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. 2017 Nov;21(11):3066-3075.
doi: 10.1111/jcmm.13218. Epub 2017 Jun 4.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus

Alessia Di Giancamillo  1 , Daniela Deponti  2 , Silvia Modina  1 , Irene Tessaro  3 , Cinzia Domeneghini  1 , Giuseppe Maria Peretti  3   4
Affiliations

Age-related modulation of angiogenesis-regulating factors in the swine meniscus

Alessia Di Giancamillo et al. J Cell Mol Med. 2017 Nov.

Abstract

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

Keywords: Meniscus; VEGF; endostatin; micro-vasculature; pig; tissue maturation.

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Figures

Figure 1
Figure 1
VEGF. Western blot and immunohistochemical appearance in pig neonatal, young and adult menisci. VEGF expression levels obtained by Western blot analysis were measured by densitometry analyses and normalized to GAPDH (housekeeping) levels. Arrows: vessels. All the figures have the same scale bar as located in Figure 3A: 100 μm.
Figure 2
Figure 2
VE‐CADHERIN. Western blot and immunohistochemical appearance in pig neonatal, young and adult menisci. VE‐CADHERIN expression levels obtained by Western blot analysis were measured by densitometry analyses and normalized to GAPDH (housekeeping) levels. Arrows: vessels. All the figures have the same scale bar as located in Figure 1A: 100 μm.
Figure 3
Figure 3
ENDOSTATIN. Western blot and immunohistochemical appearance in pig neonatal, young and adult menisci. ENDOSTATIN expression levels obtained by Western blot analysis were measured by densitometry analyses and normalized to GAPDH (housekeeping) levels. Arrowheads: fibrochondrocytes. All the figures have the same scale bar as located in Figure 2A: 100 μm.
Figure 4
Figure 4
SOX9. Western blot and immunohistochemical appearance in pig neonatal, young and adult menisci. SOX9 expression levels obtained by Western blot analysis were measured by densitometry analyses and normalized to GAPDH (housekeeping) levels. Arrows: fibres. All the figures have the same scale bar as located in (A): 100 μm.
Figure 5
Figure 5
COLL II Western blot and immunohistochemical appearance in pig neonatal, young and adult menisci. COLLAGEN‐2 expression levels obtained by Western blot analysis were measured by densitometry analyses and normalized to GAPDH (housekeeping) levels. Arrows: fibres; asterisk: matrix. All the figures have the same scale bar as located in (A) 100 μm.
Figure 6
Figure 6
Regulation of vascularization in comparison with meniscus maturation in neonatal, young and adult pigs.
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