# Nanoscale Modification of Titanium Implants Improves Behaviors of Bone Mesenchymal Stem Cells and OsteogenesisIn Vivo

Authors: Huangdi Li; Jinghui Huang; Yanpeng Wang; Ziyuan Chen; Xing Li; Qiuping Wei; Xifeng Liu; Zi Wang; Bin Wen; Yuetao Zhao; Jing Liu; Jun Zuo
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

The surficial micro/nanotopography and physiochemical properties of titanium implants are essential for osteogenesis. However, these surface characters’ influence on stem cell behaviors and osteogenesis is still not fully understood. In this study, titanium implants with different surface roughness, nanostructure, and wettability were fabricated by further nanoscale modification of sandblasted and acid-etched titanium (SLA: sandblasted and acid-etched) by H2O2 treatment (hSLAs: H2O2 treated SLA). The rat bone mesenchymal stem cells (rBMSCs: rat bone mesenchymal stem cells) are cultured on SLA and hSLA surfaces, and the cell behaviors of attachment, spreading, proliferation, and osteogenic differentiation are further analyzed. Measurements of surface characteristics show hSLA surface is equipped with nanoscale pores on microcavities and appeared to be hydrophilic. In vitro cell studies demonstrated that the hSLA titanium significantly enhances cell response to attachment, spreading, and proliferation. The hSLAs with proper degree of H2O2 etching (h1SLA: treating SLA with H2O2 for 1 hour) harvest the best improvement of differentiation of rBMSCs. Finally, the osteogenesis in beagle dogs was tested, and the h1SLA implants perform much better bone formation than SLA implants. These results indicate that the nanoscale modification of SLA titanium surface endowing nanostructures, roughness, and wettability could significantly improve the behaviors of bone mesenchymal stem cells and osteogenesis on the scaffold surface. These nanoscale modified SLA titanium scaffolds, fabricated in our study with enhanced cell affinity and osteogenesis, had great potential for implant dentistry.


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## Body

## 1. Introduction

Current tooth restoration has evolved success since the interfacial bone formation at machined endosseous implants [1]. Titanium implants, in particular, have been identified as ideal biomaterials in clinic due to their favorable physiochemical characteristics, biocompatibility, and osseointegration ability [2, 3]. Osseointegration, the direct contact between the vital bone and the implant at the microscopic level, plays an essential role in improving implants’ efficiency and reducing risk, especially in selected patient populations (e.g., smokers and diabetics) [4–7]. Complex mechanisms (e.g., local anatomic, local biologic, systemic, or functional factors) have evolved to achieve osseointegration at high rate. A major interest is increasing bone forming at the implant surface by controlling the implant surface characters [8–11].The surface micro/nanotopography and physiochemical properties of titanium implants are crucial characters for osseointegration and have recently been highlighted [12–15]. The topographical micrometric features can increase osseointegration by direct bone-to-implant contact and resistance to applied loads on titanium surfaces [16]. Further nanoscale modification of the surface affecting both topography and surface chemistry has been shown to influence adhesion, motility, and proliferation of specific cells (e.g., osteoblast) by direct (cell-surface interactions) and indirect (protein-surface interactions) mechanisms [17]. The surface energy or wettability is also reported to increase the expression of key osteogenesis genes in osteoblast and displays superior degree of early osseointegration [18–21]. Evoked by those theories, multiple surface treatment techniques, e.g., sandblasted and acid-etched (SLA), alkali and heat treatment, anodic oxidation, double-etching, and magnetron sputtering, have been developed to generate micro/nanosurface structure and better bone formation in vitro and in vivo [22–25].On the other hand, bone mesenchymal stem cells (BMSCs), the first cells to colonize the surface of the implant after its insertion, have the ability to improve bone regeneration by differentiation into parenchymal cells and the production of growth factors [26]. Controlling mesenchymal stem cells (MSCs) differentiation towards the osteogenic lineage may assist more rapid osseointegration with beneficial effects [27]. By providing topographical cues and mechanical stimuli, the micro- and nanoenvironments of stem cell differentiation can be engineered in vitro [28, 29]. Nevertheless, the effect of the titanium surface micro/nanofeatures on stem cell behaviors and osteogenesis in vivo still needs to be systematically studied.Herein, we illustrate that titanium’s topography and physiochemical property influence MSCs responses and promote osseointegration effect of the implants. Specifically, traditional titanium surface is nanoscale modified by etching the SLA titanium in hydrogen peroxide for different times to generate various micro/nanostructured topography and wettability. Subsequently, changes in surface characteristics are systematically explored, and the initial cell attachment, spreading, proliferation, and differentiation behaviors of rat bone mesenchymal stem cells (rBMSCs) are measured to evaluate the osseointegration differencein vitro. Finally, the osteogenesis effect is tested in vivo, confirming that the titanium microsurface with suitable nanostructure and wettability has the best ability to promote bone formation.


## 2. Materials and Methods

### 2.1. Sample Preparation

The samples were prepared as previously described [30]. Commercial pure TA4 titanium plate (Hunan Xiangtou Jintian Titanium Metal Co. Ltd.) was cut into disks (Φ20 mm or Φ32 mm, 2 mm thick). Then, disks were polished to Ra<0.5μm and sandblasted by Al2O3 (Φ0.25~0.5 mm) at a pressure of 0.5 mPa. After ultrasonic cleaning in acetone, absolute ethanol, and ultrapure water for 10 minutes, the etching was carried out in the boiling mixture of H2SO4 (Zhuzhou, Hunan) and HCl (Zhuzhou, Hunan) for several minutes. Finally, the SLA (sandblasted and acid-etched) group was obtained by washing with ultrapure water and drying with hot air. The SLA titanium plates were immersed in concentrated H2O2 for 1 hour and 2 hours, respectively, to form hSLA (H2O2 treated SLA) groups, named h1SLA (treating SLA with H2O2 for 1 hour) and h2SLA (treating SLA with H2O2 for 2 hours), respectively. Then, the materials were cleaned and dried by ultrasound. The preparation process is shown in supplementary material (Figure S1).


### 2.2. Surface Characterization

#### 2.2.1. Surface Morphology

Surface morphologies were observed by scanning electron microscopy (SEM, Nova NanoSEM 230, USA). At least three points of each sample were detected. The observation and photos were taken at five magnification ratios of 1000x, 2000x, 5000x, 50000x, and 100000x, respectively.


#### 2.2.2. Surface Roughness

Surface roughness was measured by surface profiler (wyko NT9100, USA) with each testing line of 1 mm. Five test points were randomly selected for each sample. Then, the average values of Ra (arithmetic mean deviation of contour: arithmetic mean of absolute value of contour offset within sampling length) and Rz (maximum height of profile: the sum of the average of the five largest contour peak heights and the average of the five largest contour valley depths within the sampling length) on each titanium plate were calculated by software.


