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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.


Quantification of cell viability as a function of culture time. Data are reported as mean values ± standard deviation for n = 3.*p < 0.05 when comparing cell numbers between time points.
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fig3-2041731414539344: Quantification of cell viability as a function of culture time. Data are reported as mean values ± standard deviation for n = 3.*p < 0.05 when comparing cell numbers between time points.

Mentions: Cell viability within 3 wt% FEFEFKFK gel samples was extended up to 14 days, and fluorescent micrographs of the live/dead assay and optical micrographs monitoring cell morphology over time are given in Figure 2. It is evident from live/dead screening that the majority of cells within each gel after 1, 7 and 14 days in culture were alive, with only a very few dead cells (Figure 2(a)–(c)). Importantly, cells were also incorporated throughout the peptide hydrogel, as evidenced by the even cell distribution in the Z-direction shown in the confocal image in Figure 2(d) (~250 µm thickness). Such entrapment and viability is similar to our previous work with bovine chondrocytes21 and demonstrates the hydrogels can support cells within 3D. The optical micrographs in Figure 2(e)–(g) reveal that the HOBs typically acquired a rounded morphology during their 2 weeks in culture within the peptide hydrogel. When HOBs are present within natural bone, and their synthesis activity is induced, they tend to acquire a cuboidal/plump 3D morphology. This is in contrast to the rather flattened morphology commonly seen for bone lining cells, and also when the cells are grown on many 2D substrates.23,24 This suggests that the HOBs within our peptide hydrogel adopt an in vivo–like morphology, suggesting the peptide hydrogel is mimicking the 3D in vivo matrix environment. Such behaviour has been observed previously when HOBs have been cultured within synthetic poly(ethylene glycol) (PEG)22 and natural alginate scaffolds.25 It was also observed that HOBs formed some clusters throughout the FEFEFKFK hydrogel. These are also naturally present during osteoid synthesis.24 To quantify the viability of HOBs within the peptide gels over their 2 weeks in culture, the percentage of viable cells was determined using the trypan blue exclusion quantification method. It is clear from these data presented in Figure 3 that the percentage viability of HOBs increases over the whole 14 days. This increase is most significant over the first 7 days.


Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro.

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

Quantification of cell viability as a function of culture time. Data are reported as mean values ± standard deviation for n = 3.*p < 0.05 when comparing cell numbers between time points.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig3-2041731414539344: Quantification of cell viability as a function of culture time. Data are reported as mean values ± standard deviation for n = 3.*p < 0.05 when comparing cell numbers between time points.
Mentions: Cell viability within 3 wt% FEFEFKFK gel samples was extended up to 14 days, and fluorescent micrographs of the live/dead assay and optical micrographs monitoring cell morphology over time are given in Figure 2. It is evident from live/dead screening that the majority of cells within each gel after 1, 7 and 14 days in culture were alive, with only a very few dead cells (Figure 2(a)–(c)). Importantly, cells were also incorporated throughout the peptide hydrogel, as evidenced by the even cell distribution in the Z-direction shown in the confocal image in Figure 2(d) (~250 µm thickness). Such entrapment and viability is similar to our previous work with bovine chondrocytes21 and demonstrates the hydrogels can support cells within 3D. The optical micrographs in Figure 2(e)–(g) reveal that the HOBs typically acquired a rounded morphology during their 2 weeks in culture within the peptide hydrogel. When HOBs are present within natural bone, and their synthesis activity is induced, they tend to acquire a cuboidal/plump 3D morphology. This is in contrast to the rather flattened morphology commonly seen for bone lining cells, and also when the cells are grown on many 2D substrates.23,24 This suggests that the HOBs within our peptide hydrogel adopt an in vivo–like morphology, suggesting the peptide hydrogel is mimicking the 3D in vivo matrix environment. Such behaviour has been observed previously when HOBs have been cultured within synthetic poly(ethylene glycol) (PEG)22 and natural alginate scaffolds.25 It was also observed that HOBs formed some clusters throughout the FEFEFKFK hydrogel. These are also naturally present during osteoid synthesis.24 To quantify the viability of HOBs within the peptide gels over their 2 weeks in culture, the percentage of viable cells was determined using the trypan blue exclusion quantification method. It is clear from these data presented in Figure 3 that the percentage viability of HOBs increases over the whole 14 days. This increase is most significant over the first 7 days.

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.