Initial interaction of U2OS cells with noncoated and calcium phosphate coated titanium substrates
J. ter Brugge, S. Dieudonne, J. A. Jansen. Journal of Biomedical Materials Research. 2002. Vol. 61, Issue 3, Pages 399-407.
Summary:
Previous studies have investigated the long-term effect of calcium phosphate coated implants and have shown that they stimulate bone formation in comparison to uncoated implants in terms of proliferation and differentiation. The initial interaction of cells with a biomaterial is also of importance however. The composition and organization of the substrate can affect the attachment and spreading of cells on its surface. The exact mechanism that dictates how cell function is affected by the surface conditions of the substrate is unclear, but it is known that they interact with the material through an intervening protein layer. Thus the composition of the protein layer can drastically affect the cellular response to the material as well as various cellular processes. This study focused on the initial effect of CaP coated and noncoated titanium substrates on the attachment, spreading, integrin expression, and shape of osteosarcoma cells. The findings can aid in the design of safe and effective implants.
U2OS cells were cultured in α minimum Eagle’s medium and supplemented with 10% fetal calf serum and 50 µg/mL gentamycin. After the cells were cultured for one week, they were trypisinized and added to various substrates. These included pure titanium disks that were machined (Ti-s) or gritblasted and were either left uncoated (Ti-r) or had a 2 µm thick RF-magnetron sputtered calcium phosphate coating (CaP-ht). Surface roughness of the substrates was measured using atomic force microscopy.
Cells were seeded onto the substrates and then trypsinized and counted after 0.5, 1, 2, 3, 4, 7, or 24 hours. The number of attached cells increased quickly within the first 90 min for all substrates. Afterwards, cell attachment continued to increase, but at a slower rate. At 90 min, the number of attached cells was different for each material with Ti-r > CaP-ht > Ti-s.
Figure 1. Attachment of U2OS cells on different substrates, at 0.5, 1, 1.5, 3, 7, and 24 h. Inset shows the percentage of attached cells at 24 h.
Scanning electron microscopy analysis showed that cells on Ti-r and CaP-ht had a smooth, round morphology and attached to the surface with lamellipodia for the first 3 hours of culture. Cells on Ti-s on the other hand were rounded but had a ruffled membrane. Both the attachment assay and SEM results could have been due to the fact that on a smooth surface, cells have a limited number of adhesion sites compared to rough substrates.
Figure 2. Attachment and spreading of U2OS on Ti-s, Ti-r, and CaP-ht.(A) SEM image of U2OS cells, after 3 h on CaP-ht. Original magnification 8000×.(B) U2OS cells, after 3 h on Ti-s. Original magnification 6000×. (C) SEM image of a number of U2OS cells on Ti-r, after 7 h. Original magnification 1000×. (D) U2OS cells cultured for 24 h on Ti-r. Original magnification 2500× α3 at T = 0, and at 3, 7, and 24 h on Ti-s, Ti-r, and CaP-ht. Values represent the mean ± SD of 5 experiments.
It has previously been determined that integrin expression can also change, depending on the substrate surface characteristics. Evaluation of the U2OS cultures using FACS showed that the cells expressed α2, α5, α6, and αv integrin subunits and had high mean fluorescent intensity for α3 and β1. These subunits combine to form receptors for various ECM proteins. The β1 subunit specifically interacts with signaling and cytoskeletal proteins and thus affects cell spreading. The expression of β1 was significantly higher for CaP-ht than Ti-s and Ti-r after 3 hours but this difference was not present after the 7 hour mark.
Figure 3. Expression of α3 and β1 subunits. (A) Mean fluorescent intensity for β1 at T = 0, and at 3, 7, and 24 h on Ti-s, Ti-r, and CaP-ht. Values represent the mean ± SD of 5 experiments. Asterisk indicates p < t =" 0,">
Fluorescent microscopy also showed that cells on the smooth surface spread out to a larger cell area than on rough substrates, with a more elongated cell shape associated with the spreading. Cell size started increasing after 3 hours of culture. After 16 hours, only cells on Ti-r continued to increase. During the entire 24 hours, cell size differed significantly between the three substrates with Ti-s > Ti-r > CaP-ht.
