Bovine Placental Cotyledon-Derived Hydrogel: Methodology to Produce a Substrate for Tissue Engineering

Tissue collection

This study was approved by the Animal Ethics Committee of São Paulo State University (UNESP-FCAV) under protocol 3236/2024. Bovine placentas (n = 10) at 4–5 months of gestation, determined by crown-rump length (Evans and Sack, 1973), were obtained from local slaughterhouses and transported on ice to the Laboratory of Regenerative and Reproductive Biotechnology (UNESP-FCAV). Decellularization and hydrogel production were repeated in triplicates using tissue from three independent animals (biological replicates), totaling, at least, three batches of biomaterials.

Placental decellularization

Umbilical arteries were cannulated with 14G catheters and perfused using a peristaltic pump. Tissues were first washed with phosphate-buffered saline (PBS; 136.9 mM NaCl, 26.8 mM KCl, 14.7 mM KH₂PO₄, and 8.1 mM Na₂HPO₄·7H₂O; pH 7.2) at 0.5 mL/min for 24 h, followed by sequential perfusion with sodium dodecyl sulfate (SDS) in increasing concentrations: 0.01% (24 h), 0.1% (48 h), 0.25% (48 h), 0.5% (72 h), and 1% (24 h). Residual lipids were removed by perfusion with 1% Triton X-100 for 3 h. The decellularized tissues were washed in sterile PBS for 24 h to eliminate detergent residues. ⁵ Cotyledons were then dissected from the allantochorionic membrane and stored at −20°C for up to 3 months in sterile, sealed polypropylene tubes until further use.

Validation of the decellularization process

ECM-reminiscent protein by mass spectrometry

According to Hedrick et al. ¹⁴ and Barreto et al., ¹⁵ native and decellularized cotyledons were sampled, lysed, urea reduced, acetone precipitated, dithiothreitol reduced, iodoacetamide alkylated, trypsin digested, and C-18 column purified. Finally, 3 µg of protein were loaded in the OrbitrapFusionLumos spectrometer (ThermoScientific). Spectra were exported to MaxQuant software (v1.6.10.43) for protein list production of each sample, and LFQ intensities were statistically (p > 0.05) analyzed by Inferno software (v.1.1.6970). After, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8.

Hydrogel preparation

Biomaterials were subjected to three cycles of 5-min ultrasonic baths, followed by incubation in sterile PBS supplemented with 2% penicillin/streptomycin (10% v/v). Dehydration was performed by critical point drying (Leica EM CPD300, Wetzlar, Germany), following protocols adapted from Nakayama et al.^16,17 Sterilized samples were stored at 4° C for up to 1 week or at −20°C for no longer than 3 months. ¹⁸

A total of 500 mg of dried biomaterial was added to 50 mL of 0.01 M HCl containing 50 mg of pepsin (1 mg/mL, Sigma-Aldrich, St. Louis, USA) and minced with sterile scissors to a paste-like consistency. Digestion occurred at 37°C under agitation for 24 h. The pH was then neutralized to 7.4 using 0.1 N NaOH.

To induce polymerization, sodium alginate (C₆H₇NaO₆, #1003669272, Sigma–Aldrich, St. Louis, USA) was added at 5%, 8%, 10%, and 12% (w/v). Cross-linking was achieved by adding 200 µL/mL of 1% calcium chloride (CaCl₂, PM 110.98, #13-10490-05, Nova Biotecnologia, Campinas, Brazil). Aliquots of 1 mL were dispensed into 12-well plates and incubated at 37°C in 5% CO₂ for 24 h. ¹⁹

After polymerization, hydrogels were fixed in 4% paraformaldehyde vapor, freeze-dried in a lyophilizer (OPERON FDB5503), sputter-coated with gold (Emitech K550), and examined using an SEM (Leo 435 VP) to evaluate porosity and three-dimensional network organization.

Cytocompatibility assay

Previously to cell culture, biomaterial and hydrogels were incubated for 7 days in Dulbecco’s modified Eagle’s medium low glucose supplemented with 10% of fetal bovine serum (FBS #12657-029, Gibco, Grand Island, USA) and 0.5% penicillin/streptomycin (10,000 U/mL and 10 mg/mL, respectively; #15070-063, Gibco, USA) at 37°C in a 5% CO₂ atmosphere. Changes in turbidity, color, and transparency of the medium were monitored daily as indicators of contamination.

