Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.

Abstract Custom‐made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA‐induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast‐activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

polymerize, during which time monomer linking and subsequent formation of solid polymer occur. The MMA monomer is known to be cytotoxic (Ansteinsson, Kopperud, Morisbak, & Samuelsen, 2013;Hattori et al., 2008;Jinno et al., 2006;Schweikl, Schmalz, & Spruss, 2001). Although the mixing and initial polymerization process occurs outside the implantation site, it is unknown whether PMMA that has not completely polymerized has adverse effects when it comes into contact with the bone tissue at the implant site.
The use of PMMA has two main disadvantages, that is, the highly exothermic reaction during the polymerization process, and the potential release of non-reacted monomers, which may cause damage to the surrounding bone tissue after PMMA implantation (Dunne & Orr, 2002). Aseptic loosening of PMMA implants caused by monomermediated bone damage has been reported previously, as well as neurotoxicity due to the monomer, and lack of osteointegration, because of the bio-inert properties of the material (Dahl, Garvik, & Lyberg, 1994;Heini & Berlemann, 2001;Jaeblon, 2010). Although implant failure has been reported to occur, the potential adverse effect of PMMA bone cement on the surrounding bone tissue has to be weighed against the advantageous high mechanical strength and stability provided by the material.
Mesenchymal stem cells (MSCs) and osteoblasts present within the tissue surrounding a bone defect are potentially exposed to cytotoxic factors leaching from the implanted PMMA. In vivo, MSCs migrate to the implant site, where they differentiate into an osteoblastic phenotype (Verrier et al., 2012). PMMA particles have been shown to inhibit osteoblast proliferation and collagen synthesis which may result in reduced periprosthetic bone formation, whereas PMMA particles stimulate osteocalcin and IL-6 synthesis, known to stimulate bone resorption (Ishida & Amano, 2004;Kudo et al., 2003;Zambonin, Colucci, Cantatore, & Grano, 1998). In addition, in a murine bone marrow cell culture in vitro, PMMA particles added to mature osteoclasts result in an increase in the number of TRAP-positive cells which persisted over a longer time period, and increased bone resorption (Zhang et al., 2008). Thus, PMMA may affect osteoblast and osteoclast activity in a way that could contribute to peri-prosthetic osteolysis.
Whether PMMA as a bulk material can adversely affect cells present in the bone tissue surrounding the implant, for example, through the leaching of non-reacted monomers, is still unknown. Knowledge about possible adverse effects is highly relevant as implanted PMMA material is in direct contact with the bone tissue where MSCs and osteoblasts are present. Therefore, the aim of our study was to investigate whether PMMA affects the osteogenic potential, and the expression of signaling molecules that regulate osteoclast activity in hASCs and/or osteoblasts. We measured lactate dehydrogenase (LDH) levels, metabolic activity, proliferation, and expression of osteogenic differentiation and osteoclast-activation markers by both cell types, in comparison to control cells without PMMA. We hypothesized that PMMA is cytotoxic and adversely affects metabolic activity, proliferation, osteogenic potential, as well as osteoclast activation potential by hASCs and hOBs.

| Preparation of PMMA
Commercially available PMMA was obtained from two companies (PMMA bone cement, Antibiotic Simplex ® ; Stryker Orthopaedics, Mahwah, New Jersey, and Rapid Simplified; Vertex dental BV, Zeist, The Netherlands). In our study we used PMMA samples polymerized at distinct temperatures and for different time periods, that is, PMMA polymerized at room temperature for 15 min, at 60 C for 5 min, or at 100 C for 20 min. Increasing polymerization temperature of PMMA has been shown to decrease residual monomer content of polymers, and to improve PMMA compatibility (Vallittu, Ruyter, & Buykuilmaz, 1998). For experiments, PMMA was prepared according to the manufacturer's instructions. Briefly, the liquid monomer (methylmethacrylate-styrene) was mixed with the powder polymer PMMA at room temperature, and molds were filled to obtain a disk-shaped material (diameter: 1 cm, height: 0.2 cm). PMMA samples were allowed to polymerize at room temperature for 15 min, at 60 C for 5 min, or at 100 C for 20 min.

