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Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro.

Castillo Diaz LA, Saiani A, Gough JE, Miller AF - J Tissue Eng (2014)

Bottom Line: The gel elasticity increased over time when cells were present - in comparison to a decrease in control samples - indicating the deposition of matrix throughout the peptide scaffold.Moreover, significant quantities of calcium phosphate were deposited.Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, UK ; Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.

ABSTRACT
Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present - in comparison to a decrease in control samples - indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

No MeSH data available.


Fluorescence micrographs of actin cytoskeleton staining (green) and collagen I production (red) by HOBs cultured in 3D within 3 wt% FEFEFKFK hydrogel over 14 days (magnification = 10×, scale bar = 500 µm).HOB: human osteoblast.
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fig5-2041731414539344: Fluorescence micrographs of actin cytoskeleton staining (green) and collagen I production (red) by HOBs cultured in 3D within 3 wt% FEFEFKFK hydrogel over 14 days (magnification = 10×, scale bar = 500 µm).HOB: human osteoblast.

Mentions: It is also important to consider that HOBs are specialised cells, and as such, their predominant role is the secretion of proteins. It is believed that their activation and proliferation are triggered mainly by systemic factors such as hormones and other local factors, which include cytokines and growth factors released after bone resorption occurs. The role of osteocytes sensing micro-alterations or mechanical loads on bone tissue is also very important to activate HOBs. Finally, the autocrine activation via bone-specific ECM proteins is fundamental to induce the proliferation of osteoblasts.24,28,29 The rate of proliferation of HOBs within 3D culture systems is, therefore, complex and clearly dependent on cell culture conditions used, such as type of scaffold, type of cells and/or the cell densities used. Here, we demonstrated that our system is able to support the viability and proliferation of HOBs, without any additional cell proliferative factor. Consequently, the next step is to test the ability of cells to produce key proteins for bone formation, including collagen-I (col-I). Figure 5 shows the fluorescence and immunocytochemistry images of stained gels for the identification of F-actin that defines the cell architecture, and col-I, at days 7 and 14. It is clear that there is significant production of col-I inside the cell cytoplasm over the first 7 days of culture. There is also some evidence of extracellular col-I staining, albeit with less intensity. After 14 days in culture, however, a high concentration of col-I was detected, not only inside the cells but also surrounding the cells, particularly in areas of the gel where cells formed clusters. The high concentration of col-I suggests that cells are functional within the gel and consequently synthesise proteins to remodel their niche. This confirms that HOBs can survive in the in vitro hydrogel and moreover produce ECM proteins that are important for their survival and bone formation.


Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro.

Castillo Diaz LA, Saiani A, Gough JE, Miller AF - J Tissue Eng (2014)

Fluorescence micrographs of actin cytoskeleton staining (green) and collagen I production (red) by HOBs cultured in 3D within 3 wt% FEFEFKFK hydrogel over 14 days (magnification = 10×, scale bar = 500 µm).HOB: human osteoblast.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4221980&req=5

fig5-2041731414539344: Fluorescence micrographs of actin cytoskeleton staining (green) and collagen I production (red) by HOBs cultured in 3D within 3 wt% FEFEFKFK hydrogel over 14 days (magnification = 10×, scale bar = 500 µm).HOB: human osteoblast.
Mentions: It is also important to consider that HOBs are specialised cells, and as such, their predominant role is the secretion of proteins. It is believed that their activation and proliferation are triggered mainly by systemic factors such as hormones and other local factors, which include cytokines and growth factors released after bone resorption occurs. The role of osteocytes sensing micro-alterations or mechanical loads on bone tissue is also very important to activate HOBs. Finally, the autocrine activation via bone-specific ECM proteins is fundamental to induce the proliferation of osteoblasts.24,28,29 The rate of proliferation of HOBs within 3D culture systems is, therefore, complex and clearly dependent on cell culture conditions used, such as type of scaffold, type of cells and/or the cell densities used. Here, we demonstrated that our system is able to support the viability and proliferation of HOBs, without any additional cell proliferative factor. Consequently, the next step is to test the ability of cells to produce key proteins for bone formation, including collagen-I (col-I). Figure 5 shows the fluorescence and immunocytochemistry images of stained gels for the identification of F-actin that defines the cell architecture, and col-I, at days 7 and 14. It is clear that there is significant production of col-I inside the cell cytoplasm over the first 7 days of culture. There is also some evidence of extracellular col-I staining, albeit with less intensity. After 14 days in culture, however, a high concentration of col-I was detected, not only inside the cells but also surrounding the cells, particularly in areas of the gel where cells formed clusters. The high concentration of col-I suggests that cells are functional within the gel and consequently synthesise proteins to remodel their niche. This confirms that HOBs can survive in the in vitro hydrogel and moreover produce ECM proteins that are important for their survival and bone formation.

Bottom Line: The gel elasticity increased over time when cells were present - in comparison to a decrease in control samples - indicating the deposition of matrix throughout the peptide scaffold.Moreover, significant quantities of calcium phosphate were deposited.Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, UK ; Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.

ABSTRACT
Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present - in comparison to a decrease in control samples - indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

No MeSH data available.