● Timing 1 week

• Print cylinder constructs under optimal conditions using desired concentrations of the prepared silk-based and dECM (bio)inks (see Procedure 1).
• Transfer each printed construct into the well of a 6-well plate. Perform this procedure with three replicates.
• Pipette 10 mL of PBS into each well to immerse the construct, and then incubate at 37 °C.
• Soak the constructs in PBS for predetermined time points, where the changes in mass signify water-uptake of the samples.
• Weigh the harvested constructs to obtain swollen masses.

  ▲CRITICAL STEP Surface water of the constructs should be removed by gently blotting with filter paper before weighing.

• The wet mass of each construct is noted as $W_S$.
• Freeze the constructs overnight at −20 °C, and lyophilize for 3 days.
• The dry mass of the corresponding construct is noted as $W_d$.
• The water-uptake (%) is calculated as follows:

  $$\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{u}\mathrm{p}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}(\mathrm{\%})=(\frac{{\mathrm{W}}_{\mathrm{S}}-{\mathrm{W}}_{\mathrm{d}}}{{\mathrm{W}}_{\mathrm{S}}})\times 100\mathrm{\%}$$

In vitro degradation assay ● Timing 6 months for silk, 28 days for dECM

• The volumetrically printed silk-based constructs are transferred into PBS, or 5 U mL⁻¹ of protease XIV PBS solution, or 40 U mL⁻¹ of α-chymotrypsin PBS solution in the wells of 48-well plates at 37 °C.
• The volumetrically printed dECM constructs are transferred into PBS, or 5 U mL⁻¹ of collagenase IV PBS solution, or 40 U mL⁻¹ of α-chymotrypsin PBS solution in the wells of 48-well plates at 37 °C.

  ▲CRITICAL STEP Perform this procedure with three replicates and measure each sample at least three times to obtain an average value.

• The incubation solution is changed with fresh solution every 2 days.

  ▲CRITICAL STEP The samples are rinsed in DW twice at desired time points.

• Harvest the samples from the solutions at predetermined time points.
• Freeze the harvested sample at −80 °C for at least 12 h.
• Lyophilize the samples until completely dry (usually 3 days).
• The remaining dry mass of the samples are measured. The dry mass of each remaining sample at day n is noted as $W_n$ and the initial dry mass of the corresponding sample at 0 day is as $W_0$. The residue mass ratio (%) is calculated as follows:

  $$\mathbf{R}\mathbf{e}\mathbf{s}\mathbf{i}\mathbf{d}\mathbf{u}\mathbf{e}\mathbf{m}\mathbf{a}\mathbf{s}\mathbf{s}\mathbf{r}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\left(\mathrm{\%}\right)=({\mathit{W}}_{\mathit{n}}/{\mathit{W}}_{\mathbf{0}})\times \mathbf{100}\mathrm{\%}$$

Procedure 2

Preparation cell-laden SS or SF bioink ● Timing 2 h

▲CRITICAL For cell culture, work in a class II biological safety hood and use sterile instruments. Clean all working surfaces with 70% (v/v) ethanol and expose to UV light for at least 20 min before use.

▲CRITICAL Use 0.22-μm syringe filters to filter all reagents before mixing with bioinks. All the reagents used need to be sterile.

• 1Dissolve 7.396 mg of Ru in 500 μL of serum-free DMEM to prepare the 20-mM Ru stock solution, and dissolve 23.875 mg of SPS in 500 μL of serum-free DMEM to prepare the 200-mM SPS stock solution. Wrap both with aluminum foil.

  ▲CRITICAL STEP Ru/SPS can be activated by light, leading to a premature gelation of the (bio)ink. Hence, always block light when preparing the (bio)ink (e.g., cover the container with aluminum foil or work in a dark room).

  ▲CRITICAL STEP Ensure Ru/SPS dissolves completely; Ru and SPS should be added to the (bio)ink at a fixed concentration ratio of 1:10 without premix. Insufficient dissolution would lead to a poor VBP quality.

  ▲CRITICAL STEP Use serum-free cell culture medium for preparing the Ru/SPS solution and SS or SF solution instead of DPBS.

• 2Filter 20 mM of Ru and 200 mM of SPS solution using 0.22-μm syringe filter.
• 3Dilute the SS or SF solution with corresponding cell culture medium to make the desired concentration.

  ▲CRITICAL STEP To obtain sterile materials, the SS or SF solution should be filtered using the 0.22-μm syringe filter, and preparation of SS or SF solution should be performed in cell culture hood.

