Spray Crosslinking Enhances Amniotic Membrane Strength

Published in the journal Collagen and Leather in April 2026, the study presents a novel protocol that addresses the long-standing limitations of amniotic membrane as a clinical biomaterial. The primary finding is that this spray-based approach increases the maximum uniaxial tensile fracture stress of the membrane by 156.1% while reducing its weight loss during enzymatic degradation from 100% to 63.6%. This method offers a practical solution for preserving the functional properties of the membrane without the drawbacks associated with traditional immersion techniques.

Human amniotic membrane is a valuable natural biomaterial widely used in clinical applications such as ophthalmology, wound healing, and surgery due to its anti-inflammatory and tissue-repair properties. However, its clinical utility has been restricted by its thin structure, which ranges from 0.02 to 0.05 mm in thickness, and its relatively low mechanical strength. In current practice, the membrane is often applied in a fresh state or preserved without chemical crosslinking, making it susceptible to rapid enzymatic degradation in vivo, often within about one week. This rapid degradation and susceptibility to tearing during handling limit its use to small-area tissue repair surgeries.

To overcome these challenges, the research team developed a spray-crosslinking technology that utilizes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). This chemical combination promotes the formation of covalent bonds within the collagen network of the membrane. This supplementary solution is designed to replace water-mediated hydrogen bonds with stable glycerol-mediated hydrogen bonds, thereby preserving the membrane’s native flexibility after rehydration.

The study compared the spray-crosslinking method with traditional immersion crosslinking to evaluate its efficacy in preserving the membrane’s biological components. Results indicated that the spray method better preserved the water-soluble total protein content compared to immersion. In the immersion group, the water-soluble total protein concentrations were significantly lower, ranging from 205.98 to 220.45 µg/mL, whereas the spray-crosslinked groups retained higher concentrations, with the highest reaching 1371.47 µg/mL. This difference is attributed to the fact that immersion requires soaking the membrane in crosslinker solutions, which leads to the loss of water-soluble proteins, while the spray method allows the reagents to penetrate via capillary action without excessive dissolution of endogenous proteins.

Furthermore, the spray method resulted in lower residual levels of the crosslinking agents. The residual EDC concentrations in spray-crosslinked samples were measured at 0.05, 0.13, and 0.22 mM for different concentrations, which were substantially lower than the 0.19, 0.87, and 1.91 mM found in immersion-crosslinked samples. This reduction in chemical residue is significant for biomedical applications, as it minimizes the potential for toxicity associated with unreacted crosslinking agents. The study confirmed the successful formation of covalent bonds through Fourier transform infrared spectroscopy, which detected a distinct C-N bond absorption peak at 1206 cm⁻¹ in the crosslinked samples, a peak absent in the uncrosslinked controls.

Mechanical testing revealed that the spray-crosslinked amniotic membrane exhibited the highest uniaxial tensile strength among all tested groups, reaching 3.15 ± 0.27 MPa. This was a marked improvement over the fresh amniotic membrane, which showed a tensile strength of 0.62 ± 0.03 MPa, and the membrane treated only with the hydrogen-bond supplementary solution, which measured 1.23 ± 0.24 MPa. The suture pull-out testing also demonstrated enhanced performance, with the crosslinked samples requiring significantly greater stress for suture pull-out compared to the fresh samples. These mechanical improvements suggest that the crosslinked membrane is more robust and better suited for surgical handling and implantation.

The study also evaluated the enzymatic stability of the membrane using pepsin degradation assays. The weight loss ratio of the crosslinked samples decreased significantly compared to the uncrosslinked materials. Specifically, the 24-hour enzyme degradation weight loss ratio for the spray-crosslinked amniotic membrane decreased by 36.37%, while the pig intestine surrogate showed a decrease of 85.55%. This enhanced resistance to enzymatic degradation is likely due to the formation of stable amide bonds that strengthen intermolecular forces and reduce the number of available enzyme binding sites. The protective effect of the crosslinking system was further confirmed in a mouse subcutaneous implantation test, where the crosslinked samples showed a lower degree of degradation compared to the fresh samples.

Biosafety evaluations were conducted to ensure the clinical viability of the spray-crosslinked membrane. Cytotoxicity tests using human embryonic kidney 293 T cells showed that the cell viability of the spray-crosslinked samples was 85.11%, which was not significantly different from the fresh control group at 87.95%. Hemocompatibility tests indicated that the hemolysis rate of the crosslinked samples was 0.82%, well below the 2% safety threshold specified in ISO 10993-1 standards. Additionally, scanning electron microscopy revealed that the crosslinked membrane had significantly lower platelet adhesion compared to the fresh membrane, suggesting superior anticoagulant performance and blood compatibility.

The research team utilized pig intestine casing as a surrogate material for most of the experiments due to the limited availability of human amniotic membrane. The pig intestine has a similar collagenous composition and thickness to human amniotic membrane, making it a suitable model for investigating the general feasibility of the spray-crosslinking method. However, the study acknowledges that the use of a surrogate material is a limitation, and further research is needed to confirm the findings in human amniotic membrane. The study also noted that the immunogenicity evaluation focused primarily on macrophage response, without investigating effects on other inflammatory cells or cytokine expressions.

The development of this spray-crosslinking technology represents a significant advancement in the preparation of dry amniotic membrane. Unlike freeze-drying, which can increase tissue brittleness and reduce mechanical strength, the spray method allows for room-temperature storage while retaining mechanical properties comparable to the wet membrane. The process is described as simple, efficient, and mild, with the advantage of maximizing the retention of water-soluble total protein while reducing chemical residues. This approach could expand the clinical applicability of amniotic membrane by providing a more durable and biocompatible biomaterial for a wider range of surgical and therapeutic applications.

The study highlights the potential of the spray-crosslinking technique as an innovative strategy for fabricating durable, biocompatible amniotic membrane-based biomaterials. By addressing the issues of poor mechanical strength, fast degradation rate, and difficulty in preserving water-soluble total protein, this method paves the way for future clinical and biomedical applications. The findings suggest that the spray-crosslinked amniotic membrane could be a promising candidate for use in ophthalmology, wound healing, and other surgical fields where the integrity and longevity of the biomaterial are critical.

The authors conclude that the synergistic strategy of covalent bond network enhancement, hydrogen bond protection, and active component retention effectively solves the problems associated with traditional amniotic membrane preparation methods. The spray-crosslinking protocol yields a mechanically robust and enzymatically stabilized membrane without compromising its flexibility. This research provides a practical and efficient solution for the preservation and enhancement of amniotic membrane, potentially leading to improved outcomes in clinical settings where this biomaterial is used.

Subject of Research: Development of a spray-crosslinking method to improve the mechanical and enzymatic properties of dry human amniotic membrane.

Article Title: Improved mechanical performance and enzymatic stability of crosslinked dry amniotic membrane by spraying

Article References: Wang, Y., Cao, L., He, H., Khabbir, A., Hu, C., Liu, R., & Lei, Y. (2026). Improved mechanical performance and enzymatic stability of crosslinked dry amniotic membrane by spraying. Collagen and Leather, 8(1), Article 17. https://doi.org/10.1186/s42825-026-00241-0

Image Credits: AI Generated

DOI: 10.1186/s42825-026-00241-0

Keywords: amniotic membrane, biomaterials, crosslinking, EDC/NHS, mechanical strength, enzymatic stability, tissue engineering, spray technology, clinical applications, collagen, Improved, mechanical