Metal-Organic Framework Functionalized Bioceramic Scaffolds with Antioxidative Activity for Enhanced Osteochondral Regeneration

Abstract

Osteoarthritis (OA) is a degenerative disease that often causes cartilage lesions and even osteochondral damage. Osteochondral defects induced by OA are accompanied by an inflammatory arthrosis microenvironment with overproduced reactive oxygen species (ROS), resulting in the exacerbation of defects and difficulty regenerating osteochondral tissues. Therefore, it is urgently needed to develop osteochondral scaffolds that can not only promote the integrated regeneration of cartilage and subchondral bone, but also possess ROS-scavenging ability to protect tissues from oxidative stress. Herein, zinc-cobalt bimetallic organic framework (Zn/Co-MOF) functionalized bioceramic scaffolds are designed for repairing osteochondral defects under OA environment. By functionalizing Zn/Co-MOF on the 3D-printed beta-tricalcium phosphate (β-TCP) scaffolds, the Zn/Co-MOF functionalized β-TCP (MOF-TCP) scaffolds with broad-spectrum ROS-scavenging ability are successfully developed. Benefiting from its catalytic active sites and degradation products, Zn/Co-MOF endows the scaffolds with excellent antioxidative and anti-inflammatory properties to protect cells from ROS invasion, as well as dual-bioactivities of simultaneously inducing osteogenic and chondrogenic differentiation in vitro. Furthermore, in vivo results confirm that MOF-TCP scaffolds accelerate the integrated regeneration of cartilage and subchondral bone in severe osteochondral defects. This study offers a promising strategy for treating defects induced by OA as well as other inflammatory diseases.

Keywords: antioxidative stress; bioceramic scaffolds; metal-organic frameworks; osteochondral regeneration.

Scheme 1

Illustration diagrams of metal‐organic framework tricalcium phosphate (MOF‐TCP) scaffolds in treating osteochondral defects caused by osteoarthritis (OA). With zinc‐cobalt bimetallic organic framework (Zn/Co‐MOF) functionalization, the scaffolds could scavenge excessive reactive oxygen species (ROS) and build an anti‐inflammatory immune microenvironment to protect cells. Meanwhile, MOF‐TCP scaffolds exhibited dual bioactivities of stimulating the cells toward specific differentiation by releasing multiple bioactive ions. Briefly, the MOF‐TCP scaffolds, which possessed ROS‐scavenging abilities, ion release properties, and immunomodulatory activities, exhibited ideal function of promoting osteochondral regeneration.

Figure 1

Characterization of the 3D‐printed beta‐tricalcium phosphate (β‐TCP) scaffolds functionalized with different amount of zinc‐cobalt bimetallic organic framework (Zn/Co‐MOF). a) Digital photographs of scaffolds with different levels of Zn/Co‐MOF functionalization. b) Scanning electron microscope (SEM) images of the surfaces of scaffolds. c) Cell viability of MOF‐TCP scaffolds prepared by Zn/Co‐MOF reaction solution with concentrations of 6.25, 12.5, 25, 50 × 10⁻³ m (n = 4). d) Cell viability of MOF‐TCP scaffolds prepared by Zn/Co‐MOF reaction solution with concentrations of 12, 17, 21, 25 × 10⁻³ m (n = 4). e) Multiple reactive oxygen species (ROS) elimination ratios of the scaffolds (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

The promoting effects of MOF‐TCP scaffolds on cell proliferation and adhesion under oxidative stress. Proliferation of a) rabbit bone marrow mesenchymal stem cells (rBMSCs) and b) chondrocytes cultured on the scaffolds with hydrogen peroxide (H₂O₂) stimulation (n = 6). c) Morphology of rBMSCs and chondrocytes cultured on the scaffolds with H₂O₂ stimulation. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

The promoting effects of MOF‐TCP scaffolds on osteogenic differentiation of rBMSCs under both normal condition and oxidative condition. The expression of a) OPN gene, b) OCN gene, c) RUNX2 gene, d) BMP2 gene of rBMSCs (n = 3). e) The alkaline phosphatase (ALP) staining of rBMSCs with different treatment. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

The promoting effects of MOF‐TCP scaffolds on mature of chondrocytes under both normal condition and oxidative condition. The expression of a) SOX9 gene, b) Aggrecan gene, c) COL II gene in chondrocytes (n = 3). d) The immunofluorescent staining of Aggrecan protein in chondrocytes with different treatment (green: Aggrecan protein, blue: cell nucleus). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

In vitro antioxidative and anti‐inflammatory activities of MOF‐TCP scaffolds. a) ROS fluorescence staining images in rBMSCs, chondrocytes, and RAW 264.7 cells cultured with scaffolds under H₂O₂ stimulation. b) The expression of proinflammatory genes in chondrocytes (n = 3). c) The expression of anti‐inflammatory genes (IL‐10 and Arg‐1) and proinflammatory genes (IL‐1β and IL‐6) in RAW 264.7 cells (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

The in vivo regeneration effects of the osteochondral defects treated with different scaffolds. Digital photographs (a1–c1), the transverse view images (a2–c2), and sagittal view images (a3–c3) of the osteochondral defects reconstructed by microcomputed tomography (Micro‐CT). The green, red, and gray‐white color in Micro‐CT images represent new bone, scaffold, and native bone, respectively. The newly formed bone assessment including d) the ratio of new bone volume to defect volume (BV/TV), e) bone surface mineral density (BS/TV), f) the trabecular number of the new bone (Tb. N) after 12 weeks of implantation (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Histological analysis of the in vivo regeneration of the osteochondral defects. Toluidine blue (TB) staining images, Safranin O staining images, Van Gieson staining images, N‐Cadherin fluorescence staining images, and Aggrecan fluorescence staining images of the defects after 12 weeks of implantation.