Development of a compartmentalized silk fibroin–reinforced GelMA hydrogel platform for kidney-inspired tissue engineering

Abstract

Chronic kidney disease (CKD) and the limited availability of donor organs continue to drive the need for advanced biomaterial systems for renal tissue engineering and in vitro kidney modeling. However, reproducing the spatial complexity and compartmentalized organization of native renal tissue remains a significant challenge. In this study, a compartmentalized silk fibroin (SF)-reinforced gelatin methacrylate (GelMA) hydrogel platform with spatially defined architectures inspired by renal tissue organization was developed. Initially, SF-reinforced GelMA hydrogels with tunable stiffness were fabricated by varying the concentration of the hydroxyethyl methacrylate (HEMA) crosslinker, while SF was incorporated at concentrations of 25 mg/mL and 50 mg/mL to systematically modulate the physicochemical properties of the hydrogels. The resulting formulations were characterized through degradation and swelling analyses, along with preliminary in vitro cytocompatibility studies. Degradation studies demonstrated that low-stiffness hydrogels exhibited more controlled degradation behavior compared to high-stiffness formulations, particularly at higher SF concentrations, indicating improved structural stability. Swelling analysis revealed that all hydrogel groups supported fluid uptake, with hydrogels containing 25 mg/mL SF exhibiting comparatively stable swelling and degradation profiles. In vitro studies confirmed that all scaffold formulations supported cellular attachment and exhibited non-toxic behavior. Based on the optimized hydrogel formulation, a two-zone compartmentalized platform was subsequently developed consisting of a central triangular-patterned region inspired by the renal medulla and a surrounding honeycomb-patterned region representing the renal cortex. Confocal imaging demonstrated spatial organization of L929 fibroblasts within the patterned hydrogel architecture, while depth-coded three-dimensional confocal imaging of nuclei-stained renal proximal tubular epithelial cells (RPTECs) at days 1, 3, and 5 enabled visualization of cell localization within the engineered patterned regions. Collectively, this study demonstrates the feasibility of developing a compartmentalized SF–GelMA hydrogel platform with tunable material properties and spatially organized architectures inspired by renal tissue organization. The platform provides a promising foundation for future studies involving kidney-specific cell populations and region-specific renal tissue engineering applications.

1. Introduction

Chronic kidney disease (CKD) affects approximately one in ten individuals worldwide and is projected to become the fifth leading cause of years of life lost by 2040 (Miranda et al., 2022). Current therapeutic strategies, including dialysis and kidney transplantation, remain limited by donor organ scarcity, high treatment costs, and significant impacts on patients’ quality of life (Nyokabi et al., 2024). The increasing global burden of CKD, coupled with associated comorbidities, underscores the urgent need for alternative and advanced therapeutic approaches such as engineered renal constructs. Renal tissue engineering seeks to recreate functional kidney tissue by investigating cellular behavior and functionality within biomimetic scaffold environments that closely replicate native extracellular matrix and microenvironmental cues (Syed Mohamed et al., 2023). The development of functional renal tissue constructs, which aim to replace, restore, or enhance the biological function of damaged renal tissue, remains a critical challenge in regenerative medicine due to the kidney’s complex architecture, functional heterogeneity and limited self-repair capacity (Rayat Pisheh et al., 2024). Successfully recapitulating this intricate renal architecture would represent a significant milestone in regenerative medicine. Achieving this goal requires a fundamental understanding of how diverse renal cell populations interact with one another and with the extracellular matrix (ECM), as these cell–cell and cell–ECM interactions play a pivotal role in guiding tissue organization, function, and regeneration (Wang et al., 2024).

In this context, insights from renal developmental biology provide a critical framework for understanding how these interactions are orchestrated in a spatiotemporally regulated manner. During renal development, particularly in branching morphogenesis, the ECM exhibits a highly dynamic and transient nature, continuously remodeling to regulate cellular behavior. The dynamic interplay of cell–cell and cell–extracellular matrix (ECM) interactions play a critical role in governing the complex biophysical processes underlying organogenesis (Lang et al., 2021). Multiple extracellular cues, including mechanical stiffness, topography, and matrix composition, collectively influence cell proliferation, migration, differentiation, and renal lineage specification (Nauryzgaliyeva et al., 2023). The integration of mechanically tunable and biocompatible scaffold systems therefore offers a powerful platform to investigate how distinct microenvironmental cues regulate structural maturation during metanephric development. Moreover, in recent years we have witnessed the emergence of kidney organoids as promising in vitro models to study renal development, cell–cell interactions and disease mechanisms, as well as to identify potential therapeutic targets (Long et al., 2024). Despite these developments, current organoid systems often lack the spatial organization and microenvironmental heterogeneity characteristic of native renal tissue. Against this backdrop, the design of multicompartment scaffolds with spatially defined stiffness gradients that mimic native renal tissue provides an appropriate biomimetic platform for various studies. Specifically, the development of a compartmentalized, two-zone macro anatomic approach based biomimetic platform represents a novel and strategic method to more effectively recapitulate renal architecture. Such systems enable the spatial segregation of distinct renal microenvironments, thereby facilitating the study of region-specific cell behavior, stage-specific cellular responses, cell–cell and cell–extracellular matrix (ECM) interactions and mechanobiological signaling. Furthermore, these platforms hold significant potential for applications in tissue regeneration to study kidney development in vitro, disease modeling, and drug screening by providing a more physiologically relevant and controllable in vitro environment.

The choice of biomaterial is as critical as the design of the platform itself. Hydrogels (Trebunova et al., 2025) have emerged as promising scaffolds for kidney tissue engineering due to their hydrated, biocompatible nature that closely mimics the native extracellular matrix and supports cellular survival, proliferation, and differentiation (Kim et al., 2022). Different biopolymers are widely used to develop smart hydrogels for tissue repair as they exhibit tunable stiffness, swellability, and controlled biodegradability, making them highly suitable for tissue engineering. Additionally, their stimuli-responsive behavior supports dynamic cell–material interactions, enabling the development of advanced biomimetic constructs (Khan et al., 2024). Among the various hydrogel systems explored for tissue regeneration, methacrylate-based hydrogels such as gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) have demonstrated notable potential in directing mesenchymal stem/stromal cell differentiation and promoting the deposition of organized, tissue-specific extracellular matrix (Natarajan et al., 2025). However, their relatively weak intrinsic mechanical stability can limit long-term structural integrity under extended culture conditions. In this context, GelMA, a photocrosslinkable derivative of gelatin, is particularly advantageous for renal regeneration owing to its tunable mechanical properties, favorable bioactivity, and inherent cell-adhesive motifs that collectively support renal lineage specification and tissue-like organization. Clerkin et al., studied the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. This work highlights the critical role of matrix stiffness in regulating the maturation and differentiation of hiPSC-derived kidney organoids, demonstrating that GelMA hydrogels can effectively recapitulate physiologically relevant mechanical cues. Notably, hydrogels with stiffness approximating that of the adult human kidney (∼5,000–10,000 Pa) promoted enhanced podocyte maturation and earlier upregulation of renal vesicle-associated genes compared to softer matrices (∼400 Pa). Furthermore, the study underscores how tunable mechanical environments influence disease modeling, as softer hydrogels attenuated TGFβ-induced fibrotic responses, emphasizing the importance of biophysical cues in guiding organoid development and pathology (Clerkin et al., 2025). However, most existing studies have not replicated the native spatial heterogeneity of the kidney, particularly the physiological gradient in stiffness from the stiff medullary region to the soft cortical region. To address this limitation, silk fibroin, a natural protein (Liu et al., 2025), was selected to engineer and modulate graded mechanical properties, enabling a more biomimetic representation of renal tissue architecture. Silk fibroin (SF) has emerged as a versatile biomaterial due to its excellent biocompatibility, biodegradability, low immunogenicity and tunable mechanical properties, along with high water absorption capacity. SF-based hydrogels are widely explored in biomedical applications, often in combination with other biomaterials through chemical, physical, or enzymatic approaches to enhance functionality. These systems have shown significant potential in drug delivery, wound healing, tissue engineering, and implantable devices, owing to their controlled release behavior, regenerative support, and adaptability to functional modifications. The incorporation of bioactive agents and nanomaterials further expands their application scope by improving mechanical and biological performance (Fernández-González et al., 2024). SF based hydrogels are widely applied in tissue engineering, particularly in cartilage, where they serve as drug delivery systems and support chondrocyte regeneration. Advanced fabrication methods such as 3D bioprinting offer improved precision and design flexibility, although challenges like limited mechanical strength and the use of toxic solvents remain. SF hydrogels also contribute to bone and skin regeneration through incorporation of growth factors, inorganic minerals, polymer blending, and surface modification. While emerging applications include flexible electronics, corneal repair, dental pulp regeneration, and artificial tympanic membranes, these areas require further investigation (Lyu et al., 2023). Thus, incorporation of silk fibroin can reinforce GelMA hydrogels, enhancing their mechanical integrity while maintaining a favorable microenvironment (Patel et al., 2025) for renal cells.

