Investigating the Vascularization of Tissue-Engineered Bone Constructs Using Dental Pulp Cells and 45S5 Bioglass(®) Scaffolds.

Identification of a suitable cell source combined with an appropriate 3D scaffold is an essential prerequisite for successful engineering of skeletal tissues. Both osteogenesis and angiogenesis are key processes for bone regeneration. This study investigated the vascularization potential of a novel combination of human dental pulp stromal cells (HDPSCs) with 45S5 Bioglass(®) scaffolds for tissue-engineered mineral constructs in vivo and in vitro. 45S5 Bioglass scaffolds were produced by the foam replication technique with the standard composition of 45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt% CaO, and 6 wt% P2O5. HDPSCs were cultured in monolayers and on porous 45S5 Bioglass scaffolds under angiogenic and osteogenic conditions for 2-4 weeks. HDPSCs expressed endothelial gene markers (CD34, CD31/PECAM1, and VEGFR2) under both conditions in the monolayer. A combination of HDPSCs with 45S5 Bioglass enhanced the expression of these gene markers. Positive immunostaining for CD31/PECAM1 and VEGFR2 and negative staining for CD34 supported the gene expression data, while histology revealed evidence of endothelial cell-like morphology within the constructs. More organized tubular structures, resembling microvessels, were seen in the constructs after 8 weeks of implantation in vivo. In conclusion, this study suggests that the combination of HDPSCs with 45S5 Bioglass scaffolds offers a promising strategy for regenerating vascularized bone grafts

Bone tissue engineering using adult mesenchymal stem cells and biomimetic P-15 scaffolds

Introduction: The increasing clinical demand for bone regeneration and repair in the context of our aging population poses a challenge both to healthcare providers and society more generally. Tissue engineering provides a promising strategy to meet this clinical demand by developing functional bone construct using stem/stromal cells, scaffolds, with/without growth factors. However, several issues remain to be addressed before clinical translation of this technology. The main challenge is to identify the most appropriate combination of the three elements that can be used to achieve optimum regeneration of damaged bone tissue. A second issue relates to cell death on exposure of the graft constructs to temporary hypoxic in vivo microenvironment such as that found at fracture sites. The aim of this thesis was to investigate the osteogenic potential of human dental pulp stem/stromal cells (HDPSCs) and to determine the possible use of these cells in combination with a biomimetic collagen peptide coated with anorganic bone mineral particles (ABM-P-15) for bone tissue engineering. The secondary aim of the study was to investigate the effect of hypoxia in the osteogenic differentiation potential of commercial human bone marrow stromal cells (HBMSCs) - MultiStem® cells, on ABM-P-15 scaffolds. Methodology: HDPSCs and HBMSCs were isolated from dental pulp and bone marrow respectively. In monolayer culture, proliferation, multilineage (osteogenic, adipogenic and chondrogenic) differentiation, time point based osteogenic gene expression profile (ALP, Ctll. I, Runx2 and OCN) and also ALPSA of HDPSCs was compared to HBMSCs. Then, HDPSCs or HBMSCs were cultured on 3D ABM-P- 15 and ABM scaffolds in basal media for up to 6 weeks and samples were analyzed using confocal microscopy, SEM and histological staining. For in vivo investigation, HDPSCs on ABM- P-15 and ABM scaffolds were sealed within diffusion chambers which were implanted intraperitoneally in MFl nu/nu rmce for 8 weeks and assessed for histological and immunohistochemical staining. Meanwhile, MultiStem® cells were cultured on ABM-P-15 and ABM scaffolds under in vitro hypoxic and normoxic conditions for up to 6 weeks and were analyzed for cell attachment, growth and ALP expression. Hypoxia expanded MultiStem® cells on ABM-P-15 and ABM scaffolds in diffusion chambers were also implanted intraperitoneally in MFl nu/nu mice for 8 weeks and samples were assessed for histological and immunohistochemical staining. Results: Similar to HBMSCs, HDPSCs were proliferative and also exhibited the potential to differentiation into the three lineages (osteogenic, adipogenic and chonderogenic) in monolayer culture. Under basal culture conditions, HDPSCs expressed the osteogenic markers (COLI, ALP, Runx2 and OCN) similar to HBMSCs. However, biochemical assays confirmed higher ALP protein expression in HDPSCs to that of HBMSCs. When cultured on 3D scaffolds (ABM-P-15 and ABM scaffolds alone), ABM-P-15 enhanced both HDPSCs and HBMSCs attachment and spreading at 24 hours and cell bridge formation after 14 days. Confocal microscopic images confirmed that more HDPSCs grew on both ABM-P-15 and ABM alone compared to HBMSCs on similar scaffold groups. Both ALP staining and quantitative assays showed high ALP expressions with both HDPSCs and HBMSCs on ABM-P-15 scaffolds compared to those cultured on ABM alone. Following 8 weeks in vivo implantation, HDPSCs on ABM-P-15 and ABM alone scaffolds revealed extracellular matrix deposition that was positive for COLI, OCN and OPN and the cells on ABM-P-15 scaffolds in particular showed extensive organised collagenous matrix formation, confirmed by Sirius red staining. Under hypoxic conditions, MultiStem® cells cultured on ABM-P-15 and ABM scaffolds appeared to have reduced . ALP expression. However, after in vivo implantation, hypoxic expanded MultiStem® cells produced ECM that stained positively for COLI, OCN and OPN. Those cells on ABM-P-15 scaffolds appeared to have more organised collagen matrix compared with those on ABM alone. Conclusion: The results suggest that HDPSCs together with ABM-P-15 scaffolds could be a useful combination for bone augmentation. These cell pre-expanded in hypoxic conditions may also be useful to reduce the cell death seen after clinical implantation at the fracture site which will be beneficial during clinical translational of tissue engineering.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Isolation and characterization of mesenchymal stem cells from human fetus heart

