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. 2021 Mar 13;8(2):rbab006.
doi: 10.1093/rb/rbab006. eCollection 2021 Mar.

Characterization and evaluation of a femtosecond laser-induced osseointegration and an anti-inflammatory structure generated on a titanium alloy

Yang Liu  1   2 , Zhongying Rui  3 , Wei Cheng  1   2 , Licheng Song  1   2 , Yunqiang Xu  1 , Ruixin Li  4 , Xizheng Zhang  2
Affiliations

Characterization and evaluation of a femtosecond laser-induced osseointegration and an anti-inflammatory structure generated on a titanium alloy

Yang Liu et al. Regen Biomater. .

Abstract

Cell-material interactions during early osseointegration of the bone-implant interface are critical and involve crosstalk between osteoblasts and osteoclasts. The surface properties of titanium implants also play a critical role in cell-material interactions. In this study, femtosecond laser treatment and sandblasting were used to alter the surface morphology, roughness and wettability of a titanium alloy. Osteoblasts and osteoclasts were then cultured on the resulting titanium alloy disks. Four disk groups were tested: a polished titanium alloy (pTi) control; a hydrophilic micro-dislocation titanium alloy (sandblasted Ti (STi)); a hydrophobic nano-mastoid Ti alloy (femtosecond laser-treated Ti (FTi)); and a hydrophilic hierarchical hybrid micro-/nanostructured Ti alloy [femtosecond laser-treated and sandblasted Ti (FSTi)]. The titanium surface treated by the femtosecond laser and sandblasting showed higher biomineralization activity and lower cytotoxicity in simulated body fluid and lactate dehydrogenase assays. Compared to the control surface, the multifunctional titanium surface induced a better cellular response in terms of proliferation, differentiation, mineralization and collagen secretion. Further investigation of macrophage polarization revealed that increased anti-inflammatory factor secretion and decreased proinflammatory factor secretion occurred in the early response of macrophages. Based on the above results, the synergistic effect of the surface properties produced an excellent cellular response at the bone-implant interface, which was mainly reflected by the promotion of early ossteointegration and macrophage polarization.

Keywords: cell–material interactions; femtosecond laser; hybrid micro-/nanostructure; macrophage polarization; osseointegration.

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Figures

Figure 1.
Figure 1.
Representative SEM images of the different polished, sandblasted, femtosecond laser-treated and sandblasted + femtosecond laser-treated titanium disk surfaces used in the preliminary experiments: (A) polished titanium (pTi) at different magnifications; (B) sandblasted titanium (STi) at different magnifications; (C) femtosecond laser-treated titanium (FTi) at different magnifications; (D) femtosecond laser-treated and sandblasted titanium (FSTi) at different magnifications. The second and third columns in each group are low-magnification images of the samples.
Figure 2.
Figure 2.
Characterization of the titanium surfaces. (A) Roughness and wettability of the titanium alloy substrates. (B) Contact angle of samples in the pTi, STi, FTi and FSTi groups. pTi: polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium. Significance: *P < 0.05 (STi, FTi, FSTi vs pTi.); #P < 0.05 (pTi, FTi, FSTi vs STi); &P < 0.05 (pTi, STi, FSTi vs FTi).
Figure 3.
Figure 3.
(A) Crystal structure of the titanium alloy surface detected by XRD. (B) XRD patterns of hydroxyapatite (HAp) deposition on the surface of samples in the different titanium alloy groups. Black dots represent characteristic peaks of titanium alloys, hollow dots represent HAp. The data were moved up equidistantly to reflect more information of the characteristic peaks and peak intensities brought about by the different processing methods. pTi: Polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium.
Figure 4.
Figure 4.
HAp Deposition on the surface of titanium alloys immersed in SBF imaged by SEM. (A) Polished titanium (pTi) at different magnifications; (B) sandblasted titanium (STi) at different magnifications; (C) femtosecond laser-treated titanium (FTi) at different magnifications; (D) femtosecond laser-treated and sandblasted titanium (FSTi) at different magnifications. The second and third columns in each group are low-magnification images of the samples.
Figure 5.
Figure 5.
Protein adsorption to different specimens incubated in BSA. pTi: Polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium significance: *P < 0.05 (STi, FTi, FSTi vs pTi.); #P < 0.05 (pTi, FTi, FSTi vs STi).
Figure 6.
Figure 6.
LDH Activity of osteoblasts on different titanium surfaces. pTi: Polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium. Significance: *P < 0.05 (compared with pTi).
Figure 7.
Figure 7.
CCK-8 Assay to detect the osteoblast adhesion and proliferation on different titanium alloy surfaces. pTi: Polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium. Significance: *P < 0.05 (STi, FTi, FSTi vs pTi.); #P < 0.05 (FSTi vs STi) ; &  P < 0.05 (FSTi vs FTi).
Figure 8.
Figure 8.
ALP Activity of osteoblasts on different titanium alloy surfaces. pTi: Polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium. Significance: * P < 0.05 (STi, FTi, FSTi vs pTi).
Figure 9.
Figure 9.
Spreading of osteoblasts on the surface of titanium alloys. (A) Polished titanium (pTi); (B) sandblasted titanium (STi); (C) femtosecond laser-treated titanium (FTi); (D) femtosecond laser-treated and sandblasted titanium (FSTi).
Figure 10.
Figure 10.
ECM Mineralization of samples stained after 21 days of culture with osteoblasts. (A) Qualitative analysis of osteoblasts cultured for 21 days (alizarin red stain, black arrows point to calcium nodules). (B) Quantitative analysis of ECM mineralization after 21 days of osteoblast culture. (a): polished titanium (pTi); (b): sandblasted titanium (STi); (c): femtosecond laser-treated titanium (FTi); (d): femtosecond laser-treated and sandblasted titanium (FSTi). Significance: *P < 0.05 (STi, FTi, FSTi vs pTi).
Figure 11.
Figure 11.
Collagen secretion after 7 and 14 days of osteoblast culture and staining by sirius red. (A) (a): Polished titanium (pTi); (b): sandblasted titanium (STi); (c): femtosecond laser-treated titanium (FTi); (d): femtosecond laser-treated and sandblasted titanium (FSTi). Qualitative analysis of osteoblasts cultured for 7 and 14 days; the first row shows osteoblasts stained after 7 days and the second row shows osteoblasts stained after 14 days. (B) Quantitative analysis of collagen after 7 and 14 days of osteoblast culture. Significance: *P < 0.05 (STi, FTi, FSTi vs pTi.); #P < 0.05 (FSTi vs STi) ; &P < 0.05 (FSTi vs FTi).
Figure 12.
Figure 12.
Inflammatory factor secretion of osteoclasts cultured on the sample surface for 1 and 3 days. pTi: polished titanium; STi: sandblasted titanium; FTi: femtosecond laser-treated titanium; FSTi: femtosecond laser-treated and sandblasted titanium. The left panel shows the release of inflammatory factors after 1 day of culture, and the right panel shows the release after 3 days. The parallel images indicate the same detection index. Significance: *P < 0.05 (STi, FTi, FSTi, vs pTi); #P < 0.05 (pTi, FTi, FSTi, vs STi); &P < 0.05 (pTi, STi, FSTi, vs FTi).
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