Enhanced osteogenic differentiation of mesenchymal stem cells by surface lithium modification in a sandblasted/acid-etched titanium implant

Abstract

This study investigated the osteogenesis-related cell functions of osteoprogenitor cells modulated by surface chemistry modification using lithium (Li) ions in a current clinical oral implant surface in order to gain insights into the future development of titanium (Ti) implants with enhanced osteogenic capacity. Wet chemical treatment was performed to modify a sandblasted/acid-etched (SLA) Ti implant surface using Li ions. The osteogenesis-related cell response to the surface Li ion-modified SLA sample was evaluated using two kinds of murine bone marrow stem cells, bipotent ST2 cells and primary multipotent mesenchymal stem cells (MSCs). The modified surface exhibited the formation of an Li-containing Ti oxide layer with plate-like nanostructures. The Li-incorporated surface enhanced early cellular events, including spreading, focal adhesion formation and integrin mRNA expression (α2, α5, αv and β3), and accelerated osteogenic differentiation of bipotent ST2 cells compared with unmodified SLA surface. Surface Li modification significantly increased GSK-3β phosphorylation and suppressed β-catenin phosphorylation, and promoted the subsequent osteogenic differentiation of primary MSCs. These results indicate that surface chemistry modification of SLA implants by wet chemical treatment with Li ions induces a more favorable osseointegration outcome through the promotion of the osteogenic differentiation of bone marrow MSCs via the positive regulation of GSK-3β and β-catenin activity.

Keywords

Titanium implants mesenchymal stem cells lithium surface modifications osteogenic differentiation glycogen synthase kinase 3β

Introduction

Surface chemistry modification using bioactive ions is promising approach in the development of endosseous titanium (Ti) implants with enhanced osteogenic capacity.^1–6 Various ions have been probed,^1–6 and some of them have been employed in surface chemistry modification of currently used clinical Ti oral implants.^1,3 A Ti implant surface modified with bioactive ions, including calcium, strontium or fluoride, is known to improve early implant osseointegration by enhancing early cellular events such as spreading and activation of integrin-mediated signaling pathways that affect osteoblast function, and promote the osteoblastic differentiation of osteoprogenitor cells.^2,5,7

Recently, several studies have explored the potential of lithium (Li) ions as a candidate element for surface chemistry modification of bone implantation materials for biomedical applications.^8–11 Li salt is nonselective inhibitor of glycogen synthase kinase 3β (GSK-3β), which indirectly inhibits GSK-3β activity by phosphorylating GSK-3β at the N-terminal Ser9.^12,13 GSK-3β is a negative regulator of the Wnt/β-catenin signaling pathway.^13,14 Deactivation of GSK-3β by Ser9 phosphorylation stabilizes cytoplasmic β-catenin and activates canonical Wnt signaling so as to initiate the osteogenesis cascade of osteoprogenitor cells.^12,13,15 The β-catenin stabilization and subsequent translocation of soluble β-catenin into the nucleus activates the transcription of target genes related to osteogenesis, such as Runx2 and osterix, by interacting with T-cell factor/lymphoid enhancer factor (TCF/LEF) family, which then promotes osteogenic differentiation of osteoprogenitor cells.^15–17 Incubation of mesenchymal stem cells (MSCs) or osteoblastic cells with Li salt promotes osteoblastic differentiation^12,18 and systemic administration of LiCl enhances the bone filling around Ti screws in the tibia of ovariectomized rats.¹⁸

However, there are only very limited studies yet reported on the beneficial osteogenic effect of Li ions when directly incorporated into the solid surface of clinical Ti implants.¹⁰ Studies have reported the beneficial effect of Li treatment of the Ti surfaces.^10,19,20 Liu et al.²⁰ prepared a Li-containing coating on entangled Ti wire scaffolds by microarc oxidation and assessed cell behavior in osteoblastic MG63 cells. They showed that Li incorporation promoted osteoblast differentiation by inhibiting GSK-3β activity. However, it is not clear that direct incorporation of Li ions into the clinically available osteoconductive Ti oral implant surface would also function as a GSK-3β inhibitor. Thus, attempts to assess the osteogenic effects of surface chemistry modification of microstructured Ti implants using Li ions would provide insight into the development of an ion-modified Ti surface with increased osteogenic capacity.