Based on this premise, we hypothesize that reinforcing gelatin methacrylate (GelMA) hydrogels with SF can yield mechanically robust and biologically supportive scaffolds with tunable properties suitable for renal tissue engineering. Most previously reported GelMA/SF renal tissue engineering platforms are primarily designed as single-phase or homogenous hydrogel systems, focusing on bulk biocompatibility and general cell support (Patel et al., 2025). In contrast, the present study introduces a kidney-inspired compartmentalized hydrogel architecture, enabling spatially controlled stiffness heterogeneity and region-specific cell–material interactions. This design better reflects the functional zonation and structural complexity of renal tissue, which is not achievable using conventional uniform GelMA/SF scaffolds. Additionally, we highlight that the dual-compartment strategy allows for independent tuning of scaffold properties within distinct regions, thereby providing a more biomimetic microenvironment for future renal tissue modeling and regeneration strategies. Accordingly, this study aims to systematically modulate hydrogel stiffness by varying the concentration of the hydroxyethyl methacrylate (HEMA) crosslinker and to investigate the influence of SF incorporation on the physicochemical and mechanical properties of the resulting hydrogels. In addition, scaffold stability, swelling behavior, degradation kinetics under physiological conditions, and cell attachment using L929 cells and cell viability with renal proximal tubule epithelial cells (RPTECs) were evaluated to identify an optimal formulation that balances mechanical strength with biological functionality. Furthermore, the central focus of this work is the design and development of patterned hydrogels with defined structural anisotropy and hierarchical organization to regulate cell behavior. The preferential spatial localization of RPTECs on patterns were also assessed. Such engineered architecture provides a platform for future investigations into matrix-guided mechanotransduction and its role in directing stem cell differentiation toward renal progenitor lineages, ultimately advancing the development of biomimetic scaffolds for functional renal tissue regeneration.

2. Experimental section

2.1. Materials

The following reagents and materials were used in this study: Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, #11965092), Antibiotic–Antimycotic (100X, #15240062), Trypsin-EDTA (0.25%, #25200056), Dulbecco’s Phosphate Buffered Saline (PBS, #14190144), and Fetal Bovine Serum (FBS, #A3840001), all purchased from Gibco™; Alexa Fluor 488 conjugated phalloidin (#A11029), DRAQ5, purchased from Invitrogen™; Triton X-100 (#A16046.AE), Invitrogen Alamar Blue Cell Viability Reagents, Bovine Serum Albumin (BSA, #B14), Methacrylated Gelatin (GelMA), PEG 300 (#192220010), 2-Hydroxy Methacrylate (HEMA), Ammonium Persulfate (APS, #17874), and Ascorbic Acid, all purchased from Thermo Scientific; Fibroin Silk Solution (Advanced Biomatrix, 50 mg/mL, # NC0944620) purchased from Fisher Scientific; and Polylactic Acid (PLA) spool for 3D printing, purchased from Flash Forge, United States. RPTECs; ATCC PCS-400-010, Renal epithelial cell basal medium (ATCC PCS-400-030), Renal epithelial cell growth kit (ATCC PCS-400-040) were purchased from ATCC. Ultrapure distilled water was used for all experiments.

2.2. Methodology

2.2.1. Fabrication of hydrogels with varying stiffness

GelMA–silk fibroin (GSF) hydrogels with tunable mechanical properties were fabricated by modulating the concentration of the hydroxyethyl methacrylate (HEMA) crosslinker and varying silk fibroin (SF) content, following the protocol already established in our lab for GelMA hydrogels (Natarajan et al., 2024). Four formulations were prepared: LS25 (low crosslinker, 25 mg/mL SF), LS50 (low crosslinker, 50 mg/mL SF), HS25 (high crosslinker, 25 mg/mL SF), and HS50 (high crosslinker, 50 mg/mL SF). Briefly, 400 µL of 0.1% GelMA precursor solution maintained at 40 °C was mixed with HEMA (200 µL for low crosslinking and 400 µL for high crosslinking), followed by the addition of 100 µL SF solution (25 or 50 mg/mL) and 50 µL PEG 300. Free radical polymerization was initiated by the sequential addition of 100 µL ammonium persulfate (APS, 0.1 M) and 100 µL ascorbic acid (0.1 M), acting as the initiator and catalyst, respectively. The precursor solutions were allowed to undergo crosslinking for 24 h at room temperature, after which the formed hydrogels were carefully retrieved from well plates.

2.2.2. Characterization of developed hydrogels

2.2.2.1. SEM

The microstructural morphology of GelMA–silk fibroin (GSF) hydrogels (LS25, LS50, HS25, and HS50) was analyzed using scanning electron microscopy (SEM). Briefly, hydrogel samples were first frozen at −80 °C and subsequently lyophilized to preserve their internal porous architecture. The dried samples were then sectioned to expose the cross-sectional morphology. Prior to imaging, the samples were mounted onto aluminum stubs using conductive carbon tape and sputter-coated with gold (5 nm thickness) using a LUXORAu sputter coater to enhance surface conductivity. SEM imaging was conducted using a Phenom XL Desktop SEM under accelerating voltage of 10 KV to visualize the pore structure and surface morphology. Images were captured at multiple magnifications to assess pore size, distribution, and interconnectivity across different hydrogel formulations.

2.2.2.2. Attenuated total reflectance—fourier transform infrared spectroscopy (ATR-FTIR)

ATR-FTIR spectroscopy was performed using Thermo Scientific Nicolet iS20 FTIR spectrometer to analyze the chemical structure and confirm the incorporation of silk fibroin (SF) within GelMA-based hydrogels (LS25, LS50, HS25, and HS50). The samples were subjected to FTIR analysis using an attenuated total reflectance (ATR) mode over a wavenumber range of 4,000–500 cm^-1. Characteristic absorption peaks corresponding to functional groups of GelMA and SF, including amide I, amide II, and amide III bands, were identified and compared across different formulations to assess molecular interactions and successful incorporation of SF within the hydrogel network.

2.2.2.3. Rheology

To evaluate the hydrogel stiffness (storage modulus (G′) across different formulations of GelMA–silk fibroin (GSF) (LS25, LS50, HS25, and HS50), a rotational rheometer equipped with a parallel-plate geometry (25 mm diameter and maintaining a controlled gap of 5 mm was used and Frequency sweep tests in oscillatory shear mode with a strain of 1% was conducted using a TA Instruments TRIOS HR30 rheometer. Hydrogel samples were prepared in disc form to ensure consistent sample thickness and, frequency sweep tests were carried out over a frequency range of 0.1 rad/s to 100 rad/s to measure the storage modulus (G′) and loss modulus (G″). The storage modulus (G′) was used as an indicator of hydrogel stiffness and elastic behavior. Data were recorded and compared across different formulations to evaluate the influence of HEMA crosslinker concentration and silk fibroin incorporation on the mechanical properties of the hydrogels.

2.2.2.4. Degradation studies

Hydrogel stability was evaluated through in vitro degradation studies in phosphate-buffered saline (PBS) under physiological conditions. The degradation behavior of GelMA–silk fibroin (SF) hydrogels was evaluated through mass loss analysis under physiological conditions. Pre-weighed hydrogel samples (initial weight, W_{dry initial}) were incubated in phosphate-buffered saline (PBS) at 37 °C over a period of 21 days. At predetermined time points (Day 7, Day 14, and Day 21), samples were retrieved, gently washed to remove residual salts, and dried to obtain the remaining weight (W_{dry final}).

The mass loss was calculated using the formula:

$$\text{Mass\hspace{0.17em}Loss\hspace{0.17em}}\left(\%\right)=\left(\frac{{\mathrm{W}}_{\left\{\text{dry}\text{initial}‐\text{dry}\text{final}\right\}}}{{\mathrm{W}}_{\left\{\text{dry}\text{initial}\right\}}}\right)*100$$

All experiments were conducted in triplicate, and results were expressed as mean ± standard deviation. Statistical analysis was performed to determine significant differences between groups, enabling assessment of the effects of hydrogel stiffness and silk fibroin concentration on degradation behavior.

2.2.2.5. Swelling studies

The swelling behavior of GelMA–silk fibroin (SF) hydrogels was evaluated to determine their water uptake and fluid retention capacity. Pre-weighed dry (lyophilized) hydrogel samples (W0) were immersed in phosphate-buffered saline (PBS) and incubated at 37 °C under physiological conditions. At predetermined time intervals (0, 24, 48, 72, and 120 h),the samples were removed, gently blotted to eliminate excess surface liquid, and weighed to obtain the swollen weight (Wt).

