Limits...
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: 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.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

a Alcian Blue and b Alizarin Red staining of all three groups on the day of implantation (following 3 weeks of in vitro priming). Images were taken at a magnification of 10× and 40×. c Representative images of CD31+ (green) stained sections in the CP21 + HUVECs:MSCs group prior to implantation. Arrows denote the presence of positive CD31+ staining. Each section is 8 μm thick and each of the images was taken at a magnification of 20× and 60×. Nuclear counterstain: DAPI (blue). CP21 Chondrogenically primedat day 21, HUVEC Human umbilical vein endothelial cell, MSC Mesenchymal stem cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: a Alcian Blue and b Alizarin Red staining of all three groups on the day of implantation (following 3 weeks of in vitro priming). Images were taken at a magnification of 10× and 40×. c Representative images of CD31+ (green) stained sections in the CP21 + HUVECs:MSCs group prior to implantation. Arrows denote the presence of positive CD31+ staining. Each section is 8 μm thick and each of the images was taken at a magnification of 20× and 60×. Nuclear counterstain: DAPI (blue). CP21 Chondrogenically primedat day 21, HUVEC Human umbilical vein endothelial cell, MSC Mesenchymal stem cell

Mentions: All three experimental groups stained positive blue for sGAG and Alizarin Red prior to implantation after 42 days of culture (Fig. 2a,b). There was no significant difference in sGAG or calcium production after the 6 weeks of culture.Fig. 2


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)

a Alcian Blue and b Alizarin Red staining of all three groups on the day of implantation (following 3 weeks of in vitro priming). Images were taken at a magnification of 10× and 40×. c Representative images of CD31+ (green) stained sections in the CP21 + HUVECs:MSCs group prior to implantation. Arrows denote the presence of positive CD31+ staining. Each section is 8 μm thick and each of the images was taken at a magnification of 20× and 60×. Nuclear counterstain: DAPI (blue). CP21 Chondrogenically primedat day 21, HUVEC Human umbilical vein endothelial cell, MSC Mesenchymal stem cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: a Alcian Blue and b Alizarin Red staining of all three groups on the day of implantation (following 3 weeks of in vitro priming). Images were taken at a magnification of 10× and 40×. c Representative images of CD31+ (green) stained sections in the CP21 + HUVECs:MSCs group prior to implantation. Arrows denote the presence of positive CD31+ staining. Each section is 8 μm thick and each of the images was taken at a magnification of 20× and 60×. Nuclear counterstain: DAPI (blue). CP21 Chondrogenically primedat day 21, HUVEC Human umbilical vein endothelial cell, MSC Mesenchymal stem cell
Mentions: All three experimental groups stained positive blue for sGAG and Alizarin Red prior to implantation after 42 days of culture (Fig. 2a,b). There was no significant difference in sGAG or calcium production after the 6 weeks of culture.Fig. 2

Bottom Line: 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.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