• 4Add the Ru stock solution with pipette to the SS or SF solution and invert the tube 3–5 times gently, and then add the SPS stock solution with pipette and invert the tube up and down 3–5 times gently to mix homogeneously in the dark at RT.

  ! CAUTION To ensure the effectiveness of bioinks containing the photoinitiator, finish the bioprinting procedure within 1 h after mixing with Ru/SPS.

  ▲CRITICAL STEP To prevent contamination, ensure that the lid is closed and wrap with aluminum foil.

  ▲CRITICAL STEP Make sure that no bubbles are induced when mixing Ru/SPS with the SS or SF solution.

• 5Use 0.25% (w/v) trypsin-EDTA solution to detach cells at 80% confluence.
• 6Terminate the cell trypsinization process with 10% (w/v) FBS in DMEM and transfer cell suspension to 15-mL conical tubes.
• 7Count the cells with a cell counter, and prepare a final resuspension at the required cell concentration.

  ▲CRITICAL STEP We recommend 5 × 10⁶ cells mL⁻¹ for C2C12 myoblasts, NIH/3T3 fibroblasts, and MDA-MB-231 breast cancer cells. Approximately 1 mL of the cell-laden bioink is needed to print five square samples with a size of 10 ×10 × 2 (W × L × H) mm³.

  ▲CRITICAL STEP The appropriate cell density depends on the type and size of cells used and should be determined experimentally. However, high cell densities (>5 × 10⁶ cells mL⁻¹) may affect the curing effect of bioink during the VBP process⁷.

• 8Centrifuge cells for 3 min at 1000 g.
• 9Aspirate the medium and re-suspend with the silk-based bioink (Fig. 9g).

  ■PAUSE POINT Keep the bioprinted samples briefly in the cell culture incubator before further use.

  ▲CRITICAL STEP For keeping high cell viabilities, print the prepared silk-based bioinks as soon as possible and then transfer the bioprinted silk-based constructs into cell culture medium quickly.

  ? TROUBLESHOOTING (Table 1)

Preparation of cell-laden dECM bioinks ● Timing 3 days

▲CRITICAL STEP For cell culture, work in a class II biological safety hood and use sterile instruments. Clean all working surfaces with 70% (v/v) ethanol and expose to UV light for at least 20 min before use.

▲CRITICAL STEP Use 0.22-μm syringe filters to filter all reagents before mixing with bioinks. All the reagents used need to be sterile.

• 10Prepare the Ru/SPS stock solutions as Procedure 2, steps 1 and 2.
• 11Place the dECM powder under UV light in the cell culture hood for 2 h.
• 12Dissolve h-dECM powder in pepsin/0.5-M acetic acid solution at 15 mg mL⁻¹ at RT for 72 h. The ratio of pepsin:h-dECM is 1:10 (w/w).
• 13Dissolve Ms-dECM powder in pepsin/0.5-M acetic acid solution at 15 mg mL⁻¹ at RT for 72 h. The ratio of pepsin:Ms-dECM is 1:10 (w/w).

  ▲CRITICAL STEP The tube needs to be placed on a shaker to ensure full dissolution of the material.

• 14Centrifuge the solution at a speed of 3000 g for 20 min to remove the undigested parts of the material or other impurities.
• 15Collect the transparent dECM supernatant from the centrifuged solution and store at 4 °C.
• 16The dECM solution is neutralized (pH=7.4) using the 10-N sodium hydroxide solution on the ice box.

  ▲CRITICAL STEP The neutralized dECM materials are thermosensitive, which may self-crosslink at RT, and therefore the materials should be kept in the ice box before the VBP process.

  ▲CRITICAL STEP The cold condition of dECM would typically not change within 2 min during the VBP process at RT, and thus the dECM materials should be printed once taking out from the ice box.

• 17Add 10× DPBS solution into the neutralized dECM solution at a ratio of 1:9 (v/v) to make sure that the pH value and osmotic pressure are suitable for cell-encapsulation and proliferation.
• 18Dilute the dECM solution from step 17 with corresponding cell culture medium to prepare the desired concentration (e.g., 1% (w/v) dECM is usually prepared).

  ▲CRITICAL STEP To obtain sterile materials, the dECM solution should be filtered using the 0.22-μm syringe filter, and preparation of dECM solution should be performed in cell culture hood.

