Z25 - Impact of Collagen Structural Variants and Media Conditions on Osteogenesis of Mesenchymal Stem Cells in 3D Fibrin Gel Systems

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Azhar Muhammad

Student
Stony Brook University
Bayside, New York, United States

Co-Author(s)

Xiangyu Gong, PhD

Post Doctoral Researcher
Stony Brook University
New Haven, Connecticut, United States

Primary Investigator(s)

MM

Michael Mak

Associate Professor
Stony Brook University, United States

Introduction: : Mesenchymal stem cells (MSCs) are key to regenerative medicine due to their osteogenic capacity, immunomodulation, and pro-angiogenic properties. While 2D cultures fail to mimic native tissue environments, 3D hydrogels provide a more biomimetic context by replicating extracellular matrix (ECM) architecture and mechanics. Prior studies have explored the role of matrix stiffness in osteogenesis, but the influence of microscale ECM structure—particularly collagen topology—remains underexplored. Here, we present a tunable 3D fibrin-collagen hydrogel platform incorporating two collagen architectures: a dense reticular meshwork and cell-scale bundles generated via macromolecular crowding (MMC). We examine how these topologies, combined with media conditions, influence MSC osteogenesis.

Materials and

Methods: : The reticular collagen network was prepared by mixing 24 μL of chilled ddH₂O, 20 μL of 10× PBS with phenol red, 150 μL of 8 mg/mL collagen I, and 6 μL of 0.5 M NaOH on ice, yielding a 200 μL neutralized solution at ~6 mg/mL, confirmed by the phenol red pH indicator. For bundle formation, 150 μL of 8 mg/mL collagen was rapidly pipetted into 3 mL of 200 mg/mL PEG8000 in PBS to initiate macromolecular crowding-induced assembly (Fig. 1a). After 20–30 vigorous pipetting cycles, the solution was diluted with 12 mL PBS and centrifuged (4000 rpm, 3 min). The pellet was resuspended in 200 μL PBS, scissor-cut for 30 s, and homogenized for 15 s, yielding bundles at ~6 mg/mL.

Three gel formulations were prepared: fibrin alone (Fig. 1b), fibrin with neutralized collagen (Fig. 1c), and fibrin with collagen bundles (Fig. 1d). In each, fibrinogen (6 mg/mL) was mixed 1:1 with a thrombin solution (4 U/mL) containing either PBS, neutralized collagen, or collagen bundles. MSCs were suspended in the fibrinogen fraction at 60,000 cells/mL to achieve 6000 cells per 40 μL dome. Gels were cultured in DMEM, DMEM + 100 ng/mL BMP-2, osteogenic medium, or osteogenic medium + BMP-2 for 7 days. Following culture, gels were fixed in 4% paraformaldehyde, dehydrated in 100% ethanol, and stained with 2% Alizarin Red S for 30 minutes to assess calcium deposition. Samples were imaged using transmission light microscopy.

Results, Conclusions, and Discussions:: The collagen-fibrin hybrid gels successfully incorporated tunable collagen architectures. Reticular collagen formed a dense submicron mesh, while MMC-induced bundles resembled native tissue-scale ECM with thicker, aligned fibers. Compared to meshwork, collagen bundles significantly enhanced MSC elongation and infiltration. Under osteogenic conditions, both collagen-containing gels (Fig. 2a) exhibited greater mineralization than fibrin alone. The combination of collagen bundles with BMP-2–supplemented osteogenic medium produced the most pronounced calcium deposition, evident as dense Alizarin Red staining (Fig. 2b).
Alizarin Red staining patterns varied distinctly across conditions, reflecting differences in calcium deposition morphology. In fibrin-only gels, mineralization appeared faint and granular, while the inclusion of collagen bundles led to dense, cloud-like regions of staining, suggesting localized calcium aggregation around osteogenic cell clusters. Neutralized collagen gels showed more diffuse and homogeneous staining, indicating widespread but less concentrated mineral deposition. The osteogenic + BMP-2 groups exhibited the most intense staining, particularly in collagen-containing gels, where mineralized regions extended radially or formed patch-like domains, potentially reflecting directed matrix remodeling or collective cell behavior. These distinct spatial patterns highlight how matrix composition influences not only the extent but also the distribution and organization of osteogenic mineralization within the 3D gels.
This study highlights the critical role of ECM microarchitecture in regulating MSC fate within 3D environments. By recapitulating the native ECM’s fibrous structure, collagen bundles enhance MSC morphogenesis and osteogenic differentiation more effectively than conventional reticular matrices. When paired with BMP-2 and osteoinductive media, these constructs significantly improve early osteogenic markers and matrix mineralization. Our platform provides a promising foundation for engineering bone tissue. Furthermore, our ongoing efforts will incorporate endothelial cells to further investigate angiogenesis-osteogenesis coupling within these hybrid constructs, aiming to recreate in-vitro vascularized osseous structures.

Acknowledgements and/or References (Optional): :

References:
Gong, Xiangyu, Zhang Wen, Zixie Liang, et al. 2025. “Instant Assembly of Collagen for Tissue Engineering and Bioprinting.” Nature Materials, June 6, 1–12. https://doi.org/10.1038/s41563-025-02241-7.