O7 - Development of a novel decellularization protocol for the fabrication of 3D-printed decellularized bone extracellular matrix biomaterials

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

AL

Ana Letca

Student (Undergraduate)
Rice University, United States

Co-Author(s)

AL

Ana Letca

Student (Undergraduate)
Rice University, United States
Vasiliki Kolliopoulos, PhD

Postdoctoral Fellow
Rice University
Houston, Texas, United States

Primary Investigator(s)

AM

Antonios Mikos

Professor of Bioengineering
Rice University, United States

Co-Author(s)

SC

Sadhana Chari

Student (Undergraduate)
Rice University, United States

Introduction: : Critical-size bone defects in which the bone does not spontaneously regenerate present a significant burden to patients and can arise from high-impact trauma, tumor resection, or congenital craniomaxillofacial defects such as cleft palate. Standards of care include autograft and allograft implantation, however these approaches face limitations related to availability, repair lifetime, and disease transmission. Demineralized bone matrix (DBM) biomaterials have been clinically approved alternatives for decades, providing degradable scaffolds with osteoinductive properties to facilitate bone regeneration. However, commercial DBM preparation involves demineralization through acid treatments that solubilizes the mineral phase of the native extracellular matrix (ECM), limiting mechanical properties and osteogenesis. An alternative approach is harnessing native ECM, retaining its mineral component and osteogenic biomolecules through gentler decellularization. Supercritical drying using CO2 (scCO2) shows promise to reduce exposure time to reagents that demineralize or denature the ECM and has been demonstrated in literature to decellularize vascular, adipose, and nervous tissue below the established biomaterial immunogenicity threshold of 50ng DNA/mg tissue. However, scCO2 in combination with other reagents has not been thoroughly investigated in bone, representing a gap in the literature. In this work, we report a novel method of bone decellularization that maintains the heterogeneous components of bone providing a more physiologically relevant biomaterial platform.

Materials and

Methods: : Porcine tissue (n=2 Duroc, n=1 Yorkshire) sourced from femurs was separated by tissue type (trabecular or cortical) and underwent a 5-day wash in PBS and 1% antibiotic-antimycotic prior to lyophilization and pulverization, with retention of particles 53 – 300 µm. This tissue underwent one of two processing protocols: standard demineralization in acid to obtain DBM, or decellularization using scCO2 to obtain bone decellularized ECM (bdECM). DBM was processed through sequential incubation in ethanol, ethyl ether, and 0.6M HCl. To obtain bdECM, tissue was washed using low concentrations of detergent (0.2% sodium dodecyl-sulfate and 2% Triton X-100), incubated in 50U/mL DNAse I, and underwent scCO2 treatment with ethanol as a co-solvent. For both trabecular and cortical tissue, DNA, sulfated glycosaminoglycan (sGAG), and collagen, and total calcium content were quantified as metrics of biomolecule retention as a function of processing protocol. DNA content was evaluated through UV spectrophotometry, sGAG content through a 1,9-dimethylmethylene blue (DMMB) assay, and collagen content through a hydroxyproline assay. For a preliminary assessment of mineral retention as a function of processing protocol, cortical tissue sourced from Sprague-Dawley rat femurs and tibiae (n=1) was processed into DBM and bdECM using the protocol described above. Total calcium content was quantified through Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). Statistics performed by two-way ANOVA using GraphPad Prism 10.2.3 (GraphPad Software, San Diego, CA).

Results, Conclusions, and Discussions:: For both trabecular and cortical tissue, DNA content did not significantly differ between native and DBM groups. In contrast, cortical bdECM demonstrated effective decellularization in range of the established biomaterial immunogenicity threshold at 68.2 ± 16.9 ng DNA/mg tissue and trabecular bdECM was significantly lower than trabecular native and trabecular DBM at 103.1 ± 17.8 ng DNA/mg tissue, as depicted in Figure 1. Trabecular tissue had significantly greater DNA content in native and DBM groups, but not bdECM. Furthermore, preliminary data using cortical tissue indicates that bdECM contains the greatest mineral content, with 11.67% total calcium content in bdECM compared to DBM (1.04%) and a gelatin control (0.095%). Together, this data suggests that compared to standard demineralization, the scCO2 bdECM protocol was more effective at decellularizing both trabecular and cortical tissue to remove residual DNA that could potentially trigger an immune response, and the bdECM protocol successfully retained the mineral phase. Next, sGAG and collagen content, components of the organic phase of the ECM that play critical roles in regulation of bone remodeling, flexibility, and stress resistance, were quantified as a function of tissue type and processing protocol. Trabecular tissue had significantly greater sGAG content than cortical tissue in native and DBM groups, but not bdECM. The bdECM protocol led to the least sGAG content in trabecular but not cortical tissue. Collagen content was not found to significantly differ among tissue types or processing protocols. Following this material fabrication and characterization, ongoing work is focused on DBM and bdECM functionalization with methacrylic anhydride for downstream fabrication of UV-crosslinkable 3D-printed scaffolds to investigate compositional effects on mechanical and osteogenic properties.

Acknowledgements and/or References (Optional): :