This study investigated whether direct Li ion doping enhances osteogenesis-related cellular functions when introduced into a clinically used oral implant surface. For this purpose, we incorporated Li ions into an SLA (sandblasted, large-grit, and acid-etched; Straumann AG, Basel, Switzerland) type implant surface by wet chemical treatment, then evaluated the osteogenic capacity of the modified surface using two kinds of murine bone marrow-derived osteoprogenitor cells. Studies have demonstrated that the SLA implant surface promotes early bone apposition and reduces the healing period for osseointegration.^21–23 Long-term clinical studies reported high success rates for SLA implants loaded with a shortened healing period.^23,24 Thus, we expected that results of the evaluation of osteogenic potential of Li ion modification in this highly osteoconductive SLA implant surface would provide a greater clinical significance for the future development of Ti oral implants with increased osteogenic capacity. We first briefly evaluated the response to the modified surface in bipotent ST2 cells, then confirmed osteogenic effect of Li modification, including the activity of GSK-3β and β-catenin using primary multipotent MSCs.

Materials and methods

Sample preparation

To prepare a microtopographically complex Ti surface similar to that of a clinical SLA implant, commercially pure Ti disks (ASTM grade 4, 15 mm in diameter and 3 mm thick) were sandblaed using large grit Al₂O₃ particles, which was followed by an acid etching process using a mixed solution of H₂SO₄ and HCl (the SLA group). Samples were washed in dionized water. Wet chemical treatment was performed to incorporate Li ions into the surface of the SLA samples. Briefly, SLA samples were treated hydrothermally in 50 mM LiOH at 220°C for 4 h in a Ti reactor (the Li-SLA group), then thoroughly cleaned and air dried.

Surface characterization

The surface morphology of the investigated samples was evaluated under field emission-scanning electron microscopy (FE-SEM; S-4800, Hitachi, Tokyo, Japan). The three-dimensional surface roughness values of the samples were determined under confocal laser scanning microscopy (CLSM, LSM700; Carl Zeiss, Oberkochen, Germany) over a 300 μm × 300 μm area (n = 5). The crystallographic structure of the surface oxide layer of the investigated sample was characterized by thin-film X-ray diffractometry with Cu-Kα radiation (XRD; X’Pert-APD, Philips, Almelo, Netherlands). The surface chemical composition of the investigated samples was evaluated with X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Scientific, East Grinstead, UK).

Cell culture

Two kinds of murine bone marrow stem cells, bipotent stem cells and primary multipotent mesenchymal stem cells (MSCs), were used for the evaluation of osteogenesis-related cellular functions to the investigated surfaces. Mouse bone marrow-derived bipotent stem cells (ST2 cell; RIKEN, Tsukuba, Japan) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL Life Technology, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco BRL Life Technologies), 100 U/mL penicillin (Keunhwa Pharmaceutical, Seoul, Korea) and 100 U/mL streptomycin (Donga Pharmaceutical, Seoul, Korea).

Primary MSCs were isolated from the bone marrow of the tibiae and femurs of 6-week-old mice. In this study, we used cell stock obtained from previous experiments performed according to the protocol approved by the Institutional Animal Care and Use Committee of Kyungpook National University, Daegu, Korea [approval no. KNU 2011-96]. Primary bone marrow MSCs were maintained in α-MEM (Gibco BRL Life Technologies) containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin.

The cells were incubated at 37°C under 100% humidity and 5% CO₂. After reaching confluence, cells were digested with 0.25% trypsin/0.02% EDTA. Cells were cultured on Ti disk samples in 24-well culture plates at an initial seeding density of 2 × 10⁴ cells/well. On day 2 post-plating of the cells on the Ti samples, osteogenic differentiation was induced by the addition of 50 μg ascorbic acid/mL and 10 mM β-glycerophosphate.

Bipotent ST2 cell behavior affected by surface Li modification

Cell attachment and proliferation assay

Early cell attachment (at 4 h) and proliferation (at 24 and 72 h) were assessed by a colorimetric assay using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan) according to a previously described method (n = 7 per group).²

Evaluation of morphology of spread cells

The morphology, cytoskeletal arrangement and focal adhesion development in adherent cells on the investigated samples were evaluated at 48 h of culture (at an initial seeding density of 1 × 10⁴ cells/well) by CSLM (LSM700; Carl Zeiss) observation. Focal adhesions (vinculin) and cytoskeletal arragement (actin filaments) of the spread cells on the investigated samples were identified following the double staining of actin (green fluorescence) and vinculin (red fluorescence) using diluted monoclonal anti-vinculin (Sigma-Aldrich, St. Louis, MO, USA), goat-anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), and fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma-Aldrich) according to a method described previously.²

