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. 2017 Nov 21;18(11):2478.
doi: 10.3390/ijms18112478.

Vascular Endothelial Growth Factor Sequestration Enhances In Vivo Cartilage Formation

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Free PMC article

Vascular Endothelial Growth Factor Sequestration Enhances In Vivo Cartilage Formation

Carolina M Medeiros Da Cunha et al. Int J Mol Sci. .
Free PMC article

Abstract

Autologous chondrocyte transplantation for cartilage repair still has unsatisfactory clinical outcomes because of inter-donor variability and poor cartilage quality formation. Re-differentiation of monolayer-expanded human chondrocytes is not easy in the absence of potent morphogens. The Vascular Endothelial Growth Factor (VEGF) plays a master role in angiogenesis and in negatively regulating cartilage growth by stimulating vascular invasion and ossification. Therefore, we hypothesized that its sole microenvironmental blockade by either VEGF sequestration by soluble VEGF receptor-2 (Flk-1) or by antiangiogenic hyperbranched peptides could improve chondrogenesis of expanded human nasal chondrocytes (NC) freshly seeded on collagen scaffolds. Chondrogenesis of several NC donors was assessed either in vitro or ectopically in nude mice. VEGF blockade appeared not to affect NC in vitro differentiation, whereas it efficiently inhibited blood vessel ingrowth in vivo. After 8 weeks, in vivo glycosaminoglycan deposition was approximately two-fold higher when antiangiogenic approaches were used, as compared to the control group. Our data indicates that the inhibition of VEGF signaling, independently of the specific implementation mode, has profound effects on in vivo NC chondrogenesis, even in the absence of chondroinductive signals during prior culture or at the implantation site.

Keywords: chondrogenesis; collagen scaffold; dendron; nasal chondrocyte; soluble VEGF receptor-2.

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study, the collection, analyses, or interpretation of data, the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
In vitro expression of CD8 and bio-activity of the sFlk-1 released by genetically modified human nasal chondrocytes. (A) Representative Fluorescence-activated cell sorting (FACS) plot shows that nasal chondrocytes (NC) had transduction efficiency above 80% (green-tinted plot) and populations of CD8-positive cells were purified after sorting (blue-tinted line). Not stained cells were acquired as control (in red). (B) Human umbilical vein endothelial cells (HUVEC) metabolic activity assay showing that sFlk-1-containing supernatants efficiently blocks Vascular Endothelial Growth Factor (VEGF). CD8-expressing NC supernatants were used as control (2 donors, n = 4). (C) HUVEC migration assay after 12 h showing that sFlk-1-expressing NC hampered HUVEC migration by blocking different gradients of VEGF, whereas CD8-only expressing NC allowed HUVEC migration. * p < 0.01.
Figure 2
Figure 2
In vitro chondrogenesis of nasal chondrocytes. (A) Representative Safranin-O staining images of NC cultured in pellets at 20% of oxygen tension. Naïve, naïve mock-sorted (Naïve sorted), CD8 (control), and sFlk-1-releasing NC were cultured in vitro in pellets and analyzed for their chondrogenic potential. (B) Glycosaminoglycan (GAG) content of pellets generated by NC cultured at either 2% or 20% of oxygen tension (2 donors, n = 9). (C) Amount of mouse sFlk-1 released by sFlk-1-expressing NC-based pellets cultured at different oxygen tensions (2 donors, n = 4). No statistical significant difference has been found.
Figure 3
Figure 3
In vivo blocking of angiogenesis. (A) Representative immunofluorescence pictures of control (naïve NC), sFlk-1, and dendron-based constructs stained either in red for PECAM (CD31), an endothelial marker, (first raw, scale bar = 100 µm) or in green for F4/80, a macrophage marker (second raw, scale bar = 50 µm). Cell nuclei were stained in blue with DAPI (A). White arrows indicate the vascular structures, whereas white dotted lines mark the boundary between the implant and the surrounding host tissue. Asterisks indicate the implant area. (B) Quantification of the vessel length density in µm/µm2 at the center or edge of the implant area of naïve, sFlk-1, and dendron-based constructs (1 donor, n = 4). * p < 0.01.
Figure 4
Figure 4
In vivo chondrogenesis of nasal chondrocytes—cell-based gene therapy approach. (A) Safranin-O staining for naïve (control) and sFlk-1-releasing NC after 8 weeks in vivo. (B) Types II and X collagen stainings for naïve and sFlk-1 NC at 8 weeks in vivo. (C) Total amount of GAG deposited in vivo at 4 and 8 weeks by naïve and sFlk-1 expressing NC (2 donors, n = 8). * p < 0.01. Scale bars = 50 µm.
Figure 5
Figure 5
In vivo chondrogenesis of nasal chondrocytes-dendron-functionalized approach. (A) Safranin-O, types II and X collagen staining for control (naïve NC) and dendron-based constructs at 8 weeks in vivo. Dotted white line indicates the cartilaginous part of the dendron based implants. Scale bar = 50 µm. (B) Total GAG production in vivo at 4 and 8 weeks by control (naïve NC) and dendron-based constructs (1 donor, n = 5). * p < 0.01.

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