  ▲CRITICAL STEP To prevent contamination, ensure that the lid is closed and wrap with aluminum foil.

• 19Add the Ru/SPS stock solution from Procedure 2, step 10 into different concentrations of dECM solutions according to the predetermined formulations.

  ! CAUTION To ensure the effectiveness of bioinks containing the photoinitiator, finish the bioprinting procedure within 1 h after mixing with Ru/SPS.

• 20Use 0.25% (w/v) trypsin-EDTA solution to detach cells at 80% confluence.
• 21Terminate the cell trypsinization with 10% (w/v) FBS in DMEM and transfer cell suspension to 15-mL conical tubes.
• 22Count the cells with a cell counter, and prepare a final resuspension at the required cell concentration.

  ▲CRITICAL STEP We recommend 1 × 10⁶ cells mL⁻¹ for NIH/3T3 fibroblasts, rCMs, and hMSCs. Approximately 1 mL of cell-laden bioink is needed to print five square samples with a dimension of 10 ×10 × 2 (W × L × H) mm³.

  ▲CRITICAL STEP The appropriate cell density depends on the type and size of cells used and should be determined experimentally. However, high cell densities (>5 × 10⁶ cells mL⁻¹) may affect the curing effect of bioink during the VBP process⁷.

• 23Centrifuge cells for 3 min at 1000 g.
• 24Aspirate the medium and re-suspend with the prepared sterilized dECM bioink (Fig. 9g).

  ■PAUSE POINT Keep the bioprinted samples briefly in the cell culture incubator before further use.

  ▲CRITICAL STEP For keeping high cell viabilities, print the prepared dECM bioinks as soon as possible and then transfer the bioprinted constructs into cell culture medium quickly.

  ? TROUBLESHOOTING (Table 1)

VBP of silk screw-like constructs ● Timing 45 s

• 25Perform Procedure 1, steps 74–76.
• 26Gently introduce 2.5% (w/v) silk (bio)ink into the printing vial.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Use a 1-mL pipette to gently remove any bubble at the surface of the (bio)ink or inside the (bio)ink in the printing vial.

• 27Place the printing vial onto the printing stage of the VBP system.

  ▲CRITICAL STEP The screw-like construct is printed using 2.5% (w/v) SF with 0.25/2.5-mM Ru/SPS for 45 s of printing time or 2.5% (w/v) SS with 0.5/5-mM Ru/SPS for 45 s of printing time.

• 28After printing, remove the printing vial from the stage and rinse the printed construct twice for 2 min with growth medium to remove the un-crosslinked SF (bio)ink.
• 29The SF screw-like constructs (without cells) with a single photocrosslinked network are immersed in 70% (v/v) ethanol aqueous solution to induce the formation of β-sheet conformation within the same SF prints for 2 h at RT.
• 30The same SF screw-like constructs with double-crosslinked networks are left in a chemical fume hood to evaporate the remaining ethanol overnight for further use.

Cell seeding and cell proliferation assessments ● Timing 1 day (with additional 7 days of cell proliferation)

▲CRITICAL The cells are seeded on the surface of printed SF screws, because the double-crosslinked networks of SF screws are induced by immersing in 70% (v/v) ethanol aqueous solution after printing, which is fatal to living cells.

• 31Use 0.25% (w/v) trypsin-EDTA solution to detach hMSCs at 80% confluence from culture dish.
• 32Count the cell umber using a cell counter, and prepare a final suspension with hMSC growth medium at the required cell concentration.
• 33Place the SF screw-like constructs in the wells of 48-well plates and cover them with hMSC suspension (1×10⁷ cells mL⁻¹). Keep the plates in a cell incubator for 3 h.
• 34Use autoclaved tweezer to move the SF screw-like constructs to new wells and turn them over. Add hMSC suspension (1×10⁷ cells mL⁻¹) to seed the cells again. Keep the plates in a cell incubator overnight.
• 35Transfer the SF screw-like constructs to new wells and wash twice with DPBS.
• 36Add 1 mL of hMSC growth medium, and culture for 7 days.

  ▲CRITICAL STEP Change the medium every 24 h.

