Unlike previous methods used to prepare microscale porous TiO2
in the present study, F+
-based electrolyte anodization was used to obtain the TiO2
nanotubular surface on dental implants. However, in the transgingival part of a Ti dental implant, a smooth surface helps to maintain cleanliness. It has been shown that various types of cells have dissimilar reactions to different nanotubular surfaces of disparate tube sizes.14
Sterilization also affects the cytocompatibility of the TiO2
surface, and ultraviolet irradiation is one of the methods to improve the bioactivity of TiO2
without unexpected biological contamination.35
Application of FGF2 to periodontal tissue regeneration has been previously reported.36
FGF2 can be immobilized on to Ti surface24
and hydroxyapatite-chitosan scaffolds.38
The immobilization procedure used in this study as well as the chemical inertness and stability of TiO2
ensure that there is very little possibility for the FGF2 to react with the TiO2
NT array. Besides, using a TiO2
nanotubular surface for long-term drug elution is feasible due to its multitubular-like structure and ability to trap relatively small amounts of drug.25
FGF2 (17 kDa) is approximately 1.45 nm39
in hydrodynamic radius, and a biomolecule with such a small size is more likely to enter the inner space of the TiO2
NTs (). Intratube FGF2 storage may provide the possibility for an NT-based control release system. Although the total amount of immobilized FGF2 is difficult to quantify accurately, the elution kinetics of dissolvable immobilized FGF2 can be monitored with an enzyme-linked immunosorbent assay. The elution curves in this study are similar to those reported previously by Peng et al,25
indicating that the length of the NT mainly affects the controlled release of immobilized FGF2.
Pattern of nanotube-based FGF2 control release system: (A) initial stage, (B) in culture medium, FGF2 is gradually released from the nanotubes.
Compared with PT, NT and NT-F-H exhibit significantly suppressed cell adhesion probably because of the surface physicochemical properties and nanotopography. The TiO2
NT array has a multiringed shape with small initial cell- substrate contact areas. The hollow structure of the TiO2
NT array may also inhibit the formation of focal adhesion. Integrin clustering and focal adhesion reinforcement are not influenced by the nanoscale pits with a diameter of <70 nm irrespective of pit depth. However, increasing the size of the central lumen to >70 nm in these vertically aligned NTs significantly reduces cellular adhesion.15
In this study, the attachment of HGFs is better on NT-F-L and NT-F-M than on NT, probably due to FGF2 immobilization. This offers functional regions to accelerate cell attachment and increases the contact area between the cells and substrate. The main reason for the suppressed HGF attachment on NT-F-H may be the direct contact with the clumps of FGF2 on NT-F-H with over FGF2 immobilization. According to FE-SEM results and FGF2 elution kinetics, when the samples are immersed in the culture medium, the clumps of FGF2 dissolve rapidly, leaving the HGFs with a relatively unstable environment and making HGFs hard to attach.
Inverse concentration-dependent effect has been observed in the proliferation of HGFs on NT-F-H, which was much less than that on PT on Days 1 and 3, but gradually increased by Days 6 and 9. On the contrary, both NT-F-L and NT-F-M demonstrated excellent performance in terms of proliferation at all time points. Our results illustrate the enhanced behavior of fibroblasts in a medium with the appropriate concentration of eluted FGF2. Referring to the results in and , the activity and concentrations of eluted FGF2 from NT-F-L, -M, and -H are all suitable for cell proliferation within 3 days (10–100 ng/mL),22
whereas NT-F-H shows extremely low proliferation. An unstable microenvironment is probably the main reason for this suppressive effect. On the NT-F-H surface, FGF2 dissolves within the first day and impedes the attachment of HGFs, therefore delaying their proliferation when compared with those on NT-F-L and NT-F-M. In all organ systems, the normal mammalian response to injury occurs in three overlapping but distinctive stages: (1) inflammation, (2) new tissue formation, and (3) remodeling. Stage 1 lasts for about 2 days after injury, whereas Stage 2 occurs 2–10 days after injury and is characterized by cellular proliferation.41
Therefore, early proliferation of HGFs after implantation is crucial to healthy soft tissue sealing, and concentration-related impeded proliferation should be avoided.
