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Manufacturing micropatterned collagen scaffolds with chemical-crosslinking for development of biomimetic tissue-engineered oral mucosa

Medicine and Health

Manufacturing micropatterned collagen scaffolds with chemical-crosslinking for development of biomimetic tissue-engineered oral mucosa

A. Suzuki, Y. Kodama, et al.

This study by Ayako Suzuki and colleagues explores the development of biomimetic tissue-engineered oral mucosa using innovative micropatterned collagen scaffolds. By creating negative molds with various patterns, they significantly enhanced the mechanical properties of these scaffolds, paving the way for improved epithelial regeneration and tissue grafting applications.

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Playback language: English
Introduction
The interface between epithelium and connective tissue in oral mucosa, characterized by rete ridges, is crucial for tissue integrity and nutrient supply. Current tissue constructs lack this 3D undulating structure. This study focuses on creating biomimetic oral mucosa using micropatterned collagen scaffolds. The lack of clinically relevant constructs mimicking the dermal-epidermal junction (DEJ) structure, particularly the rete ridges, presents a significant challenge in regenerative medicine. Rete ridges are essential for maintaining epithelial integrity, nutrient transport, and stem cell niche function. Existing methods for scaffold creation have limitations in precisely reproducing the complex 3D architecture of the DEJ, particularly in oral mucosa, small intestinal mucosa, gastric mucosa, and corneal limbus. Previous attempts by the authors to create microstructured collagen scaffolds resulted in flattening and expansion of the microstructures, diminishing the biomimetic cues. To improve upon this, this study investigates the use of chemical crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to enhance the mechanical properties of the collagen scaffold, aiming for better preservation of the micropatterned features and improved tissue regeneration. The study hypothesizes that chemically crosslinked micropatterned collagen scaffolds will provide a superior microenvironment for oral mucosa regeneration, leading to the formation of a well-stratified epithelium with rete ridge-like structures.
Literature Review
Several nano- and micro-fabrication techniques have been used to mimic the DEJ microstructure. However, there is a lack of studies focusing on tissue-engineered oral mucosa constructs with geometric features mimicking the cell-scaffold interface. Previous research has demonstrated the benefits of micropatterning on various biomaterials, showing improved cell responses and tissue integration. For example, micropatterned poly-vinyl alcohol vascular grafts have shown enhanced patency and endothelialization compared to non-patterned grafts. The use of reconstituted collagen is widespread, but it often lacks the physical properties of native tissues due to incomplete crosslinking. Chemical crosslinking, particularly with EDC, is a well-established method to improve the mechanical integrity and stability of collagen-based biomaterials. However, the existing methods for creating these complex microstructures, including photolithography, electrospinning, and 3D bioprinting, have their own limitations and challenges in replicating the intricacies of the DEJ structure.
Methodology
The study employed a combination of microelectromechanical systems (MEMS) processes and soft lithography to fabricate negative molds with 15 different micropattern designs, featuring grid and pillar configurations with varying dimensions and aspect ratios. These molds were used to create micropatterns on 1% type I tilapia scale atelocollagen matrices. Half of the collagen gels were then chemically crosslinked using 1% EDC. The physical properties of the resulting collagen scaffolds, with and without EDC crosslinking, were characterized using rheometry and tensile testing to measure storage modulus (G'), loss modulus (G''), and Young's modulus. The handling properties were assessed via suturing tests using 4-0 braided silk. The ability of the micropatterned scaffolds to support the growth and differentiation of human oral mucosa keratinocytes was evaluated by creating tissue-engineered oral mucosa equivalents (EVPOMEs). Human oral mucosa keratinocytes were seeded onto the scaffolds and cultured in a submerged condition followed by an air-liquid interface culture. The diameter of the EVPOMEs was measured daily to assess contraction. Finally, histological analysis (hematoxylin and eosin staining) was conducted to assess the preservation of the micropatterns and the resulting epithelial structure. Scanning electron microscopy (SEM) was also used to examine the collagen fibril network structure in the crosslinked and non-crosslinked scaffolds.
Key Findings
Image analysis confirmed successful transfer of the micropatterns onto the scaffold surfaces. Rheological tests showed that the collagen scaffold exhibited viscoelastic properties characteristic of an ideal gel, with EDC crosslinking increasing the Young's modulus (approximately 48.65 kPa vs. 29.10 kPa at 37°C). EDC crosslinking significantly improved the mechanical properties of the scaffolds, making them more durable and easier to handle during suturing. Macroscopic observation revealed that EDC crosslinking significantly reduced the contraction of EVPOMEs during the culture period. Histological examination showed that the grid-type micropatterns, particularly those with smaller dimensions, were better preserved than pillar-type micropatterns, even with EDC crosslinking. A well-stratified epithelial layer with rete ridge-like structures was observed on the scaffolds with grid-type micropatterns, especially those crosslinked with EDC. In contrast, pillar-type micropatterns showed significant collapse, even after EDC crosslinking. The SEM images showed that the diameters of fibrils in the 1% EDC crosslinked collagen matrices ranged from 40 to 120 nm, approximately 1.2 times thicker than those without EDC crosslinking.
Discussion
The findings demonstrate the successful fabrication of micropatterned collagen scaffolds with improved mechanical properties due to EDC crosslinking. The grid-type micropatterns proved superior in supporting epithelial regeneration and rete ridge formation. The improved mechanical strength afforded by EDC crosslinking is crucial for handling and suturing the scaffold during surgical procedures. The results highlight the importance of careful consideration of micropattern design, specifically choosing grid-type configurations for better maintenance of the microstructure and consequently, better tissue regeneration. Although EDC crosslinking enhanced the scaffold’s properties, some gaps between cells and the scaffold remained at the corners of the micropatterns, suggesting the need for further optimization of the design, particularly considering curved surfaces for better cell adhesion. The use of tilapia-derived collagen provides a sustainable alternative to mammalian collagen, expanding the possibilities for biomaterial production. The study’s limitations, especially in terms of incomplete shape fidelity, necessitates ongoing optimization to achieve a more precise replication of the in vivo DEJ architecture.
Conclusion
This study successfully demonstrated a method for manufacturing micropatterned collagen scaffolds with enhanced mechanical properties using EDC crosslinking. The grid-type micropatterns supported the formation of a well-stratified epithelium with rete ridge-like structures. This improved biomimetic scaffold holds promise for tissue engineering applications, particularly in oral mucosa regeneration. Future research should focus on addressing the limitations of the current scaffold design to further improve the replication of the in vivo DEJ structure and enhancing cell adhesion to the scaffold surface, potentially by incorporating basement membrane components or exploring alternative crosslinking strategies. In vivo studies are needed to evaluate the long-term biocompatibility and efficacy of these scaffolds.
Limitations
The study showed incomplete shape fidelity of the micropattern, particularly with pillar-type designs, highlighting the need for further optimization of the fabrication process and material selection. The study mainly focused on in vitro analysis, and in vivo studies are essential to validate the findings and assess long-term biocompatibility, biodegradability, and the scaffold's effect on epithelial regeneration in a living organism. The study only assessed a limited number of micropattern designs and aspect ratios; more extensive exploration of design parameters might reveal even better configurations for promoting epithelial regeneration. The use of human cells from only five individuals might influence the overall generalizability of the obtained results.
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