miRNA induced 3D bioprinted-heterotypic osteochondral interface

Abstract

The engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on the three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach to inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for the chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using a commercially-available differentiation medium. Upon confirmation of the osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with a stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise for the biofabrication of stratified tissues, not only for the osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as, but not limited to, the bone-tendon-muscle interface and craniofacial tissues.

Keywords: bioprinting; bone; cartilage; miRNA; spheroids; stem cells.

Fig 1.

A schematic diagram describing osteogenic and chondrogenic differentiation for 4 weeks (Note: BM stands for basal medium, CM stands for chondrogenic medium, and OM stands for osteogenic medium).

Fig 2.

A schematic diagram describing the timeline for the bioprinting process for fabrication of the osteochondral interface using osteogenic and chondrogenic spheroids differentiated via two different approaches.

Fig 3.

Chondrogenic differentiation of ADSC spheroids. (a) Immunostaining images and (b) SOX9, COL2A1, COL1A1, and ACAN expression of spheroids for the negative control, miR-(140+21) transfected, and chondrogenic control groups at Weeks 1, 2, 3, and 4. Scale bars in insets correspond to 100 μm (n=3; p^* < 0.05; p^** < 0.01; p^**** < 0.0001).

Fig 4.

Chondrogenic spheroids: (a) H&E images, (b1) sGAG, and (b2) total DNA content of single spheroids from the negative control, miR-(140+21) transfected, and chondrogenic control groups at Weeks 1, 2, 3, and 4 (n=3; p^* < 0.05; p^** < 0.01).

Fig 5.

Toluidine Blue staining of chondrogenic spheroids at Week 1, 2, 3, and 4.

Fig 6.

Osteogenic differentiation of ADSC spheroids: (a) immunostaining images and (b) COL1, RUNX2, BSP, BMP-4, and OSTERIX expression of spheroids for the negative control, miR-148b transfected, and osteogenic control groups at Weeks 1, 2, 3, and 4. Scale bars in insets correspond to 100 μm (n=3; p^* < 0.05; p^** < 0.01; p^*** < 0.001; p^**** < 0.0001).

Fig 7.

Morphological analysis, ALP activity, and cell proliferation of osteogenic spheroids; (a) H&E images, (b1) ALP activity, and (b2) DNA content of single spheroids from negative control, miR-148b transfected, and osteogenic control groups at Week 1, 2, 3, and 4 (n=3; p^* < 0.05; p^** < 0.01).

Fig 8.

Alizarin red staining of osteogenic spheroids at Week 1, 2, 3, and 4.

Fig 9.

A schematic showing 3D bioprinting of the OC interface using osteogenic and chondrogenic spheroids and the corresponding images demonstrating steps involved in the physical process.

Fig 10.

Histochemical staining of bioprinted Osteochondral interfaces for a) Toluidine Blue, Alizarin Red, H&E, and OsteoImage, b) IHC results for Coll2 and Osterix markers for both differentiation medium and miR transfection groups, c) qRT-PCR gene expression results for osteogenic and chondrogenic gene markers at Week 4 (n=3; p^* < 0.05; p^** < 0.01).