| TheraCal LC ®
TheraCal LC ® (Bisco, Schaumburg, Illinois) consists of tri-calcium silicate particles in a hydrophilic monomer such as hydroxyethyl methacrylate (HEMA) and polyethylene glycol dimethacrylate (PEGDMA) (Cannon, Martin, Suh, & Yin, n.d.). These monomers have been reported to be cytotoxic (Chang et al., 2005;Orimoto et al., 2013). Therefore, we included TheraCal LC ® in our experiments as an internal positive control. TheraCal LC ® was used according to the manufacturer's instructions. Briefly, TheraCal LC ® consisting of a single paste was placed into a mold to obtain a disk-shaped material (diameter: 1 cm, height: 0.2 cm), and light cured using an UV-lamp (3 M ESPE Elipar™ S10, St. Paul, Minnesota).  (Varma et al., 2007). Adipose tissue represents a promising source of MSCs, as liposuction can be performed with minimal patient discomfort and yields higher numbers of MSCs than bone marrow. In addition, a previous study by our group has demonstrated the safety, feasibility, and efficacy of the use of hASCs in human maxillary sinus floor elevation, and the pro-angiogenic effect of ASC-containing stromal vascular fraction (Farre-Guasch et al., 2018).

| Isolation and culture of hOBs
For all experiments, a pool of cells obtained from two donors at P2 was used.

| Platelet lysate
Pooled platelet products from five donors were obtained from the Bloodbank Sanquin (Sanquin, Amsterdam, The Netherlands) and contained approximately 1 × 10 9 platelets per ml (Prins et al., 2009). PL was obtained by lysing the platelets through temperature shock at −80 C. Before usage PL was thawed and centrifuged at 600g for 10 min to eliminate remaining platelet fragments. The supernatant was added at 2% (v/v) to the medium.

| Cytotoxicity assay
The potential cytotoxic effect of PMMA on hASCs and/or hOBs was assessed by measuring LDH (Roche, Mannheim, Germany). After 1 day of culture of hASCs and hOBs with PMMA, supernatants were collected and incubated with reaction mixture for 30 min at room temperature. The LDH catalyzed conversion results in reduction of tetrazolium salt to formazan, which was measured at 490 nm in a Synergy HT ® spectrophotometer (BioTek Instruments, Winooski, Vermont). The amount of LDH release is proportional to the number of lysed cells. Cytotoxicity of PMMA on hASCs and hOBs expressed as percent LDH activity was determined relative to controls as described by the manufacturer. Cells treated with 1% Triton-X100 were used as positive (maximum) LDH release control.