#### 2.2.3. Wettability

The water contact angles (WCA) were measured by contact angle measuring instrument (SDC-100, China) to evaluate the hydrophilicity of the sample. Three sites were randomly selected on each fresh sample surface to calculate the mean and standard deviation of water contact angle on each sample surface.


#### 2.2.4. Surface Chemistry

Surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, USA) with Al Kα irradiation and X-ray energy dispersive spectroscopy (EDS, Nova NanoSEM 230, USA). In XPS detection, three sites were randomly selected for each sample, and the obtained detection data were further used for spectral peak calibration, peak separation fitting, and element calibration. The spectral peak corresponding to the C1s spectral peak was 285.0 eV for calibration. The valence states of O element and Ti element were analyzed.


### 2.3. Isolation and Culturing of Rat BMSCs

Three-week-old male Sprague-Dawley rats were used for the isolation and culture of bone mesenchymal stem cells (BMSCs) as formerly described [31]. Briefly, the femur of rats was taken, and the bone marrow cavity was continuously rinsed with a mixture of high-glucose DMEM complete medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 IU/mL penicillin-streptomycin solution (Gibco, USA) until it turned white. The morphology of cells was observed by inverted microscope (Figure S3) [32], and the cells were used for subsequent experiments when the state and quantity of cells were appropriate. Cells of passage 2-5 were seeded on titanium samples in either growth medium or osteogenic medium, which was growth medium with the addition of 50 μM ascorbic acid (Sigma, USA), 10 mM β-glycerophosphate (Sigma, USA), and 100 nM dexamethasone (Sigma, USA).


### 2.4. Cell Behaviors on the Surface

#### 2.4.1. Cell Attachment

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. Rat bone mesenchymal stem cells (rBMSCs) were inoculated on the titanium plates with a density of4×104/well. After incubating for 4 hours, cells were fixed in 4% paraformaldehyde (Sigma, USA) followed by staining with acridine orange (A.O., Sigma, USA). After observing under fluorescence microscope (Nikon Eclipse TE300, Japan) and capturing the images, the number of cells was counted from 5 random fields for each plate and three plates for each group.


#### 2.4.2. Cell Spreading

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. Cells were fixed in 4% paraformaldehyde (Sigma, USA) to explore cell spreading after incubating for 12 hours. Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan). At a magnificence of 20x, images of 5 random fields for each plate were captured to calculate their spreading areas by Image J.


#### 2.4.3. Cell Proliferation

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. After incubating for 24 h, 48 h, and 72 h, respectively, cell proliferation was measured by cell staining and CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the method previously described [33]. All these experiments were conducted in triplicates.In addition, the expression of the marker of proliferation Ki-67 (Mki67) marker at each time point was observed by immunofluorescence assay. Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 30 min. Subsequently, they were incubated with the primary antibody of Mki67 (Abclonal, China) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 15 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


### 2.5.In Vitro Osteogenesis Ability

#### 2.5.1. Cell Attachment and Spreading

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d, cells were fixed in 4% paraformaldehyde (Sigma, USA). Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan).


#### 2.5.2. Bone-Related Gene Expression of rBMSCs

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d and 7 d in an osteogenic medium, RNA was extracted and reverse transcribed according to manufacturer’s instructions. The mRNA levels of bone-related genes alkaline phosphatase (ALP, Sangon Biotech, China), RUNX family transcription factor 2 (RUNX2, Sangon Biotech, China), collagen type I alpha 1 chain (COL1A1, Sangon Biotech, China), bone morphogenetic protein 2 (BMP2, Sangon Biotech, China), and integrin-binding sialoprotein (BSP, Sangon Biotech, China) in rBMSCs were evaluated by RT-PCR (Eppendorf AG 22331 Hamburg, Germany). The mRNA levels were normalized for glyceraldehyde-3-phosphate dehydrogenase mRNA (Gapdh, Sangon Biotech, China). The primers used are shown in supplemental instrument (Table S1).


#### 2.5.3. Alkaline Phosphatase (ALP) Activity Assay

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 7 d and 14 d in osteogenic medium, the ALP activity of the adherent cells was detected using an Alkaline Phosphatase assay kit (Beyotime, China). The absorbance was measured at 405 nm by Microplate Reader (BioTek, USA), and values of ALP activity were read off according to a standard curve based on standard samples provided by the kit itself. In addition, three pieces of material were taken from each group for ALP staining experiment at 14 d. After cleaning with DPBS for 3 times, cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min, incubated with BCIP/NBT ALP Color Development Kit (Beyotime, China) for 10~30 minutes, and photographed.


#### 2.5.4. Immunofluorescence Staining

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, immunofluorescence staining was carried out to detect the expressions of osteocalcin (OCN). Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 1 h. Subsequently, they were incubated with the primary antibody of OCN (Santa, USA) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, they were stained with rhodamine-phalloidin (Cytoskeleton, USA) for 1 h, and cell nuclei were stained with DAPI (Beyotime, China) for 10 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


#### 2.5.5. Alizarin Red Staining for Mineralization

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, cells on samples were fixed and stained with the Alizarin red S (ARS) reagent (Cyagen, China) for 15 min at room temperature. After the samples were washed with DPBS three times, images were taken. Then, the mineralized nodules were eluted with 10% hexadecylpyridinium chloride monohydrate (Sigma, USA), and the O.D. value at 570 nm was measured for quantitative detection.


### 2.6.In Vivo Osteogenesis Study

One-year-old male Beagles were purchased from Fuzhou Zhenhe Experimental Animal Technology Development Co., Ltd., weighing 10-13 kg, and all thein vivo experiments followed the protocols approved by the Animal Care and Use Committee of the Xiangya School of Stomatology, Central South University. To establish the osteogenesis model, SLA implants and h1SLA implants were separately implanted on the normal alveolar bone between the two adjacent teeth after removing the crowns from bilateral mandibular premolars of beagles (Figure S8). Six weeks later, the beagles were killed, and the rotation torque value of each implant was measured.To determine the new bone formation on the implants, fixed sample was measured by the Cone Beam Computer Tomography (CBCT, ProMax 3D, Helsinki), and the images were generated from Planmeca Romexis. After performing the CBCT, hard tissue sections were made and stained with methylene blue magenta to observe the osteogenesis.


### 2.7. Statistical Analysis

Data from cellular experiments on the substrates are presented as the mean error and standard deviation of the mean error of all the measurements. Data were evaluated by analysis of significant differences among different groups, which were determined using one-way ANOVA andt-test by “GraphPad Prism 8.0.” Asterisk (∗) represents significant differences between different substrates. ∗ indicates 0.01≤p<0.05, ∗∗ indicates 0.001≤p<0.01, ∗∗∗ indicates 0.0001≤p<0.001, and ∗∗∗∗ indicates 0.00001≤p<0.0001.