Real-time polymerase chain reaction (PCR) analysis of integrin and osteogenesis-related gene expression

ST2 cells were cultured for 24 h for the evaluation of the mRNA levels of critical integrin genes (α2, α5, αν, β1, and β3) that are known to affect subsequent osteogenic functionality of anchorage dependent bone forming cells.^2,5,25,26 The mRNA expression levels of osteogenesis-related genes—critical transcription factor genes regulating osteogenic differentiation (Runx2 and osterix) and osteoblast phenotype genes [type I collagen (COL), alkaline phosphatase (ALP) and osteocalcin (OC)]—in cells grown on the SLA and Li-SLA samples were evaluated at 7 days of culture. Reverse transcription was performed with total RNA extracted from the cultured cells on ten (at 24 h) or five (at 7 days) Ti disks, and real-time PCR was performed as described previously² using the primers shown in Table 1. Data were analyzed with the 2^−ΔΔCT method, normalizing it against GAPDH. The expression levels of target genes were expressed as fold differences of the gene expression relative to the results of a control SLA surface.

Table 1.

Primer sequences for real-time polymerase chain reaction.

Target	Forward primer sequence (5^′–3^′)	Reverse primer sequence (5^′–3^′)
Integrin α2	GGA CTG CAG AAC CAC TTC CT	AGC GGC AGA GAT CGA TAC AC
Integrin α5	GGC AGA AGG CAG CAA TGG TG	AGG CAT CTG AGG TGG CTG GA
Integrin αν	CAT CTT GGC AGT TCT CGC AG	GCG CCA CTT AAG AAG CAC CT
Integrin β1	TTA TTG GCC TTG CCT TGC TG	CCG CCT GAG TAG GAT TCA TT
Integrin β3	ATG AAT GCG CAG CAC AGA GC	CAG GAA GGC GCG TAA GCA AT
Runx2	TAA GAA GAG CCA GGC AGG TG	TGG CAG GTA CGT GTG GTA GT
Osterix	TCA CTT GCC TGC TCT GTT CC	GCG GCT GAT TGG CTT CTT CT
β-catenin	GCT GTA GTG TCT GAA CGT GC	TTC ACT GCG TGA GTC ACT CC
COL	ATC CAA CGA GAT CGA GCT CA	GGC CAA TGT CTA GTC CGA AT
ALP	CTT GAC TGT GGT TAC TGC TG	GAG CGT AAT CTA CCA TGG AG
BSP	GGA GGA GAC AAC GGA GAA GA	CCA TAC TCA ACG GTG CTG CT
OC	TGC TTG TGA CGA GGT ATC AG	GTG ACA TCC ATA CTT GCA GG
GAPDH	GGC ATT GCT CTC AAT GAC AA	TGT GAG GGA GAT GCT CAG TG

COL: type I collagen, ALP: alkaline phosphatase, BSP: bone sialoprotein, OC: osteocalcin, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Total cellular ALP activity and OC protein production

Total cellular ALP activity in the cell lysates was measured in 2-amino-2-methyl-1-propanol buffer, at pH 10.3 and 37°C with p-nitrophenyl phosphate as the substrate at 7 days of culture. The absorbance change at 405 nm was measured using a microplate reader (n = 7 per group). Total protein was extracted from the cell lysates using a protein extraction solution (Thermo Fisher Scientific, Rockford, IL, USA) and quntified with Pierce^TM BCA Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions (n = 5 per group). ALP activity was expressed as the nanomoles of p-nitrophenol (PNP) liberated per μg of total cellular protein.

Enzyme-linked immunosorbent assay (ELISA) was performed to detect OC protein production by bipotent ST2 cells. The protein concentration of OC secreted by adherent ST cells on the investigated samples into cell culture media was measured with a commercially available OC ELISA kit (R&D Systems, Minneapolis, MN, USA) at 7 and 14 days of culture according to the manufacturer’s instructions. The OC protein levels in the supernatant were measured at 450 nm (n = 7 per group).

