Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach

Anne-Marie Galow; Philipp Wysotzki; Werner Baumann; Jan Gimsa

doi:10.1515/cdbme-2016-0121

Article Open Access

Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach

Anne-Marie Galow , Philipp Wysotzki , Werner Baumann and Jan Gimsa

Published/Copyright: September 30, 2016

Published by

Become an author with De Gruyter Brill

Author Information Explore this Subject

From the journal Current Directions in Biomedical Engineering Volume 2 Issue 1

Abstract

The effects of alkaline pH on the initial adhesion of osteoblasts to titanium surfaces was analyzed by single cell force microscopy (SCFM). In the SCFM measurements, the same cells were used to compare their unspecific adhesion to uncoated titanium with their specific adhesion to collagen coated titanium. When the maximum detachment forces (MDFs) were compared at pH 7.4 and 8.0, only slight differences were found on pure titanium, while the MDFs were significantly increased at collagen coated surfaces at pH 8.0. Effects on the subsequent proliferation and gene expression were investigated in an in vitro model system consisting of an alkalizing polyvinyl alcohol (PVA) matrix and a perforated titanium disc. The sodium hydroxide releasing matrix maintained the medium pH between pH 7.6 and pH 8.4 during the entire experiment. Under these conditions, cell counts were significantly increased with respect to the control system after 7 days in culture. These results were supported by gene expression analyses, which showed an upregulation of proliferation-controlling genes of the EGFR1 and PI3K/AKT pathways after 14 days in culture. The SCFM data were complemented by findings of an intensive regulation of genes known to be associated with focal adhesion such as Itga8 and Tnn.

Keywords: alkalosis; osseointegration; osteoblast

1 Introduction

Development and screening of new biomaterials are a focus of orthopaedic implant technology. Besides the mechanical properties of the materials, the interactions of the living cells with the implant surface are crucial for the successful integration of the implant. To this end, modifications of the implant components and functionalization of their surfaces became pivotal for implant technology [1].

It is well known that pH plays a substantial role in the control of bone remodelling [2]. For example, metabolic alkalosis is inhibiting bone resorption and stimulates osteoblastic collagen synthesis [3]. Nevertheless, the development of implants that modify the local pH environment are not yet focus of research. Recently, we could show that osteoblast in vitro proliferation was severely increased at alkaline pH [Galow et al. submitted]. Our findings let us conclude that implants, which alkalize the peri-implant zone will be beneficial for bone regeneration and improve osseointegration. However, before animal testing or clinical studies in human patients more thorough in vitro studies are required.

2 Material and methods

2.1 Cell culture

Osteoblast-like MC3T3-E1 cells were obtained from the German collection of microorganisms and cell culture (DSMZ, Braunschweig, Germany) and cultured at 37°C in alpha MEM, supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (all purchased from Biochrom AG, Berlin, Germany) in 25 cm² cell-culture flasks (Greiner bio-one, Frickenhausen, Germany).

For determining the initial adhesion, cells were trypsinated and diluted in PBS to approximately 10.000 cell per milliliter. For the determination of cell growth on the modified implant surface, 50.000 cells per well of a 12-well plate were seeded onto perforated titanium discs and cultured in alpha MEM over an experimental period of 7 days. Half of the medium was exchanged after 4 days in culture to guarantee for sufficient nutrient supply and to remove accumulating waste products.

2.2 Determination of initial adhesion

Four titanium spots (5-mm diameter) were sputtered onto round 32 mm glass cover slips (Menzel-Gläser, Braunschweig, Germany). One spot per cover slip was coated with collagen A by applying a 10-μl droplet of a collagen solution before incubating the cover slips at 37°C for 30 min. Prior to the SCFM measurements, Arrow TL-1 cantilevers (Nanoworld, Switzerland) were functionalized with poly-dopamine. This was archived by carefully submerging each cantilever in a 100 μl droplet of PBS. Finally, 2 μl of a dopamine hydrocloride (Sigma Aldrich, St. Louis, USA) solution (2 mg/ml DOPA-HCl, 5% acetic acid) was added. The formation of poly-dopamine was induced by adding 2 μl of sodium hydroxide (2 M) and incubation at room temperature for 30 min. Cantilever calibration was performed by the thermal noise method above non-coated areas of the cover slips before transferring the cell suspension. Immediately after transfer, the cantilever was aligned above a single cell and approached in the cell-capture mode. After 30 sec, the cantilever was fully retracted and kept in this position for 10 min to ensure a firm contact of the cell. The MDF measurements were started by aligning the cantilever above a randomly chosen surface. For the measurements, a set-point of 1 nN was chosen. Approach and retract velocities were set to 5 μm/s together with a contact time of 5 s. At least 20 MDFs were determined for each surface. To modify the pH for measurements with the same cell, the petri-dish was rinsed with PBS of pH 7.4 and 8.0, respectively.

2.3 Makeup of the implant model

Titanium discs with a diameter of 21 mm were cut out from a 150 μm thick titanium sheet to fit 12-well plates. Fifty micrometer holes were processed at rectangular distances of 500 μm with an ultrashort-pulsed IR laser. In our system, the cells were cultured on the discs, which were placed onto alkalizing matrices.

