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Dental follicle stem cells in bone regeneration on titanium implants.

Lucaciu O, Soriţău O, Gheban D, Ciuca DR, Virtic O, Vulpoi A, Dirzu N, Câmpian R, Băciuţ G, Popa C, Simon S, Berce P, Băciuţ M, Crisan B - BMC Biotechnol. (2015)

Bottom Line: Differentiation into bone cells was induced in presence or absence of BMP-2 and TGFβ1.Additional BMP-2 in the medium did not allow DF stem cells to develop a more mature phenotype, leaving them in a pre-osteogenic stage.Long term culturing (70 days) on titanium surfaces of DF stem cells in standard medium without soluble osteogenic inducers, indicated that HA coating is more favorable, with the acquisition of a more mature osteoblastic phenotype as shown by immunocytochemical staining.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Rehabilitation, "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, 15 Victor Babeș Street, 400012, Cluj-Napoca, Cluj, Romania. ondineluc@yahoo.com.

ABSTRACT

Background: We aimed to demonstrate that DF stem cells from impacted molars and canines can be used to improve bone regeneration on titanium implants surfaces. This study highlights the presence of stem cells in DF, their potential to adhere and differentiate into osteoblasts on different types of titanium surfaces.

Results: Isolated cells from the harvested DF tissue from impacted canine/molars, expressed stem cells markers. Differentiation into bone cells was induced in presence or absence of BMP-2 and TGFβ1. The presence of growth factors until 28 days in medium maintained the cells in an earlier stage of differentiation with a lower level of specific bone proteins and a higher expression of alkaline phosphatase (ALP). Influence of titanium implants with different bioactive coatings, hydroxyapatite (TiHA) and with silicatitanate (TiSiO2), and porous Ti6Al7Nb implants as control (TiCtrl), was studied in terms of cell adhesion and viability. Ti HA implants proved to be more favorable for adhesion and proliferation of DF stem cells in first days of cultivation. The influence of titanium coatings and osteogenic differentiation mediums with or without growth factors were evaluated. Additional BMP-2 in the medium did not allow DF stem cells to develop a more mature phenotype, leaving them in a pre-osteogenic stage. The best sustained mineralization process evaluated by immuno-cytochemical staining, scanning electron microscopy and Ca(2+) quantification was observed for TiHA implants with a higher expression of ALP, collagen and Ca(2+) deposition. Long term culturing (70 days) on titanium surfaces of DF stem cells in standard medium without soluble osteogenic inducers, indicated that HA coating is more favorable, with the acquisition of a more mature osteoblastic phenotype as shown by immunocytochemical staining. These findings demonstrated that even in absence of exogenous osteogenic factors, TiHA implants and in a lesser extent TiCtrl and TiSiO2 implants can induce and sustain osteogenic differentiation of DF stem cells, by their chemical and topographical properties.

Conclusions: Our research demonstrated that DF stem cells have a spontaneous tendency for osteogenic differentiation and can be used for improving bone regeneration on titanium implants surfaces.

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Related in: MedlinePlus

a DF stem cells adhesion on titanium implants after 1 h and cell viability at 48 h evaluated by fluorescein diacetate (FDA) test (area scan) (b) Fluorescence microscopy images of FDA stained DF stem cells cultivated 7 days on titanium surfaces in standard stem cells medium (Legend: TiCtrl- Ti6Al7Nb alloy porous titanium, TiHA-titanium infiltrated with hydroxyapatite, TiSiO2-titanium infiltrated with silicatitanate) (magnification ×100)
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Fig7: a DF stem cells adhesion on titanium implants after 1 h and cell viability at 48 h evaluated by fluorescein diacetate (FDA) test (area scan) (b) Fluorescence microscopy images of FDA stained DF stem cells cultivated 7 days on titanium surfaces in standard stem cells medium (Legend: TiCtrl- Ti6Al7Nb alloy porous titanium, TiHA-titanium infiltrated with hydroxyapatite, TiSiO2-titanium infiltrated with silicatitanate) (magnification ×100)