## 2.1. Sample Preparation

The samples were prepared as previously described [30]. Commercial pure TA4 titanium plate (Hunan Xiangtou Jintian Titanium Metal Co. Ltd.) was cut into disks (Φ20 mm or Φ32 mm, 2 mm thick). Then, disks were polished to Ra<0.5μm and sandblasted by Al2O3 (Φ0.25~0.5 mm) at a pressure of 0.5 mPa. After ultrasonic cleaning in acetone, absolute ethanol, and ultrapure water for 10 minutes, the etching was carried out in the boiling mixture of H2SO4 (Zhuzhou, Hunan) and HCl (Zhuzhou, Hunan) for several minutes. Finally, the SLA (sandblasted and acid-etched) group was obtained by washing with ultrapure water and drying with hot air. The SLA titanium plates were immersed in concentrated H2O2 for 1 hour and 2 hours, respectively, to form hSLA (H2O2 treated SLA) groups, named h1SLA (treating SLA with H2O2 for 1 hour) and h2SLA (treating SLA with H2O2 for 2 hours), respectively. Then, the materials were cleaned and dried by ultrasound. The preparation process is shown in supplementary material (Figure S1).


## 2.2. Surface Characterization

### 2.2.1. Surface Morphology

Surface morphologies were observed by scanning electron microscopy (SEM, Nova NanoSEM 230, USA). At least three points of each sample were detected. The observation and photos were taken at five magnification ratios of 1000x, 2000x, 5000x, 50000x, and 100000x, respectively.


### 2.2.2. Surface Roughness

Surface roughness was measured by surface profiler (wyko NT9100, USA) with each testing line of 1 mm. Five test points were randomly selected for each sample. Then, the average values of Ra (arithmetic mean deviation of contour: arithmetic mean of absolute value of contour offset within sampling length) and Rz (maximum height of profile: the sum of the average of the five largest contour peak heights and the average of the five largest contour valley depths within the sampling length) on each titanium plate were calculated by software.


### 2.2.3. Wettability

The water contact angles (WCA) were measured by contact angle measuring instrument (SDC-100, China) to evaluate the hydrophilicity of the sample. Three sites were randomly selected on each fresh sample surface to calculate the mean and standard deviation of water contact angle on each sample surface.


### 2.2.4. Surface Chemistry

Surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, USA) with Al Kα irradiation and X-ray energy dispersive spectroscopy (EDS, Nova NanoSEM 230, USA). In XPS detection, three sites were randomly selected for each sample, and the obtained detection data were further used for spectral peak calibration, peak separation fitting, and element calibration. The spectral peak corresponding to the C1s spectral peak was 285.0 eV for calibration. The valence states of O element and Ti element were analyzed.


## 2.2.1. Surface Morphology

Surface morphologies were observed by scanning electron microscopy (SEM, Nova NanoSEM 230, USA). At least three points of each sample were detected. The observation and photos were taken at five magnification ratios of 1000x, 2000x, 5000x, 50000x, and 100000x, respectively.


## 2.2.2. Surface Roughness

Surface roughness was measured by surface profiler (wyko NT9100, USA) with each testing line of 1 mm. Five test points were randomly selected for each sample. Then, the average values of Ra (arithmetic mean deviation of contour: arithmetic mean of absolute value of contour offset within sampling length) and Rz (maximum height of profile: the sum of the average of the five largest contour peak heights and the average of the five largest contour valley depths within the sampling length) on each titanium plate were calculated by software.


## 2.2.3. Wettability

The water contact angles (WCA) were measured by contact angle measuring instrument (SDC-100, China) to evaluate the hydrophilicity of the sample. Three sites were randomly selected on each fresh sample surface to calculate the mean and standard deviation of water contact angle on each sample surface.


## 2.2.4. Surface Chemistry

Surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, USA) with Al Kα irradiation and X-ray energy dispersive spectroscopy (EDS, Nova NanoSEM 230, USA). In XPS detection, three sites were randomly selected for each sample, and the obtained detection data were further used for spectral peak calibration, peak separation fitting, and element calibration. The spectral peak corresponding to the C1s spectral peak was 285.0 eV for calibration. The valence states of O element and Ti element were analyzed.


## 2.3. Isolation and Culturing of Rat BMSCs

Three-week-old male Sprague-Dawley rats were used for the isolation and culture of bone mesenchymal stem cells (BMSCs) as formerly described [31]. Briefly, the femur of rats was taken, and the bone marrow cavity was continuously rinsed with a mixture of high-glucose DMEM complete medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 IU/mL penicillin-streptomycin solution (Gibco, USA) until it turned white. The morphology of cells was observed by inverted microscope (Figure S3) [32], and the cells were used for subsequent experiments when the state and quantity of cells were appropriate. Cells of passage 2-5 were seeded on titanium samples in either growth medium or osteogenic medium, which was growth medium with the addition of 50 μM ascorbic acid (Sigma, USA), 10 mM β-glycerophosphate (Sigma, USA), and 100 nM dexamethasone (Sigma, USA).


## 2.4. Cell Behaviors on the Surface

### 2.4.1. Cell Attachment

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. Rat bone mesenchymal stem cells (rBMSCs) were inoculated on the titanium plates with a density of4×104/well. After incubating for 4 hours, cells were fixed in 4% paraformaldehyde (Sigma, USA) followed by staining with acridine orange (A.O., Sigma, USA). After observing under fluorescence microscope (Nikon Eclipse TE300, Japan) and capturing the images, the number of cells was counted from 5 random fields for each plate and three plates for each group.


### 2.4.2. Cell Spreading

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. Cells were fixed in 4% paraformaldehyde (Sigma, USA) to explore cell spreading after incubating for 12 hours. Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan). At a magnificence of 20x, images of 5 random fields for each plate were captured to calculate their spreading areas by Image J.


### 2.4.3. Cell Proliferation

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. After incubating for 24 h, 48 h, and 72 h, respectively, cell proliferation was measured by cell staining and CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the method previously described [33]. All these experiments were conducted in triplicates.In addition, the expression of the marker of proliferation Ki-67 (Mki67) marker at each time point was observed by immunofluorescence assay. Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 30 min. Subsequently, they were incubated with the primary antibody of Mki67 (Abclonal, China) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 15 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


## 2.4.1. Cell Attachment

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. Rat bone mesenchymal stem cells (rBMSCs) were inoculated on the titanium plates with a density of4×104/well. After incubating for 4 hours, cells were fixed in 4% paraformaldehyde (Sigma, USA) followed by staining with acridine orange (A.O., Sigma, USA). After observing under fluorescence microscope (Nikon Eclipse TE300, Japan) and capturing the images, the number of cells was counted from 5 random fields for each plate and three plates for each group.


## 2.4.2. Cell Spreading

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. Cells were fixed in 4% paraformaldehyde (Sigma, USA) to explore cell spreading after incubating for 12 hours. Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan). At a magnificence of 20x, images of 5 random fields for each plate were captured to calculate their spreading areas by Image J.


