Connexin 43 as a signaling platform for increasing the volume and spatial distribution of regenerated tissue

Abstract

Gap junction intercellular communication (GJIC) is ubiquitous in the majority of vertebrate cells and is required for the proper development of most tissues. The loss of gap junction-mediated cell-to-cell communication leads to compromised development in many tissues and organs. Because cells constantly interact through gap junctions to coordinate tissue functions and homeostasis, we hypothesized that increasing cell-to-cell communication, via genetically engineering cells to overexpress gap junction proteins, could enhance cell differentiation in the interior regions of 3D tissue equivalents, thereby increasing the ability to regenerate larger and more uniform volumes of tissue. To test this hypothesis, we used bone as a model tissue because of the difficulty in achieving spatially uniform bone regeneration in 3D. In bone marrow stromal cells (BMSC), GJIC and osteogenic differentiation were compromised in 3D cultures relative to 2D monolayers and in the core of 3D cultures relative to the surface. Overexpression of connexin 43 (Cx43) via transduction of BMSCs with a lentivirus overcame this problem, enhancing both the magnitude and spatial distribution of GJIC and osteogenic differentiation markers throughout 3D constructs. Transplantation of cells overexpressing Cx43 resulted in an increased volume fraction and spatial uniformity of bone in vivo, relative to nontransduced BMSCs. Increased GJIC also enhanced the effect of a potent osteoinductive agent (BMP-7), suggesting a synergism between the soluble factor and GJIC. These findings present a platform to improve cell-to-cell communication in 3D and to achieve uniformly distributed tissue regeneration in 3D.

Fig. 1.

BMSCs transduced with LV-Cx43-GFP. Expression of the Cx43-GFP (A) in transduced BMSCs under phase contrast and fluorescent microscopy indicates that a high percentage of cells express the Cx43-GFP fusion gene. Quantification of Western blot band intensities showed a significant increase (3.74- ± 0.49-fold increase; P < 0.001) in Cx43 expression in cells that were transduced compared to nontransduced BMSCs and BMSCs transduced with GFP only (B). * indicates P < 0.001 versus BMSC and BMSC-LV-GFP control groups.

Fig. 2.

GJIC in BMSCs is enhanced with Cx43 overexpression. Images demonstrate the transfer of calcein in BMSCs (A), BMSC-Cx43 (B), BMSC-BMP7 (C), and mutant BMSC-Cx43Δ7 cells (D) after 5 h of exposure to donor cells. Arrows indicate donor cells (DiI, wide field inverted) overlaid in the images. The transfer fraction of calcein-AM was measured for cells cultured in monolayer and 3D (E). Cells overexpressing Cx43 had a significantly higher transfer fraction than non-Cx43-transduced cells. Transfer was significantly enhanced in 3D when cells overexpressed Cx43 relative to 2D. Cells that did not overexpress Cx43 showed significantly less dye transfer in the core of the scaffolds compared to the periphery, whereas no significant difference was evident in cells overexpressing Cx43 (F). Horizontal bars represent pairs that are significantly different; * indicates groups that are significantly greater than BMSCs; ** indicates groups that are significantly less.

Fig. 3.

Cx43 overexpression is associated with higher levels of OCN mRNA expression (A), suggesting that increased GJIC results in an enhanced capacity of cells to undergo osteogenic differentiation. Increased cell-cell communication also increases the production of osteoinductive soluble factors (D and E). Overexpression of Cx43 also had significant effects on OCN expression when comparing 2D to 3D cultures (B). OCN mRNA expression was significantly greater in 2D for cells that did not overexpress Cx43, while cells that overexpressed Cx43 exhibited no significant difference in level of OCN expression between 2D and 3D cultures. Analysis of the surface versus core of the scaffolds (C) suggests that increased GJIC in the interior regions of a tissue equivalent can enhance the magnitude and reduce spatial gradients in differentiation. Significant increases are indicated by * versus Cx43Δ7, ** vs. BMSC, *** vs. Cx43, # vs. BMP-7. Horizontal bars indicate pairs that are significantly different.

Fig. 4.

MicroCT renderings and histological sections (taken from regions of microCT images highlighted by red rectangle) of bone regenerated following transplantation of BMSCs (A–L). Different patterns of bone regeneration between BMSCs and BMSCs overexpressing Cx43 are observed. BVF quantifies these differences (M). Cells transduced with Cx43 and cotransduced with BMP-7 form larger volumes of tissue compared to BMSCs. Contrast image (N), image of GFP fluorescence (O), and co-localized image (P) of the same section of tissue regenerated 12 weeks after transplantation of BMSCs overexpressing Cx43. The continued extent of the GFP-Cx43 fusion gene expression is observed. Significant increases are indicated by ** vs. BMSC, *** vs. Cx43, # vs. BMP-7.

Fig. 5.

Cortical-like bone thickness and trabecular-like bone volume fraction of tissue engineered bone (A). Cx43-transduced cells produced an average cortical thickness that was significantly greater than the cortical thickness of bone produced by BMSCs (B). The trabecular-like BVF (C) was also significantly greater in bone regenerated from cells overexpressing Cx43 compared to BMSCs at 4 and 12 weeks. Cotransduced cells led to a significantly larger fraction of trabecular-like bone at 8 and 12 weeks compared to all other groups. Significant increases are indicated by ** vs. BMSCs, *** vs. Cx43, # vs. BMP-7, and ## vs. Cx43-BMP7.