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Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

Freeman FE, Allen AB, Stevens HY, Guldberg RE, McNamara LM - Stem Cell Res Ther (2015)

Bottom Line: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days.Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation.In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomechanics Research (BMEC), Biomedical Engineering, College of Engineering and Informatics, National University of Ireland Galway, Galway, Ireland. f.freeman1@nuigalway.ie.

ABSTRACT

Introduction: During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo.

Methods: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo.

Results: Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups.

Conclusions: Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemical staining of the groups after 4 weeks implantation. Boxes denote area of magnification. Images were taken at 10× and 60×. Schematic of the plane in which the section was taken is in the middle of the image. CD31 stained in green, nucleus stained in blue, smooth actin stained in red. Arrows denote presence of CD31 (green) within vessel formation
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Fig9: Immunohistochemical staining of the groups after 4 weeks implantation. Boxes denote area of magnification. Images were taken at 10× and 60×. Schematic of the plane in which the section was taken is in the middle of the image. CD31 stained in green, nucleus stained in blue, smooth actin stained in red. Arrows denote presence of CD31 (green) within vessel formation

Mentions: μCT reconstruction of the explant vasculature illustrated the presence of host blood vessels surrounding the construct and infiltrating the construct through the holes present within the nanofibre mesh (Fig. 7a). Vessel volume was quantified in two ways: 1) total vessel volume; and 2) average vessel diameter. After 4 weeks in vivo there was no significant difference in total vessel volume or average vessel diameter between any of the groups (Fig. 8a,b). To further analyse the data a vessel diameter of 0.15 mm (150 μm) was chosen as a threshold to distinguish between thick and thin vessels and preclude smaller structures (that were unlikely to be mature vessels) from obscuring the results of the analysis. The threshold was chosen on the basis of our immunostaining (described in detail below), which revealed that positively stained α-smooth actin and CD31 and structures with a visible lumen had diameters in the range of 150 μm (see Figs. 9 and 10, described in detail below). Moreover, the majority of vessels in a Sprague–Dawley rat femora are in the range of 120–150 μm [61] and the average vessel diameter achieved within a bone tissue-engineering scaffold implanted in a rabbit alveolar bone defect was 152 μm by 4 weeks [62]. For both the Cartilage Template and the Pre-vascularised Cartilage template groups a large proportion of the vessels were thicker than 0.15 mm. When only vessels with a diameter greater than 0.15 mm are considered, in both the alginate and the Co-culture Cartilage Template group only three out of eight rats had vessels greater than 0.15 mm. These vessels only accounted for 7 % of the overall vessels within the construct. However, in both the Cartilage Template and Pre-vascularised Cartilage Template group five out of eight rats had vessels present with a greater diameter than 0.15 mm and these vessels accounted for up to 14 % of the overall vessels seen within the construct (see Fig. 7b).Fig. 7


Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

Freeman FE, Allen AB, Stevens HY, Guldberg RE, McNamara LM - Stem Cell Res Ther (2015)

Immunohistochemical staining of the groups after 4 weeks implantation. Boxes denote area of magnification. Images were taken at 10× and 60×. Schematic of the plane in which the section was taken is in the middle of the image. CD31 stained in green, nucleus stained in blue, smooth actin stained in red. Arrows denote presence of CD31 (green) within vessel formation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4635553&req=5

Fig9: Immunohistochemical staining of the groups after 4 weeks implantation. Boxes denote area of magnification. Images were taken at 10× and 60×. Schematic of the plane in which the section was taken is in the middle of the image. CD31 stained in green, nucleus stained in blue, smooth actin stained in red. Arrows denote presence of CD31 (green) within vessel formation
Mentions: μCT reconstruction of the explant vasculature illustrated the presence of host blood vessels surrounding the construct and infiltrating the construct through the holes present within the nanofibre mesh (Fig. 7a). Vessel volume was quantified in two ways: 1) total vessel volume; and 2) average vessel diameter. After 4 weeks in vivo there was no significant difference in total vessel volume or average vessel diameter between any of the groups (Fig. 8a,b). To further analyse the data a vessel diameter of 0.15 mm (150 μm) was chosen as a threshold to distinguish between thick and thin vessels and preclude smaller structures (that were unlikely to be mature vessels) from obscuring the results of the analysis. The threshold was chosen on the basis of our immunostaining (described in detail below), which revealed that positively stained α-smooth actin and CD31 and structures with a visible lumen had diameters in the range of 150 μm (see Figs. 9 and 10, described in detail below). Moreover, the majority of vessels in a Sprague–Dawley rat femora are in the range of 120–150 μm [61] and the average vessel diameter achieved within a bone tissue-engineering scaffold implanted in a rabbit alveolar bone defect was 152 μm by 4 weeks [62]. For both the Cartilage Template and the Pre-vascularised Cartilage template groups a large proportion of the vessels were thicker than 0.15 mm. When only vessels with a diameter greater than 0.15 mm are considered, in both the alginate and the Co-culture Cartilage Template group only three out of eight rats had vessels greater than 0.15 mm. These vessels only accounted for 7 % of the overall vessels within the construct. However, in both the Cartilage Template and Pre-vascularised Cartilage Template group five out of eight rats had vessels present with a greater diameter than 0.15 mm and these vessels accounted for up to 14 % of the overall vessels seen within the construct (see Fig. 7b).Fig. 7

Bottom Line: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days.Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation.In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomechanics Research (BMEC), Biomedical Engineering, College of Engineering and Informatics, National University of Ireland Galway, Galway, Ireland. f.freeman1@nuigalway.ie.

ABSTRACT

Introduction: During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo.

Methods: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo.

Results: Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups.

Conclusions: Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

No MeSH data available.


Related in: MedlinePlus