Osteogenesis-related MSC functions affected by surface Li modification

Assessment of phosphorylated GSK-3β and β-catenin expression at the protein and mRNA level

To investigate whether surface Li modification exerts an inhibitory effect on GSK-3β activity when applied to the SLA type implant surface, the protein expression levels of the total cellular GSK-3β and phosphorylated GSK-3β (phosphorylated at Ser 9; pS9) in cells grown on the investigated samples were semi-quantitively measured using a commercially available GSK-3β ELISA kit (ab205711; Abcam, Cambridge, UK) at 3 and 7 days of culture according to the manufacturer’s instructions.

In addition, the expression of total cellular β-catenin protein and phosphorylated β-catenin protein (phosphorylated at Ser 45; pS45) in adherent MSCs was also measured using a β-catenin ELISA kit (ab205705; Abcam) at 3 and 7 days of culture. Studies have demonstrated that a multi-protein destruction complex composed of scaffold protein Axin, casein kinase 1α (CK1α), adenomatous polyposis coli (APC) and GSK-3β is involved in the regulation β-catenin activity and degradation through a sequential and complicated interactions between components.^15,27–31 However, exact mechanisms on the biochemical and molecular level have not been fully understood. GSK-3β dependent phosphorylation of β-catenin requires prior priming through phosphorylation of Ser45.^27–29 Axin is known to recruit CK1 to phosphorylate β-catenin at Ser45.^27–29 Gavagan et al.³¹ recently reported that Axin increases β-catenin phosphorylation rate (pS45) when a competing substrate for GSK-3β is present. Thus, we sought to additionally evaluate whether the modified Ti surface would also affect GSK-3β-independent β-catenin activity by examining the expression of phosphorylated Ser45 β-catenin protein.

The protein levels of GSK-3β and β-catenin in the cell lysates were measured at 600 nm (n = 7 per group). The data were normalized to the total protein content. The expression levels of the pS9 GSK-3β and pS45 β-catenin protein were expressed as fold differences relative to the results from the unmodified SLA surface. The mRNA expression levels of GSK-3β and β-catenin in cells grown on the SLA and Li-SLA samples were evaluated by real-time PCR analysis at 3 and 7 days of culture using the primers shown in Table 1.

Osteogenesis-related gene expression and OC protein production

The mRNA expression levels of osteoblast phenotype genes—marker genes for the early (COL), intermediate (bone sialoprotein, BSP) and terminal (OC) stages of osteoblast differentiation in MSCs grown on the investigated samples were evaluated by real-time PCR analysis at 3, 7 or 14 days of culture. Real-time PCR was performed using the primers shown in Table 1.

Statistical analysis

Three independent cell culture experiments were performed. Statistical analysis was performed using nonparametric Mann-Whitney U test to evaluate differences between groups. p < .05 was considered statistically significant.

Results

Surface characteristics of the samples

Figure 1 shows the surface morphologies of the investigated samples. Both the SLA and Li-SLA surfaces displayed the charcteristic surface morphology of the SLA type implants, i.e. hierarchically complex surface structures composed of macro-rough and micro-rough pits (at a magnification of 3,000×). No notable difference was observed in surface morphology between the two samples at the micro-scale. At higher magnifications (×10,000 and 80,000), the investigated samples exhibited markedly different nano-scale surface morphologies. The surface of the SLA sample was smooth at the nanometer level (× 80,000). In contrast, the Li-SLA surface displayed the formation of plate-like surface nanostructures superimposed on the micro-pits, which slightly the obscured original micron-scale concavities caused by the acid etching process, but roughly preserved the primitive micro-pit geometry (at a magnification of 10,000×). The thickness of nanoplates of the Li-SLA sample was approximately 20 nm.

Topographical measurement images and surface roughness values of the investigated samples determined by CLSM are shown in Figure 2. There was no notable difference in the surface micro-topographies or roughness values beween the SLA and Li-SLA samples. Thus, wet chemical treatment produced evident surface nanostructures on the SLA samples and roughly maintained the surface features of the SLA implant at the micrometer level.

