Photocrosslinked Human Amniotic Membrane Hydrogel for Spinal Cord Injury Repair

Academic Background

Spinal Cord Injury (SCI) is a severe neurological disorder that often leads to loss of motor function and a decline in the quality of life for patients. Despite significant advancements in tissue engineering and regenerative medicine in recent years, functional recovery after spinal cord injury remains a global challenge. The primary issues lie in the difficulty of axonal regeneration in the injured area and the formation of scar tissue, which hinders nerve repair. The Human Amniotic Membrane (HAM), as a biological material, offers advantages such as protecting nerve growth, inhibiting scar formation, and promoting neovascularization. However, its weak physical properties limit its application in spinal cord injury treatment.

To address this issue, researchers have attempted to enhance the mechanical properties of the human amniotic membrane through chemical modification and photocrosslinking techniques while preserving its biological activity. In this study, researchers decellularized the human amniotic membrane and chemically grafted it with Methacrylic Anhydride (MA), followed by photocrosslinking with Gelatin Methacrylate (GelMA) to prepare a novel composite hydrogel scaffold for spinal cord injury repair.

Source of the Paper

This paper was co-authored by Tao Xu, Changwei Yang, Yang Lu, Heng Wang, Cheng Chen, Yuchen Zhou, and Xiaoqing Chen, all affiliated with the Department of Spine Surgery, Affiliated Hospital of Nantong University Medical School. The paper was published online on November 12, 2024, in the journal Bio-design and Manufacturing, with the DOI 10.1007/s42242-024-00318-x.

Research Process

1. Decellularization and Chemical Modification of Human Amniotic Membrane

First, researchers obtained fresh human amniotic membranes from the Department of Obstetrics at the Affiliated Hospital of Nantong University. After washing with phosphate-buffered saline (PBS), the membranes underwent freeze-thaw cycles and were treated with trypsin-EDTA solution for 12 hours to remove epithelial cells, resulting in decellularized human amniotic membrane (HAAM). Subsequently, HAAM was chemically grafted with Methacrylic Anhydride (MA) to form the HAAM-MA composite. Successful grafting of MA was confirmed through Fourier Transform Infrared Spectroscopy (FTIR) analysis.

2. Preparation of GelMA-HAAM-MA Composite Hydrogel

Lyophilized GelMA was dissolved in PBS and heated to 70°C until fully dissolved. HAAM-MA and the photoinitiator Lithium Phenyl(2,4,6-trimethylbenzoyl) Phosphinate were then added, and the mixture was photocrosslinked under ultraviolet light (395-480 nm) to form a 2-mm-thick gel. Scanning Electron Microscopy (SEM) revealed that the GelMA-HAAM-MA composite hydrogel had a uniform porous structure with an average pore size of 26±9 µm, suitable for the growth and migration of nerve cells.

3. Mechanical Properties and Degradation Testing of the Composite Hydrogel

Researchers conducted mechanical tensile tests on HAAM, HAAM-MA, GelMA, and GelMA-HAAM-MA. The results showed that GelMA-HAAM-MA had a maximum load value of 4.18±0.06 MPa and an elastic modulus of 19.69±0.52 MPa, significantly outperforming the other materials. Additionally, GelMA-HAAM-MA completely degraded within 15 days, with a moderate degradation rate suitable for spinal cord injury repair applications.

4. In Vitro Cell Experiments

Neurons were extracted from neonatal Sprague-Dawley (SD) rats and co-cultured with the GelMA-HAAM-MA composite hydrogel. The results demonstrated that the composite hydrogel could maintain neuronal viability and significantly improve neuronal survival under low-nutrient conditions. Furthermore, the composite hydrogel promoted the proliferation and migration of Human Umbilical Vein Endothelial Cells (HUVECs), indicating excellent angiogenic potential.

5. In Vivo Animal Experiments

In an SCI model using SD rats, researchers implanted the GelMA-HAAM-MA composite hydrogel at the injury site. After four weeks, pathological sections and immunofluorescence staining revealed that the composite hydrogel significantly reduced scar tissue formation and promoted the growth of new neurons. Additionally, the motor function recovery score (BBB score) of the rats improved significantly, demonstrating the composite hydrogel’s effectiveness in promoting spinal cord injury repair.

Key Findings

1. Successful Preparation of HAAM-MA: Through chemical grafting, HAAM-MA retained the biological activity of the human amniotic membrane while enhancing its mechanical properties.
2. Superior Mechanical Properties of GelMA-HAAM-MA: The composite hydrogel’s maximum load value and elastic modulus were significantly better than those of pure GelMA and HAAM, making it suitable for long-term stability at the spinal cord injury site.
3. Support for Neurons: GelMA-HAAM-MA maintained neuronal viability and significantly improved neuronal survival under low-nutrient conditions.
4. Angiogenic Potential: The composite hydrogel promoted the proliferation and migration of HUVECs, demonstrating excellent angiogenic potential.
5. In Vivo Repair Effects: In the SD rat SCI model, GelMA-HAAM-MA significantly reduced scar tissue formation and promoted the growth of new neurons, with a notable improvement in the rats’ motor function recovery scores.

Conclusion and Significance

This study successfully prepared a novel photocrosslinked GelMA-HAAM-MA composite hydrogel. This material not only exhibits excellent mechanical properties but also effectively mimics the extracellular matrix microenvironment of the spinal cord injury site, promoting nerve regeneration and angiogenesis. Both in vitro and in vivo experiments demonstrated the composite hydrogel’s good biocompatibility and repair effects, offering a promising biomaterial for spinal cord injury treatment.

Research Highlights

1. Innovative Material Design: By combining chemical grafting and photocrosslinking techniques, the human amniotic membrane was successfully integrated with GelMA to create a composite hydrogel with superior mechanical properties and biological activity.
2. Multifunctional Repair Effects: The composite hydrogel not only promotes nerve regeneration but also inhibits scar formation and enhances angiogenesis, demonstrating multifunctional repair capabilities.
3. Significant In Vivo Repair Effects: In the SD rat SCI model, the composite hydrogel significantly improved motor function recovery scores, showing great potential for clinical applications.

Additional Valuable Information

The success of this study provides a new approach for spinal cord injury repair. Future research could further optimize the degradation rate and scar-inhibiting effects of the composite hydrogel to better meet clinical needs. Additionally, the preparation method of this material offers a reference for the development of other tissue engineering materials.