Based on these results, the study concluded that the initial interaction U2OS cells with a material in terms of attachment and spreading is dependent on the surface characteristics. Integrin expression can also be influenced by substrate surface composition such as a CaP coating.
Critique: One important factor the study fails to address is the viability of the U2OS cell culture used. With the attachment assays, for instance, the percent of attached cells was calculated as the number of attached cells divided by the number of seeded cells. There is a chance however that some of these cells are no longer alive, thus diminishing the credibility of the results drawn from the assay. Additionally, although the amount of β1 expression was obtained, it does not necessarily directly correlate with the observed attachment and spreading of cells. It would be of greater use to determine the distribution of β1 subunits throughout the surface of each material. The relative expression of β1 in a given area can then be compared against the corresponding changes in attachment and spreading.
7 comments:
When looking at the integrin expression by the cells when exposed to the CaP-coated surface, do the authors give any indication as to why the cells express significantly more of the beta1 subunit at the 3 hour mark but not at the 7 hour mark or afterwards? This seems like a strange result to me.
After seeding the cells, did the authors perform tests to check if they were still proliferating properly?
The results show that a rough Ti substrate stimulates more adhesion than the calcium phosphate coated implants. Furthermore, although the B1 integrin expression was higher for the CaP substrate initially, it evens out within a few hours. How does this correlate to the idea that in the lond-term, CaP coated implants stimulate more bone formation than uncoated implants?
Furthermore, how exactly was cell attachment measured? The paper could have also tested to determine patterns of upregulation in proteins that correlate with ECM cues and stimulate cell proliferation and adhesion pathways such as EGFR. This would help us understand how the cells are responding internally to the external substrate.
It is definitely interesting to see that surface modifications to the titanium disks lower cellular adhesion. If these implants are implicated for use in stimulating bone growth, do you think that a decrease in cellular adhesion would be optimal, especially if stem cells need very stiff matrices to become osteoblasts? Also, as you mentioned, U2OS cells are derived from osteosarcomas, which is a type of bone cancer. Do you expect that the results would be the same for healthy bone cells?
Do you happen to know what motif on the metal substrates allows for the integrins to bind? Do they mention the surface area of each substrate perchance?
Joe:
Although the paper does not mention specifically why the beta1 subunit expression decreases, it does say that cell spreading appears to depend on activity of the beta1 subunit. Thus the increased expression at 3hrs could be due to an increase in cell spreading at that time, during the more initial interaction of the cells and substrates.
Michelle:
No specific tests were mentioned in the paper. However, I believe in performing the attachment assay and trypsinizing and counting the cells after various timepoints, the authors were able to tell whether or not the cells were proliferating. It would be useful if they included specific observations about this though.
Aishwarya:
The aim of the paper was not to prove that CaP coated implants stimulate more bone formation. Rather, it just studied the initial interactions of cells with the mentioned substrates. The studies done previously proving that CaP coats lead to more bone formation were long-term investigations (1-2 wks). Thus the increase in stimulation probably occurs after the 24h mark. Cell attachment was measured by trypsinizing and counting the cells. Then the percentage of attached cells was calculated as the number of attached cells divided by the number of seeded cells.
Johntus:
Seeing as how the smooth titanium disks promote the least cell attachment, I would think that smooth titanium would be the least ideal candidate for an implant. However more long-term studies, including in vivo studies, would have to be done. In terms of attachment studies, the effect of surface roughness has already been shown before on rat and human osteoblast-like cells. Because of this, I believe that similar results would arise from healthy bone cells.
Andrew:
I do not know what motif then allows the integrins to bind. The paper does mention that the diameter of the Ti disks were 25mm. The CaP coating was 2um thick.
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