## 2.4.3. Cell Proliferation

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of2×104/well. After incubating for 24 h, 48 h, and 72 h, respectively, cell proliferation was measured by cell staining and CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the method previously described [33]. All these experiments were conducted in triplicates.In addition, the expression of the marker of proliferation Ki-67 (Mki67) marker at each time point was observed by immunofluorescence assay. Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 30 min. Subsequently, they were incubated with the primary antibody of Mki67 (Abclonal, China) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 15 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


## 2.5.In Vitro Osteogenesis Ability

### 2.5.1. Cell Attachment and Spreading

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d, cells were fixed in 4% paraformaldehyde (Sigma, USA). Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan).


### 2.5.2. Bone-Related Gene Expression of rBMSCs

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d and 7 d in an osteogenic medium, RNA was extracted and reverse transcribed according to manufacturer’s instructions. The mRNA levels of bone-related genes alkaline phosphatase (ALP, Sangon Biotech, China), RUNX family transcription factor 2 (RUNX2, Sangon Biotech, China), collagen type I alpha 1 chain (COL1A1, Sangon Biotech, China), bone morphogenetic protein 2 (BMP2, Sangon Biotech, China), and integrin-binding sialoprotein (BSP, Sangon Biotech, China) in rBMSCs were evaluated by RT-PCR (Eppendorf AG 22331 Hamburg, Germany). The mRNA levels were normalized for glyceraldehyde-3-phosphate dehydrogenase mRNA (Gapdh, Sangon Biotech, China). The primers used are shown in supplemental instrument (Table S1).


### 2.5.3. Alkaline Phosphatase (ALP) Activity Assay

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 7 d and 14 d in osteogenic medium, the ALP activity of the adherent cells was detected using an Alkaline Phosphatase assay kit (Beyotime, China). The absorbance was measured at 405 nm by Microplate Reader (BioTek, USA), and values of ALP activity were read off according to a standard curve based on standard samples provided by the kit itself. In addition, three pieces of material were taken from each group for ALP staining experiment at 14 d. After cleaning with DPBS for 3 times, cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min, incubated with BCIP/NBT ALP Color Development Kit (Beyotime, China) for 10~30 minutes, and photographed.


### 2.5.4. Immunofluorescence Staining

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, immunofluorescence staining was carried out to detect the expressions of osteocalcin (OCN). Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 1 h. Subsequently, they were incubated with the primary antibody of OCN (Santa, USA) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, they were stained with rhodamine-phalloidin (Cytoskeleton, USA) for 1 h, and cell nuclei were stained with DAPI (Beyotime, China) for 10 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


### 2.5.5. Alizarin Red Staining for Mineralization

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, cells on samples were fixed and stained with the Alizarin red S (ARS) reagent (Cyagen, China) for 15 min at room temperature. After the samples were washed with DPBS three times, images were taken. Then, the mineralized nodules were eluted with 10% hexadecylpyridinium chloride monohydrate (Sigma, USA), and the O.D. value at 570 nm was measured for quantitative detection.


## 2.5.1. Cell Attachment and Spreading

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d, cells were fixed in 4% paraformaldehyde (Sigma, USA). Staining with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and rhodamine-phalloidin (Cytoskeleton, USA), cells were observed under fluorescence microscope ((Nikon eclipse TE300, Japan).


## 2.5.2. Bone-Related Gene Expression of rBMSCs

Put three pieces for each group Ф 20 mm titanium plate and four pieces Ф32 mm titanium plate into a large dish together. The rBMSCs were inoculated on the titanium plates, and the inoculation density was about6×105 per plate. After incubating for 4 d and 7 d in an osteogenic medium, RNA was extracted and reverse transcribed according to manufacturer’s instructions. The mRNA levels of bone-related genes alkaline phosphatase (ALP, Sangon Biotech, China), RUNX family transcription factor 2 (RUNX2, Sangon Biotech, China), collagen type I alpha 1 chain (COL1A1, Sangon Biotech, China), bone morphogenetic protein 2 (BMP2, Sangon Biotech, China), and integrin-binding sialoprotein (BSP, Sangon Biotech, China) in rBMSCs were evaluated by RT-PCR (Eppendorf AG 22331 Hamburg, Germany). The mRNA levels were normalized for glyceraldehyde-3-phosphate dehydrogenase mRNA (Gapdh, Sangon Biotech, China). The primers used are shown in supplemental instrument (Table S1).


## 2.5.3. Alkaline Phosphatase (ALP) Activity Assay

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 7 d and 14 d in osteogenic medium, the ALP activity of the adherent cells was detected using an Alkaline Phosphatase assay kit (Beyotime, China). The absorbance was measured at 405 nm by Microplate Reader (BioTek, USA), and values of ALP activity were read off according to a standard curve based on standard samples provided by the kit itself. In addition, three pieces of material were taken from each group for ALP staining experiment at 14 d. After cleaning with DPBS for 3 times, cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min, incubated with BCIP/NBT ALP Color Development Kit (Beyotime, China) for 10~30 minutes, and photographed.


## 2.5.4. Immunofluorescence Staining

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, immunofluorescence staining was carried out to detect the expressions of osteocalcin (OCN). Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 1 h. Subsequently, they were incubated with the primary antibody of OCN (Santa, USA) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, they were stained with rhodamine-phalloidin (Cytoskeleton, USA) for 1 h, and cell nuclei were stained with DAPI (Beyotime, China) for 10 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).


## 2.5.5. Alizarin Red Staining for Mineralization

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of6×104/well. After incubating for 14 d in osteogenic medium, cells on samples were fixed and stained with the Alizarin red S (ARS) reagent (Cyagen, China) for 15 min at room temperature. After the samples were washed with DPBS three times, images were taken. Then, the mineralized nodules were eluted with 10% hexadecylpyridinium chloride monohydrate (Sigma, USA), and the O.D. value at 570 nm was measured for quantitative detection.


## 2.6.In Vivo Osteogenesis Study

One-year-old male Beagles were purchased from Fuzhou Zhenhe Experimental Animal Technology Development Co., Ltd., weighing 10-13 kg, and all thein vivo experiments followed the protocols approved by the Animal Care and Use Committee of the Xiangya School of Stomatology, Central South University. To establish the osteogenesis model, SLA implants and h1SLA implants were separately implanted on the normal alveolar bone between the two adjacent teeth after removing the crowns from bilateral mandibular premolars of beagles (Figure S8). Six weeks later, the beagles were killed, and the rotation torque value of each implant was measured.To determine the new bone formation on the implants, fixed sample was measured by the Cone Beam Computer Tomography (CBCT, ProMax 3D, Helsinki), and the images were generated from Planmeca Romexis. After performing the CBCT, hard tissue sections were made and stained with methylene blue magenta to observe the osteogenesis.


## 2.7. Statistical Analysis

Data from cellular experiments on the substrates are presented as the mean error and standard deviation of the mean error of all the measurements. Data were evaluated by analysis of significant differences among different groups, which were determined using one-way ANOVA andt-test by “GraphPad Prism 8.0.” Asterisk (∗) represents significant differences between different substrates. ∗ indicates 0.01≤p<0.05, ∗∗ indicates 0.001≤p<0.01, ∗∗∗ indicates 0.0001≤p<0.001, and ∗∗∗∗ indicates 0.00001≤p<0.0001.