The swelling ratio was calculated using the following equation:

$$\left[\frac{\mathrm{W}\left\{\mathrm{t}\right\}‐\mathrm{W}\left\{0\right\}}{\left.\mathrm{W}\{0\right)}\right]*100$$

All measurements were performed in triplicate, and the average values were reported to assess the influence of hydrogel composition and stiffness on swelling behavior.

2.2.2.6. In vitro studies

2.2.2.6.1. Evaluation of cytocompatibility

In-vitro cell attachment and biocompatibility of the fabricated scaffolds were evaluated using L929 mouse fibroblast cells. Prior to cell seeding, scaffolds (LS 25, LS 50, HS 25, and HS 50) were sterilized by UV exposure and subsequently washed with sterile phosphate-buffered saline (PBS). The scaffolds were then preconditioned in complete culture medium for 3 h to facilitate cell attachment. L929 cells were seeded onto each scaffold at density 1 × 10⁵ cells per scaffold and incubated under standard culture conditions (37 °C, 5% CO₂). After 72 h of incubation, samples were washed with PBS to remove non-adherent cells and fixed using 4% paraformaldehyde. For visualization of cell attachment and morphology, the actin cytoskeleton was stained using phalloidin conjugated to Alexa Fluor 488, while cell nuclei were counterstained with DRAQ5. Stained samples were imaged using Nikon confocal laser scanning microscopy. Z-stack images were acquired to assess cell distribution, morphology, and infiltration within the scaffold architecture. Maximum intensity projections and 3D reconstructed images were generated using image analysis software.

2.2.2.6.2. Cell viability assessment (alamar blue assay)

To evaluate the viability of renal-specific cells on the fabricated scaffolds (LS25, LS50, HS25, and HS50), human renal proximal tubular epithelial cells (RPTECs; ATCC PCS-400-010) were seeded onto preconditioned scaffolds at a density of 1 × 10⁵ cells per scaffold. The constructs were maintained in complete renal epithelial cell culture medium consisting of renal epithelial cell basal medium (ATCC PCS-400-030) supplemented with the renal epithelial cell growth kit (ATCC PCS-400-040), under standard culture conditions (37 °C, 5% CO₂) to facilitate cell attachment and growth. Cell viability was assessed at predetermined time points (Day 1, Day 3, Day 5 and Day 7) using the Alamar Blue assay. At each time point, the culture medium was removed, and the scaffolds were gently washed with Dulbecco’s phosphate-buffered saline (dPBS) to remove non-adherent cells. Subsequently, Alamar Blue reagent was added to each sample at a 1:9 ratio with phenol red-free culture medium and incubated for 4 h at 37 °C. Following incubation, the reagent-containing medium was collected and transferred to a black 96-well plate with a clear bottom. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The measured fluorescence values, corresponding to metabolic activity, were analyzed and plotted graphically to compare cell viability across different scaffold groups. Statistical analysis was performed to determine significant differences among the groups.

2.2.3. Design and fabrication of patterned hydrogels for mimicking the renal tissue architecture

Patterned GelMA–Silk fibroin (SF) hydrogels were designed and fabricated to recapitulate the anisotropic and hierarchical architecture of native renal tissue. The construct was engineered with two distinct regions representing the renal medulla and cortex. The central region consisted of a triangular pattern, designed to mimic the medullary architecture and fabricated using a relatively higher stiffness GelMA–SF formulation (HS 50). In contrast, the surrounding outer region represented the cortical compartment and was fabricated using a comparatively lower stiffness GelMA–SF hydrogel (LS 50). However, to identify an optimal scaffold architecture for efficient cell seeding and subsequent culture, multiple patterned designs with varying geometric features were systematically evaluated. Three scaffold designs were developed using nTopology (nTop) software and exported as stereolithography (STL) files. All designs were engineered as a multi-compartment construct incorporating defined geometric features to mimic renal architecture. In design 1, the central medullary region consisted of triangular patterns with a characteristic dimension of 6 mm and a height of 1.7 mm. The height of the triangular region was deliberately designed to be greater than the surrounding areas to facilitate the formation of central triangular depressions within the hydrogel, enabling spatial confinement and guided cell localization within the patterned medullary compartment. This region was surrounded by a cortical compartment featuring a honeycomb pattern with an overall diameter of 10 mm, designed to provide a porous and structurally organized microenvironment. An additional outer ring of 2 mm thickness was incorporated to enhance structural integrity, resulting in a total construct diameter of 18 mm. The entire assembly was supported by a baseplate with a thickness of 2 mm, ensuring mechanical stability during fabrication and handling. Design 2 incorporated triangular features with a height of 1.2 mm, engineered to produce hydrogels with elevated (protruding) triangular patterns, thereby enabling distinct surface topography for studying cell–material interactions. Design 3 incorporated a hierarchical architecture comprising 1.2 mm triangular features surrounded by a peripheral ring of 1.8 mm height to introduce spatial complexity and enable compartmentalization. The increased height of the ring relative to the triangular region was intentionally designed to generate protruding triangular patterns enclosed by a recessed annular space. This configuration facilitates localized retention of cell-laden media within the peripheral compartment while minimizing mixing with cells seeded in the central medullary region. Such geometric confinement is expected to promote spatial segregation and establish gradients in cell distribution and organization across the construct.

Following the design phase, the models were exported and converted into G-code, and corresponding polylactic acid (PLA) templates were fabricated using a Creator Max 3D printer. The templates were printed using optimized parameters (Table 1) and were subsequently utilized as molds for hydrogel casting, enabling reproducible fabrication of the patterned constructs. During fabrication, the higher-stiffness GelMA–SF prepolymer solution containing all the components was first introduced into the central triangular region, ensuring confinement within the patterned compartment. The central compartment was then partially crosslinked for 15 min to achieve sufficient structural integrity and initial gelation while maintaining interfacial compatibility with the subsequently added hydrogel phase. Following this partial crosslinking step, the lower-stiffness GelMA–SF solution was introduced into the remaining template space to form the outer cortical compartment. The entire construct was then subjected to complete crosslinking and stabilization. Following gelation, the hydrogels were transferred to refrigerated conditions to facilitate complete curing and structural stabilization. The constructs were carefully demolded the following day and washed thoroughly with ultrapure distilled water to remove any residual solvents or unreacted components, yielding a structurally stable compartmentalized hydrogel platform.

TABLE 1.

Optimized printing parameters for fabrication of templates with three designs.

Printing parameters	For design1/2/3
Shell count (Nos.)	5
Shell thickness (mm)	2
Overlap perimeter (%)	30
Top solid layers (Nos.)	0
Bottom solid layers (Nos.)	3
Infill density (%)	0
Fill pattern	No
Overlap perimeter (%)	15
Solid speed (%)	70
Raft (Yes/No)	No

2.2.4. Selection of patterned hydrogels from three designs for cell distribution studies

To rationally select the optimal patterned hydrogel design from the three scaffold designs for evaluating subsequent cell distribution studies, following screening experiments were performed.

2.2.4.1. Media retention and fluid stability assessment

For visualization of spread and mixing, different predefined volumes of media (with or without dye) were carefully dispensed to the triangular region of each scaffold using a micropipette. The retention and spread of the liquid within patterned regions were visually monitored using stereomicroscopy. The observation time points are immediate (0 min) and subsequent intervals (5, 10, 30, 60 min). The parameters evaluated are retention within the patterned region, extent of fluid spread, leakage outside defined regions, overflow beyond scaffold boundaries and mixing between adjacent compartments and were recorded to assess the ability of each design to maintain compartmentalization. A assessment criteria where a qualitative scoring or descriptive analysis based on visible retention, containment, and compartment integrity (Table 2) and a semiquantitative scoring was also performed based on Table 3.

TABLE 2.

Media retention and fluid stability assessment at different volumes.

Media (µL)	Presence of dye	Initial retention (0 min)	Spread within region	Leakage observed	Overflow observed	Mixing between compartments	Overall retention performance
10 µL	With/Without	Yes/No	None/Minimal moderate/Extensive	Yes/No	Yes/No	Yes/No	Good/Moderate/Poor
15 µL	With/Without	Yes/No	None/Minimal moderate/Extensive	Yes/No	Yes/No	Yes/No	Good/Moderate/Poor
20 µL	With/Without	Yes/No	None/Minimal moderate/Extensive	Yes/No	Yes/No	Yes/No	Good/Moderate/Poor
25 µL	With/Without	Yes/No	None/Moderate/Extensive	Yes/No	Yes/No	Yes/No	Good/Moderate/Poor
35 µL	With/Without	Yes/No	None/Minimal moderate/Extensive	Yes/No	Yes/No	Yes/No	Good/Moderate/Poor

TABLE 3.