For biomaterials testing, fragments of approximately 1 cm³ were cultured in the complete medium (DEMEM Low Glucose + 10% FBS + 0.5% penicillin/streptomycin) (#10-013-CV, Corning, NY, USA) with 2.6 × 10⁵ canine adipose-derived mesenchymal stem cells (MSCs) per fragment. The MSCs used were isolated from healthy adult donor dogs under approved ethical protocol. Cells were used between passages 4 and 6, and were previously characterized for surface markers and trilineage differentiation potential. Culture was maintained at 37°C and 5% CO₂, with medium changes every 2–3 days for 25 days. On days 15 and 25, fragments were collected for analysis of cell viability, distribution, and scaffold integration.

For hydrogel formulation, 1 mL was dispensed into 12-well plates and seeded with 5 × 10⁵ MSCs suspended in 1 mL of complete medium. Cultures were maintained at 37°C and 5% CO₂ for 7 days, a period chosen based on preliminary optimization assays indicating this as the optimal timeframe for initial cell adhesion, viability assessment, and early matrix remodeling without compromising hydrogel integrity or inducing significant degradation.

Biomaterial production and validation

Throughout the decellularization process, a progressive reduction in tissue volume and a distinct whitening of the bovine cotyledons were observed (Fig. 2A and 2B). These macroscopic alterations are indicative of effective removal of cellular components. Notably, the tissue maintained the gross anatomical integrity and elasticity, suggesting preservation of major ECM constituents. The whitening effect may be attributed to the removal of blood and chromophore cellular components, further evidencing the efficacy of the protocol in preparing a suitable acellular scaffold (Fig. 2A).

OPEN IN VIEWER

FIG. 2.

Decellularization of bovine cotyledons. Bovine allantochorionic sac at 4 months gestation before (A) and after the decellularization process (B). In (B), the allantochorionic sac was opened and the fetus was removed; there is a change in the color of the tissue, which becomes whitish and translucent. (C) Histological analysis by Hematoxylin and Eosin (HE) staining of the native cotyledon, showing the presence of cell nuclei and extracellular matrix; scale bar = 100 µm. (D) Histological analysis by HE of the decellularized cotyledon, showing the preservation of the architecture of the extracellular matrix and the absence of cell nuclei; scale bar = 100 µm. (E) DAPI staining of the control cotyledon, showing cell nuclei (blue fluorescence). (F) DAPI staining of the decellularized cotyledon, showing the absence of cell nuclei (no blue fluorescence). (G) Scanning electron microscopy (SEM) analysis: in (E), native cotyledon, the presence of cells embedded in the extracellular matrix is seen; in (H), decellularized cotyledon, only the interweaving of extracellular matrix fibers is seen, with a complete absence of cells.

Histological analysis using H&E staining revealed a complete absence of nuclear basophilia, with tissue architecture maintained and devoid of cellular remnants (Fig. 1C and 1D). DAPI staining confirmed the lack of nuclear DNA, supporting the H&E observations (Fig. 2E and 2F). These findings collectively validate the efficiency of the decellularization process in eliminating nuclear material, while preserving key ECM structures, including the vascular framework.

Quantification of residual DNA revealed an average concentration of 9.1 ng/mg of dry tissue. This value is significantly below the widely accepted immunogenicity threshold of 50 ng of gDNA per mg of dry tissue, reinforcing the potential of the scaffold for biomedical applications. The low DNA content further corroborates the histological and structural analyses and indicates a minimal risk of host immune activation upon implantation (Fig. 3).

OPEN IN VIEWER

FIG. 3.

Quantification of genomic DNA. Concentration of genomic DNA (gDNA) in native and decellularized bovine cotyledon. Decellularization led to a significant reduction in the residual gDNA, resulting in an average of 9.1 ng gDNA/mg of dry material, which is lower than the established immunogenic limit (<50 ng gDNA/mg of dry material). The dashed line represents the reference value considered safe for tissue bioengineering applications.

SEM demonstrated a fibrillar ECM with well-preserved collagen bundles and an absence of cellular debris (Fig. 2G and 2H). The structural integrity of the decellularized tissue suggests that the protocol preserved the native architecture essential for cell adhesion and migration. The open and organized fiber arrangement supports the potential for subsequent recellularization and nutrient diffusion.

