Associate Professor New Jersey Institute of Technology Newark, New Jersey, United States
Introduction: The treatment of large or critical-sized bone defects poses significant clinical challenges, often resulting from diseases, traumatic injuries, infections, tumor removal, or birth defects. 3D printing has emerged as a revolutionary method for fabricating scaffolds with complex shapes and material compositions for bone tissue engineering. One of the main advantages of 3D printing is its ability to utilize a patient's medical image to design and fabricate a scaffold tailored to the specific shape of the tissue or defect. However, there remains a demand for functional ink formulations that better mimic the native bone tissue microenvironment to enhance bone formation and the functional integration of newly formed tissue into the defect site. This study aims to address this need by developing biocomposite ink formulations comprising methacrylated alginate (MeALG) hydrogel incorporated with human bone allograft (HBA), hydroxyapatite (HA), or tricalcium phosphate (TCP) particles. Detailed rheological studies are conducted to correlate particle loading with ink flow properties and printability. Selected ink formulations are then utilized to fabricate 3D scaffolds, and their efficacy is assessed by studying seeded stem cell behavior. Overall, this research aims to advance the field of bone tissue engineering by developing innovative ink formulations that promote enhanced tissue integration and bone formation within large bone defects.
Materials and
Methods: Materials: Methacrylated alginate (MeALG) was synthesized per protocol, while cancellous allograft bone chips were milled into particles ranging from ~4 µm for subsequent analysis and cell studies. TCP and HA powders were purchased from Sigma with size particle of 4 µm and 2µm respectively. Rheology: Viscosity measurements were conducted across shear rates (0.01 to 1000 s-1) for different composite inks with various bioceramic particle concentration (HBA: 0-50%, TCP: 0-30%, HA: 0-60% w/v), with maximum volume fraction determining suspension behavior. 3D printing: Ink formulations comprising 3%w/v MeAlg and various concentrations of each TCP, HA and HBA in PBS with 0.5% photoinitiator were utilized. Grid pattern is designed to print scaffolds. HMSCs (passage 4, Lonza) were seeded onto printed scaffolds (50K/cm2) using an extrusion based BioX bioprinter. In-vitro studies (biocompatibility of bone particles): 2D cell culture examined cell-bone particle interactions over 21 days, with fluorescent microscopy at Day 21. AlamarBlue assay assesses cell growth on 3D printed scaffolds for up to 14 days, while ALP activity and alizarin red assay evaluate hMSC osteogenic differentiation.
Results, Conclusions, and Discussions: In this study, composite bioink formulations were developed by combining HBA, TCP or HA particles with MeALG hydrogel. All formulations showed shear thinning bahavior and increasing particle loading led to a significant increase in zero shear viscosity of the composite inks (Fig. 1A). Our results further indicated that the maximum achievable particle concentration for workable inks was determined by the particles, such that TCP allowed up to 30% (w/v) whereas HA and HBA reached up to 60% and 50% (w/v), respectively (Fig 1B). An extrusion-based 3D printer was employed to fabricate grid patterns using different particle concentrations for each type of composite ink (Fig. 1C). The images illustrated that as the concentration of particles increased, the printed struts exhibited enhanced structural integrity and fidelity, well-correlated with increasing ink viscosity. Preliminary in vitro studies with larger HBA particles (35-100 m) indicated strong interaction with hMSCs, cells attaching and covering the particles (Fig. 1D). For 3D printed scaffolds (Fig. 1C), hMSCs showed high cell retention and increased metabolic activity during short-term culture (4-days). Ongoing studies aim to determine the osteogenic differentiation of hMSCs with respect to ink composition during long term culture up to 28 days. Overall, this ink platform will enable direct comparison on the effects of HBA, TCP and HA on stem cell osteogenesis.
Acknowledgements (Optional): We acknowledge funding from NSF (CAREER Award #2044479), MTF Biologics, and New Jersey Health Foundation.