Thin-film XRD patterns of the investigated surfaces are shown in Figure 3(a). The SLA sample exhibited the presence of hydrogenated Ti (δ-TiH₂, JCPDS #25–982), which is typical characteristic found in the surface of acid-etched commercially pure Ti implants.^32–34 After high temperature wet chemical treatment, Ti hydride peaks disappeared from the surface of the Li-SLA sample (Figure 3(a)). This finding is somewhat consistent with the result of other studies reporting that a thermal treatment removes the Ti hydrides from the surface of SLA implants.^32,35 Thin film XRD analysis revealed the formation of Li-containing Ti oxide layer on the surface of sandblasted/acid-etched Ti sample after wet chemical treatment (Figure 3(a)). The Li-SLA sample displayed small peaks of lithium titanium oxide hydrate, (Li_1.81H_0.19)Ti₂O₅∙xH₂O (JCPDS #47–0123). The chemical compositions of the SLA and Li-SLA samples are shown in Table 2. XPS analysis revealed the presence of Li ions (6%) on the surface of the Li-SLA sample. The atomic percentages of Ti2p and O1s for the SLA surface were 19.8% and 48.8%, respectively. The atomic percentages of Ti2p and O1s for the Li-SLA surface were 21.1% and 51.5%, respectively. A small amount of N, Na, and Al were detected as surface contaminants in the SLA and Li-SLA samples. XPS high resolution spectra of Ti2p, O1s and Li1s obtained from the Li-SLA sample are shown in Figure 3(b). The value of 285.0 eV for the C1s line was used as a charge correction reference. In the Ti2p region of the Li-SLA sample, doublet peak of Ti2p_1/2 and Ti2p_3/2 were detected at 464.6 and 458.8 eV with binding energy separation (Δ2p_1/2–2p_3/2) of 5.8 eV. The XPS O1s peak of the Li-SLA sample was deconvoluted into three component peaks assigned to anhydrous oxide (O^2–), hydroxyl group (OH^–), and hydrate (anhydrous solid ∙ xH₂O) and/or adsorbed water (H₂O) at 530.3, 532.1, and 533.1 eV, respectively.^36,37 Relative ratio of O^2–, OH^–, and hydrate (and/or H₂O) content in O1s region of the Li-SLA sample were 83 mol%, 8.2 mol%, and 8.8 mol%, respectively. Li1s peak was detected approximately at 55 eV.

Table 2.

Chemical composition of investigated surfaces determined by XPS analysis (atomic %).

Group	Ti2p	O1s	C1s	Li1s	Al2p	N1s	Na1s
SLA	19.8	48.8	24.3		2.8	2.5	1.8
Li-SLA	21.1	51.5	16.3	6	1.8	1.8	1.5

SLA: sandblasted/acid-etched.

Osteogenesis functions of bipotent ST2 cells

Cell attachment, proliferation and integrin mRNA expression

Surface Li modification resulted in a lower level of early cell attachment at 4 h (p < .05; Figure 4(a)). This lower early attachment on the Li-modified SLA surface resulted in a subsequent decrease in early cell proliferation at 24 h (p < .05; Figure 4(a)), but no difference was found in terms of cell proliferation between the SLA and Li-SLA surfaces at prolonged incubation time (at 72 h).

The mRNA expression levels of integrin genes in the ST2 cells grown on the investigated surfaces were determined by real-time PCR analysis and are shown in Figure 4(b). At 24 h of incubation, the expression of α2, α5, αν and β3 integrin genes were notably unregulated in cells grown on the Li-SLA surface compared with the SLA surface (p < .05).

Cellular spreading and focal adhesion formation

Morphologies of spread cells and distribution of focal adhesion evaluated by CLSM observation are shown in Figure 5. Cells grown on both the unmodified SLA and Li-incorporated SLA samples exhibited well spread morphology, i.e. polygonal cell shape and cytoplasmic extensions. However, surface Li incorporation supported better cell spreading and more focal adhesions. Cells on the Li-SLA surface exhibited a more intense vinculin expression alongside the filopodial attachments compared with those on the SLA surface.

Osteogenic differentiation of ST2 cells

Figure 6(a) shows the results of mRNA expression levels of osteogenesis-related genes in the ST2 cells grown on the investigated samples. The mRNA expression levels of key transcription factors regulating osteoblast differentiation (Runx2 and osterix) were notably increased in cells grown on the Li-incorporated SLA surface compared with the unmodified SLA surface at 7 days of culture (p < .05; Figure 6(a)). The mRNA expression of osteoblast phenotype genes were upregulated in cells grown on the Li-SLA surface (Figure 6(a)). Surface Li incorporation markedly increased the expression of the early (COL and ALP) and terminal (OC) marker genes for osteoblast differentiation in bipotent ST2 cells (p < .05; Figure 6(a)).