## 3. Results and Discussion

### 3.1. Surface Characteristics

It is reported that hierarchical micro/nanotopography contributed to cell adhesion and bone integrationin vivo [34]. Observed from the scanning electron microscopy (SEM) images in Figure 1, it can be seen both of the SLA (sandblasted and acid etched) and hSLA (H2O2 treated SLA) groups have uniform honeycomb-like holes ranging from 1 μm to 3 μm at 5k× magnification (Figures 1(g)–1(i)). At the magnification of 50k× (Figures 1(j)–1(l)) and 100k×(Figures 1(m)–1(o)), for the SLA group, the wall is smooth, and the edge of the holes is sharp, with no nanostructure found. In contrast, the cavity walls of the h1SLA (treating SLA with H2O2 for 1 hour) and h2SLA (treating SLA with H2O2 for 2 hour) groups are rough, and the edges of the cavity are round and blunt. Moreover, many nanoparticles were evenly distributed between and on the cavity wall.Figure 1
Scanning electron microscopy images of SLA, h1SLA, and h2SLA surface at different magnifications. (a–c) SLA, h1SLA, and h2SLA surfaces at 1k× magnification. (d–f) SLA, h1SLA, and h2SLA surfaces at 2k× magnification. (g–i) SLA, h1SLA, and h2SLA surfaces at 5k× magnification. (j–l) SLA, h1SLA, and h2SLA surfaces at 50k× magnification. (m–o) SLA, h1SLA, and h2SLA surfaces at 100k× magnification.We find no significant difference in nanomorphology between h1SLA and h2SLA groups, indicating the longer etching time of H2O2 does not influence the nanostructure of surface. However, the microstructure and the surface roughness might be affected by the treatment time. Titanium surface with increased roughness that resulted from microscale topography is more beneficial for cells and bone tissue attachment [35]. There is no obvious difference between SLA and h1SLA samples in the 3D vision (Figures 2(a) and 2(b)) because of the short etching time, and similar profile peaks and valleys are further confirmed in the 2D analysis (Figures 2(d) and 2(e)). However, more protrusions with lower height are observed on h2SLA samples (Figure 2(c)), with lower profile peaks (Figure 2(f)), indicating longer H2O2 treating time may eliminate the microcavities. From the analysis of Ra (arithmetic mean deviation of contour) and Rz (maximum height of profile) (Table 1), we find no significant difference between SLA and h1SLA groups, while each parameter of h2SLA is, respectively, lower. The results indicate that the longer treatment of H2O2 leads to flatter microstructures without obvious changes on nanostructures.Figure 2
Roughness and wettability results of SLA, h1SLA, and h2SLA surface. (a–c) 3D surface profile of SLA, h1SLA, and h2SLA surface. (d–f) 2D analysis of SLA, h1SLA, and h2SLA surface. (g–i) Water contact angles of SLA, h1SLA, and h2SLA surface.
(a)(b)(c)(d)(e)(f)(g)(h)(i)Table 1
Roughness parameters (Ra and Rz) of SLA, h1SLA, and h2SLA surfaces. Ra: arithmetic mean deviation of contour. Rz: maximum height of profile.
SLA (μm)h1SLA (μm)h2SLA (μm)Ra2.193±0.0452.198±0.0502.003±0.037Rz23.323±0.85923.763±0.69221.573±1.178The water contact angles (WCA) of both h1SLA and h2SLA samples are less than 10° (Figures2(h) and 2(i)), significantly lower than that of SLA, which is about 70° (Figure 2(g)). This may be attributed to the O22- group formed during the etching process of H2O2 [30]. Surface hydrophilicity is crucial in recovering the effect of micro/nanotopography on promoting osteoblastic differentiation of MSCs (mesenchymal stem cells) [36]. The combination of nanostructures and hydrophilicity could improve protein adsorption levels, leading to early and most significant blood coagulation [37] and the strongest bone response [38].Surface chemistry is also related to the bioactivity of materials [39]. From the XPS (X-ray photoelectron spectroscopy) analysis (Figure 3) and EDS (X-ray energy dispersive spectroscopy) detection (Figure S2), we find that the content of titanium and oxygen of h1SLA and h2SLA is changed. The Ti2P spectra (Figures 3(d)–3(f)) show the vanishment of Ti0 peak at 453.5 eV and increase of titanium oxide peak, which may be due to the oxidation of titanium with treatment of H2O2. The presence of metal Ti0 on SLA samples (Figure 3(d)) suggests the oxide layer is either thin or heterogeneous [40]. In contrast, the oxide layers on the surface of the h1SLA and h2SLA groups are either thicker or more homogeneous for the absence of metal Ti0 (Figures 3(e) and 3(f)). More details in the O1S spectra show an additional peak at 533.5 eV (Figures 3(g)–3(i)), which can be postulated to be O22− group because of the formation of Ti(H2O2)24+ during etching [41].Figure 3
X-ray photoelectron spectroscopy results of SLA, h1SLA, and h2SLA surfaces. (a–c) X-ray photoelectron spectroscopy survey spectrums. (d–f) Ti2p valence analysis. (g–i) O1s valence analysis.


### 3.2. Cell Behaviors of rBMSCs on Different Titanium Surface

We first checked the cell behaviors (e.g., attachment, spreading, and proliferation) of rBMSCs (rat bone mesenchymal stem cells) on different titanium surface. In the initial cell-implant interactions, cell adhesion is one of the most critical processes [42]. We find that both h1SLA and h2SLA surfaces display more cell attachment in comparison with SLA surfaces in Figures 4(a)–4(c). The number of adherent rBMSCs (Figure 4(d)) on h1SLA and h2SLA surfaces are both higher than that on SLA surface, with statistically significant differences.Figure 4
(a–c) Fluorescence microscopy images of rBMSCs attached to SLA (a), h1SLA (b), and h2SLA (c) surfaces stained by AO. (d) Quantitative analysis of cell attachment. (e–g) Fluorescence microscopy images of rBMSCs spreading on SLA (e), h1SLA (f), and h2SLA (g) surfaces. Orange red: rhodamine-phalloidin. Blue: DAPI. (h) Quantitative analysis of spreading areas. (i–k) Fluorescence microscopy images of Mki67 expression on SLA (i), h1SLA (j), and h2SLA (k) surfaces at 3 d. Green: Mki67. (l) Quantitative analysis of fluorescence intensity (n=40).The cytoskeletons of cells are more extended, and polygonal osteoblastic-like shapes with more obvious filopodia are observed on h1SLA and h2SLA surfaces in Figures4(e)–4(g), which have been proved to be closely related to MSC osteogenic activity [43, 44]. As presented in Figure 4(h), the cell spreading areas on h1SLA and h2SLA surfaces are significantly larger than SLA.Cell proliferation is observed by Mki67 immunofluorescence assay (Figures4(i)–4(l)), cell staining (Figure S4), and CCK-8 experiment (Figure S5). By immunofluorescence detection (Figures 4(i)–4(k)) and fluorescence intensity quantitative detection (Figure 4(l)), we observed that the expression of Mki67 was more robust in the treatment group, especially in h1SLA group. The cell staining further confirmed the increase of cell numbers on the h1SLA surface. From the CCK-8 test results, the cell numbers on the three surfaces have no obvious difference at 1 d. However, with the increase of time, the h1SLA surface shows significant increase in cell numbers. This may be explained by the relatively higher hydrophilicity [45] of group h1SLA. Overall, these data demonstrate that h1SLA surface is most biocompatible among them. The excellent biocompatibility of h1SLA surface for rBMSCs may be benefit for osteogenesis.


