Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Abstract

The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.

Keywords: biomaterials; chondrocyte; gene therapy; genetic engineering; regenerative medicine.

Fig. 1.

(A) Representative histogram showing green fluorescence intensity of nontransduced cells (black line) or cells after LVG immobilization to a 2D PCL film and subsequent transduction (green line). Flow cytometry was performed 5 days posttransduction. (B) Scanning electron micrograph showing the architecture of the 3D orthogonal woven PCL scaffold. (Scale bar, 500 µm.) (C) Representative histogram showing fluorescence intensity distribution of cells isolated from 3D woven PCL scaffolds 14 days after seeding scaffolds. The nontransduced population is represented by the black curve, and the green curve corresponds to a representative iLVG sample. An average of 81.6 ± 4.36% of cells were eGFP⁺ (n = 3). (D) Confocal microscopy image showing eGFP expression of hMSCs transduced via LV immobilization to the 3D woven PCL scaffold 14 days after seeding. (Scale bar, 100 µm.)

Fig. 2.

(A) Schematic of the viral genome used to produce the LVT vector. LTR, long terminal repeat; Ψ, psi packaging signal sequence; RRE, Rev response element; cPPT/CTS, central polypurine tract/central termination sequence; EF1α, internal promoter from the Elongation Factor 1α gene; TGF-β3, coding sequence for human TGF-β3; IRES, internal ribosome entry site; dsRed, coding sequence for the red fluorescent protein from Discosoma sp.; WPRE, woodchuck hepatitis posttranscriptional regulatory element; U3PPT, U3 polypurine tract. (B) An ELISA was performed to compare the level of TGF-β3 production between constructs containing cells pretransduced with the TGF-β3 lentivirus (pLVT) and cells transduced via immobilization of the virus to the 3D woven scaffold (iLVT). Levels were comparable between the two groups (P > 0.05). (C) Sulfated glycosaminoglycan (sGAG) per double-stranded DNA measured via the dimethylmethylene blue assay in nontransduced (NT), pLVT, or iLVT constructs. Bars represent the mean ± SEM (n = 4). (D–F) Safranin-O/fast green/hematoxylin histology from NT (D), pLVT (E), and iLVT (F) constructs harvested 14 days after chondrogenic induction. (Scale bar, 500 µm.) (G–I) Fluorescence microscopy from NT (G), iLVG (H), or iLVT (I) constructs after 28 days in chondrogenic culture. No fluorescence was observed in NT constructs, whereas cells within iLVG constructs fluoresced and retained an elongated fibroblast-like morphology spanning along PCL fibers. Cells in the iLVT constructs fluoresced due to the IRES-dsRed component of the TGF-β3 expression cassette. These cells adopted a rounded morphology, indicated by white arrows, suggestive of a chondrocyte phenotype. (Scale bar, 100 µm.)

Fig. 3.

(A) Quantitative RT-PCR assessing expression of the TGF-β3 transcript from samples in the iLVT group at D14 and D28. Bars represent the mean fold change in expression ± SEM (n = 4) compared with matched, nontransduced controls and normalized by the r18S reference gene. (B and C) ELISA results quantifying the levels of (B) total and (C) free TGF-β3 protein in culture media at various time points after seeding scaffolds in the iLVT group. Total levels represent the sum of protein bound in the latent complex as well as protein free to bind the ALK5 receptor and initiate signaling. Bars represent the mean ± SEM (n = 4).

Fig. 4.

(A and B) Relative expression levels of aggrecan (ACAN) and the α1 chain of type II collagen (COL2A1) comparing the hMSC response to rhTGF-β3– or iLVT-mediated differentiation at D14 (A) or D28 (B) after chondrogenic induction. Bars represent the mean fold change in expression ± SEM (n = 4) compared with matched, nontransduced controls and as normalized by the r18S reference gene. (C and D) Quantification of the development of cartilaginous ECM components in the nontransduced (NT), rhTGF-β3 (rhT), and immobilized lentiviral TGF (iLVT) groups. Sulfated glycosaminoglycan content and total collagen content were normalized to DNA content. Bars represent means ± SEM (n = 6). Groups not sharing the same letter or symbol are statistically different (P < 0.05). (E–H) Safranin-O/fast green/hematoxylin staining of D28 rhTGF-β3 (E and F) and iLVT (G and H) constructs. [Scale bars, 500 µm (E and G) and 100 µm (F and H).] (I–L) Immunohistochemistry probing for type II collagen in D28 rhTGF-β3 (I and J) and iLVT (K and L) constructs. [Scale bars, 500 µm (I and K) and 100 µm (J and L).]

Fig. 5.

(A and B) Relative expression levels of the type I chains of collagens 1 (COL1A1) and 10 (COL10A1), comparing the hMSC response to rhTGF-β3– or iLVT-mediated differentiation at D14 (A) or D28 (B) after chondrogenic induction. Bars represent the mean fold change in expression ± SEM (n = 4) compared with matched, nontransduced controls and as normalized by the r18S reference gene. Groups not sharing the same letter or symbol are statistically different (P < 0.05). (C and D) Immunohistochemistry probing type I collagen in D28 rhTGF-β3 (C) and iLVT (D) samples. (E and F) Immunohistochemistry probing type X collagen in D28 rhTGF-β3 (E) and iLVT (F) samples. (Scale bars, 100 µm.)

Fig. 6.

(A and B) Aggregate modulus (H_A) and hydraulic permeability (k) of nontransduced (NT), rhTGF-β3 (rhT), and immobilized lentiviral TGF (iLVT) constructs cultured for 14 days or 28 days in chondrogenic induction medium. Dotted lines represent D0 values.