In the morphogenesis of virtually all tissues, the ECM is essential and forms a structural framework possessing signal-transducing receptors that interact with cells. VEGFA enhances blood vessel growth and permeability and is a powerful mitogen for endothelial cells, fulfilling an important role in angiogenesis.42
Hence, an FGF2-immobilized TiO2
nanotubular surface may promote vascularization and further accelerate soft tissue regeneration around dental implants. In this work, the VEGFA
expressions of NT, NT-F-L, -M, and -H are all upregulated, and NT-F-H shows the highest expression on Days 3 and 6. Overexpression of VEGFA
may produce tissue inflammation or edema, thereby delaying tissue regeneration.
Integrins constitute the primary family of cell-surface receptors that mediate attachment to the ECM and substrates44
and play a key role in early signal transduction.45
In the integrin family, β-integrin is important in cellular binding on coated or textured Ti implants.46
The total ITGB
expression levels are concentration-dependent and increased by FGF2.47
In this study, NT-stimulated ITGB
is overexpressed at all time points, because the nanotubular surface hinders cellular attachment in the early stage but improves ITGB
expression due to reverse compensation. NT-F-L, -M, and -H significantly enhance ITGB
expression at all time intervals, and NT-F-M shows the highest gene expression among them on Days 3 and 6. However, this trend is not observed at Day 9. The probable reason may be that ITGB
is richly expressed during the early stages. After cell-material integration has occurred, the expression of ITGB
is less sensitive to the various concentrations of FGF2. Unlike VEGFA
expression, excessive FGF2 does not give rise to a reverse effect but cannot fully exert its bioactivity. The TiO2
nanotubular surface is probably the main factor regulating ITGB
expression after Day 9.
ICAM1, which is a widely expressed cell adhesion molecule48
and belongs to the immunoglobulin super family, plays an important physiological role in routing polymorphonuclear neutrophils to the gingival sulcus efficiently and initiating a host response toward the implant. The expression of endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM1 is significantly upregulated in inflamed tissues by FGF2.51
Our results reveal that the TiO2
nanotubular surface does not affect ICAM1
expression, and even the NT-F-L’s efficacy is nil. It is only when the concentration of FGF2 reaches a certain threshold that the effect is observed, thereby impairing (NT-F-H, , Day 3) or enhancing (NT-F-M , Days 3, 6, and 9; NT-F-H, , Days 6 and 9) ICAM1
expression. This effect may result in the increase of intercellular adhesion in the early stage as well as recruitment of polymorphonuclear neutrophils to produce anti-infective activity in the long term,50
thereby improving the microecological environment around the dental implant.
Laminins are CAMs found predominantly in basement membranes52
and may help to form a base for cell-substrate adhesion and improve the interface between cells (even epithelium) and dental implants. The mRNA levels of laminin in periodontal ligament cells are specifically upregulated by FGF2 stimulation.36
In this study, both the TiO2
nano-tubular surface and FGF2 exhibit stimulated the LAMA1
expression. The inverse concentration-dependent effect was significantly observed on NT-F-H on Days 3 and 6 but diminished on Day 9.
From the perspective of cell adhesion, proliferation, and ECM-related gene expression, our results reveal that the functions of FGF2 immobilized on TiO2 nanotubular surface are definitely bidirectional as well as concentration- and time-dependent. The FGF2 solution with a concentration of 500 ng/mL is optimal for repeated lyophilization in spite of some loss of bioactivity with time. The TiO2 nanotubular surface might be ideal because its microstructure facilitates FGF2 immobilization, storage, and controlled release within 9 days. However, overimmobilization of FGF2 could lead to unwanted impairment of tissue healing and must therefore be avoided. Although the NT-based drug control release system needs further research for optimization, this FGF2/TiO2 nanotubular surface (NT-F-M) modification may lead to excellent gingival tissue-implant integration in vivo and has promising applications in dentistry and other biomedical devices.