### 3.3.In Vitro Study of rBMSC Osteogenic Differentiation

We first observed the cell morphology on the titanium surfaces at 4 d after osteogenesis induction. As shown in Figures5(d)–5(f), there are more cell accounts of rBMSCs on the h1SLA surfaces than the h2SLA and SLA groups, and the cells are more extended on h1SLA. This cell spreading trend is consistent with which in Figure 4 without osteogenesis induction, preliminarily indicating that h1SLA group is more conducive to osteogenic differentiation.Figure 5
In vitro rBMSC osteogenic differentiation on different surfaces. (a) Expression of osteogenic index of rBMSCs on SLA, h1SLA, and h2SLA at 7 d. (b) Relative ALP activity of rBMSCs on different samples at 7 d and 14 d. (c) Quantitative detection by Alizarin red staining at 14 d. (d–f) Adhesion and spreading of cells on each surface after osteogenesis induction for 4 d. Red: rhodamine-phalloidin. Blue: DAPI. (g–i) Staining of ALP in SLA, h1SLA, and h2SLA groups at 14 d. (j–l) The expression of OCN (green) explored by immunofluorescence assay at 14 d. (m–o) Pictures of mineralization nodules deposited on different surfaces at 14 d.Real-time PCR detects the mRNA expression level of osteogenic indexes at 4 d (FigureS6) and 7 d (Figure 5(a)). ALP and RUNX2 play an important role in early osteogenesis. The expression of these two genes in h1SLA group is significantly higher than that in the other two groups, followed by h2SLA group. The significant increase of other osteogenesis-related genes such as COL1A1, BMP2, and BSP also shows that the osteogenic properties of the materials are significantly improved after hydrogen peroxide treatment. The ALP activity at the protein level is measured at 7 d and 14 d, respectively (Figure 5(b)). Compared with SLA, the ALP protein expression in both h1SLA and h2SLA are significantly increased, whereas the h1SLA shows the highest level at 14 d. By carefully observing the ALP staining on the materials surface directly (Figures 5(g)–5(i)), it can also be observed that the ALP staining in h1SLA group is deeper than the other two groups.We also explore the expression of osteocalcin, which is a late osteogenic marker, by immunofluorescence assay at 14 d. The green-stained protein is observed to be stronger on h1SLA and h2SLA surfaces, revealing better osteogenic effect of h1SLA and h2SLA surfaces than SLA, whereas the h1SLA shows the highest level (Figures5(j)–5(l)). By quantitative analysis of fluorescence intensity (Figure S7), the expression of OCN in h1SLA group increased significantly. Alizarin Red staining was carried out at 14 d (Figures 5(m)–5(o)). The results show that the h1SLA has the most obvious mineralization nodules, followed by the h2SLA group, and the SLA group has the least mineralized nodules. This difference is reflected in the quantitative test results (Figure 5(c)). These results further confirm that the h1SLA and h2SLA surface can promote bone formation than SLA, and the effect on the h1SLA surface is most significant.


### 3.4.In Vivo Osteogenesis Study

Inspired by rBMSCs differentiation result, we further performed the osteogenesis experiments of h1SLAin vivo using SLA as control. The CBCT (Cone Beam Computer Tomography) image (Figures 6(a)–6(d)) 6 weeks after implantation shows more obvious white projection around the h1SLA implants, indicating more bone formation around h1SLA than in the SLA group, and more abundant deep red staining areas around h1SLA could be observed (Figures 6(e) and 6(f)) through methylene blue acid magenta staining of hard tissue sections, which is consistent with the trend indicated by CBCT results. Meanwhile, the torque value of implant rotation from h1SLA group is greater than that of SLA group (Table 2), in line with CBCT and staining trend. Hard tissue section staining data show that BIC%, which represents the ratio of bone to implant contact, in h1SLA group is nearly 2 times higher than that in SLA group (Table 2). Animal experiments further confirm that nanostructured and hydrophilicity are more suitable for osteogenic differentiation of rBMSCs, which is very important for further clinical promotion. From those results, it could be inferred that biopotential mimicking surface, which is modified in structures, wettability, and indirectly in surface chemistry, plays a key role in the improvement of osteogenesis performance.Figure 6
In vivo osteogenesis: (a–d) Cone Beam Computer Tomography images of SLA group and h1SLA group 6 weeks after implant surgery. (e–f) Methylene blue acid fuchsin dyeing results of hard tissue sections 6 weeks after implantation in SLA group and h1 group. Red: bone tissues. Blue arrow: new bone formation region.
(a)(b)(c)(d)(e)(f)Table 2
The analysis of implant rotation torque values (the second column) and hard tissue section data (between three and six columns) 6 weeks after implantation in SLA group and h1SLA group. Im.Pm: perimeter of implants. Tb.In.Pm: length of contact between trabecula and implant. Ct.In.Pm: length of contact between cortical bone and implant. BIC%: ratio of bone to implant contact.
M±SD (N/cm)Im.PmTb.In.PmCt.In.PmBIC%SLA71.8±25.6131.40046.2621.3610.362h1SLA84.2±18.4134.99371.17911.1190.610The surface properties of titanium implants such as topography, roughness, wettability, and chemistry have a significant impact on biological activity [46–50]. In our study, experimental results in vitro and in vivo confirm that the nanoscale modification, which brings nanostructure morphology, hydrophilicity, and chemistry change together to the titanium implants, is positive to rat bone mesenchymal stem cell responses. However, the use of rBMSCs and the rat model may still face some limitations, whether this material is suitable for human-related cell needs to be further studied. Therefore, in future research, in addition to continuing to study the decisive factors affecting material properties, human cells such as human gingival mesenchymal stem cells or human periodontal ligament stem cells will be used for osteogenesis verification [32, 51]. Besides, it has been reported that the presence of O22− group and nanoscale modification can significantly improve the antibacterial and immunity properties to promote wound healing [52, 53], so we will also conduct relevant experiments for further studies.


