Poster M14 - 3D-Printed Osteochondral Platform Reveals New Biomechanical Insights in Tidemark of Bone-Articular Regions

Introduction: Osteochondral fractures resulting from trauma, aging, or surgical interventions disrupt the homeostatic interactions between the articular and subchondral compartments. Current therapeutic options may involve the use of anti-inflammatory medications. In more severe cases, surgical intervention to restore both cartilage and bone is considered a more effective solution than solely focusing on cartilage repair. To overcome these limitations, it is crucial to create innovative treatments for osteochondral fractures through the development and implementation of new in vitro techniques utilizing advanced manufacturing methods. This study presents the development of a bilayer platform, referred to as a "tidemark," which has been strongly linked with the onset and advancement of osteochondral fractures and can indicate the progression of osteoarthritis. The utilization of 3D bioprinting in creating a bilayer platform enables the investigation of the interfacial biomechanical properties of the complex osteochondral junction region.

Materials and

Methods: To test this hypothesis, we developed and employed a 3D printed bilayer scaffold consisting of two different layers. The first layer mimics the subchondral tissue, consisting of a hard material, while the second layer recapitulates the articular part. The 3D-printed bilayer units shown in Figure 1(a) were formed by using composite inks containing hydroxyapatite (HA) and polyvinyl butyral (PVB). Subsequently, collagen (type I) bioinks with concentrations of 1.5, 3, 4, 6, and 8 mg/mL, containing human articular chondrocytes (HAC) at a concentration of 0.1 million, were used for 3D printing on the top of the pre-printed rigid compartments. The rheological properties of different collagen concentrations were measured using a rheometer and atomic force microscopy (AFM). Following the creation of the bilayer structure, the interactions between the collagen matrix and the hydroxyapatite scaffolds were analyzed using scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). The adhesion strength between the two compartments with varying stiffness was quantified using the pressurized blister test method (Figure 1(b-d)). This technique involves introducing a gas between a thin collagen layer and a substrate, resulting in the formation of a blister. The gas pressure is then gradually increased until the collagen film separates from the substrate. Ultimately, the viability and functionality of the HAC were assessed using a live/dead assay and Alcian Blue staining, respectively.

Results, Conclusions, and Discussions: Our rheological measurements revealed that the complex shear modulus at angular frequency (w) 1 of collagen gel at a concentration of 8 mg/mL exhibited approximately 1.3 times greater stiffness (p < 0.05) compared to collagen gel at a concentration of 6 mg/mL and approximately 126 times greater stiffness (p < 0.05) compared to collagen gel at a concentration of 1.5 mg/mL. Furthermore, our scanning electron microscopy (SEM) results showed that the thickness of the 0.5 mm collagen layer changed shape and broke at concentrations of 1.5 and 3 mg/mL before it separated from the hard compartment. Our blister measurements showed that once the pressure was raised to over 5 mbar, the layer with a thickness of 0.5 mm at collagen concentration (4, 6, and 8 mg/mL) separated from the HA compartment. The bilayer adhesion energy of the soft and hard compartments was quantified between 0.063 and 0.106 J/m2. This suggests that a high concentration of collagen helps maintain the interactions and mechanical strength of the bilayer in the interfacial region. Furthermore, in those particular circumstances, the Alcian blue staining revealed HAC functionality that was six times greater (p < 0.05) in collagen gels with a concentration of 8 mg/mL as compared to the other conditions. In the end, we found that increasing the thickness of the collagen film to 1 mm and the polymerization time to 24 hours made the adhesion strength 10 percent and 35 percent higher, respectively (p < 0.05). Through the utilization of 3D printing technology, this study in its entirety serves as a foundation for a better understanding of the mechanisms involved in the initiation and progression of osteochondral defects in the osteochondral junctional region.

Acknowledgements (Optional): Georgetown University has provided support for this project (Assignee: 91252). The facilities furnished by NIST and ISMSM for the execution of this project are greatly appreciated. Dr. Stella Alimperti, Dr. Xinran Zhang (ISMSM), Dr. Jonathan Seppala (NIST), Dr. Paul Salipante (NIST), Dr. Anthony Kotula (NIST), and the entire Alimperti Lab are individuals to whom I extend my sincere appreciation. I also extend my gratitude to Muhammad Zakaria Khan, our high school intern from Bellarmine Preparatory School.