• 37Fix the printed SF screw-like constructs at days 1, 4, and 7 with 10% (w/v) formaldehyde solution for 20 min.
• 38Permeabilize with 0.1% (w/v) Triton X-100 in DPBS for 10 min at RT.
• 39After blocking with 1% (w/v) BSA in DPBS at 4 °C overnight, the samples are separately incubated with the Alexa Fluor 488-phalloidin DPBS solution at a ratio of 1:1000 (v/v) at 4 °C overnight.
• 40Wash the sample twice and stain with the DAPI DPBS solution at a ratio of 1:5000 (v/v) for 15 min at RT.
• 41Capture the fluorescence micrographs using an inverted fluorescence microscope to check the cell morphologies (Fig. 9h).

  ? TROUBLESHOOTING (Table 1)

Cell differentiation and immunostaining ● Timing 4 weeks

• 42Change the hMSC growth medium to the hMSC osteogenic differentiation medium after day 7.
• 43Fix the SF screw-like constructs at days 1, 7, 14, 21, and 28 with 10% (w/v) formaldehyde solution for 20 min and permeabilizes with 0.1% (w/v) Triton X-100 in DPBS for 10 min at RT.
• 44After blocking with 1% (w/v) BSA in DPBS at 4 °C overnight, the samples are separately incubated with the mouse anti-RUNX2 antibody and mouse anti-osteocalcin antibody solutions at 4 °C overnight.

  ▲CRITICAL STEP The mouse anti-RUNX2 antibody DPBS solution is prepared at a ratio of 1:500 (v/v) and the mouse anti-osteocalcin antibody DPBS solution is prepared at a ratio of 1:1000 (v/v), or according to manufacturers’ instructions.

• 45The samples that stained with primary antibodies are then stained with the secondary antibody (goat anti-mouse IgG H&L) DPBS solution at a ratio of 1:400 (v/v) after washing with DPBS for three times.
• 46The samples are incubated with the DAPI DPBS solution at a ratio of 1:5000 (v/v) for 15 min.
• 47Capture the fluorescence micrographs using an inverted fluorescence microscope to check the cell differentiation status.

RNA-isolation and real-time reverse transcription qPCR ● Timing 4 weeks

• 48RNAs of differentiated hMSC at days 1, 7, 14, 21, and 28 are extracted using the RNeasy mini kit.
• 49All the extracted RNAs are reversed-transcribed into complementary DNAs (cDNAs) by using the QuantiTect reverse transcription kit.
• 50qPCR is performed using the SYBR Green qPCR Master Mix.
• 51
  The expressions of each target messenger RNA (mRNA) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in different days are calculated based on the threshold cycle (Ct) as 2-Δ(ΔCt), where:

  $$\mathbf{\Delta }\mathbf{C}\mathbf{t}=\mathbf{C}\mathbf{t}\left(\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\right)-\mathbf{C}\mathbf{t}\left(\mathbf{G}\mathbf{A}\mathbf{P}\mathbf{D}\mathbf{H}\right)$$

  $$\mathbf{\Delta }\left(\mathbf{\Delta }\mathbf{C}\mathbf{t}\right)=\mathbf{\Delta }\mathbf{C}\mathbf{t}\left(\mathbf{c}\mathbf{e}\mathbf{l}\mathbf{l}\mathbf{s}\mathbf{u}\mathbf{n}\mathbf{d}\mathbf{e}\mathbf{r}\mathbf{g}\mathbf{o}\mathbf{i}\mathbf{n}\mathbf{g}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{e}\mathbf{o}\mathbf{g}\mathbf{e}\mathbf{n}\mathbf{i}\mathbf{c}\mathbf{d}\mathbf{i}\mathbf{f}\mathbf{f}\mathbf{e}\mathbf{r}\mathbf{e}\mathbf{n}\mathbf{t}\mathbf{i}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}\right)-\mathbf{C}\mathbf{t}\left(\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}\right)$$
• 52Quantitative gene-expression levels are measured from three replicate samples at days 1, 7, 14, 21, and 28.

  ▲CRITICAL STEP The expression at day 1 is set as the control group for each gene.

Ex vivo implantation ● Timing 1 week

• 53Fresh porcine femur is purchased from a local supermarket.
• 54Remove the meat from the femur.

  ▲CRITICAL STEP Make sure that the meat is properly removed, and the bones are exposed to the air.

  ! CAUTION When using a knife, be careful to avoid cutting.