## 3.1. Surface Characteristics

It is reported that hierarchical micro/nanotopography contributed to cell adhesion and bone integrationin vivo [34]. Observed from the scanning electron microscopy (SEM) images in Figure 1, it can be seen both of the SLA (sandblasted and acid etched) and hSLA (H2O2 treated SLA) groups have uniform honeycomb-like holes ranging from 1 μm to 3 μm at 5k× magnification (Figures 1(g)–1(i)). At the magnification of 50k× (Figures 1(j)–1(l)) and 100k×(Figures 1(m)–1(o)), for the SLA group, the wall is smooth, and the edge of the holes is sharp, with no nanostructure found. In contrast, the cavity walls of the h1SLA (treating SLA with H2O2 for 1 hour) and h2SLA (treating SLA with H2O2 for 2 hour) groups are rough, and the edges of the cavity are round and blunt. Moreover, many nanoparticles were evenly distributed between and on the cavity wall.Figure 1
Scanning electron microscopy images of SLA, h1SLA, and h2SLA surface at different magnifications. (a–c) SLA, h1SLA, and h2SLA surfaces at 1k× magnification. (d–f) SLA, h1SLA, and h2SLA surfaces at 2k× magnification. (g–i) SLA, h1SLA, and h2SLA surfaces at 5k× magnification. (j–l) SLA, h1SLA, and h2SLA surfaces at 50k× magnification. (m–o) SLA, h1SLA, and h2SLA surfaces at 100k× magnification.We find no significant difference in nanomorphology between h1SLA and h2SLA groups, indicating the longer etching time of H2O2 does not influence the nanostructure of surface. However, the microstructure and the surface roughness might be affected by the treatment time. Titanium surface with increased roughness that resulted from microscale topography is more beneficial for cells and bone tissue attachment [35]. There is no obvious difference between SLA and h1SLA samples in the 3D vision (Figures 2(a) and 2(b)) because of the short etching time, and similar profile peaks and valleys are further confirmed in the 2D analysis (Figures 2(d) and 2(e)). However, more protrusions with lower height are observed on h2SLA samples (Figure 2(c)), with lower profile peaks (Figure 2(f)), indicating longer H2O2 treating time may eliminate the microcavities. From the analysis of Ra (arithmetic mean deviation of contour) and Rz (maximum height of profile) (Table 1), we find no significant difference between SLA and h1SLA groups, while each parameter of h2SLA is, respectively, lower. The results indicate that the longer treatment of H2O2 leads to flatter microstructures without obvious changes on nanostructures.Figure 2
Roughness and wettability results of SLA, h1SLA, and h2SLA surface. (a–c) 3D surface profile of SLA, h1SLA, and h2SLA surface. (d–f) 2D analysis of SLA, h1SLA, and h2SLA surface. (g–i) Water contact angles of SLA, h1SLA, and h2SLA surface.
(a)(b)(c)(d)(e)(f)(g)(h)(i)Table 1
Roughness parameters (Ra and Rz) of SLA, h1SLA, and h2SLA surfaces. Ra: arithmetic mean deviation of contour. Rz: maximum height of profile.
SLA (μm)h1SLA (μm)h2SLA (μm)Ra2.193±0.0452.198±0.0502.003±0.037Rz23.323±0.85923.763±0.69221.573±1.178The water contact angles (WCA) of both h1SLA and h2SLA samples are less than 10° (Figures2(h) and 2(i)), significantly lower than that of SLA, which is about 70° (Figure 2(g)). This may be attributed to the O22- group formed during the etching process of H2O2 [30]. Surface hydrophilicity is crucial in recovering the effect of micro/nanotopography on promoting osteoblastic differentiation of MSCs (mesenchymal stem cells) [36]. The combination of nanostructures and hydrophilicity could improve protein adsorption levels, leading to early and most significant blood coagulation [37] and the strongest bone response [38].Surface chemistry is also related to the bioactivity of materials [39]. From the XPS (X-ray photoelectron spectroscopy) analysis (Figure 3) and EDS (X-ray energy dispersive spectroscopy) detection (Figure S2), we find that the content of titanium and oxygen of h1SLA and h2SLA is changed. The Ti2P spectra (Figures 3(d)–3(f)) show the vanishment of Ti0 peak at 453.5 eV and increase of titanium oxide peak, which may be due to the oxidation of titanium with treatment of H2O2. The presence of metal Ti0 on SLA samples (Figure 3(d)) suggests the oxide layer is either thin or heterogeneous [40]. In contrast, the oxide layers on the surface of the h1SLA and h2SLA groups are either thicker or more homogeneous for the absence of metal Ti0 (Figures 3(e) and 3(f)). More details in the O1S spectra show an additional peak at 533.5 eV (Figures 3(g)–3(i)), which can be postulated to be O22− group because of the formation of Ti(H2O2)24+ during etching [41].Figure 3
X-ray photoelectron spectroscopy results of SLA, h1SLA, and h2SLA surfaces. (a–c) X-ray photoelectron spectroscopy survey spectrums. (d–f) Ti2p valence analysis. (g–i) O1s valence analysis.


## 3.2. Cell Behaviors of rBMSCs on Different Titanium Surface

We first checked the cell behaviors (e.g., attachment, spreading, and proliferation) of rBMSCs (rat bone mesenchymal stem cells) on different titanium surface. In the initial cell-implant interactions, cell adhesion is one of the most critical processes [42]. We find that both h1SLA and h2SLA surfaces display more cell attachment in comparison with SLA surfaces in Figures 4(a)–4(c). The number of adherent rBMSCs (Figure 4(d)) on h1SLA and h2SLA surfaces are both higher than that on SLA surface, with statistically significant differences.Figure 4
(a–c) Fluorescence microscopy images of rBMSCs attached to SLA (a), h1SLA (b), and h2SLA (c) surfaces stained by AO. (d) Quantitative analysis of cell attachment. (e–g) Fluorescence microscopy images of rBMSCs spreading on SLA (e), h1SLA (f), and h2SLA (g) surfaces. Orange red: rhodamine-phalloidin. Blue: DAPI. (h) Quantitative analysis of spreading areas. (i–k) Fluorescence microscopy images of Mki67 expression on SLA (i), h1SLA (j), and h2SLA (k) surfaces at 3 d. Green: Mki67. (l) Quantitative analysis of fluorescence intensity (n=40).The cytoskeletons of cells are more extended, and polygonal osteoblastic-like shapes with more obvious filopodia are observed on h1SLA and h2SLA surfaces in Figures4(e)–4(g), which have been proved to be closely related to MSC osteogenic activity [43, 44]. As presented in Figure 4(h), the cell spreading areas on h1SLA and h2SLA surfaces are significantly larger than SLA.Cell proliferation is observed by Mki67 immunofluorescence assay (Figures4(i)–4(l)), cell staining (Figure S4), and CCK-8 experiment (Figure S5). By immunofluorescence detection (Figures 4(i)–4(k)) and fluorescence intensity quantitative detection (Figure 4(l)), we observed that the expression of Mki67 was more robust in the treatment group, especially in h1SLA group. The cell staining further confirmed the increase of cell numbers on the h1SLA surface. From the CCK-8 test results, the cell numbers on the three surfaces have no obvious difference at 1 d. However, with the increase of time, the h1SLA surface shows significant increase in cell numbers. This may be explained by the relatively higher hydrophilicity [45] of group h1SLA. Overall, these data demonstrate that h1SLA surface is most biocompatible among them. The excellent biocompatibility of h1SLA surface for rBMSCs may be benefit for osteogenesis.