<div><p>Background</p><p>Mesenchymal stem cells (MSCs) are promising cells for cardiovascular regenerative medicine. However, their potential may be limited, because of their restricted cardiovascular differentiation potential and decline in their number and functional characteristics with increasing donor age. We have previously shown that rat fetus heart harbors primitive MSCs and administration of these cells improved left ventricular (LV) function after ischemia/reperfusion injury in rats. To evaluate their potential as a new cell type for clinical cardiovascular cell therapy, we have undertaken this study on the isolation and characterization of human fetal cardiac MSCs (hfC-MSCs).</p><p>Methods</p><p>MSCs were isolated from the heart of five 14-16-week-old aborted human fetuses and studied for their growth characteristics, karyotypic stability and senescence over successive passages, expression of mesenchymal and embryonal markers by flow cytometry and immunocytochemistry, constitutive expression of cardiovascular genes by RT-PCR, differentiation into cells of the cardiovascular lineage and their immunomodulatory properties.</p><p>Results</p><p>The hfC-MSCs grew as adherent monolayer with spindle shaped morphology and exhibited rapid proliferation with an average population doubling time of 34 hours and expansion to up to more than 80 population doublings with maintenance of a normal karyotype and without senescence. Immunophenotyping showed that they had similar phenotype as human bone marrow mesenchymal stem cells (hBM-MSCs) expressing CD73, CD90, CD105 and lacking expression of CD31, CD34, CD45, HLA-DR. However, hfC-MSCs expressed significantly higher levels of CD117 and SSEA-4 compared to hBM-MSCs. In addition, hfC-MSCs expressed the embryonal markers Oct-4, Nanog and Sox-2 as compared to hBM-MSCs. Further, hfC-MSCs had significantly higher expression of the cardiovascular genes viz. ISL-1, flk-1, GATA-4, NKX2.5 and MDR-1 as compared to hBM-MSCs, and could be differentiated into major cardiovascular cells (cardiomyocytes, endothelial cells, smooth muscle cells). Interestingly, hfC-MSCs markedly reduced T-lymphocyte proliferation with an increased secretion of TGF-β and IL-10.</p><p>Conclusions</p><p>Our results show that human fetus heart is a novel source of primitive MSCs with cardiovascular commitment which may have a potential therapeutic application in cardiovascular regenerative medicine.</p></div

Differentiation of hfC-MSCs into cardiovascular cells.

<p>Representative immunocytochemistry images (40X, 20μm) showing differentiation of human fetal cardiac stem cells (hfC-MSCs) into Cardiomyocytes (B: Troponin-T (cTnT); Endothelial cells (D: CD31); Smooth Muscle Cells (F: Smooth Muscle- myosin heavy chain (SM-MHC); (A, C and E, were control cells without induction medium showing only hoechst dye). Data shown are from three independent experiments at passage 3–5.</p

Phenotypic characteristics of hfC-MSCs compared to BM-MSCs.

<p>Representative bar graphs showing a comparison of the expression of CD73, CD90, CD105, SSEA-4, CD117, CD34, CD45, HLA-DR and CD31 on hfC-MSCs and hBM-MSCs as demonstrated by flow cytometry. Values are mean ± SE of three independent experiments of both the cell types at passage-5.</p

Morphology and Growth Kinetics of hfC-MSCs compared to hBM-MSCs.

<p>Representative photomicrographs (10X, 20μm) of (A) Human fetal cardiac mesenchymal stem cells (hfC-MSCs); (B) Bone marrow mesenchymal stem cells (hBM-MSCs) showing spindle shaped morphology at 5th passage; (C) Growth kinetics of hfC-MSCs and hBM-MSCs seeded at a density of 1,000 cells per cm².</p

hfC-MSCs inhibit PHA-induced proliferation of lymphocytes.

<p><b>(A)</b> PBMCs (1 × 10⁵ cells) stimulated with or without PHA (5 μg/ mL) in the presence or absence of irradiated hfC-MSCs (1 × 104–5 × 10⁴ cells). Data are expressed as the mean ± SE of three independent experiments. *p<0.05 (Control PBMCs vs. PBMCs+ MSCs), NS: Not significant. <b>(B)</b> TGF-β levels were analyzed in the culture supernatants of co-cultured hfC-MSCs and PBMCs stimulated with or without PHA. PBMCs (1 × 10⁵ cells) cultured with PHA (5 μg/mL) in the presence or absence of hfC-MSCs (1 × 104–5× 10⁴ cells). Data are expressed as the mean ± SE of three independent experiments. *p<0.05 (Control PBMC vs. PBMC+ MSC), NS: Not significant. <b>(C)</b> IL-10 levels were analysed in the culture supernatants of co-cultured hfC-MSCs and PBMCs stimulated with or without PHA. PBMCs (1 × 10⁵ cells) cultured with PHA (5 μg/mL) in the presence or absence of hfC-MSCs (1 × 104–5× 10⁴ cells). Data are expressed as mean ± SE of three independent experiments. *p<0.05 (Control PBMCs vs. PBMCs+ MSCs), NS: Not significant.</p

Primers used in this study.

<p>Primers used in this study.</p