(B-61) A Biofabrication Approach to Investigate the Effects of Locally Disrupted Collagen Fiber Alignment on Cell Motility

Introduction:: The extracellular matrix (ECM) is a complex fibrous network that functions as a structural support for cells and provides guidance cues that affect cell phenotype and motility [1]. Furthermore, the ECM responds to biochemical and biophysical stimuli by remodeling its microarchitecture. For instance, Type-1 collagen fibers (COL1) are organized into aligned domains by cell-generated traction forces [2]. This alignment of fibers plays a key role in the formation of hierarchal tissues, guiding morphogenesis, and directing cell migration [2]. While it is well-known that cells can sense and respond to mechanical stimuli transmitted through the aligned fibrous microarchitecture [3], it remains unclear how local disruptions in fiber alignment affect cell motility and long-distance cellular communication. Answering these questions is critical to understanding tissue remodeling and cellular dynamics, which are relevant to a wide range of areas from developmental biology to cancer metastasis.

Despite the availability of various biofabrication methods to engineer collagen fiber alignment, none can engineer local discontinuities in fiber alignment. This experimental capability is necessary to explore how various cell types perceive and respond to the heterogeneous fiber structures present in the ECM. We have recently shown that extensional flow within a microchannel can be used to control the degree of fiber alignment and create long-range (5 mm scale) fiber alignment [4] (Fig. 1A). Here, we utilize fluid dynamics principles to create an abrupt disruption in the extensional flow to create local disorder in the alignment of fibers.


Materials and Methods:: A silicon mold was fabricated using standard soft lithography techniques [4]. Degassed PDMS prepolymer (10:1 base to catalyst ratio) was poured over the mold and cured for 1 h at 70 °C on a hotplate. The PDMS chip was cored using a 1mm biopsy punch and bonded to a coverslip that was treated with poly (octadecene maleic-alt-anhydride) (POMA) to promote collagen attachment [5].

Methacrylated Type I bovine collagen with a concentration of 2.5 mg/ml was neutralized and injected into the chips at a flow rate of 150 µl/min. After gelation, collagen fibers were imaged using confocal reflectance microscopy (CRM) with a Leica SP5 confocal microscope with a 40× water objective. At the region of interest, 2 μm tall z-stacks comprised of 13 slices were captured. For collagen fiber quantification, a maximum intensity projection image was created using FIJI. Subsequently, LOCI CT-FIRE software was employed to detect the collagen fibers present in the projected CRM images and determine the Coefficient of Alignment (CoA).

All experiments were independently performed in triplicate, and the results were reported as mean ± SD. For all statistical analyses, p < 0.05 was considered to be significant. GraphPad Prism 9.5.1 was used for analysis and plotting.


Results, Conclusions, and Discussions:: Results: The increase of flow velocity in a constricting microchannel resulted in an extensional strain rate that can be utilized to enhance the alignment of collagen fibers [4] (Fig. 1A). To locally disrupt alignment, we added a cross channel perpendicular to the main channel (Fig. 1B). The collagen solution accelerates as it flows through the main constricting channel until it reaches a branching intersection, where its velocity abruptly drops and then increases after the intersection. This abrupt decrease and increase of velocity at the intersection results in the local disruption of fiber alignment (Fig. 1C). As shown in Fig. 1D, the coefficient of fiber alignment at the intersection is 0.39 ± 0.05 while it is 0.62 ± 0.07 before and 0.59 ± 0.11 after the intersection­.

Conclusion: Gaining knowledge about the influence of local disruptions in fiber alignment on cell motility and long-distance communication can offer valuable insights into how cells navigate through the 3D ECM across various length scales. In this work, we developed a new biofabrication method for creating controlled local disruptions in a continuous alignment landscape. Our approach represents the first demonstration of such a method, and it allows us to investigate how cells interpret and respond to non-uniform features in their environment.


Acknowledgements (Optional): :

References (Optional): : [1] Ahmed, Adeel, et al. "Engineering fiber anisotropy within natural collagen hydrogels." American Journal of Physiology-Cell Physiology 320.6 (2021): C1112-C1124.

[2] Sonbol, Hala Salim. "Extracellular matrix remodeling in human disease." Journal of microscopy and ultrastructure 6.3 (2018): 123.

[3] Wang, Hailong, et al. "Long-range force transmission in fibrous matrices enabled by tension-driven alignment of fibers." Biophysical journal 107.11 (2014): 2592-2603.

[4] Ahmed, Adeel, et al. "Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels." Biofabrication 14.3 (2022): 035019.

[5] Ahmed, Adeel, et al. "Microengineered 3D collagen gels with independently tunable fiber anisotropy and directionality." Advanced materials technologies 6.4 (2021): 2001186.