The pattern of ALP and OC expression at the protein level was mostly similar to that at the mRNA level. Cells grown on the Li-incorporated SLA surface displayed significantly greater ALP activity than those on the unmodified SLA surface at 7 days (p < .05; Figure 6(b)). We then investigated whether Li incorporation promotes the terminal osteoblast differentiation of bipotent ST2 cells in the SLA implant surface. For this purpose, OC protein production by adherent cells was measured by ELISA at 7 and 14 days of culture. The ST2 cells grown on the Li-SLA surface secreted significantly greater OC protein into the cell culture media than those on the unmodified SLA surface at both 7 and 14 days (p < .05; Figure 6(c)).

Osteogenesis of primary bone marrow MSCs

GSK-3β and β-catenin phosphorylation and mRNA expression

The results of semi-quantitative measurement of the protein amount of phosphorylated GSK-3β (pS9) and phosphorylated β-catenin (pS45) in the cell lysates of mouse bone marrow MSCs were determined by ELISA and are shown in Figure 7(a). Li incorporation significantly increased GSK-3β phosphorylation at 3 days (p < 0.05), but no difference was found for the pS9 GSK-3β protein level between the two surfaces at 7 days. The relative percentage values of the amount of the phosphorylated GSK-3β protein compared to the amount of total GSK-3β protein was calculated. The Li-SLA surface showed a greater value in the relative percentage of pS9 GSK-3β/total GSK-3β protein compared with the SLA surface at 3 days (p < 0.05; Figure 7(a)).

Surface Li incorporation notably decreased β-catenin phosphorylation (pS45) in the cells grown on the SLA type implant sample at 3 days (p < .05; Figure 7(a)). The Li-SLA surface exhibited a significantly lower value in the relative percentage of pS45 β-catenin/total β-catenin protein compared with the SLA surface at both 3 and 7 days (p < .05; Figure 7(a)).

In addition, surface Li modification of the SLA implant sample significantly downregulated GSK-3β mRNA expression and upregulated β-catenin mRNA expression in MSCs at both 3 and 7 days of culture (p < .05; Figure 7(b)).

Osteogenic differentiation of MSCs

We evaluated the osteogenic functionality of MSCs by assessing osteoblast phenotype gene expression and OC protein production. MSCs grown on the Li-SLA surface displayed notably increased COL mRNA expression compared with those on the SLA surface at 3 (4.2-fold) and 7 (2.2-fold) days (p < .05; Figure 8(a)). At 7 days, BSP mRNA expression was significantly higher for the Li-SLA surface than the SLA surface (2.3-fold, p < .05; Figure 8(b)). The mRNA expression of a phenotype gene for mature osteoblasts, i.e., OC was notably increased in MSCs by surface Li modification at 14 days (3-fold, p < .05; Figure 8(c)). Surface Li incorporation also increased OC expression at the protein level. MSCs grown on the Li-SLA surface secreted markedly more OC protein into the cell culture media compared with those on the unmodified SLA surface (2.7-fold, p < .05; Figure 8(d)).

Discussion

We investigated whether surface chemistry modification using Li ions enhanced the osteogenic capacity of SLA implant surface by examining osteogenesis activity in two kinds of mouse bone marrow stem cells. We first briefly compared the cellular response in the investigated surfaces using bipotent ST2 stem cells, and then assessed the GSK-3β activity and osteogenic differentiation affected by surface Li modification in primary MSCs. The results demonstrate that surface Li modification by wet chemical treatment notably enhances osteogenic differentiation in both ST2 cells and primary MSCs in an SLA type implant surface. Terminal markers of osteoblast differentiation such as OC expression were notably increased in cells with Li incorporation at both the mRNA and protein level. These results indicate that surface Li ion chemistry in an SLA type implant surface promotes the full differentiation of osteoprogenitor cells into mature osteoblasts.