• 55A transparent cap with a notch is attached onto the head of each printed SF screw-like construct to assist implantation.
• 56The femur is pre-drilled using a drill bit (1-mm-wide) to create holes (1-mm-deep) that are slightly smaller than the diameter of the SF screw-like constructs.
• 57Use a screwdriver to implant the printed SF screw-like constructs into the femur.
• 58The SF screw-like construct-implanted porcine femur is examined with a micro-CT X-ray imaging system.
• 59Micro-CT scanning is performed using a microfocus reflection tungsten X-ray source with tube voltage of 70 kV and target current of 180 μA (12.6 W).
• 603142 projection images are captured per sample on a 16-bit 2000 × 2000 flat panel detector with each exposure time at 1.0 s. The achievable voxel resolution is 24 μm.

VBP of heart-like constructs with cell-laden h-dECM bioink ● Timing 45 s

• 61Perform Procedure 1, steps 74–76.
• 62Gently introduce rCM-laden or hiPSC-CM-laden 1% (w/v) h-dECM bioink into the printing vial.

  ▲CRITICAL STEP rCMs or hiPSC-CMs are embedded at 1 × 10⁶ cells mL⁻¹.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Use a 1-mL pipette to gently remove any bubble at the surface of the (bio)ink or inside the (bio)ink in the printing vial.

  ▲CRITICAL STEP To prevent premature thermo-crosslinking of the dECM bioinks at RT, we recommend maintaining the bioinks at a low temperature (~4 °C) by chilling them on ice prior to the printing process.

  ▲CRITICAL STEP After thawing, hiPSC-CMs are centrifuged and immediately mixed with 1% (w/v) h-dECM bioink and used for bioprinting without additional culturing.

• 63Place the printing vial onto the printing stage of the VBP system.

  ▲CRITICAL STEP The heart-like construct is volumetrically bioprinted with 0.5/5-mM Ru/SPS for 45 s of printing time.

• 64After printing, remove the printing vial from the stage and rinse the printed construct twice with growth medium to remove the uncrosslinked cell-laden bioink.

  ? TROUBLESHOOTING (Table 1)

Live/dead assay ● Timing 1 h, culture up to 2 weeks

• 65The volumetrically bioprinted rCM-laden heart-like constructs are cultured in the growth medium for 2 weeks.
• 66Harvest samples from the growth medium at predetermined time points (e.g., days 1, 3, 5, 7, 10, and 14).
• 67The samples are rinsed twice with DPBS and places in the 24-well plate.
• 68Add the staining solution in DPBS containing 2 μL mL⁻¹ of ethidium homodimer and 1 μL mL⁻¹ of calcein-AM to the samples in the 24-well plate.
• 69Place the samples in the incubator at 37 °C for 30 min.
• 70The samples are rinsed with DPBS twice and fluorescence images are taken on an inverted fluorescence microscope.

  ▲CRITICAL STEP The numbers of live and dead cells are quantified with ImageJ.

Cell viability assay ● Timing 1 h

• 71Export the detected image to ImageJ.
• 72Count the number of live and dead cells through ImageJ by clicking on Plugins, Analyze, Cell Counter and Initialize.
• 73
  Calculate the percentage viability (%V) as follows:

  $$\mathrm{\%}\mathrm{V}=\frac{\mathrm{L}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\mathrm{*}100\mathrm{\%}$$

  ■PAUSE POINT The samples can be stored in DPBS for approximately 5 days at 4 °C. However, the fluorescence signal of samples would reduce with time.

Proliferation assay ● Timing 4 h, culture up to 2 weeks

• 74The volumetrically bioprinted rCM-laden heart-like constructs are cultured in the growth medium for 2 weeks.
• 75Harvest samples from the growth medium at predetermined time points (e.g., days 1, 3, 5, 7, 10, and 14).
• 76The samples are rinsed twice with DPBS and placed individually in the wells of a 24-well plate.
• 77Add the staining solution in culture medium containing the MTS reagent into each well.
• 78Place the samples in the incubator at 37 °C for 3 h.
• 79Set the UV-vis spectrophotometry at 490 nm.
• 80Harvest the supernatants of each well to quantify from the UV-vis spectrophotometry.

  ▲CRITICAL STEP Similarly, the metabolic activities of samples can be also measured by the PrestoBlue reagent according to the manufacturer’s instructions.

F-actin staining ● Timing 2 days, culture up to 2 weeks

• 81The volumetrically bioprinted rCM-laden heart-like constructs are cultured in the growth medium for 2 weeks.
• 82Harvest samples from the growth medium at predetermined time points (e.g., days 1, 3, 5, 7, 10, and 14).

  ▲CRITICAL STEP The incubation solution is changed with fresh solution every 2 days.