A semiquantitative scoring criteria.

Score	Interpretation
0	Complete leakage/mixing
1	Partial retention with noticeable leakage
2	Mostly retained with minimal leakage
3	Complete retention with no mixing

2.2.4.2. Structural stability under culture conditions

To assess the structural stability under culture conditions, scaffolds with Design 1, 2 and 3, were incubated in culture media over 7 days. Structural integrity and pattern retention were evaluated using stereomicroscopy. Any deformation, swelling-induced distortion or collapse of features was documented and compared across designs as per Table 4.

TABLE 4.

Structural integrity and pattern retention assessment of scaffolds during incubation.

Time point (Days)	Scaffold design	Structural integrity	Pattern retention	Swelling/Distortion	Feature collapse	Microscopy observation	Stability (0–3) score
Day 3 and 7	Design 1	Intact/Partially intact/Degraded	Clear/Slightly distorted/Lost	None/Mild/Moderate/Severe	Yes/No	Descriptive notes	0–3
Day 3 and 7	Design 2	Intact/Partially intact/Degraded	Clear/Slightly distorted/Lost	None/Mild/Moderate/Severe	Yes/No	Descriptive notes	0–3
Day 3 and 7	Design 3	Intact/Partially intact/Degraded	Clear/Slightly distorted/Lost	None/Mild/Moderate/Severe	Yes/No	Descriptive notes	0–3

2.2.5. Cell seeding efficiency, retention, distribution study on patterned hydrogel

For studying cell seeding efficiency, its retention and distribution, L929 cells at cell seeding density 1 × 10⁴/20 µL media, were seeded onto triangular region of the patterned scaffold which was selected based on screening tests. After 60 min, cells at density 5 × 10⁵/100 µL were seeded on to the cortical region having honeycomb patterns. After 72 h, samples were fixed with 4% PFA and stained (DRAQ5/Phalloidin AF 488). Confocal fluorescence microscopic images were taken to assess cell attachment and spatial cell localization and distribution within the patterns with particular attention to differences in cell density between the edges and central regions.

To further investigate the preferential spatial localization of cells within the patterned hydrogels, nuclear staining was performed using DRAQ5. Renal proximal tubular epithelial cells (RPTECs, passage 4) were seeded onto the constructs in a region-specific manner, with a density of 2 × 10⁴ cells/20 µL applied to the triangular regions and 1 × 10⁶ cells/100 µL seeded onto the surrounding cortical region. At predetermined time points (24 h, 72 h, and day 5), samples were fixed with 4% paraformaldehyde (PFA) and stained with DRAQ5 to visualize cell nuclei. Confocal fluorescence microscopy was employed to capture high-resolution images, and Z-stack acquisition was performed. Three-dimensional reconstructions with depth coding were generated to assess cell distribution and infiltration within the patterned hydrogel architecture.

2.3. Statistical analysis

All experimental data were analyzed for statistical significance using appropriate statistical methods. The number of biological and technical replicates used for each experiment (n = 3). The t-test was employed to compare the means between the two groups, determining significant differences in measured parameters with statistical significance set at p < 0.05. The F-test was used to evaluate the equality of variances between datasets to ensure the robustness of the t-test results. One-way ANOVA was used to analyze the data and to determine statistically significant differences among hydrogel groups and all group-wise comparisons reported in the figures correspond to the stated statistical approach (significance threshold (typically p < 0.05). Data are presented as mean ± standard deviation (SD). All analyses were performed using statistical software to ensure accuracy and reliability.

3. Results and discussion

3.1. Fabrication of hydrogels with varying stiffness

GelMA–SF (GSF) hydrogels were fabricated with tunable stiffness by varying HEMA concentrations and incorporating SF at 25 mg/mL or 50 mg/mL (Figures 1a,b). The fabrication strategy enabled the successful generation of structurally stable GSF hydrogels across all four formulations. Upon addition of APS and ascorbic acid, the precursor solutions exhibited a gradual transition from a clear to a turbid state, indicating the onset and progression of free radical-mediated crosslinking (Natarajan et al., 2024). This turbidity is attributed to network formation and phase organization within the polymer matrix. The effect of varying HEMA concentration markedly influenced the crosslinking density of the hydrogels, with high-crosslinker groups (HS25 and HS50) expected to exhibit increased network compactness compared to low-crosslinker groups (LS25 and LS50). Concurrently, incorporation of SF contributed to enhanced intermolecular interactions within the matrix, likely through physical entanglement and β-sheet formation, thereby reinforcing the hydrogel structure (Patel et al., 2025b). Initial hydrogel formation occurred through APS/ascorbic acid-mediated chemical crosslinking within approximately 20–30 min following addition of the crosslinking components, producing constructs with sufficient structural integrity for handling and subsequent processing. Following this initial gelation stage, the hydrogels were maintained under refrigerated conditions for 24 h to facilitate secondary network stabilization and maturation. Thus, the 24 h period represents a post-gelation stabilization phase rather than the primary gelation process. Notably, all formulations maintained structural integrity following this stabilization period, indicating effective polymer network formation irrespective of composition.

FIGURE 1.

Photomicrographs of hydrogels and surface characterization by Scanning electron microscopy (SEM). (a,b) represents the images of GelMA-based hydrogels. (c–j) represents the SEM images of GelMA–silk fibroin (SF) hydrogels showing microstructural morphology across different formulations. (c,d) LS25, (e,f) LS50, (g,h) HS25, and (i,j) HS50, at 1000x, 2500x respectively. All hydrogel groups exhibit a highly porous, sponge-like architecture with interconnected pore networks. The scale bar lengths of panels (a,c,e,g,i) and (b,d,f,h,j) are 150 μm and 50 μm respectively.

Qualitative observations indicated that hydrogels with higher crosslinker and SF content (particularly HS50) exhibited improved handling properties and mechanical robustness. These hydrogels did not tear easily when handled with forceps, maintained their shape without noticeable sagging or collapse, and demonstrated improved structural integrity during transfer and washing. In contrast, lower crosslinked systems (LS25) appeared comparatively softer and more compliant. A qualitative comparison of the handling characteristics of the hydrogel formulations is provided in Supplementary Video S1. These findings demonstrate that the combined modulation of HEMA and SF concentrations provides a versatile approach for tuning hydrogel stiffness and structural stability. Such tunability is critical for replicating the mechanical heterogeneity of native renal tissue and establishes a foundation for subsequent evaluation of physicochemical properties and cell–material interactions.

3.2. Characterization of developed hydrogels

The Scanning electron microscopy (SEM) analysis revealed a highly porous architecture, characteristic of GelMA hydrogels subjected to lyophilization. The addition of silk fibroin (100 μL, 25 mg/mL and 50 mg/mL) appears to enhance structural complexity, resulting in a scaffold with heterogeneous pore sizes ranging from small micropores to larger macropores (Figures 1c–j). Quantitative pore size distribution analysis was performed from SEM micrographs, and the results are presented in Figure 2a. All hydrogel formulations exhibited a porous microarchitecture with interconnected pores; however, notable differences in pore size distribution were observed among the groups. The mean pore sizes were 31.5 ± 10.37 µm, 22.9 ± 9.3 µm, 28.0 ± 8.5 µm, and 17.3 ± 3.2 µm for LS25, LS50, HS25, and HS50 hydrogels, respectively. Overall, hydrogels with higher crosslinking density exhibited smaller and more uniform pore structures, indicating the formation of a denser polymer network. The pore walls were relatively thin and exhibited a fibrillar like morphology, suggesting a loosely crosslinked polymer network. The presence of these thin pore walls for LS 25 and slightly thicker for LS 50, and multiple junctions indicate open porosity rather than closed cavities which is ideal for efficient mass transport, enhanced cell migration and potential vascularization support (Mukasheva et al., 2024). The surface appears rough, fibrous, and wrinkled, with visible silk fibroin fibrillar features embedded within the GelMA matrix, whereas some regions show nodular or aggregated domains, likely due to partial SF phase organization (Figures 1c–f). These roughness can enhance protein adsorption and promote cell attachment (Cui et al., 2021). The SEM images of HS25 and HS50 reveals a highly porous, sponge-like microarchitecture with pore sizes ranging approximately from 10 to 35 µm (Figures 1g–j). The walls of the pores appear smooth, thick and continuous, suggesting effective blending and crosslinking between GelMA and silk fibroin (SF). Both rounded and polygonal pore geometries are observed, demonstrating a well-defined microstructure suitable for supporting cell infiltration and nutrient transport. The incorporation of silk fibroin at 25 and 50 mg/mL in high stiff hydrogels appears to contribute to the formation of a polygonal pores without significantly densifying the network, thereby maintaining high porosity. This structural organization likely influences swelling capacity and fluid retention, as observed in the swelling studies. However, the relatively thin pore walls and irregular architecture of low stiff hydrogels may also contribute to reduced mechanical strength and potential structural instability over prolonged incubation, aligning with the degradation behavior observed for low-stiffness hydrogels. Despite this, such a porous and compliant structure can be advantageous for applications requiring enhanced cell infiltration and soft tissue mimicry.

