ASSOCIATE PROFESSOR & GRADUATE PROGRAM CHAIR Rowan University, United States
Introduction: Extracellular matrix (ECM) hydrogels, derived from decellularized tissues, are an appropriate option for engineering biomimetic scaffolds due to their rich array of biochemical signals essential for cell differentiation and proliferation[1]. Previous studies have utilized various biomaterials, such as collagen and synthetic polymers, to engineer scaffolds that support cell growth[2]. However, ECM hydrogels offer distinct advantages due to their complex composition and bioactivity[1]. One limitation of ECM hydrogels is the lack of ordered structures to promote cell alignment for regeneration of aligned tissues.
In this study, we aim to engineer a robust 3D decellularized ECM hydrogel embedded with aligned polycaprolactone (PCL) nanofibers. PCL is a biodegradable polymer known for its mechanical strength, biodegradability, and biocompatibility[2]. By integrating PCL nanofibers into the scaffolds, we aim to provide a framework that supports the ECM hydrogel, improving its cell-supportive properties, including guiding cell alignment and migration. Our approach involves creating a composite scaffold through a three-step process: (1) layer-by-layer additive manufacturing to engineer a 3D gelatin-PCL nanofiber composite scaffold; (2) infiltration of the gelatin scaffold with alginate with subsequent removal of the gelatin; and (3) infiltrating the alginate with ECM with subsequent removal of the alginate. The resulting 3D structures will be characterized by their mechanical properties, biocompatibility, and ability to support cell growth, highlighting their potential to guide cell alignment and migration for aligned tissue regeneration.
Materials and
Methods: Layer-by-layer additive manufacturing
PCL nanofibers were made using the method of electrospinning. Spun PCL nanofibers were collected and placed in plastic frames Figure 1A. These plastic frames were then dipped completely in 10% (w/v) gelatin liquid solution at 40-50°C. The dipped frames were then stacked vertically to create the desired 3D shape. The stacked frames were then transferred to a refrigerator and subjected to a sol-gel transition for 5 minutes to solidify the gelatin Figure 1B. Once the gelatin had solidified, a 3D scaffold with the desired length and width was carefully removed from the plastic frames using a clean blade Figure 1C.
Alginate Infiltration
Gelatin-PCL composite scaffolds Figure 1D were gently immersed in a microcentrifuge containing 1 ml of 2% (w/v) sodium alginate solution. The infiltration process was conducted at room temperature on a shaker for 10 days to allow for sufficient diffusion of alginate. After complete infiltration, the scaffolds were crosslinked with 2% (w/v) calcium chloride (CaCl2) solution for 24 hours. Finally, the alginate scaffolds were placed in water to remove excess CaCl2.
ECM Hydrogel Infiltration
Decellularized ECM was prepared from cow tail tissue and dissolved in a pepsin solution[1]. The alginate scaffolds were infiltrated with the ECM solution for 10 days. After 10 days, the ECM solution was neutralized to form an ECM hydrogel. Finally, the composite alginate/ECM/PCL nanofiber scaffold was cut out of the ECM hydrogel and placed in 9% NaCl to dissolve the alginate resulting in an ECM-nanofiber composite scaffold.
Results, Conclusions, and Discussions: By using a step-by-step infiltration method, we created a scaffold that combines the highly desirable biochemical properties of ECM with aligned nanofibers, providing physical contact guidance for cell growth, including nerve cells, while maintaining biocompatibility.
This study is pioneering in the use of continuous nanofibers in the creation of ECM-nanofiber composite scaffolds. Previous studies have typically utilized short fibers or particles that are usually not aligned, but this approach of integrating continuous fibers provides superior mechanical properties and better mimics the natural extracellular matrix structure [2]. A crucial feature of the embedded aligned nanofibers is their ability to guide cell growth through the hydrogel with the nanofibers providing a fixed pathway through the hydrogel. This method offers versatile applications across various types of gels, fibers, and surface coatings. This adaptability makes our approach a promising strategy for developing scaffolds tailored to the specific regenerative needs of different types of aligned tissues.
Dissolving the leftover sacrificial scaffold is a crucial step for obtaining the desired independent scaffold after infiltration. The temperature-dependent sol-gel transition of gelatin allows us to dissolve leftover gelatin by raising the temperature in our gelatin-alginate scaffold [3]. However, gelatin is unsuitable for direct ECM infiltration because it dissolves at body temperature (37°C) and in the presence of pepsin [3], [4], while both conditions are needed for ECM hydrogel preparation [1]. To address this, we use an additional infiltration with alginate, which remains stable under these conditions.
Currently, we dissolved the alginate scaffold by immersing it in a saline (9% NaCl) solution[5]. We concluded through experiments that the saline solution does not affect the ECM gel, making it a viable method. An infiltration experiment has been performed that displayed promising results of adequate infiltration of ECM into the alginate hydrogel. The ECM composition of the final scaffold was confirmed by the dissolving of the scaffold when submerged in pepsin.
Future steps include further assessing the infiltration rate of ECM into alginate. If successful, the subsequent step will involve fabricating a scaffold with embedded aligned nanofibers using our method and incorporating the scaffold into in-vivo and in-vitro studies.
Acknowledgements (Optional): [1] B. A. Flynn, H. J. Woodhouse, E. H. Cerra, J. M. Barker, and M. T. Wolf, "Decellularized ECM Hydrogels Using Pepsin and Neutralization: Methods and Applications," Acta Biomaterialia, vol. 73, pp. 20-31, Mar. 2018.
[2] L. A. Smith and P. X. Ma, "Nano-fibrous scaffolds for tissue engineering," Colloids and Surfaces B: Biointerfaces, vol. 39, no. 3, pp. 125-131, 2004.
[3] M. Nakayama, K. Takahashi, T. Matsuura, A. Nagata, and R. Sakaue, "Temperature-Responsive Hydrogels Based on Gelatin and Poly(N-isopropylacrylamide) for Controlled Drug Release," Journal of Biomedical Materials Research Part A, vol. 102, no. 5, pp. 346-352, May 2014.
[4] S. E. Freeman, C. D. Lupton, and J. L. Wilkins, "Enzymatic Degradation of Crosslinked Gelatin Scaffolds for Tissue Engineering," Tissue Engineering Part B: Reviews, vol. 21, no. 6, pp. 555-564, Dec. 2015.