Mentions: We investigated the behavior of DF stem cells cultivated on surfaces of titanium implants, in order to lay the foundation for finding a new way to induce bone regeneration on the titanium implant surface. The adhesion process was evaluated after 1 h of cultivation of DF stem cells in standard stem cells medium on three types of titanium implants (TiCtrl, TiHA and TiSiO2) using the fluorescein diacetate test (FDA). The highest fluorescence values were found for TiHA and Ti Ctrl implants with statistically different values comparing with TiSiO2 implants (statistical analysis was performed using One-way analysis of variance; p value < 0.001). The most favorable substrate was proved to be titanium implants infiltrated with HA, especially in the first hour of cell adhesion process. The differences were statistically significant at 1 h after seeding the cells. At 48 h and at 7 days of cultivation the HA infiltrated titanium implants preserved the advantages for cell proliferation, but the differences were not statistically significant (Fig. 7). Microscopical analysis of FDA stained DF stem cells confirmed the increased number of cells after 48 h and 7 days for DF stem cells cultivated on Ti Ctrl and TiHA implants (Additional file 2: Figure S2). Cell viability and subsequent cell proliferation were evaluated by an additional viability test (Alamar blue) in two conditions: (1) in standard stem cells medium and (2) in a comparative study between stem medium and differentiation medium OS and OC. Alamar test revealed as FDA test that in the first day of cultivation the Ti HA offers slightly increased DF stem cells adhesion, but there are no differences between implants after 4 and 12 days in terms of viability and proliferation (Additional file 3: Figure S3). These findings are strengthened by the results obtained for the cells cultivated with stem cell medium and osteogenic medium for 4 and 12 days. The differences appeared between stem cell medium and osteogenic differentiation medium, as inducing the osteogenic differentiation had caused, as expected, a decrease in cell numbers after 4 and 12 days of cultivation (Additional file 4: Figure S4).Fig. 7


Dental follicle stem cells in bone regeneration on titanium implants.

Lucaciu O, Soriţău O, Gheban D, Ciuca DR, Virtic O, Vulpoi A, Dirzu N, Câmpian R, Băciuţ G, Popa C, Simon S, Berce P, Băciuţ M, Crisan B - BMC Biotechnol. (2015)

a DF stem cells adhesion on titanium implants after 1 h and cell viability at 48 h evaluated by fluorescein diacetate (FDA) test (area scan) (b) Fluorescence microscopy images of FDA stained DF stem cells cultivated 7 days on titanium surfaces in standard stem cells medium (Legend: TiCtrl- Ti6Al7Nb alloy porous titanium, TiHA-titanium infiltrated with hydroxyapatite, TiSiO2-titanium infiltrated with silicatitanate) (magnification ×100)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4697321&req=5

