Development of a Multilayered Osteochondral Tissue Construct Using 3D Bioprinting

Abstract

Osteochondral defects can arise from trauma, age-related degenerative changes, or pathological conditions, leading to pain, impaired mobility, and a reduced quality of life. Current clinical treatments have significant limitations, rendering them inadequate for full-thickness osteochondral defects. Although tissue engineering represents a promising strategy for osteochondral tissue regeneration, the complex, depth-dependent architecture of native tissue presents a major challenge to achieve functional repair. Moreover, conventional tissue engineering strategies remain limited in their ability to replicate the structural and functional characteristics of native tissue. Previous studies have explored osteochondral tissue engineering using two-region constructs comprising cartilage and subchondral bone. Nevertheless, despite these efforts, engineering a biologically functional osteochondral tissue construct that closely resembles native tissue remains elusive. The overall objective of this thesis was to engineer a multilayered osteochondral tissue construct, fabricated using 3D printing with region-specific bioinks to mimic the organization of native tissue. To fulfill this objective, the specific aims were to: 1. develop and characterize specific bioinks for the chondral, calcified, and osseous regions of the multilayered osteochondral tissue construct; and 2. fabricate a full-thickness, multilayered osteochondral tissue construct using 3D bioprinting and characterize its physicochemical, mechanical, and biological properties. The chondral- and calcified-region bioinks were formulated using alginate, heparinized alginate, gelatin methacryloyl (GelMA), and methacrylated hyaluronic acid (MeHA), with hydroxyapatite nanopowder (HAnp) incorporated only into the calcified-region bioink. Both bioinks were supplemented with transforming growth factor β1 (TGF β1). Compared with the calcified-region bioink, the osseous-region bioink included fibrinogen instead of MeHA, a lower concentration of GelMA, and a higher concentration of HAnp. This bioink was supplemented with bone morphogenetic protein 2 (BMP 2). The three inks were characterized in terms of rheological properties and printability. All exhibited shear-thinning behavior and demonstrated good printability, while enabling sustained release of TGF β1 and BMP 2 from 3D-printed region-specific scaffolds. Full-thickness, multilayered constructs were fabricated through sequential extrusion-based 3D bioprinting of region-specific bioinks laden with normal human bone marrow-derived mesenchymal stem cells (hBM-MSC). The compressive modulus of the constructs, determined from unconfined compression testing, remained low. Immunohistochemical analysis of constructs cultured for up to 21 days revealed the presence of osteogenic (type I collagen) and chondrogenic (type II collagen) markers in the osseous and chondral regions of the constructs, respectively, as well as a marker of hypertrophic chondrogenic differentiation (type X collagen) in the calcified region. Histochemical analysis further demonstrated increased alkaline phosphatase (ALP) activity in the osseous region, consistent with early osteogenic differentiation. While a reinforcement strategy remains to be implemented, collectively, these findings demonstrate that the multilayered construct, fabricated using regionally-defined bioinks, has significant potential for full-thickness osteochondral tissue regeneration.