For the matrices, a new polymer electrolyte was used, which was originally established for battery cells by Wu et al. [4]. For biocompatibility, sodium hydroxide (NaOH) was used instead of the high original potassium hydroxide concentration in the synthesis. For preparing the gel, 6 g poly vinyl alcohol, 3 g titanium dioxide, 2.39 g NaOH and 60 ml destilled water were mixed to obtain 1-M NaOH electrolyte gel sufficient for a 60 cm² petri dish. The gel was dried in the petri dish at 40°C for 24 h. Before used in cell culture, the gels were removed from the petri dishes and either soaked with 1 M NaOH solution or distilled water (control). To remove the vast majority of NaOH, each control gel was submerged in 1 l distilled water, which was replaced twice a day for at least 5 days. Prior to cell culture experiments, the NaOH-loaded and control gels were rinsed with PBS and incubated in cell culture medium overnight, to remove excess NaOH and to equilibrate the osmolarities at their surfaces. To evaluate the stability of the systems during the experiments, the degree of alkalization was checked by measuring the pH prior to every proliferation test (n = 15) at days 1, 3, 5 and 7 (Figure 1).

Figure 1:

Medium pH in the in vitro systems during cell culture.

2.4 Determination of proliferation

To evaluate cell proliferation, cell counts were determined microscopically with a Neubauer cell-counting chamber. For cell trypsinizing, the titanium discs were transferred into gel-free 12-well plates to avoid adverse pH effects on the enzyme.

2.5 Determination of gene expression

After 7 days in culture, media were supplemented with 10 mg/ml β-glycerol phosphate and 10 ng/ml calcitriol in order to restrict proliferation and to support the differentiation of the osteoblasts [5], [6]. For gene expression analysis, the cells were harvested after 14 days in culture, lysed and further processed for GeneChip Mouse Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturers protocol. Five cell samples each were measured from the alkalizing systems and the non-alkalizing controls. The raw data was analyzed with the Transcriptome Analysis Console v3.0 software of Affymetrix.

3 Results

3.1 pH-dependent initial adhesion

The MDFs of individual cells were measured at pH 7.4 and 8 on collagen A-coated and non-coated titanium. Scatter was reduced by normalising the MDF data of each individual cell measured at pH 8.0 to its MDF data measured at pH 7.4 for coated and non-coated surfaces. At pH 8, the mean MDF was insignificantly increased to 164 ± 86% (Student’s t-test: p = 0.087) on pure titanium, while it was significantly increased to 284 ± 194% (p < 0.05) on collagen A (Figure 2).

Figure 2:

MDFs of eight cells, each of which were measured on pure and collagen-coated titanium at pH 7.4 and 8.0. The MDFs are plotted relative to their values at pH 7.4 (p < 0.05).

3.2 Modification of medium pH in the in vitro model

In the alkalizing model systems, the pH was stabilized around pH 8.05 after 1 day in culture. In the control systems, the pH was slightly elevated with respect to pH 7.4, stabilizing around pH 7.5. In the control system, cell metabolism lead to a steady decrease of pH. In the alkalizing model system, the additional pH-decrease induced by the exchange of the alkalized medium with fresh standard culture medium at day 4 could be compensated by the matrix at day 7 (Figure 1). As a result, the pH was maintained above pH 7.8 throughout the entire experimental period, proving the usability of our new alkalizing matrix for stabilizing the pH in biological systems.

3.3 Cell counts in the implant model

Cell counts were determined at days 1, 3, 5 and 7. In the alkalizing implant model system, higher cell counts were recognizable at day 3 and became significant at day 7. Beyond the biocompatibility of our new system, these results prove its beneficial properties (Figure 3).

Figure 3:

Relative cell counts normalized to the counts in the control system at day 3 (n = 15; p < 0.05).

3.4 Gene expression in modified implant model

Genes were considered regulated when their fold changes were higher than two with respect to the non-alkalizing control systems. In the alkalizing systems, we found 252 out of approximately 30,000 detected genes to be regulated, with 169 genes being upregulated and 83 genes being down-regulated. The regulated genes could be related to pathways of three main areas: adhesion, proliferation, and DNA repair. For example Proteins involved in the formation of focal adhesions such as Integrin alpha 8 (fold change: 2.81; p < 0.001) were upregulated. Upregulated genes upstream of the PI3K-Akt pathway are related to proliferation, such as the c-fos induced growth factor (fold change: 2.64; p < 0.001). Proliferation-related are also other upregulated gene products, which belong to the EGFR1 signalling pathway. EGFR1 itself, which had a fold change of 2.44, is a receptor for various growth factors, influencing cell proliferation and cell survival. Besides these proliferation supporting gene products, genes known to be involved in DNA repair were upregulated, including members of the p53 pathway such as cyclins, which act at the control points of mitosis. Upregulation of these pathways hints at mechanisms preventing malignant degeneration during enhanced cell proliferation.