FIGURE 2.

Characterization of GelMA–silk fibroin (SF) hydrogels. (a) Pore-size distribution graph (bin center vs. number of values) for each scaffold (b) ATR FTIR spectra of different formulations of Gelma-SF hydrogels (c) Rheological analysis showing the storage modulus (G′) of hydrogels with varying stiffness and SF concentrations: LS25 (low stiffness, 25 mg/mL SF), LS50 (low stiffness, 50 mg/mL SF), HS25 (high stiffness, 25 mg/mL SF), and HS50 (high stiffness, 50 mg/mL SF), (d) Degradation profile represented as mass loss (mg) of hydrogels over time under physiological conditions and (e,f) Swelling behavior of the hydrogels, illustrating their water uptake capacity and equilibrium swelling characteristics across different formulations. Data are presented as mean ± SD (n = 3) (***p < 0.001, **p < 0.01, *p > 0.05, ns: nonsignificant).

The ATR-FTIR spectra of the different hydrogels were obtained to evaluate their chemical composition and molecular interactions. The ATR-FTIR spectra of GelMA–silk fibroin (GSF) hydrogels (Figure 2b) exhibited characteristic absorption bands corresponding to the constituent protein-based materials, confirming the incorporation of SF within the hydrogel network. A broad band observed around 3,300 cm^-1 is attributed to overlapping O–H and N–H stretching vibrations, indicating the presence of hydroxyl groups from GelMA and amide A vibrations from protein chains (Pilley et al., 2022). Prominent peaks observed within the 1,624–1,646 cm^-1 region (amide I) and around 1,455–1,456 cm^-1 (amide II) correspond to C=O stretching and N–H bending vibrations, respectively, confirming the proteinaceous nature of the hydrogel formulations. The amide III band observed around 1,250 cm^-1 further supports the presence of polypeptide structures. Peaks in the 1,030–1,100 cm^-1 region are assigned to C–O stretching vibrations associated with the methacrylate backbone and crosslinking components (Khosravimelal et al., 2021).

Since both GelMA and silk fibroin contain protein-derived amide functionalities, substantial overlap is expected within the amide I, II, and III regions. Nevertheless, incorporation of SF resulted in noticeable changes in the amide I region. While GelMA controls exhibited amide I bands at 1,646 cm^-1 (GelMA LS) and 1,635 cm^-1 (GelMA HS), the GSF formulations displayed bands within the 1,624–1,636 cm^-1 range, with several formulations exhibiting peaks at 1,624–1,626 cm^-1. These lower-wavenumber bands are characteristic of β-sheet-rich silk fibroin structures and suggest enhanced intermolecular interactions within the composite hydrogel network. Furthermore, slight variations in peak intensity and sharpness were observed with increasing SF concentration and crosslinking density, indicating interactions between GelMA and SF chains. The absence of new characteristic peaks indicates that the incorporation of SF occurs primarily through physical interactions, such as hydrogen bonding and chain entanglement, rather than covalent bonding. These results support the successful formation of a composite GelMA–SF hydrogel system while preserving the fundamental molecular structures of both GelMA and silk fibroin.

Rheological analysis was performed to evaluate the viscoelastic properties of the GelMA–silk fibroin (SF) hydrogels, with particular emphasis on the storage modulus (G′), which reflects the elastic or solid-like behavior of the material. The results demonstrate a clear and statistically significant increase in storage modulus (p < 0.01) across the different hydrogel formulations, indicating that both crosslinker concentration and SF content play a role in modulating mechanical properties (Figure 2c). Among the low-stiffness groups, LS25 exhibited the lowest storage modulus, indicating a relatively soft and compliant network. Increasing the SF concentration to 50 mg/mL (LS50) resulted in a moderate increase in stiffness, suggesting that SF incorporation slightly enhances network integrity even at lower crosslinking densities. In contrast, the high-stiffness formulations displayed a substantial increase in storage modulus, with HS25 showing significantly higher values than both LS groups, confirming the dominant role of crosslinker concentration in governing hydrogel stiffness. Notably, HS50 exhibited the highest storage modulus among all groups, indicating a synergistic effect of increased crosslinking density and higher SF content in reinforcing the hydrogel network. This progressive enhancement in mechanical stiffness can be attributed to increased crosslink density and improved intermolecular interactions between GelMA and SF, leading to a more tightly packed and elastically stable polymeric network. The observed mechanical enhancement arises from the combined influence of crosslinking density and SF incorporation. The incorporation of SF likely contributes to additional physical interactions and structural reinforcement, thereby enhancing the load-bearing capacity of the hydrogels. The ability to finely tune hydrogel stiffness is critical for mimicking the mechanical microenvironment of native renal tissue. The observed range of storage modulus in KPa (LS 25: 0.73 ± 0.10; LS 50: 0.93 ± 0.19; HS 25: 2.14 ± 0.15; HS 50: 2.94 ± 0.14) indicates that these hydrogels can be tuned to achieve stiffness values spanning those associated with softer cortical and relatively stiffer medullary regions of the kidney. Such stiffness variation is relevant for studies of mechanotransduction, as cells are known to respond to substrate stiffness, which can influence their proliferation, differentiation, and functional behavior. The rheological findings therefore demonstrate that the developed GelMA–SF hydrogels offer a two zone platform with adjustable stiffness. While this tunability suggests potential for eliciting region-specific cellular responses through the use of distinct hydrogel formulations, such outcomes remain to be experimentally validated in the future study along with identifying the individual effects through systematically varying each parameter independently to decouple their respective contributions. The absence of compressive or tensile modulus data is acknowledged as a limitation, as such measurements can provide additional validation of the bulk mechanical behavior and tissue-mimetic properties of hydrogel systems. Future studies will incorporate bulk mechanical testing, particularly compressive modulus evaluation, to complement the rheological data and provide a more comprehensive mechanical characterization of the compartmentalized hydrogel system.

The degradation profile of GelMA–silk fibroin (SF) hydrogels was evaluated by quantifying mass loss (%) over a period of 21 days in phosphate-buffered saline (PBS). All hydrogel formulations exhibited a time-dependent increase in mass loss from Day 7 (D7) to Day 21 (D21), indicating progressive scaffold degradation under physiological conditions (Figure 2d). Among the low-stiffness groups, LS25 demonstrated a minimal and gradual increase in mass loss over time, with no statistically significant differences observed across the evaluated time points, suggesting relatively stable degradation behavior. In contrast, LS50 exhibited a comparatively higher mass loss, with statistically significant changes over time, indicating that an increased SF concentration within low-stiffness matrices accelerates degradation. For the high-stiffness formulations, HS25 showed moderate mass loss with no significant temporal variation, reflecting a controlled and stable degradation profile. However, HS50 displayed a pronounced increase in mass loss by Day 21, accompanied by strong statistical significance, suggesting enhanced degradation in this group. The degradation behavior thus highlights the combined influence of hydrogel stiffness and SF concentration on scaffold stability. Increasing SF content (50 mg/mL) appears to promote more pronounced degradation, particularly at later time points. Notably, high-stiffness hydrogels exhibited a more consistent and controlled degradation pattern compared to low-stiffness counterparts, indicating improved structural stability. From the tissue engineering perspective, such controlled degradation is critical for ensuring a balance between scaffold resorption and new tissue formation (Umemori and Little, 2025). Hydrogels with higher SF concentration and stiffness, particularly HS50, may support gradual scaffold remodeling and facilitate tissue integration. These properties are especially relevant for renal tissue engineering, where scaffold degradation must be synchronized with cellular organization and functional tissue maturation.

