In vitro evaluation of osteoblast responses to carbon nanotube-coated titanium surfaces
Biological response of osteoblasts to an implant substrate is influenced by both surface roughness and surface chemistry [31]. Relatively rougher surfaces have been reported to be more advantageous for osteoblast proliferation, differentiation, and formation of bone matrix since rough surface substrates have more surface area than smoother surface substrates and the osteoblasts adhere to rougher surfaces more than smoother surfaces [32]. Numerous studies have found MWCNTs to be cyto-compatible [11–13, 15–19]. These studies were conducted on neuronal cells [11, 13], osteoblasts [12], and fibroblasts [15] from human and rat sources. When the MWCNTs were functionalized, the presence of COOH groups enhances cell proliferation, viability, and adhesion due to the -COOH groups rendering the surface hydrophilic and wettable [14]. Carboxyl functionalized CNTs (CNT–COOH) have been used as an effective template to chemically synthesize HAp [33]. This is due to the capability of carboxylate ions (COO–) to adsorb calcium ions (Ca2+) and contribute to HAp crystallization as a result of exposure to phosphate ions (PO43). Initiation of HAp nucleation was shown to take place within carboxyl group [34]. Thus, the presence of carboxyl groups on MWCNTs facilitated HAp formation and mineralization.
In this study, MWCNT and MWCNT-COOH were selected to evaluate their effect on MC3T3-E1 osteoblast-like cell line. It was expected that the surface roughness of the MWCNT-coated discs would influence cell proliferation, differentiation, and matrix mineralization; and there would be a difference between these two groups due to the presence or absence of -COOH functional groups. MC3T3 is an osteoblast precursor cell line derived from mouse calvaria [35]. The MC3T3-E1 sub-line is a physiologically relevant cell line for study of transcriptional control in calvarial osteoblasts, which is an appropriate and relevant osteoblast model for in vitro studies and similar to human osteoblasts in terms of proliferation, differentiation, and matrix mineralization [36]. Hence, this cell line was chosen for this in vitro study.
The MC3T3-E1 cells follow a two-stage developmental process including a 1–2-week initiation phase during which cells slowly proliferate, express ALP activity and other bone specific genes, resulting in the formation of collagen matrix [37]. During the second maturation phase of MC3T3-E1 cells occurring from weeks 2–4, matrix mineralization is observed [38, 39]. Matrix mineralization is considered a functional in vitro endpoint reflecting advanced cell differentiation. Interpreting from the MTT, ALP and ARS assays, it can be understood that the presence of MWCNTs in both CNT groups influenced the rate of cell proliferation from day 3 to 7. In a recently published study, which evaluated the effect of MWCNTs coatings deposited on titanium discs on osteoblast growth, it was shown that there was a strong dependence of the extent of osteoblast proliferation and differentiation on the presence of MWCNTs in the coatings [20]. It can be inferred from this study [20] that higher surface roughness due to MWCNTs was responsible for the relatively higher extent of MC3T3-E1 cell proliferation and differentiation.
CRL-11372 osteoblasts synthesized more alkaline phosphatase and mineralized matrix on the surfaces of MWCNTs grown from anodized nanotubular Ti than on anodized nanotubular Ti without MWCNTs [17]. MWCNTs have also been used as additives to enhance the structural properties of biocompatible scaffolds. When MWCNTs were mixed with poly(lactic-co-glycolic acid) (PLGA) microsphere scaffolds, it was shown that the addition of MWCNT made the PLGA scaffold mechanically stronger and elicited enhanced cellular responses from MC3T3-E1 cells in terms of cell proliferation, differentiation and mineralized matrix formation [40].
The cross-section of the coatings showed that there was no topographical difference as both the MWCNTs were similar in dimension and exhibited same surface roughness. The results from our study confirm that the presence of MWCNTs influenced proliferation from day 3 to 7 as suggested by these studies [17, 20, 40]. However, it was lower than the tissue culture plastic group. Tissue culture plastic which is surface treated elicited higher cell proliferation and the presence of MWCNTs as foreign bodies could have influenced the amount of cell proliferation initially. When cell differentiation and matrix mineralization were evaluated after days 7 and 28, the MWCNT-COOH group showed increased cell differentiation and matrix mineralization, when compared to the MWCNT group. The reason for this could be that the presence of -COOH groups influenced differentiation and mineralization. As discussed earlier, COOH group promotes matrix mineralization. The presence of -COOH groups likely increased differentiation on day 7 [14] and matrix mineralization after day 28 due to increased hydrophilicity, surface wettability of MWCNTs and facilitating mineral matrix deposition [33, 34].
The results of our study concur with previously published literature [14, 17, 20, 33, 34, 40]. MWCNT and MWCNT-COOH coatings can be utilized for future in vivo studies to evaluate the effect of these coatings on titanium MSI osseointegration. One of the limitations of our study is the use of collagen to coat CNTs onto Ti discs and no collagen coating on the uncoated Ti group. However, in a recent study to evaluate the effect on MC3T3-E1 growth from collagen MWCNT composite coating deposited on Ti, the control groups included Ti discs coated with only collagen and MWCNTs as controls [20]. Although this study did not use MWCNT-COOH coatings, the results suggested a strong dependence of the extent of cell proliferation, differentiation, and mineralization on the amount of MWCNTs incorporated in the composite compared to the control groups. Another limitation was the limited number of samples (n=3/group) in ALP assay and the data was analyzed taking this limitation into consideration. In an in vivo setting, it is unknown whether a similar result can be seen from the coatings as an animal model is a living-three dimensional system. Host factors (local and systemic) could influence the outcome (adhesion, proliferation, differentiation, and bone formation). However, these coatings on implants should to be evaluated for bone to MSI contact, inflammatory response, and bone remodeling and for reverse torque to unscrew the MSIs; the CNT coating which can influence sufficient initial osseointegration. It is unknown if they will hamper the removal of MSIs or what effect they would have on the interfacial bond strength.