The expression of typical osteoblast markers, like those of the bone morphogenetic proteins (BMPs) family, was largely unchanged, with significant differences only for BMP4 and BMP6. While BMP6 was minimally downregulated in the alkalizing system (fold change: −1.07; p = 0.041), BMP4 was slightly upregulated (fold change: 1.3; p = 0.023). The Runt-related transcription factor 2 (Runx2), which is of major relevance for osteoblast differentiation, was minimally upregulated (fold change: 1.07; p = 0.047). Nevertheless, some genes regulated by Runx2 were down-regulated, such as Bglap and Alpl. However, other gene products reported to enhance the osteoblastic phenotype and mineralization, such as OGN [7] were upregulated. OGN (fold change: 2.02; p < 0.001) belongs to class III of the small leucine-rich proteoglycans. It was initially isolated from bovine bone as an inducer of matrix mineralization [8]. Because bone regeneration does not solely rely on osteoblasts, but rather on the balanced interaction of osteoblasts and osteoclasts, also factors had to be included in the analysis, which act on osteoclasts but are secreted by osteoblasts, namely RANKL and OPG. Their ratio determines bone formation and degradation [9]. While the gene regulation of OPG was insignificant, the RANKL gene was downregulated (fold change: −1.31; p = 0.003) in the alkalizing model system resulting in a shift in the RANKL-OPG ratio.

4 Conclusion

We could demonstrate the beneficial effects of alkalizing the peri-implant zone by three independent in vitro approaches. Under alkaline conditions, SCFM measurements showed a significantly increased specific adhesion to collagen surfaces and a tendency of enhanced unspecific adhesion to pure titanium. These findings suggest an enhanced receptor-mediated specific adhesion. In our in vitro model, the NaOH release was sufficient to alkalize the medium for up to 7 days, showing the feasibility of stable alkalinization of the peri-implant zone in vivo.

Proliferation was significantly enhanced under alkaline in vitro conditions, suggesting an improved osseointegration in vivo.

Gene expression analysis showed the pathways for DNA repair, as well as adhesion and proliferation control to be most severely regulated, thereby supporting the data gained by SCFM measurements and the cell number determination. In vivo, the observed shift in the RANKL-OPG ratio would suppress osteoclast activity, thus reducing matrix degradation.

Our results suggest an improved bone regeneration and osseointegration in possible medical applications of our system.

Acknowledgement

We thank Paul Oldorf (SLV GmbH, Rostock) for processing the titanium discs and Dr. Dirk Koczan (Institute für Immunlogie, Rostock) for preparing the GeneChip arrays.

Author’s Statement

Research funding: The work was funded by DFG, Graduate School “welisa” DFG 1504/2. Conflict of interest: The authors state no conflict of interests. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.

References

[1] Wang W, Ouyang Y, Poh CK. Orthopaedic implant technology: biomaterials from past to future. Ann Acad Med Singap. 2011;40:237–44.10.47102/annals-acadmedsg.V40N5p237Search in Google Scholar

[2] Arnett TR. Extracellular pH regulates bone cell function. J Nutr. 2008;138:415S–8S.10.1093/jn/138.2.415SSearch in Google Scholar

[3] Bushinsky DA. Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Physiol Ren Fluid Electrolyte Physiol. 1996;40:F216.10.1152/ajprenal.1996.271.1.F216Search in Google Scholar

[4] Wu Q, Zhang J, Sang S. Preparation of alkaline solid polymer electrolyte based on PVA–TiO2–KOH–H2O and its performance in Zn–Ni battery. J Phys Chem Solids. 2008;69:2691–5.10.1016/j.jpcs.2008.06.132Search in Google Scholar

[5] Fratzl-Zelman N, Fratzl R, Hörandner H, Grabner B, Varga F, Ellinger A, et al. Matrix mineralization in MC3T3-E1 cell cultures initiated by β-glycerophosphate pulse. Bone. 1998;23:511–20.10.1016/S8756-3282(98)00139-2Search in Google Scholar

[6] Jørgensen NR, Henriksen Z, Sørensen OH, Civitelli R. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids. 2004;69:219–26.10.1016/j.steroids.2003.12.005Search in Google Scholar

[7] Tanaka K, Matsumoto E, Higashimaki Y, Katagiri T, Sugimoto T, Seino S, et al. Role of osteoglycin in the linkage between muscle and bone. J Biol Chem. 2012;287:11616–28.10.1074/jbc.M111.292193Search in Google Scholar

[8] Bentz H, Nathan RM, Rosen DM, Armstrong RM, Thompson AY, Segarini PR, et al. Purification and characterization of a unique osteoinductive factor from bovine bone. J Biol Chem. 1989;264:20805–10.10.1016/S0021-9258(19)47133-0Search in Google Scholar

[9] Khosla S. Minireview: the OPG/RANKL/RANK system. Endocrinology. 2001;142:5050–5.10.1210/endo.142.12.8536Search in Google Scholar PubMed

Published Online: 2016-9-30

Published in Print: 2016-9-1

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Articles in the same Issue

https://doi.org/10.1515/cdbme-2016-0121

Keywords for this article

alkalosis; osseointegration; osteoblast

Creative Commons

BY-NC-ND 4.0