The swelling behavior of low-stiffness (LS) and high-stiffness (HS) hydrogels, formulated with different silk fibroin concentrations (25 mg/mL and 50 mg/mL), was evaluated over 5 days (Figures 2e,f). The swelling trends showed clear differences between the two groups. HS hydrogels (HS 25 and HS 50) exhibited significantly higher swelling by weight (Wt vs. time) compared to LS hydrogels throughout the study (Figure 2e). Both HS groups showed a rapid increase in weight from day 1 to day 2, reaching a maximum swelling peak at approximately 48 h, followed by a plateau with minimal decline up to day 5. This behavior indicates that HS hydrogels possess a stable network. In contrast, LS hydrogels (LS 25 and LS 50), formulated with lower crosslinker (HEMA) content, demonstrated reduced swelling by weight (Wt) when compared to HS scaffolds. However, LS hydrogels showed enhanced swelling ratio (%) which indicates it has more capability of retaining large amounts of fluid which is attributed to the lower crosslinking density. But, the swelling ratio of LS hydrogels declines as time progresses which indicate initial mass loss due to leaching/degradation. This suggests weaker network formation and presence of pores of varying sizes limited fluid retention which leads to possible structural relaxation or compaction during incubation. The absence of a pronounced swelling peak further supports the formation of a less stable polymer network. The increased stiffness in HS is typically associated with higher crosslinking density that restricts swelling. However, the presence of silk fibroin likely promotes interconnected porous architecture and introduces hydrophilic amino acid segments which contribute to moisture absorptions, thereby facilitating enhanced water uptake while preserving mechanical integrity (Johari et al., 2020). This could be the reason for stable swelling ratio though less than LS. Between the concentrations, both HS 25 and HS 50 showed similar swelling trends, with only marginal differences in peak swelling. This indicates that within the tested range, silk fibroin concentration (25 vs. 50 mg/mL) modifies overall network stiffness, although slight variations may arise due to differences in fiber entanglement and matrix density. The results thus demonstrate that high-stiffness hydrogels (HS) exhibit stable swelling capacity and fluid retention compared to low-stiffness (LS) hydrogels, highlighting their suitability for applications requiring sustained hydration and structural stability.

In vitro cell attachment and biocompatibility of the scaffolds were evaluated using L929 fibroblast cultures. Upon confocal imaging, cells seeded on all four scaffold groups (LS 25, LS 50, HS 25, and HS 50) demonstrated successful attachment, indicating that all formulations support basic cell adhesion and are cytocompatible (Figures 3a–p)). However, clear differences in cell morphology and spreading were observed among the groups. Cells cultured on HS 25 and HS 50 scaffolds exhibited a well-spread, elongated morphology with prominent cytoskeletal organization, which is characteristic of healthy and actively adhering fibroblasts (D’Urso et al., 2024). In contrast, cells on LS 25 and LS 50 scaffolds appeared comparatively less spread, with a more rounded or less defined morphology, suggesting relatively weaker cell–material interactions. The improved cellular response on HS scaffolds can be attributed to their enhanced structural properties, including increased stiffness and the presence of higher silk fibroin content. These factors are likely to contribute to a more favorable microenvironment by providing improved mechanical support and potentially better surface characteristics for cell anchorage. Substrate stiffness is known to influence fibroblast adhesion and spreading (Tiskratok et al., 2025), and the higher stiffness of HS scaffolds appears to promote stronger cell attachment and cytoskeletal development. Thus, while all scaffold groups supported cell attachment, HS 25 and HS 50 scaffolds demonstrated superior performance in terms of cell morphology and spreading. These findings suggest that high-stiffness scaffolds provide a more conducive environment for cell attachment and may be better suited for tissue engineering applications requiring robust cell–material interactions.

FIGURE 3.

In vitro cell attachment on LS and HS scaffolds. Confocal microscopy images showing L929 fibroblast attachment on LS 25, LS 50, HS 25, and HS 50 scaffolds. F-actin cytoskeleton was stained with phalloidin conjugated to Alexa Fluor 488 (green), and cell nuclei were stained with DRAQ5 (red). (a,e,i,m) represent F-actin staining (Alexa Fluor 488), highlighting cytoskeletal organization. (b,f,j,n) show nuclear staining (DRAQ5). (c,g,k,o) correspond to maximum intensity projections, illustrating overall cell distribution and spreading across the scaffold surface. (d,h,l,p) present 3D volume-rendered views, providing insight into cell infiltration and spatial organization within the scaffold architecture. (q) represents the cell metabolic activity of RPTECs cultured on different hydrogels at different time points using the Alamar Blue assay. Data are presented as mean ± SD (n = 3). (***p < 0.001, **p < 0.01, *p > 0.05, ns: nonsignificant).

Further, the metabolic activity of RPTECs seeded on different hydrogel formulations (LS25, LS50, HS25, and HS50) was evaluated using the Alamar Blue assay at days 1, 3, 5, and 7 (Figure 3q). Altogether, all hydrogel systems supported cell viability across the assessed time points, indicating good cytocompatibility. At early time points (day 1 and day 3), LS25 exhibited higher metabolic activity compared to LS50. However, this trend shifted at later time points, with LS50 demonstrating greater metabolic activity than LS25 on days 5 and 7. Hydrogels with higher stiffness (HS group) consistently showed enhanced metabolic activity compared to the LS group at all time points. Within the HS group, HS25 exhibited higher metabolic activity at early time points (day 1 and day 3), whereas HS50 showed increased activity by day 5. By day 7, HS25 maintained relatively stable metabolic activity, while HS50 showed a slight decrease. A general reduction in metabolic activity was observed across all groups by day 7, particularly in the LS groups where the decline was more pronounced.

The Alamar Blue assay results demonstrate that all GelMA–SF hydrogel formulations provide a supportive environment for RPTEC viability, confirming their cytocompatibility. The observed differences in metabolic activity between formulations may be attributed to variations in hydrogel stiffness and network structure. At early time points, the relatively higher metabolic activity in lower stiffness hydrogels (LS25 and HS25) may reflect favorable initial cell attachment and spreading due to a more compliant substrate. As culture progressed, hydrogels with higher silk fibroin content and/or crosslinking density (LS50 and HS50) exhibited increased metabolic activity, potentially due to improved structural stability and sustained support for cell proliferation. The consistently higher metabolic activity observed in HS hydrogels compared to LS formulations suggests that increased stiffness may provide a more supportive microenvironment for RPTEC growth over time. However, the slight reduction in metabolic activity at day 7 across most groups is likely associated with increased cell density and the onset of confluency, which can limit proliferation and metabolic output. These findings indicate that hydrogel composition and stiffness influence temporal cell behavior, although further studies incorporating functional assays and longer culture durations are required to better understand their impact on renal cell phenotype and function.

3.3. Design and fabrication of patterned hydrogels inspired by renal tissue architecture

Three designs were successfully fabricated, yielding structurally stable and well-defined patterned hydrogels (Figures 4a–c) to mimic the nephron segmentation depicted in Figure 5. Design 1 produced distinct central depressions that enabled effective confinement of the hydrogel within the triangular medullary region. The triangular patterns remained well-defined, and no evidence of strctural collapse or geometric distortion was observed. Design 2 resulted in protruding triangular features, providing increased surface topography. Design 3 demonstrated a clear hierarchical structure, with protruding triangular domains surrounded by a recessed annular region capable of retaining fluid. The compartmentalized architecture was maintained post-fabrication, and the dual-stiffness hydrogel integration between medullary and cortical regions was achieved without structural compromise. The partial crosslinking time of 12 min is critical for achieving the desired structural integrity and interfacial stability between compartments, while still allowing effective integration during the subsequent crosslinking steps. The stereomicroscopic images illustrating the spatial differentiation of the patterned hydrogel constructs.

FIGURE 4.

A comparison of all design iterations. From left to right, STL file, PLA template and demolded hydrogels of (a) design 1 with 1.8 mm height triangles, (b) design 2 with 1.2 mm height triangles and (c) design 3 with 1.2 mm height triangles surrounded by a 1.8 mm ring.

FIGURE 5.

Conceptual illustration of kidney-inspired compartmentalized organization within the patterned hydrogel platform. The schematic depicts spatially distinct cortical- and medullary-inspired regions designed to support future investigations into region-specific renal cell organization.

The construct was developed using silk fibroin (SF)-reinforced gelatin methacrylate (GelMA) hydrogels engineered into a spatially patterned architecture containing central triangular domains and surrounding peripheral regions. The triangular regions were designed as medullary-inspired compartments incorporating comparatively stiffer GelMA–SF matrices, while the surrounding peripheral regions represented cortex-inspired compartments composed of softer hydrogel formulations. Based on rheological characterization, HS 50 hydrogels (storage modulus: 2.94 ± 0.14 kPa) were selected for the central compartment, whereas LS 50 hydrogels (storage modulus: 0.93 ± 0.19 kPa) were utilized for the surrounding region. The incorporation of SF improved the structural integrity and mechanical tunability of the hydrogel system while supporting preliminary cell attachment. The spatial variation in stiffness, architecture, and compartmentalization was designed to establish distinct microenvironments that may support future investigations involving region-specific renal cell culture and spatial organization. Collectively, these findings demonstrate the feasibility of developing compartmentalized hydrogel systems inspired by renal tissue organization for advanced in vitro bioengineering applications. The different nephron segments, along with their corresponding material properties, representative cell types, and biochemical cues potentially relevant for future studies, are summarized in Table 5.

TABLE 5.

Different nephron segments and required material properties, cell types and biochemical cues.