Fig7: a DF stem cells adhesion on titanium implants after 1 h and cell viability at 48 h evaluated by fluorescein diacetate (FDA) test (area scan) (b) Fluorescence microscopy images of FDA stained DF stem cells cultivated 7 days on titanium surfaces in standard stem cells medium (Legend: TiCtrl- Ti6Al7Nb alloy porous titanium, TiHA-titanium infiltrated with hydroxyapatite, TiSiO2-titanium infiltrated with silicatitanate) (magnification ×100)
Mentions: We investigated the behavior of DF stem cells cultivated on surfaces of titanium implants, in order to lay the foundation for finding a new way to induce bone regeneration on the titanium implant surface. The adhesion process was evaluated after 1 h of cultivation of DF stem cells in standard stem cells medium on three types of titanium implants (TiCtrl, TiHA and TiSiO2) using the fluorescein diacetate test (FDA). The highest fluorescence values were found for TiHA and Ti Ctrl implants with statistically different values comparing with TiSiO2 implants (statistical analysis was performed using One-way analysis of variance; p value < 0.001). The most favorable substrate was proved to be titanium implants infiltrated with HA, especially in the first hour of cell adhesion process. The differences were statistically significant at 1 h after seeding the cells. At 48 h and at 7 days of cultivation the HA infiltrated titanium implants preserved the advantages for cell proliferation, but the differences were not statistically significant (Fig. 7). Microscopical analysis of FDA stained DF stem cells confirmed the increased number of cells after 48 h and 7 days for DF stem cells cultivated on Ti Ctrl and TiHA implants (Additional file 2: Figure S2). Cell viability and subsequent cell proliferation were evaluated by an additional viability test (Alamar blue) in two conditions: (1) in standard stem cells medium and (2) in a comparative study between stem medium and differentiation medium OS and OC. Alamar test revealed as FDA test that in the first day of cultivation the Ti HA offers slightly increased DF stem cells adhesion, but there are no differences between implants after 4 and 12 days in terms of viability and proliferation (Additional file 3: Figure S3). These findings are strengthened by the results obtained for the cells cultivated with stem cell medium and osteogenic medium for 4 and 12 days. The differences appeared between stem cell medium and osteogenic differentiation medium, as inducing the osteogenic differentiation had caused, as expected, a decrease in cell numbers after 4 and 12 days of cultivation (Additional file 4: Figure S4).Fig. 7

Bottom Line: Differentiation into bone cells was induced in presence or absence of BMP-2 and TGFβ1.Additional BMP-2 in the medium did not allow DF stem cells to develop a more mature phenotype, leaving them in a pre-osteogenic stage.Long term culturing (70 days) on titanium surfaces of DF stem cells in standard medium without soluble osteogenic inducers, indicated that HA coating is more favorable, with the acquisition of a more mature osteoblastic phenotype as shown by immunocytochemical staining.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Rehabilitation, "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, 15 Victor Babeș Street, 400012, Cluj-Napoca, Cluj, Romania. ondineluc@yahoo.com.

ABSTRACT

Background: We aimed to demonstrate that DF stem cells from impacted molars and canines can be used to improve bone regeneration on titanium implants surfaces. This study highlights the presence of stem cells in DF, their potential to adhere and differentiate into osteoblasts on different types of titanium surfaces.

Results: Isolated cells from the harvested DF tissue from impacted canine/molars, expressed stem cells markers. Differentiation into bone cells was induced in presence or absence of BMP-2 and TGFβ1. The presence of growth factors until 28 days in medium maintained the cells in an earlier stage of differentiation with a lower level of specific bone proteins and a higher expression of alkaline phosphatase (ALP). Influence of titanium implants with different bioactive coatings, hydroxyapatite (TiHA) and with silicatitanate (TiSiO2), and porous Ti6Al7Nb implants as control (TiCtrl), was studied in terms of cell adhesion and viability. Ti HA implants proved to be more favorable for adhesion and proliferation of DF stem cells in first days of cultivation. The influence of titanium coatings and osteogenic differentiation mediums with or without growth factors were evaluated. Additional BMP-2 in the medium did not allow DF stem cells to develop a more mature phenotype, leaving them in a pre-osteogenic stage. The best sustained mineralization process evaluated by immuno-cytochemical staining, scanning electron microscopy and Ca(2+) quantification was observed for TiHA implants with a higher expression of ALP, collagen and Ca(2+) deposition. Long term culturing (70 days) on titanium surfaces of DF stem cells in standard medium without soluble osteogenic inducers, indicated that HA coating is more favorable, with the acquisition of a more mature osteoblastic phenotype as shown by immunocytochemical staining. These findings demonstrated that even in absence of exogenous osteogenic factors, TiHA implants and in a lesser extent TiCtrl and TiSiO2 implants can induce and sustain osteogenic differentiation of DF stem cells, by their chemical and topographical properties.

Conclusions: Our research demonstrated that DF stem cells have a spontaneous tendency for osteogenic differentiation and can be used for improving bone regeneration on titanium implants surfaces.

Show MeSH
Related in: MedlinePlus