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Biomaterials

Biomaterials - Poster Session E

O23 - Fabrication and 3D Printing of Decellularized Bone Extracellular Matrix and Gelatin Nanoparticles for Improved Bone Repair

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Bone is a hard tissue, providing structural support and protecting vital organs in the human body.(1) Its extracellular matrix (ECM), including a mineral phase (60-70% mass), organic phase (30% mass), cells, and water, provides a microenvironment critical for joint homeostasis.(2) Thus, critical-sized bone defects are especially debilitating. Current treatments like autografts and allografts vary in efficacy and may cause donor-site morbidity. Surgical intervention for geometrically complex defects further complicates healing.(3)
Biomaterials offer long-term alternatives, retaining cues for targeted regeneration and mimicking complex native tissue architecture when 3D-printed.(4) ECM materials like demineralized bone extracellular matrix (DBM) have been clinically approved as injectables, putties, and powders. However, a mineral phase has been show to be critical in enhancing osteogenesis resulting in the need for decellularized extracellular matrix (bdECM) biomaterials.(5) Still, ECM materials have limited printability due to the lack of viscoelastic properties required. Previous research in our lab has showcased the use of gelatin nanoparticles (GNPs) in scaffold printing as an additive to dECM materials.(6-8)
Thus, this study aims to combine bdECM’s biocompatibility and GNPs’ printability to form composite inks for a bone-specific reparative platform. Specifically, this work will explore the effect of bdECM inclusion in printability and scaffold physicochemical properties. We will assess the mechanical stability of printed GNP:bdECM scaffolds at various crosslinking densities. We hypothesize that scaffold compositions with greater ECM content and interparticle crosslinking density will display lower swelling and degradation over time.

Materials and

Methods: : Bone femurs and tibiae were isolated from Sprague-Dawley rats (female, aged 12-16 weeks) donated by Rice University under Rice University Institutional Animal Care and Use Committee guidelines. Bone was subsequently demineralized following previous work, or decellularized through a 5-day PBS and 1% antibiotic-antimycotic wash, followed by washes in sodium dodecyl sulfate, Triton X-100, and DNAse I, before undergoing supercritical CO2 drying (Leica EM CPD300). Biomolecular signal retention was assessed through characterization of collagen (hydroxyproline assay), sGAGs (DMMB assay), and calcium (ICP-OES) content between native bone, bdECM, and DBM. GNPs were fabricated through two-step desolvation, following previously published work (6), after which GNPs and bdECM were methacrylated to introduce networks for controlled, interparticle crosslinking post-printing. GNP:bdECM ink ratios were determined through previously described rheological assessments on the Discovery Hybrid Rheometer®.(6) Tests included shear-thinning, self-recovery, and UV crosslinking characterizations to mimic scaffold extrusion. Dried GNPs and bdECM were then combined at viable ratios from rheology (100:0 and 67:33 ratios of GNP:bdECM) to form composite inks, which swelled over 72 hours before printing. The BioAssemblyBot® 3D printer was used to extrude 7 mm x 7 mm, two-layered gridded scaffolds that were UV crosslinked at 0 mJ/cm^2 (uncrosslinked) and 3000 mJ/cm^2 (crosslinked). After being freeze-dried, scaffolds underwent swelling (PBS) and degradation (PBS with collagenase) studies for 12 days each across the varying compositions and crosslinking degrees.