## 3.3.In Vitro Study of rBMSC Osteogenic Differentiation

We first observed the cell morphology on the titanium surfaces at 4 d after osteogenesis induction. As shown in Figures5(d)–5(f), there are more cell accounts of rBMSCs on the h1SLA surfaces than the h2SLA and SLA groups, and the cells are more extended on h1SLA. This cell spreading trend is consistent with which in Figure 4 without osteogenesis induction, preliminarily indicating that h1SLA group is more conducive to osteogenic differentiation.Figure 5
In vitro rBMSC osteogenic differentiation on different surfaces. (a) Expression of osteogenic index of rBMSCs on SLA, h1SLA, and h2SLA at 7 d. (b) Relative ALP activity of rBMSCs on different samples at 7 d and 14 d. (c) Quantitative detection by Alizarin red staining at 14 d. (d–f) Adhesion and spreading of cells on each surface after osteogenesis induction for 4 d. Red: rhodamine-phalloidin. Blue: DAPI. (g–i) Staining of ALP in SLA, h1SLA, and h2SLA groups at 14 d. (j–l) The expression of OCN (green) explored by immunofluorescence assay at 14 d. (m–o) Pictures of mineralization nodules deposited on different surfaces at 14 d.Real-time PCR detects the mRNA expression level of osteogenic indexes at 4 d (FigureS6) and 7 d (Figure 5(a)). ALP and RUNX2 play an important role in early osteogenesis. The expression of these two genes in h1SLA group is significantly higher than that in the other two groups, followed by h2SLA group. The significant increase of other osteogenesis-related genes such as COL1A1, BMP2, and BSP also shows that the osteogenic properties of the materials are significantly improved after hydrogen peroxide treatment. The ALP activity at the protein level is measured at 7 d and 14 d, respectively (Figure 5(b)). Compared with SLA, the ALP protein expression in both h1SLA and h2SLA are significantly increased, whereas the h1SLA shows the highest level at 14 d. By carefully observing the ALP staining on the materials surface directly (Figures 5(g)–5(i)), it can also be observed that the ALP staining in h1SLA group is deeper than the other two groups.We also explore the expression of osteocalcin, which is a late osteogenic marker, by immunofluorescence assay at 14 d. The green-stained protein is observed to be stronger on h1SLA and h2SLA surfaces, revealing better osteogenic effect of h1SLA and h2SLA surfaces than SLA, whereas the h1SLA shows the highest level (Figures5(j)–5(l)). By quantitative analysis of fluorescence intensity (Figure S7), the expression of OCN in h1SLA group increased significantly. Alizarin Red staining was carried out at 14 d (Figures 5(m)–5(o)). The results show that the h1SLA has the most obvious mineralization nodules, followed by the h2SLA group, and the SLA group has the least mineralized nodules. This difference is reflected in the quantitative test results (Figure 5(c)). These results further confirm that the h1SLA and h2SLA surface can promote bone formation than SLA, and the effect on the h1SLA surface is most significant.


## 3.4.In Vivo Osteogenesis Study

Inspired by rBMSCs differentiation result, we further performed the osteogenesis experiments of h1SLAin vivo using SLA as control. The CBCT (Cone Beam Computer Tomography) image (Figures 6(a)–6(d)) 6 weeks after implantation shows more obvious white projection around the h1SLA implants, indicating more bone formation around h1SLA than in the SLA group, and more abundant deep red staining areas around h1SLA could be observed (Figures 6(e) and 6(f)) through methylene blue acid magenta staining of hard tissue sections, which is consistent with the trend indicated by CBCT results. Meanwhile, the torque value of implant rotation from h1SLA group is greater than that of SLA group (Table 2), in line with CBCT and staining trend. Hard tissue section staining data show that BIC%, which represents the ratio of bone to implant contact, in h1SLA group is nearly 2 times higher than that in SLA group (Table 2). Animal experiments further confirm that nanostructured and hydrophilicity are more suitable for osteogenic differentiation of rBMSCs, which is very important for further clinical promotion. From those results, it could be inferred that biopotential mimicking surface, which is modified in structures, wettability, and indirectly in surface chemistry, plays a key role in the improvement of osteogenesis performance.Figure 6
In vivo osteogenesis: (a–d) Cone Beam Computer Tomography images of SLA group and h1SLA group 6 weeks after implant surgery. (e–f) Methylene blue acid fuchsin dyeing results of hard tissue sections 6 weeks after implantation in SLA group and h1 group. Red: bone tissues. Blue arrow: new bone formation region.
(a)(b)(c)(d)(e)(f)Table 2
The analysis of implant rotation torque values (the second column) and hard tissue section data (between three and six columns) 6 weeks after implantation in SLA group and h1SLA group. Im.Pm: perimeter of implants. Tb.In.Pm: length of contact between trabecula and implant. Ct.In.Pm: length of contact between cortical bone and implant. BIC%: ratio of bone to implant contact.
M±SD (N/cm)Im.PmTb.In.PmCt.In.PmBIC%SLA71.8±25.6131.40046.2621.3610.362h1SLA84.2±18.4134.99371.17911.1190.610The surface properties of titanium implants such as topography, roughness, wettability, and chemistry have a significant impact on biological activity [46–50]. In our study, experimental results in vitro and in vivo confirm that the nanoscale modification, which brings nanostructure morphology, hydrophilicity, and chemistry change together to the titanium implants, is positive to rat bone mesenchymal stem cell responses. However, the use of rBMSCs and the rat model may still face some limitations, whether this material is suitable for human-related cell needs to be further studied. Therefore, in future research, in addition to continuing to study the decisive factors affecting material properties, human cells such as human gingival mesenchymal stem cells or human periodontal ligament stem cells will be used for osteogenesis verification [32, 51]. Besides, it has been reported that the presence of O22− group and nanoscale modification can significantly improve the antibacterial and immunity properties to promote wound healing [52, 53], so we will also conduct relevant experiments for further studies.


## 4. Conclusions

In this study, titanium implants with different surficial features are fabricated by simple and eco-friendly H2O2 treatment of SLA (sandblasted and acid-etched) titanium to improve the behaviors of stem cells and osteogenesis in vitro and in vivo. The nanoscale modification of SLA endows nanostructure, enhanced roughness, and wettability on titanium surface, which significantly promotes the attachment, spreading, proliferation, and osteogenic differentiation of rat bone mesenchymal stem cells in vitro. The in vivo experiments further confirmed the osseointegration improvement. These results indicate that the nanoscale-modified SLA titanium may have great potential in better osteogenesis and higher implant success rate.



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*Source: 2235335-2022-01-04.xml*