• 83Fix the cardiac samples with 10% (v/v) formalin for 20 min at RT.
• 84The samples are permeated with the 0.1% (w/v) Triton X-100 DPBS solution for 15 min at RT.
• 85Block the samples with the 1% (w/v) BSA DPBS solution at 4 °C overnight.
• 86The samples are incubated with the Alexa Fluor 488-phalloidin DPBS solution at a ratio of 1:1000 (v/v) at 4 °C overnight.
• 87The samples are stained with the DAPI DPBS solution at a ratio of 1:5000 (v/v) for 15 min at RT and use an inverted fluorescence microscope to capture the fluorescence micrographs.

Maturation and synchronized contractions of hiPSC-CMs ● Timing 10 days

• 88Relace the growth medium with cardiomyocyte medium and culture the samples for up to 10 days.
• 89Observe the spontaneous beating movements of hiPSC-CMs every day under bright-filed microscopy (Fig. 9i).

  ▲CRITICAL STEP Since the qualities of hiPSC-CMs in different batches are not consistent, the exact start day of the spontaneous beating could be different. Check all cell clusters in the heart-like construct under microscope everyday carefully. Usually, the first spontaneous beating movement would happen at approximately day 5.

  ? TROUBLESHOOTING (Table 1)

Immunostaining of differentiated hiPSC-CMs ● Timing 2 days

• 90After observing spontaneous beating movements, fix the heart-like construct at day 10 with 10% (w/v) formaldehyde solution for 20 min and permeabilized with 0.1% (w/v) Triton X-100 in DPBS for 10 min at RT.
• 91After blocking with 1% (w/v) BSA in DPBS at 4 °C overnight, the samples are separately incubated with the anti-sarcomeric-α-actinin antibody solutions at 4 °C overnight.

  ▲CRITICAL STEP The anti-sarcomeric-α-actinin DPBS solution is prepared at a ratio of 1:500 (v/v), or according to manufacturer’s instructions.

• 92The samples that are stained with primary antibodies are then stained with the secondary antibody (goat anti-mouse IgG H&L) DPBS solution at a ratio of 1:400 (v/v) after washing with DPBS for three times.
• 93The samples are incubated with the DAPI DPBS solution at a ratio of 1:5000 (v/v) for 15 min.
• 94Capture the fluorescence micrographs using an inverted fluorescence microscope to check the cell differentiation status.

VBP of meniscus-like constructs with Ms-dECM bioink ● Timing 30 s

• 95Perform Procedure 1, steps 74–76.
• 96Gently transfer the hMSC-laden 1% (w/v) Ms-dECM bioink into the printing vial.

  ▲CRITICAL STEP hMSCs are embedded within 1% (w/v) Ms-dECM at 5 × 10⁶ cells mL⁻¹.

  ▲CRITICAL STEP The volume of (bio)ink loaded into the printing vial is typically 1.5 mL.

  ▲CRITICAL STEP Use a 1-mL pipette to gently remove any bubble at the surface of the (bio)ink or inside the (bio)ink in the printing vial.

  ▲CRITICAL STEP To prevent premature thermo-crosslinking of dECM bioink at RT, we recommend maintaining the bioinks at a low temperature (~4 °C) by chilling them on ice prior to the printing process.

• 97Place the printing vial onto the printing stage of the VBP system.

  ▲CRITICAL STEP The artificial meniscus-like construct is volumetrically bioprinted with 0.25/2.5-mM Ru/SPS for 30 s of printing time.

• 98After printing, remove the printing vial and rinse the printed hydrogel twice with growth medium to remove the uncrosslinked cell-laden bioink.

  ? TROUBLESHOOTING (Table 1)

Live/dead assay ● Timing 1 h, culture up to 2 weeks

• 99The volumetrically bioprinted hMSC-laden meniscus constructs are cultured in the growth medium up to 2 weeks.
• 100Perform Procedure 2, steps 66–70.

Cell viability assay ● Timing 1 h

• 101Export the detected image to ImageJ.
• 102Perform Procedure 2, steps 72–73.

Proliferation assay ● Timing 4 h, culture up to 2 weeks

• 103The volumetrically bioprinted hMSC-laden meniscus constructs are cultured in the growth medium up to 2 weeks.
• 104Perform Procedure 2, steps 75–80.

F-actin staining ● Timing 2 days, culture up to 2 weeks

• 105The volumetrically bioprinted hMSC-laden meniscus constructs are cultured in the growth medium up to 2 weeks.
• 106Perform Procedure 2, steps 82–87.