Our findings are in partial agreement with the results of other studies reporting osteogenic effects of Li treatment in the SLA type implant surface.^10,19 LiCl supplement increased ALP activity and osteogenesis-related gene expression of murine osteoprogenitor cells in the SLA implant surface.¹⁹ Huang et al.¹⁰ immersed SLA-treated Ti samples in LiOH solution, and then evaluated the osteogenic effect using rat bone marrow MSCs. They showed that the modified Ti sample exhibited sustained Li ion release and increased the proliferation and osteoblast differentiation of rat MSCs, which in turn increased bone formation around the implants in rabbit trabecular bone.¹⁰

Studies have demonstrated that Li salt inhibits GSK-3β activity and activates canonical Wnt signaling, both of which are known to play a central role in the osteogenesis cascade.^12,13 LiCl treatment increases GSK-3β phosphorylation at Ser9 and stabilizes cytoplasmic β-catenin, which in turn facilitates translocation of soluble β-catenin into the nucleus and triggers osteogenesis-related signaling cascades.^12,18 In this study, we asked if surface Li modification would also exert an effect of Li salt acting as a GSK-3β inhibitor. This has been documented in cell culture experiments when LiCl was added to the cell culture media. We found that Li modification definitely enhances the osteogenic differentiation of MSCs when introduced into an oral implant surface, in this study, an SLA-type implant. Surface Li modification significantly increased the phosphorylation of GSK-3β Ser9 in primary bone marrow MSCs. To the best of our knowledge, this is the first demonstration that direct incorporation of Li ions into the solid surface of microstructured clinical Ti implants may affect the GSK-3β activity of MSCs by promoting the phosphorylation of GSK-3β. In addition, MSCs grown on the Li-incorporated SLA sample exhibited notably decreased β-catenin phosphorylation (Ser45).

It has been shown that β-catenin phosphorylation at Ser45 is not affected by GSK-3β itself,²⁷ which is mediated by CK1α.^28,29 Phosphorylation of β-catenin at Ser45 is required for initiating subsequent GSK-3β mediated phosphorylation of Thr41, Ser37, and Ser33 in a sequential manner.^27,28 Although further detailed studies are needed to elucidate the molecular mechanisms underlying these findings (including the link between the Li-incorporated nanostructured Ti implant surface and GSK-3β-independent β-catenin activity), it is expected that surface Li modification using a high temperature wet chemical treatment will be found to promote osteogenic differentiation of bone marrow stem cells by affecting GSK-3β activity through Ser9 phosphorylation and stabilizing β-catenin when applied to the SLA type implant.

The expression of certain integrin genes (α2, α5, αv and β3) was upregulated in bipotent ST2 cells grown on the Li-incorporated SLA surface at an early incubation time point. Overexpression of these integrin subunits is believed to induce subsequently favorable osteogenesis-related cell functions.^2,5,25,26 Studies have demonstrated that nano-topographical modification increases expression of the certain integrin genes and favors the subsequent osteoblast differentiation of osteoprogenitor cells on a Ti implant surface.^2,26,38,39 Thus, the formation of favorable surface nanofeatures is yet another advantage of Li-incorporated SLA samples, a result which is commonly observed in the ion-modified Ti surface obtained by wet chemical treatment^2,3,5,10,40 Together with upregulated integrin gene expression, cellular spreading and focal adhesion formation were enhanced in ST2 cells grown on the Li-SLA surface. The plate-like surface nanostructures of the Li-incorporated SLA sample seems to increase focal adhesions and filopodial attachment by providing more optimal sites for fine cellular anchorage.^41,42 Surface charge alteration has been proposed as another advantage of Li ion modification.⁴³ Isoshima et al.⁴³ reported that the attraction of Ti to cell adhesion proteins, including fibronectin, was enhanced by changing the surface charge with Li ions. We cannot rule out a possible contribution of surface Li chemistry in the early cellular functions of osteoprogenitor cells, but further detailed investigation is needed on this. Thus, we suppose that surface nanostructure of the Li-SLA sample favored early cellular events of bone marrow stem cells, then Li chemistry activated osteogenesis-related signaling cascade by suppressing GSK-3β activity of adherent cells.

Our results suggest that surface Li modification of SLA type implants by wet chemical treatment is beneficial for inducing a more favorable osteogenesis-related environment required for implant osseointegration, an effect achieved by enhancing the osteogenic differentiation of bone marrow MSCs into mature osteoblasts through positive regulation of GSK-3β and β-catenin activity. These findings provide insight into the surface engineering of Ti implants, suggesting potential application of Li ions as a candidate element in the surface chemistry modification of microstructured oral implants.

Footnotes

Acknowledgements

The authors wish to thank Dr Youn-Jeong Kim and Je-Hee Jang, School of Dentistry, Kyungpook National University, Korea, for their assistance in cell culture experiments.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2020R1A2B5B01001581).

ORCID iD

Jin-Woo Park

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