Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs

Abstract

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.

Keywords: Cartilage repair; Gene activated matrix; Gene therapy; Immunoengineering; Osteoarthritis; Regenerative medicine.

Figure 1

Low (A) and high (B) magnification scanning electron microscope images of a CDM hemisphere (blue) with immobilized eGFP lentivirus 1 hr after seeding MSCs (yellow). (A) Scale: 1mm. (B) Scale: 2 μm.

Figure 2

Protein secretion of CDM constructs expressing dox-inducible (A) TGF-β3, (B) BMP-2, or (C) IL-1Ra. Constructs were cultured for 28 days with 1 μg/mL doxycycline added at day 0 in (A) basal chondrogenic media (without exogenous rhTGF-β3); (B) basal osteogenic media (without exogenous rhBMP-2); (C) either chondrogenic (with rhTGF-β3) or osteogenic (with rhBMP-2) media conditions in the presence or absence of rhIL-1. Mean ± SEM (n=4). Transduction efficiency was 57 ± 5% eGFP+ MSCs (mean ± SEM, n=3).

Figure 3

Total specific MMP activity of non-transduced (NT), eGFP-transduced, or IL-1Ra-transduced constructs cultured in chondrogenic media containing rhTGF-β3 either without or with rhIL-1 added beginning at day 2. Bars represent mean ± SEM (n=4). Groups not sharing same letter have p-values < 0.05.

Figure 4

Day 28 collagen (left), GAG (middle), and DNA (right) contents of (Row A) non-transduced (NT), eGFP-transduced, or TGF-β3-transduced constructs cultured in basal chondrogenic media (without exogenous rhTGF-β3), and NT constructs fed exogenous rhTGF-β3; (Row B) NT, eGFP-transduced, and IL-1Ra-transduced constructs cultured in chondrogenic media containing rhTGF-β3 with and without rhIL-1; (Row C) NT, eGFP-transduced, or BMP-2-transduced constructs cultured in basal osteogenic media (without exogenous rhBMP-2), and NT constructs fed exogenous rhBMP-2; (Row D) NT, eGFP-transduced, and IL-1Ra-transduced constructs cultured in osteogenic media containing rhBMP-2 with and without rhIL-1. Mean ± SEM (n=6). Groups not sharing same letter have p-values < 0.05. At day 0, Collagen = 8.24mg, GAG = 2.11mg, DNA = 4.36 μg.

Figure 5

Gross morphology; histology (Safranin-O and Fast Green staining); immunohistochemistry for type II collagen (COL II); type I collagen (COL I); type X collagen (COL X) on human osteochondral sections and non-transduced (NT), eGFP-transduced, or TGF-β3-transduced constructs cultured in basal chondrogenic media (without exogenous rhTGF-β3), NT constructs fed exogenous rhTGF-β3 with and without rhIL-1, and IL-1Ra-transduced constructs fed exogenous rhTGF-β3 and rhIL-1 after 28 days of culture in chondrogenic media. All images are transverse sections, showing full-thickness of each construct. Aminoethyl carbazone (AEC) produces red color. Gross picture scale: 2mm. Histology scale: 500 μm.

Figure 6

Gross morphology; histology (Safranin-O and Fast Green staining); immunohistochemistry for type II collagen (COL II); type I collagen (COL I); type X collagen (COL X) on human osteochondral sections and non-transduced (NT), eGFP-transduced, or BMP-2-transduced constructs cultured in basal osteogenic media (without exogenous rhBMP-2), and NT constructs fed exogenous rhBMP-2 with and without rhIL-1, and IL-1Ra-transduced constructs fed exogenous rhBMP-2 and rhIL-1 after 28 days of culture in osteogenic media. All images are transverse sections, showing full-thickness of each construct. Aminoethyl carbazone (AEC) produces red color. Gross picture scale: 2mm. Histology scale: 500 μm.

Figure 7

Day 28 (A&B) total volume of mineralization and (C&D) calcium content in (A&C) non-transduced (NT), eGFP-transduced, or BMP-2-transduced constructs cultured in basal osteogenic media (without exogenous rhBMP-2), and NT constructs fed exogenous rhBMP-2; (B&D) NT, eGFP-transduced, and IL-1Ra-transduced constructs cultured in osteogenic media containing rhBMP-2 with and without rhIL-1. (A&B) Bars represent mean ± SEM (n=6). Groups not sharing same letter have p-values < 0.05. (C&D) n=2. (E) Ratio of mineral distribution between the bottom and top of BMP-2-transduced constructs or NT constructs fed exogenous rhBMP-2 (bars represent mean ± SEM, n=6). Representative μCT images of constructs showing (F) CDM, (G) merged, (H) mineral content. Scale: 1mm. There was no detectable mineralization or calcium content in any of the chondrogenic samples.

Figure 8

Osteochondral constructs after 22 days of respective osteogenic or chondrogenic differentiation followed by 28 days of co-culture. (A) Illustration of chondrogenic outer hemispherical shell with immobilized TGF-β3 lentivirus and osteogenic inner hemisphere core with immobilized BMP-2 lentivirus. (B) Day 50 gross morphology of the top, side, and bottom of eGFP-transduced or TGF-β3 + BMP-2-transduced constructs. Scale: 2 mm. (C) Confocal image of eGFP-transduced MSCs in the inner hemisphere core and outer hemispherical shell at Day 50 of differentiation. Interface denoted by dotted line. Scale: 500 μm. (D) Growth factor secretion of TGF-β3 + BMP-2-transduced osteochondral constructs combined at Day 22 (mean ± SEM, n=8).

Figure 9

Histology (Safranin-O and Fast Green staining); immunohistochemistry for type II collagen (COL II); type I collagen (COL I); on human osteochondral sections and eGFP-transduced or TGF-β3 + BMP-2-transduced constructs after 50 days of differentiation. For CDM constructs, first column taken at 4x, second column taken at 10x. All images are transverse sections, showing full-thickness of each construct. Aminoethyl carbazone (AEC) produces red color. Scale: 500 μm.

Figure 10

Day 50 (A) collagen; (B) GAG; (C) DNA; (D) total volume of mineralization of eGFP-transduced (n=3) or TGF-β3 + BMP-2-transduced constructs (n=6). Mean ± SEM. Groups not sharing same letter have p-values < 0.05. Representative μCT images of constructs showing (E) CDM, (F) merged, (G) mineral content. Scale: 1mm.