RNA-isolation and real-time reverse transcription qPCR ● Timing 4 weeks

• 107The volumetrically bioprinted hMSC-laden meniscus constructs are cultured in chondrogenic differentiation medium for 21 days.
• 108Harvest the meniscus samples at days 1, 7, 14, and 21 from the chondrogenic differentiation medium.

  ▲CRITICAL STEP The incubation solution is changed with fresh solution every 2 days.

• 109Incubate the meniscus samples with collagenase IV solution at 37 °C for 30 min for digestion of the dECM.
• 110Centrifuge the digestion solution at 1000 g for 5 min and collect the cells.
• 111Use RNeasy mini kit to extract the mRNAs of the cells according to the manufacturer’s instructions.
• 112Perform Procedure 2 steps 48–52.

Histological evaluations ● Timing 24 h, culture up to 3 weeks

• 113The volumetrically bioprinted hMSC-laden meniscus-like constructs are cultured in the growth medium for 14 days and in the chondrogenic differentiation medium for 21 days.
• 114Harvest the samples at day 14 from growth medium and at days 7, 14, and 21 from the chondrogenic differentiation medium.

  ▲CRITICAL STEP The incubation solution is changed with fresh solution every 2 days.

• 115Fix the meniscus samples in 10% (v/v) formalin for 24 h.
• 116The fixed samples are dehydrated and embedded in paraffin wax.
• 117The paraffin wax is serially sectioned (5 mm in thickness) for histological analyses.
• 118The sections are rehydrated and stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Sirius red.
• 119Use a bright-field microscope to take images of the sections.

Troubleshooting

Troubleshooting advice can be found in Table 1.

Timing

Timing for Procedure 1.

Steps 1–16, Preparation of SS ● 3 days

Steps 17–36, Preparation of SF ● 3 days

Steps 37–46, Preparation of h-dECM ● 2 weeks

Steps 47–55, Preparation of Ms-dECM ● 2 weeks

Steps 56–59, Preparation of silk-based (bio)inks ● 30 min to 1 h,

Steps 60–66, Preparation of dECM (bio)ink ● 3 days

Steps 67–69, Preparation of gelatin (bio)inks ● 30 min

Steps 70–73, D_p assessment ● 1.5 h

Steps 74–79, VBP of silk-based (bio)inks ● 30–120 s

Steps 80–83, VBP of dECM (bio)inks ● 30–120 s

Steps 84–87, VBP of gelatin (bio)inks ● 30–120 s

Steps 88–89, Printability assessment ● 3 h

Steps 90–91, Printing resolution assessment ● 3 h

Steps 92–94, Mechanical property measurement ● 2–3 h

Steps 95–100, Raman spectroscopy ● 2–3 h

Steps 101–106, FTIR spectroscopy ● 3 h

Steps 107–113, SEM imaging ● 1.5–3 h

Steps 114–116, Post-printing processing for inducing the double-crosslinked network of printed SF constructs ● 3–4 days

Steps 117–120, Post-printing processing for shrinkage and expansion property evaluations of SS constructs ● 2 days

Steps 121–129, In vitro water-uptake tests ● 1 week

Steps 130–136, In vitro degradation assay ● 6 months for silk, 28 days for dECM

Timing for Procedure 2

Steps 1–9, Preparation cell-laden SS or SF bioink ● 2 h

Steps 10–24, Preparation of cell-laden dECM bioinks ● 3 days

Steps 25–30, VBP of silk screw-like construct ● 45 s

Steps 31–41, Cell seeding and cell proliferation assessments ● 1 day (with 7-days cell proliferation)