By mass spectrometry, 144 unique proteins (related to ECM or cell adhesion ontologies) were identified in the biomaterials in comparison with native bovine cotyledon. From those proteins, majorly several adhesive glycoproteins (fibronectins, laminins, and vitronectin), cell adhesion proteins (integrins, dystroglycan, epithelial cell adhesion molecule, and thigh junction proteins), collagen types (I, III, IV, V, VI, VIII, XII, and XVIII), cytoskeletal proteins (actins and myosin), growth factors (tumoral growth factor beta, von Willebrand factor A, and von Willebrand factor), and GAGs (biglycan, decorin, heparan sulfate, nidogens, and tenascin) were detected. Highlighting that even after the decellularization process, not only were the ECM structures maintained, but several pivotal ECM components for cell adhesion, maintenance, and proliferation.

Hydrogel production and validation

Following enzymatic digestion of the decellularized ECM, a viscous, collagen-rich solution was obtained. This material was then blended with sodium alginate at concentrations of 5%, 8%, 10%, and 12% (w/v) and crosslinked with calcium chloride to form hydrogels. All formulations polymerized; however, distinct macroscopic differences were observed (Fig. 4). The 12% alginate formulation yielded a hydrogel with enhanced cohesiveness, greater structural uniformity, and superior mechanical integrity, whereas lower concentrations resulted in less stable and more fragile constructs (Fig. 5A and 5B).

OPEN IN VIEWER

FIG. 4.

Polymerization test of hydrogel from bovine decellularized cotyledon. Concentration test (5%, 8%, 10%, and 12%) of sodium alginate in digested bovine cotyledon extracellular matrix solution. Representation of the concentrations, respectively. It is possible to observe the difference in viscosity between the formulations, evidenced by the variation in the color and firmness of the gels. Also noticeable is the formation of bubbles due to the cross-linking process with calcium chloride (CaCl₂).

OPEN IN VIEWER

FIG. 5.

Hydrogel structural validation. (A) In the 8% sodium alginate hydrogel, a decrease in viscosity can be seen, making it liquefied after solubilization in culture medium. (B) 12% alginate hydrogel solubilized in the culture medium, showing maintenance of shape and greater viscosity compared to the 8% one. (C) Scanning electron microscopy (SEM) image of the hydrogel showing a compacted structure without the presence of pores. (D) SEM image showing the distribution of fibers between the lamellar structures and the presence of intercalated pores formed because of the cross-linking process.

SEM imaging of the hydrogel surfaces revealed clear differences in microarchitecture across formulations. The 12% alginate hydrogel displayed a highly porous and interconnected network, characterized by uniformly distributed pores conducive to cell infiltration and nutrient diffusion (Fig. 5C). In contrast, the 8% formulation exhibited reduced porosity and irregular pore distribution, suggesting limited spatial organization and compromised structural functionality for tissue engineering purposes (Fig. 5D).

Biomaterial and hydrogel cytocompatibility assay

The combination of antibiotic immersion, ultrasonic cleaning, and critical point drying sterilization procedure does not compromise the gross integrity of the biomaterials and hydrogels. After 7 days, under standard cell culture incubation, the culture medium from the biomaterial or hydrogel do not presented turbidity or color change, and no evidence of contamination was observed under microscopy.

The cytocompatibility assay showed cells all over the biomaterial surface, such as cells that were able to migrate into the central areas (Fig. 6A and 6B). Visually, by days 15 and 25 of the culture, the number increased, indicating that cells were capable of adhering, maintaining, and proliferating themselves. In the hydrogel, the cells also migrate through central areas and are able to adhere and proliferate (Fig. 6C and 6D).

OPEN IN VIEWER

FIG. 6.

Cytocompatibility assay on biomaterial and hydrogel. (A) Decellularized biomaterial without visible nuclei, confirming the effectiveness of the decellularization protocol. (B) Biomaterial after 25 days of cell culture, showing widespread distribution of nuclei stained in blue (DAPI), indicating successful cell survival. (C) Acellular hydrogel with no evidence of nuclear staining. (D) Hydrogel after 7 days of cell culture, showing nuclei (blue) within cells exhibiting preserved cytoplasm (arrowheads), as well as cellular debris distributed throughout the matrix (arrows). These findings demonstrate that both biomaterial and hydrogel supported cell retention and interaction, suggesting favorable microenvironments for cell survival and migration. Scale bar: 100 µm for all panels.