Results, Conclusions, and Discussions:: Bone was successfully decellularized, yielding DNA concentrations of 53 ± 1.5 ng/mg pre-methacrylation, which is at the standard 50 ng/mg of dry material. bdECM successfully retained collagen, sGAGs, and calcium, exhibiting a significant increase in hydroxyproline concentration and similar sGAG concentrations compared to native tissue (Fig. 1). Furthermore, bdECM showcased significantly greater calcium content compared to DBM, indicating greater mineral presence in bdECM.  
Overall, 100:0 GNP:bdECM and 67:33 GNP:bdECM ink compositions resulted in shear-thinning inks with high print fidelity. Rheological evaluations of self-healing behavior in 100:0 and 67:33 GNP:bdECM inks showcased recovery of storage and loss moduli post-extrusion conditions (Fig. 2). Both compositions exhibited shear-thinning through a linear, non-Newtonian decrease in viscosity with increasing shear rate. Under UV crosslinking assessments, the inks displayed significantly increased storage and loss moduli, with the 67:33 ink moduli exhibiting greater moduli than its 100:0 counterpart post UV-exposure.
Altogether, uncrosslinked scaffolds displayed significantly higher swelling and degradation rates compared to crosslinked scaffolds, as indicated by their change in area and mass, respectively (Fig. 3). Notably, within the uncrosslinked groups, 100:0 GNP:bdECM scaffolds lost more mass than 67:33 GNP:bdECM scaffolds, with a 100% mass loss in 100:0 GNP:bdECM scaffolds by Day 12.   
Ultimately, this work showcases the inclusion of bdECM in GNP:bdECM scaffolds, with tunable swelling and degradation profiles. Primarily, interparticle crosslinking density influenced scaffold swelling and degradation, while the presence of mineral in the ECM may have improved uncrosslinked scaffold stability under degradation conditions. Given the successfully retained biochemical cues provided by ECM, higher ECM content may guide cell differentiation and proliferation in in vitro assessments. Thus, future work is focused on increasing ECM content in scaffolds to enhance osteogenesis.

Acknowledgements and/or References (Optional): : References
1. Baig, M. A.; Bacha, D. Histology, Bone. In StatPearls; StatPearls Publishing: Treasure Island (FL), 2023.
2. Clarke, B. Normal Bone Anatomy and Physiology. Clin J Am Soc Nephrol 2008, 3 Suppl 3 (Suppl 3), S131-139. https://doi.org/10.2215/CJN.04151206.
3. Ninarello, D.; Ballardini, A.; Morozzi, G.; La Barbera, L. A Comprehensive Systematic Review of Marketed Bone Grafts for Load-Bearing Critical-Sized Bone Defects. Journal of the Mechanical Behavior of Biomedical Materials 2024, 160, 106782. https://doi.org/10.1016/j.jmbbm.2024.106782.
4. Stevens, M. M. Biomaterials for Bone Tissue Engineering. Materials Today 2008, 11 (5), 18–25. https://doi.org/10.1016/S1369-7021(08)70086-5
5. Zhou, Q.; Ren, X.; Bischoff, D.; Weisgerber, D. W.; Yamaguchi, D. T.; Miller, T. A.; Harley, B. A. C.; Lee, J. C. Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells. Adv Healthc Mater 2017, 6 (23). https://doi.org/10.1002/adhm.201700641.
6. Diba, M.; Koons, G. L.; Bedell, M. L.; Mikos, A. G. 3D Printed Colloidal Biomaterials Based on Photo-Reactive Gelatin Nanoparticles. Biomaterials 2021, 274, 120871. https://doi.org/10.1016/j.biomaterials.2021.120871.
7. Hogan, K. J.; Perez, M. R.; Öztatlı, H.; Si, S.; Wang, Z.; Jiang, E. Y.; Diba, M.; Garipcan, B.; Mikos, A. G. Development of 3D-Printing Composite Inks Based on Photoreactive Cartilage Extracellular Matrix and Gelatin Nanoparticles. Bioprinting 2023, 36, e00317. https://doi.org/10.1016/j.bprint.2023.e00317.
8. Hogan, K. J.; Öztatlı, H.; Perez, M. R.; Si, S.; Umurhan, R.; Jui, E.; Wang, Z.; Jiang, E. Y.; Han, S. R.; Diba, M.; Jane Grande-Allen, K.; Garipcan, B.; Mikos, A. G. Development of Photoreactive Demineralized Bone Matrix 3D Printing Colloidal Inks for Bone Tissue Engineering. Regen Biomater 2023, 10, rbad090. https://doi.org/10.1093/rb/rbad090.
Acknowledgements: Thank you to Dr. Kolliopoulos for mentoring me through this project. Thank you to Dr. Mikos and the Mikos Lab for the opportunity to perform this research. I also thank my collaborators, Ana Letca and Ellena Jeon, for contributing to data collection, and Rice University’s Shared Equipment Authority, for access to lab space and instruments.