T19 - Evaluating the osteogenic potential of bone decellularized extracellular matrix dose in 3D-printed scaffolds

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Vasiliki Kolliopoulos, PhD

Postdoctoral Fellow
Rice University
Houston, Texas, United States

Co-Author(s)

Sadhana Chari

Undergraduate
Rice University
Fremont, California, United States
PK

Priya S. Kumar

Undergraduate
Rice University, United States
AL

Ana Letca

Student (Undergraduate)
Rice University, United States

Last Author(s)

AM

Antonios Mikos

Professor of Bioengineering
Rice University, United States

Primary Investigator(s)

AM

Antonios Mikos

Professor of Bioengineering
Rice University, United States

Introduction: : Critical-sized defects in bone remain a clinical challenge as they cannot heal naturally by the body requiring surgical intervention. The gold standard for repairing such defects include autografts and allografts however these are associated with secondary site morbidity and limited availability. Decellularized extracellular matrix (dECM) biomaterials have surfaced as an attractive graft alternative as they maintain a significant portion of the native ECM composition while minimizing immunogenicity associated with cellular debris. Demineralized bone matrix (DBM) is a clinically used ECM-derived material that has enhanced osteogenic potential however, in large defects it can lead to tissue collapse due to its rapid degradation and lack of mineral content. We report the development of a decellularization approach to remove cellular debris while maintaining the native mineral content resulting in bone dECM (bdECM). We build upon previous work in the lab to develop colloidal 3D-printing inks containing bdECM and gelatin nanoparticles (GNPs). The objective of this study is to evaluate the effect of bdECM dose on the scaffold physicochemical and osteogenic properties. We describe the fabrication of 3D-printed scaffolds with varying bdECM content and degree of crosslinking and evaluate the effect of bdECM dose on the materials swelling and degradation kinetics. Finally, we investigate the dose-dependent effect of bdECM on mesenchymal stem cell (MSC) osteogenic differentiation.

Materials and

Methods: : Femurs and tibias were harvested from female Duroc pigs donated from UT Health Houston. The bones were cut into small pieces 2-3 cm and washed in PBS with 1% anti-anti for 5 days to wash off the bone marrow. The bone was pulverized into a powder and subsequently decellularized by sequential overnight soaks in 0.2% w/v EDTA, 0.2% w/v SDS, 2% v/v Triton X-100, and 100 U/ml DNAse I solutions with 3 washes in MilliQ water in between each reagent. To remove any cell remnants, the decellularized powder underwent supercritical drying. Finally, the powdered bone underwent pepsin digestion and was subsequently methacrylated (bdECM-MA) to allow for post-printing crosslinking. The composition of each of these inks was quantified for their collagen content via a hydroxyproline assay, glycosaminoglycan content via 1,9-dimethylmethylene blue assay, and calcium and phosphorous via Inductively Coupled Plasma-Optical Emission Spectrometry. Inks were fabricated by combining GNPs, bdECM, in an Irgacure in PBS solution. The shear thinning and recovery behavior of the inks were evaluated rheologically prior to printing. Scaffolds were printed in a 3x3 lattice structure and crosslinked using 2 levels of UV exposure (0, 3000 mJ/cm2). A swelling study was conducted to evaluate the effect of the degree of crosslinking on the swelling behavior of these scaffolds across 12 days. A degradation study in either PBS or PBS+collagenase was used to assess the stability and degradation profile of the scaffolds. Finally, MSCs were seeded on the scaffolds and evaluated for their osteogenic differentiation via Arsenazo III staining.

Results, Conclusions, and Discussions:: Decellularization of bone into bdECM results in the maintenance of key bioactive ECM molecules such as collagen and sulfated glycosaminoglycans while adequately removing DNA below the 50 ng/mg standard reported in the literature. Most importantly, our protocol results in the maintenance of native minerals as measured by the calcium remaining in the material. We demonstrate the successful fabrication of bdECM containing 3D-printed scaffolds at two crosslinking levels. Varying bdECM content and the degree of crosslinking have a significant effect on swelling and degradation behavior as observed by decreased swelling and slower degradation kinetics compared to the GNP only control. The parameter that drives swelling and degradation patterns is the degree of crosslinking which results in a tunable property that can be modulated both via the extent of methacryloylation and the degree of UV exposure. Interestingly, the introduction of bdECM stabilizes the scaffold by restricting the swelling of the GNPs entrapped in the bdECM network and retards the dissolution of GNPs in the surrounding medium. Osteogenic differentiation was observed in a bdECM dose-dependent manner. Further evaluation of osteogenesis characterizing gene and protein expression profiles will be conducted to further investigate the effect of bdECM dose and degree of crosslinking.

This study aimed to develop a first of its kind bone decellularized extracellular matrix (bdECM) scaffold for improved bone repair. We fabricated scaffolds with varying amounts of bdECM and degrees of crosslinking and evaluated the effects of composition and crosslinking on the physicochemical and osteogenic properties of the biomaterial. This work will result in a greater understanding of the effect of ECM components and their dose on the induction of osteogenesis and a highly tunable bdECM-based scaffold with clinically translatable potential.

Acknowledgements and/or References (Optional): :