Poster O18 - Collagen and Cellular Infiltration Profile and Characterization in Tissue Remodeling Induced by a Microgel Composite

Introduction: Soft tissue injuries caused by external trauma, congenital defects, or surgical operations present significant challenges for clinicians. These injuries can lead to functional problems, psychological burdens, and even morbidity in patients. Current treatment options, such as prosthetics or autologous transplants, often result in complications like tissue necrosis, fibrosis, and infections at the injury site, and they fail to promote long-term tissue healing and remodeling effectively. Our laboratories have recently developed a novel nanofiber hydrogel composite (NHC) material that addresses these challenges. The NHC is designed to maintain sufficient integrity and porosity to encourage cell infiltration and vascularization without the need for exogenous growth factors, leading to effective remodeling of the injected area. This biostimulatory NHC material is composed of a hyaluronic acid (HA) hydrogel, which provides porosity for cellular infiltration, covalently bound to electrospun polycaprolactone (PCL) nanofibers that offer structural integrity. In the context of tissue remodeling and wound healing, collagen deposition plays an integral role. The process of collagen deposition is dynamic and time-dependent, critical for restoring the structure and function of damaged tissues. This study aims to explore and understand the progression and mechanisms of collagen deposition in the NHC-injected site, providing insights into how this material supports tissue regeneration and healing.

Materials and

Methods: NHC materials were prepared by covalently binding HA hydrogels to PCL nanofibers, following a previously established protocol with modifications for increased tunability and reproducibility. The NHC materials were processed into gel microparticles, which were then subcutaneously injected into C57BL/6 mice. Retrieved samples were subjected to comprehensive histological analysis. This included hematoxylin and eosin (H&E) staining to evaluate general tissue architecture and cellular infiltration, Masson's trichrome (MAS) staining to assess collagen deposition, and picrosirius red (PSR) staining for detailed visualization of collagen fibers. Additionally, second harmonic generation (SHG) imaging was used to observe fibrillar collagen structures in the NHC material. To analyze collagen expression at the molecular level, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed. For image analysis, Python (cv2 package) was utilized for pixel intensity quantification, and a FIJI macro was employed for cell counting in the stained samples. A hand-drawn mask was applied to each image to focus exclusively on the microgel of interest, ensuring precise evaluation of the material's effects.

Results, Conclusions, and Discussions: Results &

Conclusions:
The NHC material demonstrated substantial collagen deposition at day 35, with networks of blue collagen strands visible throughout the remodeling areas (Fig. 1A). In contrast, the control HA material showed a collagen capsule, indicative of a fibrotic response (Fig. 1A). This distinct host response highlights the NHC material's favorable environment for tissue remodeling compared to the HA control. Under polarized light, PSR staining revealed birefringent collagen within the NHC material, showcasing different types of collagens (Fig. 1B). SHG imaging provided a detailed view of fibrillar collagen distribution inside the NHC material (Fig. 1C), confirming the presence of structured collagen networks. Further molecular analysis through RT-qPCR revealed varied expression levels of different collagen isoforms, as shown in the heatmap (Fig. 1D). NHC 1 is 150 Pa, NHC 2 is 450 Pa. Preliminary image analysis results have shown significant infiltration, suggesting that while the NHC material promotes favorable collagen deposition and cellular infiltration, further refinement of evaluation methods is needed for more accurate assessment. Future goals include enhancing mask drawing accuracy and increasing the organizational and directional information about collagen strands to better understand the NHC material's impact on tissue remodeling.

Conclusion:
The NHC material facilitates tissue remodeling with a unique collagen deposition profile, distinguishing it from the HA control gel. A thorough quantitative study of collagen distribution through histological staining and SHG imaging, combined with qPCR data on collagen isoforms, has provided valuable insights into the specific mechanisms of collagen synthesis and remodeling promoted by the NHC material. Initial findings indicate the soft NHC material’s enhanced efficacy in supporting soft tissue healing. Further investigation using synthesized results from various imaging techniques will continue to reveal the underlying mechanisms at play and help refine the material for optimal clinical application.

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