Nephron segment	Material properties	Cells	Biochemical cues
Glomerulus	Softer GelMA, high porosity	Endothelial, podocytes	Laminin
Proximal tubule	Moderate stiffness, dense	Tubular epithelial cells	Egf, HGF
Loop of henle	Gradient gel	Collecting duct cells	AVP analogs
Collecting duct	Stiffer GelMA	Intercalated cells	Aldosterone, vasopressin

The systematic variation in scaffold geometry significantly influenced the structural characteristics and compartmentalization behavior of the hydrogels. Design 1 facilitated spatial confinement within recessed regions, which is advantageous for localized cell seeding and media retention (Figure 6). Design 2 introduced elevated topographical features that may influence cell–material interactions, while Design 3 provided compartmentalized regions capable of reducing fluid mixing between adjacent domains. Such spatial organization is particularly relevant for the development of kidney-inspired in vitro culture systems, where distinct microenvironments exist in close proximity. These findings highlight the importance of integrating microarchitecture and mechanical gradients to guide localized cell distribution and compartmentalization, thereby supporting the future development of region-specific renal tissue engineering and disease modeling platforms.

FIGURE 6.

Stereomicroscopic images illustrating the fabrication and spatial differentiation of the patterned hydrogel constructs. (a,d,g) represent the 3D-printed PLA templates of Design 1, Design 2, and Design 3, respectively. (b,e,h) show the corresponding patterned hydrogels fabricated from the respective templates. (c,f,i) represent selectively dye-stained hydrogels corresponding to Designs 1, 2, and 3, respectively, where the dye was introduced to enhance visualization of the patterned architecture and spatial compartmentalization. The images demonstrate the successful formation of spatially distinct hydrogel regions, with the central high-stiffness compartment surrounded by a comparatively lower-stiffness peripheral region, highlighting the structural and compositional compartmentalization achieved within the engineered hydrogel platform. The scale bar lengths of all panels are 1,000 µm.

3.4. Selection of patterned hydrogels from three designs for cell distribution studies

The media retention and fluid stability assessment revealed distinct differences in the compartmentalization efficiency among the three scaffold designs. The semi-quantitative analysis of leakage demonstrated a clear volume-dependent decline in retention performance across all scaffold geometries, with notable differences in their ability to maintain compartmentalization (Figure 7a). At lower volumes (10 and 15 µL), all designs (Design 1, Design 2, and Design 3) exhibited high retention scores (∼3), indicating negligible leakage and effective fluid confinement within the predefined triangular regions. Upon increasing the volume to 20 μL, Design 1 and Design 3 maintained high retention efficiency, whereas Design 2 showed an initial reduction in performance, suggesting the onset of fluid leakage. Moderate fluid spreading within the patterned regions was observed across all designs at this volume, indicating the beginning of capacity limitations in retaining larger fluid volumes.

FIGURE 7.

Evaluation of media retention, fluid stability, and structural integrity of patterned hydrogel scaffolds. (a) Semi-quantitative assessment score for leakage, illustrating the retention efficiency and fluid confinement ability of different scaffold designs across increasing media volumes. (b) Stability score representing structural integrity and pattern retention of the scaffolds during incubation under culture conditions at defined time points.

At higher loading volumes (25 and 35 µL), more pronounced differences in compartmentalization behavior became evident. Design 1 exhibited only a gradual decline in retention performance and maintained relatively effective fluid confinement, whereas Design 3 demonstrated moderate leakage. In contrast, Design 2 showed a substantial reduction in retention efficiency, accompanied by extensive fluid spreading and overflow beyond the scaffold boundaries. Leakage in Design 2 became apparent at 20 µL and progressively worsened at higher volumes, resulting in compromised compartmental integrity and inter-region fluid mixing at 25 μL and 35 µL. Design 3 demonstrated intermediate performance, maintaining better compartmentalization than Design 2 but not achieving the retention efficiency observed in Design 1.

The superior retention behavior of Design 1 can likely be attributed to its recessed triangular geometry, which facilitated localized fluid confinement and minimized overflow. In contrast, the elevated or less confined geometries present in Design 2 and partially in Design 3 contributed to increased fluid spreading and reduced compartmental stability. These findings demonstrate that scaffold geometry plays a critical role in governing fluid retention and compartmental integrity within patterned hydrogel systems. An effective media retention and compartmentalization are essential for maintaining localized microenvironments, especially in multicellular systems where spatial segregation of cell types is required which further favour lineage specific differentiation (Truong et al., 2026).

Structural integrity and pattern retention assessments further revealed clear differences in the stability of the scaffold designs during incubation under culture conditions (Figure 7b; Table 6). At Day 3, Design 1 maintained excellent structural integrity with well-defined triangular features and no observable swelling-induced deformation. Design 2 exhibited slight edge rounding and deformation of the elevated features, indicating early-stage structural instability, while Design 3 retained its overall geometry with minor distortion observed in the peripheral regions. By Day 7, these differences became more pronounced. Design 1 continued to preserve its geometric fidelity with minimal structural alteration, demonstrating strong resistance to prolonged incubation conditions. In contrast, Design 2 exhibited partial collapse of patterned features and loss of geometric definition, indicating reduced mechanical stability and susceptibility to swelling-induced deformation. Design 3 demonstrated intermediate structural stability, retaining portions of the triangular features while exhibiting noticeable deformation within the surrounding regions. These observations thus indicate that scaffold geometry critically influences both compartmentalization efficiency and structural stability during prolonged incubation. The recessed architecture of Design 1 provided enhanced resistance to deformation while maintaining localized fluid confinement and pattern fidelity over time. These results identify Design 1 as the most suitable architecture for maintaining compartmental integrity and localized culture environments, thereby supporting its selection for subsequent cell distribution studies.

TABLE 6.

Structural integrity and pattern retention assessment of scaffolds.

Time point (Days)	Scaffold design	Structural integrity	Pattern retention	Swelling/Distortion	Feature collapse	Microscopy observation	Stability (0–3) score
Day 3	Design 1	Intact	Clear	None	No	Excellent structural integrity, well-defined triangular features	3
Day 7	Design 1	Intact	Clear	Mild	No	Minimal structural alteration	2
Day 3	Design 2	Intact	Slightly distorted	Mild	No	Slight deformation of the elevated features	2
Day 7	Design 2	Partially intact	Slightly distorted	Moderate	Yes	Partial collapse of patterned features, loss of geometric definition	1
Day 3	Design 3	Intact	Slightly distorted	Mild	No	Slight distortion in the peripheral regions	2
Day 7	Design 3	Intact	Slightly distorted	Moderate	No	Partial retention of the triangular features, noticeable deformation in the surrounding regions	2

3.5. Cell seeding efficiency, retention, distribution study on patterned hydrogel

Phalloidin actin staining performed on Day 5 demonstrated appreciable attachment of L929 fibroblasts cultured on the patterned GelMA–SF hydrogels with Design 1, as indicated by the predominance of Alexa Fluor 488 (green) cytoskeletal staining together with DRAQ5 nuclear staining (red) (Figure 8). Large-area confocal imaging enabled visualization of cell distribution across the engineered microarchitecture, with cells observed within both the central triangular compartment and the surrounding honeycomb-patterned regions. The well-defined F-actin organization further confirmed favorable cell attachment and spreading on the hydrogel surface. The recessed triangular geometry facilitated spatial confinement of cells within the central compartment while maintaining separation between the central and peripheral regions. In addition, the recessed architecture promoted localized retention of cell-laden media during culture and media exchange, thereby minimizing fluid mixing between compartments and preserving the overall pattern fidelity during the culture period.

FIGURE 8.

Cell distribution analysis using Phalloidin Actin/DRAQ5 staining of L929 fibroblasts cultured on patterned GelMA–SF hydrogels at Day 5. Cells exhibited appreciable attachment and spatial organization within the engineered microarchitecture, with preferential localization observed within the central triangular regions and surrounding honeycomb-patterned domains. (a) Confocal microscopic image showing the overall scaffold area; (b) Alexa Fluor 488 staining of F-actin cytoskeleton; (c) DRAQ5 staining of nuclei; and (d) merged fluorescence image demonstrating cell attachment and spatial distribution within the patterned hydrogel system.

Further, to biologically validate the intended renal tissue engineering application using a kidney-relevant cell type, RPTECs were cultured on the patterned hydrogels and analyzed using nuclear staining combined with depth-coded three-dimensional confocal imaging. While the L929 fibroblast studies (Figure 8) were primarily performed to evaluate cell attachment and cytoskeletal organization, the RPTEC studies were designed to assess the spatial distribution of renal epithelial cells within the compartmentalized hydrogel architecture over time.

