X10 - TRACE Bioprinting of Cell-Laden Unmodified Collagen Constructs for Spatially Controlled Bone Development

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

DM

Dominic Mak

Research Assistant
Stony Brook University
Boston, Massachusetts, United States

Co-Author(s)

ZL

Zixie Liang

Graduate Student
Yale University
New Haven, Connecticut, United States
Xiangyu Gong, PhD

Research Scientist
Stony Brook University, United States
SW

Shue Wang

Associate Professor
University of New Haven
West Haven, Connecticut, United States
MM

Michael Mak

Associate Professor
Stony Brook University, United States

Primary Investigator(s)

MM

Michael Mak

Associate Professor
Stony Brook University, United States

Introduction: : Damage to bones due to trauma, tumors, and infectious diseases affects the quality of life for patients and is associated with high clinical burden and limited therapeutic options. Current treatments, such as autologous or allogeneic bone grafts, face challenges including donor site morbidity, immune rejection, and variable healing outcomes. Engineered bone grafts hold promise for overcoming these issues by recapitulating the native extracellular matrix (ECM) and promoting functional regeneration. However, realizing this potential requires biomaterials and fabrication techniques that support high cell viability, osteogenic differentiation, and geometrically defined architectures.
Type I collagen, the predominant ECM component in bone, supports mesenchymal stem cell (MSC) adhesion, alignment, and osteogenic signaling. Yet, traditional collagen extrusion bioprinting is hindered by slow gelation and the inability to maintain structural fidelity or encapsulate cells at physiological densities. To address these limitations, we employed TRACE (Tunable Rapid Assembly of Collagenous Elements), a nozzle-embedded bioprinting method that accelerates collagen fiber assembly using macromolecular crowding (MMC) induced by polyethylene glycol (PEG) within a support bath. This platform enables high-resolution printing of viable, MSC-laden collagen constructs with tunable geometry.
The study focused on how TRACE bioprinting enhances MSC viability, alignment, and osteogenic differentiation compared to bulk collagen gels. We further explore how design parameters, such as wall thickness and construct geometry, influence tissue compaction, mineralization, and spatial patterning of differentiation. By combining biologically relevant matrix cues with architectural control, TRACE bioprinting presents a promising strategy for fabricating patient-specific, cell-based bone grafts to treat long-bone nonunion defects.

Materials and

Methods: : To fabricate 3D bioprinted bone constructs, we utilized the TRACE method, which enables rapid type I collagen fibrillogenesis via macromolecular crowding. The TRACE support bath was prepared by mixing polyethylene glycol (PEG-8000) solution with agarose microparticles. Bioink was prepared by neutralizing 4mg/mL type I collagen and incorporating mouse MSCs (4×10⁶ cells/mL).
Bioprinting was performed using the Lulzbot syringe-based printer with customized temperature-controlled printheads (8 °C). A grid pattern was designed and sliced via Cura Lulz Edition. Bioink was printed with a 25-gauge nozzle at speeds of 5–10 mm/s, and left to set for a few minutes, then released into culture media.
Culture medium was changed to differentiation medium 24 hours after printing, and cultured for 14 days. Three groups were compared, including bulk gel (molded collagen), TRACE-printed collagen without bundles, and TRACE-printed collagen with bundles. Compaction of each construct was closely monitored daily via brightfield imaging, and percent area change was calculated and compared to Day 0. Cell viability was assessed through live/dead staining immediately post-printing and after 14 days of culturing. Osteogenic differentiation was evaluated through Alizarin Red (ALR) staining at Days 7 and 14. Cytoskeletal organization and alignment were assessed at Day 14 using F-actin (phalloidin) and DAPI staining, followed by quantitative image analysis of actin fiber directionality.

Results, Conclusions, and Discussions:: Bioprinted MSC-laden constructs were successfully fabricated and released, with the resulting geometries closely matching the original CAD designs. Compaction analysis showed that constructs printed with only cells underwent significant area reduction until day 5, after which compaction stabilized through day 14. Incorporating structural features such as walls and introducing assembled collagen bundles modulated the compaction rate, providing enhanced mechanical support and preserving geometry over time. Live/dead staining revealed high cell viability immediately post-printing, which was maintained throughout the 14-day culture period. Notably, bioprinted constructs exhibited higher cell survival compared to bulk-encapsulated gels. Alizarin Red staining demonstrated mineral deposition on day 7, with visibly intensified red staining by day 14, indicating progressive osteogenic differentiation. Quantification of DAPI-stained nuclei showed a higher cell density in the bioprinted constructs relative to bulk controls, suggesting enhanced cell retention and proliferation. Phalloidin staining at day 14 revealed well-organized actin cytoskeletal structures with significantly greater alignment and intensity in the bioprinted groups compared to bulk gels. Image analysis confirmed improved fiber orientation, suggesting a more biomimetic, structurally aligned microenvironment conducive to osteogenic maturation.
Collectively, these results highlight the advantages of bioprinting for controlling construct geometry, maintaining cell viability, and promoting osteogenic outcomes. This approach holds great promise for spatially guided bone tissue engineering, as it enables the accurate deposition of cells within designed 3D matrices that replicate complex tissue microenvironments, which drive functional bone regeneration.
Future work will focus on optimizing the bioprinting workflow by developing more efficient and gentle methods for support-bath removal, thereby streamlining post-processing and preserving construct fidelity. Additionally, the transition from in vitro models to in vivo applications using animal models enables the assessment of biocompatibility, structural integration, and regenerative capacity of the constructs in a physiologically relevant environment. In vivo investigations will provide essential validation of long-term cell survival, osteogenic functionality, and overall construct performance, supporting the translational potential of this bioprinting approach for clinical bone regeneration.

Acknowledgements and/or References (Optional): : X. Gong, Z. Wen, Z. Liang, H. Xiao, S. Lee, A. Rossello, Q. Xing, T. Wright, R. Y. Nguyen, A. Rossello, M. Mak, Instant Assembly of Collagen for Tissue Engineering and Bioprinting. Nature Materials, in press.