Steps 42–47, Cell differentiation and immunostaining ● 4 weeks

Steps 48–52, RNA-isolation and real-time reverse transcription qPCR ● 4 weeks

Steps 53–60, Ex vivo implantation ● 1 week

Steps 61–64, VBP of heart-like constructs with cell-laden h-dECM bioink ● 45 s

Steps 65–70, Live/dead assay ● 1 h, culture up to 2 weeks

Steps 71–73, Cell viability assay ● 1 h

Steps 74–80, Proliferation assay ● 4 h, culture up to 2 weeks

Steps 81–87, F-actin staining ● 2 days, culture up to 2 weeks

Steps 88–94, Maturation and synchronized contractions of hiPSC-CMs ● 10 days

Steps 95–98, VBP of meniscus-like constructs with Ms-dECM bioink ● 30 s

Steps 99–100, Live/dead assay ● 1 h, culture up to 2 weeks

Steps 101–102, Cell viability assay ● 1 h

Steps 103–104, Proliferation assay ● 4 h, culture up to 2 weeks

Steps 105–106, F-actin staining ● 2 days, culture up to 2 weeks

Steps 107–112, RNA-isolation and real-time reverse transcription qPCR ● 4 weeks

Steps 113–119, Histological evaluations ● 24 h, culture up to 3 weeks

Anticipated results

This protocol outlines a method for rapid VBP of unmodified protein-based (bio)inks with tyrosine groups, including those based on silk, dECM, and gelatin, using Ru/SPS photoinitiator system to form sophisticated shapes and architectures. We anticipate that these methods will be useful for bioprinting of in vitro models embedded with living cells.

The results in Fig. 7 show that both 2.5% SS and 2.5% SF constructs support better proliferation rates and viabilities than constructs with higher silk concentrations (5% SS, or 5% and 10% SF) (Fig. 7g, h, k, l). The cytocompatibility of low-concentration (2.5%) SS and SF bioinks makes them suitable for VBP applications where living cells are encapsulated. Of note, unlike SF, the SS constructs support desired activities (such as spreading) of encapsulated cells, implying a more promising biomedical potential than SF for cell culture-related applications. The cell proliferation rates and metabolic activities within both 1% h-dECM and 1% Ms-dECM with 0.25/2.5-mM Ru/SPS increase over 14 days, while the metabolic activities of cells with 0.5/5-mM Ru/SPS remain steady in the late stage (Fig. 7i, j), suggesting that the cells stop proliferating or reduce their metabolic activities after 7 days. Additionally, the viabilities of cells embedded in both 1% h-dECM and 1% Ms-dECM bioprinted with 0.25/2.5-mM Ru/SPS are higher than those for the cells in dECM bioprinted with 0.5/5-mM Ru/SPS (Fig. 7m, n), likely due to the reduced phototoxcity when a lower concentration of photoinitiator is used.

hMSCs exhibit nearly 100% proliferation and viability over 7 days, indicating the cytocompatibility of the double-crosslinked SF screw-like constructs. Extensive cell spreading is observed on the screw surfaces. These SF screw-like constructs, treated with 70% (w/v) ethanol and air-dried, are successfully tightened into the cortical bone without breakage or deformation, demonstrating their potential for bone implantation and device fixation. These observations highlight the feasibility and functionality of double-crosslinked SF screw-like constructs for biomedical applications (Fig. 8a). The protocol further integrates hiPSC-CMs into the bioprinted h-dECM constructs, which demonstrates structural maturation and spontaneous beating, suggesting the functional potential of these bioinks for cardiac tissue engineering (Fig. 8b). High cell viability (~95%) is sustained over 14 days, revealing the cytocompatibility of Ms-dECM. Constructs bioprinted with 0.25/2.5-mM Ru/SPS show nearly double the metabolic activities compared to those at 0.5/5 mM. Extensive cell spreading (~100%) is observed in both constructs. Further evaluation reveals successful chondrogenic differentiation, with increased expressions of collagen and aggrecan, supported by immunostaining and quantitative polymerase chain reaction (qPCR) results. Histological staining confirms the presence and intensification of cartilage extracellular components over time, suggesting potential applications in cartilage tissue engineering (Fig. 8c).

Furthermore, our experience indicates that these protein-based (bio)inks should be prepared and treated very carefully to avoid unsatisfied results (Fig. 9a–i). Therefore, we recommend keeping biomaterial sources such as cocoons, dECM, and gelatin as much consistent as possible between batches (or sourcing/preparing a larger batch for all key direct-comparison experiments), and quantifying the critical components in these proteins before use (Table 1, Procedure 1, steps 16, 35, 46, 55). There are some challenges that might occur during the VBP process, such as inhomogeneous crosslinking of (bio)printed construct, crosslinking on the surface of the printing vial, misalignment or distortion of the (bio)printed construct, and low resolution of bioprinting with cells, for which some possible solutions have been presented (Table 1, Procedure 1, steps 74–87; Procedure 2, steps 9, 24, 64, 98). Overall, this protocol aims to provide step-by-step instructions to maximize the success of VBP of pristine protein-based (bio)inks that contain tyrosine groups.