The depth-coded three-dimensional confocal reconstructions enabled visualization of RPTEC distribution within the patterned hydrogel constructs throughout the culture period (Figure 9). At 24 h, RPTECs were observed across both patterned and surrounding regions of the construct, consistent with the initial seeding distribution (Figures 9a,b). At 72 h, the triangular patterned domain remained clearly populated, and cells appeared broadly distributed throughout the engineered microarchitecture (Figures 9c,d). Similar observations were noted at day 5, where RPTECs continued to occupy both patterned and surrounding regions while maintaining substantial coverage within the triangular compartment (Figures 9e,f). The pseudo-color depth maps represent the relative z-position of RPTEC nuclei within the imaging volume and demonstrate that cells were present across multiple z-planes within the patterned constructs. Collectively, these observations indicate that the compartmentalized hydrogel architecture supported sustained RPTEC attachment and spatial organization throughout the culture period. It should be noted that the purpose of this analysis was to evaluate three-dimensional cell localization within the engineered microarchitecture rather than to quantify cellular infiltration or migration.

FIGURE 9.

Confocal fluorescence images showing nuclear-stained RPTECs cultured on patterned hydrogels to assess temporal changes in spatial cell localization. (a,b) Depth-coded three-dimensional reconstructions acquired at 24 h showing the initial distribution of RPTECs across the patterned constructs. (c,d) Images acquired at 72 h demonstrating increased localization of RPTECs within the triangular patterned regions. (e,f) Day 5 images showing continued enrichment of RPTECs within the patterned domains and reduced cell density in the surrounding inter-pattern regions. Depth coding represents the relative z-position of RPTEC nuclei within the confocal imaging volume, illustrating the three-dimensional spatial distribution of cells across multiple z-planes within the patterned hydrogel architecture.

The continued presence of RPTECs within the patterned domains throughout the culture period suggests that the compartmentalized architecture provides a supportive microenvironment for renal epithelial cell attachment and retention. Such spatial organization is relevant to renal tissue engineering applications, where cellular organization varies across distinct anatomical regions. While the mechanisms governing cell distribution within the patterned constructs remain to be determined, these findings provide an initial proof-of-concept demonstrating the feasibility of using compartmentalized GelMA–SF hydrogels to support spatially organized cultures of kidney-relevant cells. Future studies incorporating quantitative image analysis, additional kidney-relevant cell types, co-culture systems, and functional assessments will be necessary to further elucidate the influence of architectural cues on cellular compartmentalization and renal cell behavior.

To illustrate the conceptual framework of the study, Figure 10 presents a schematic representation of the relationship between engineered patterned hydrogels and the spatial organization of native kidney anatomy.

FIGURE 10.

Conceptual illustration of the relationship between the engineered patterned hydrogels and native kidney-inspired architecture. (a) Fabricated dual-zone hydrogel constructs showing distinct compartmentalization, with the central high-stiffness region selectively stained to highlight spatial differentiation. (b) Conceptual visualization of the engineered hydrogel platform in a kidney-inspired geometry generated using DALL·E. (c) Representative anatomical depiction of native kidney structure highlighting cortical and medullary regions for comparison with the engineered construct. (d) Representative imaging of cell distribution within the patterned hydrogels, illustrating spatial organization of cells within distinct hydrogel compartments.

The figure highlights the design rationale underlying the development of a compartmentalized hydrogel system with distinct central and peripheral regions inspired by cortical and medullary organization. By integrating tunable material properties with spatial patterning, the engineered platform establishes a foundation for future investigations into spatially organized renal cell culture, kidney-inspired tissue engineering, and advanced in vitro disease modeling applications. These findings indicate that scaffold topography and compartmentalized geometry play important roles in regulating localized cell organization and spatial distribution within the engineered hydrogel platform.

In the context of existing literature, recent advances in silk fibroin (SF)-based injectable and multifunctional hydrogels highlight the material’s exceptional versatility in matrix engineering, living cell integration, and stimuli-responsive design. SF has been widely adapted into hybrid systems through combinations with natural polymers (e.g., gelatin), modified biomolecules (e.g., carboxymethyl chitosan and tannic acid), and synthetic networks such as PEG-based hydrogels, as well as via chemical modifications like methacrylation and bioactive functionalization. These approaches have enabled the development of biomimetic matrices that recapitulate key features of the native extracellular matrix, including biochemical signaling, mechanical integrity, and dynamic remodeling, while also supporting applications across wound healing, musculoskeletal repair, neural regeneration, and organoid culture platforms (Mushtaq et al., 2025). Most GelMA/SF-based renal tissue engineering systems have predominantly been developed as homogeneous, single-phase hydrogels, primarily emphasizing bulk cytocompatibility and general cell support. In contrast to these predominantly homogeneous or bulk-engineered systems, the present study introduces a kidney-inspired compartmentalized GelMA–SF hydrogel strategy that emphasizes spatial organization as an additional design dimension. By enabling region-specific tuning of stiffness and localized cell–material interactions, this approach extends beyond conventional SF-based platforms that primarily focus on compositional or functional modifications. Such spatially defined architectures are particularly relevant for tissues like the kidney, where intrinsic heterogeneity and functional zonation are critical, thereby offering a complementary and potentially more physiologically relevant framework for next-generation tissue engineering applications. However, the current work thus primarily focuses on hydrogel development and proof-of-concept demonstration of dual-compartment hydrogel design to direct cell distribution for zonal tissue engineering applications and its validation using surface-seeded cells. Although fibroblasts and RPTECs were utilized to evaluate cytocompatibility and spatial localization, comprehensive assessments such as long term functional and phenotypic analyses using additional kidney-relevant cell types and co-culture systems will be required to establish how these spatial cues influence cell behavior and tissue-specific outcomes. Also, achieving fully homogeneous cell distribution across confined and unconfined regions requires further optimization of cell seeding density, hydrogel surface conditioning, and adhesion kinetics (extended time-course analysis) Secondly, the study does not incorporate cell-laden hydrogel formulations. As a result, the system does not yet fully recapitulate the three-dimensional cellular microenvironment characteristic of native renal tissue. Hence, the future studies will aim to address these limitations and will further support the feasibility of utilizing compartmentalized patterned hydrogels for region-specific tissue engineering applications.

4. Conclusion and future perspectives

The present study establishes the feasibility of developing compartmentalized GelMA–SF hydrogel systems with tunable mechanical properties and patterned architectures for kidney-inspired tissue engineering applications. Beyond demonstrating material performance, structural stability, and proof-of-concept cellular compatibility using fibroblast cultures, RPTEC metabolic activity, and spatial localization analyses, the engineered platform provides a promising foundation for the development of spatially organized renal tissue models. Future studies will focus on incorporating a broader range of renal-specific cell types to further evaluate long-term spatial organization and tissue-specific functionality within the compartmentalized hydrogel system. In parallel, the integration of cell-laden hydrogel formulations will be pursued to more closely mimic the native three-dimensional renal microenvironment. The development of such cell-encapsulating systems will require further optimization of parameters including cell encapsulation density, crosslinking conditions, hydrogel maturation behavior under physiologically relevant culture conditions, and nutrient transport within the dual-compartment architecture. These investigations will also provide insight into potential cell sedimentation effects and strategies for maintaining homogeneous cellular distribution during hydrogel fabrication. Additionally, advanced characterization approaches, including region-specific mechanical characterization and functional cellular assays, will be employed to further elucidate structure–function relationships within the engineered platform. Extended investigations focusing on long-term culture, biochemical signaling, multicellular interactions, and renal-specific functionality are expected to further enhance the physiological relevance of the system and support its application in kidney tissue engineering, disease modeling, and advanced in vitro culture platforms.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. The authors would like to thank the National Science Foundation, CREST HBCU RISE (2332041) and NSF RUI (2330023) for their financial support.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions

AN: Formal Analysis, Writing – original draft, Data curation, Investigation, Software, Writing – review and editing, Conceptualization, Visualization, Methodology, Validation. GS: Formal Analysis, Writing – review and editing, Software, Methodology. AS: Writing – review and editing, Visualization, Methodology. DD: Visualization, Project administration, Funding acquisition, Writing – review and editing, Validation, Formal Analysis. VV: Conceptualization, Supervision, Funding acquisition, Project administration, Validation, Writing – original draft, Writing – review and editing, Visualization.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was used in the creation of this manuscript. Generative AI (DALL·E) was used solely for the creation of conceptual schematic illustrations in Figures 10b,c to visually represent the kidney-inspired design rationale of the hydrogel platform. The AI-generated images were used only for illustrative purposes and did not contribute to data generation, analysis, interpretation, or scientific conclusions. All scientific content, experimental results, and manuscript preparation were reviewed and verified by the authors.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2026.1876387/full#supplementary-material

Supplementary Video

Qualitative comparison of handling properties of GelMA–silk fibroin hydrogel formulations, highlighting variations structural integrity and shape retention during manual manipulation.