| Metabolic activity assay
Metabolic activity of hASCs and hOBs cultured with PMMA was assessed by using AlamarBlue™ Cell viability reagent (Invitrogen, Rockford, IL, USA). Metabolic activity of hASCs and hOBs was analyzed at 1 and 7 days. Cells were incubated with medium containing 10% AlamarBlue for 4 hr at 37 C. Following incubation, 100 μL supernatant was transferred into a black 96-well plate and fluorescence was measured at 530-560 nm wavelength in a Synergy HT ® spectrophotometer. Medium without cells containing 10% AlamarBlue was placed in an autoclave container, and used as positive control of 100% chemically reduced AlamarBlue solution.
2.9 | DNA content quantification hASCs and hOBs cultured for 1 or 7 days with PMMA were washed with PBS, and lysis buffer was added. DNA content as a measure of total cell number was determined using the Cyquant Cell Proliferation Assay Kit (Molecular Probes, Leiden, The Netherlands). Absorption was read at 485 nm excitation and 528 nm emission with a Synergy HT ® spectrophotometer. With LightCycler ® (version 1.2), crossing points were assessed and plotted versus the serial dilution of known concentrations of the internal standard. mRNA preparations of hASCs and hOBs were used as a reference and internal standard in each assay. PCR efficiency (E) was obtained by using the formula E = 10 -1/slope . Data were used only if E = 1.85-2.00. For gene expression analysis, the values of target gene expression were normalized to reference genes GUSB, TBP, and YWHAZ based on Bestkeeper software (Pfaffl, Tichopad, Prgomet, & Neuvians, 2004). The relative expression of a gene of interest was calculated in relation to the reference gene on basis of the crossing point (Cp) deviation (delta Cp). qPCR was used to assess expression of the following genes: KI67, RUNX2, COL1, osteocalcin, alkaline phosphatase (ALP), OPG, and RANKL. All primers used were from Life Technologies (Invitrogen, Thermo fisher Scientific, Eugene, OR, USA). Primer sequences are listed in Table 1. 2.11 | ALP activity hASC and hOBs cultured for 1 or 7 days with PMMA were lysed with 500 μL lysis buffer and stored at −20 C prior to use. ALP activity was measured in the cell lysate using 4-nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3 as a substrate, according to the manufacturer's instructions. The absorbance was read at 405 nm with a Synergy HT ® spectrophotometer. ALP activity was T A B L E 1 List of primer sequences used for analysis of proliferation, expression of osteogenic markers by hASCs and hOBs, and signaling markers for osteoclast differentiation by PCR Abbreviations: ALP, alkaline phosphatase; COL1, collagen-type 1; GUSB: β-glucuronidase; KI67, proliferation marker; OC, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand; RUNX2, runt-related transcription factor-2; TBP, TATA-box binding protein; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta.
quantified against a standard curve of 4-nitrophenol solution and expressed as μmol 4-nitrophenol per ng DNA.

| Mineralization
Matrix mineralization was analyzed by alizarin red staining of hASCs or hOBs cultured with or without PMMA at day 14, by using 2%

| Expression of osteogenic markers by hASCs and hOBs
The effect of PMMA on the osteogenic differentiation potential of  Quantification of the mineralized matrix showed that PMMA did not inhibit hASCs mineral deposition (Figure 5d).
In hOBs, PMMA did not inhibit ALP gene expression at day 1 and 7, neither ALP activity at day 7 (Figure 6a,b). Mineral deposition was enhanced in hOBs cultured with PMMA, polymerized at distinct temperatures at day 14 ( Figure 6c). Quantification of mineralized matrix did not show increased mineral deposition by hOBs cultured with PMMA compared to controls where mineralization was low or not detected (Figure 6d).

| Markers of osteoblast-to-osteoclast communication
To study the effect of PMMA, polymerized at distinct temperatures, on osteoclast-activation markers by hASCs or hOBs, gene expression analysis of RANKL and OPG was performed. An increased RANKL/ OPG ratio is considered to favor bone resorption (Lacey et al., 1998).
PMMA polymerized at room temperature enhanced RANKL expression by hASCs by 2.9-fold at day 1. There were no significant differ-

| DISCUSSION
Custom-made PMMA bone cement is frequently used in craniomaxillofacial surgery for the treatment of large cranial defects due to its high mechanical strength and stability. Despite positive clinical outcomes, there is still ongoing debate whether the potential release of non-reacted monomers by PMMA cause adverse, cytotoxic effects on the bone tissue surrounding the implanted PMMA (Belkoff & Molloy, 2003;Dahl et al., 1994;Urrutia, Bono, Mery, & Rojas, 2008).
MSCs recruited to the implant site, and osteoblasts in the surrounding bone tissue may be affected by the release of non-reacted monomers.
Thus, MSCs and osteoblasts are highly relevant for the in vitro evaluation of possible cytotoxic effects of PMMA. We hypothesized that PMMA is cytotoxic by releasing non-reacted monomers thereby adversely affecting metabolic activity, proliferation, osteogenic and osteoclast activation potential by human hMSCs and hOBs. We found that PMMA did not cause a biological relevant reduction in viability, metabolic activity, proliferation, and expression of osteogenic differentiation markers in hASCs and hOB. PMMA also did not enhance expression of osteoclast-activation markers in hASCs that were grown in the presence of discs of PMMA, polymerized at distinct temperatures, in order to manipulate the potential release of nonreacted monomers.
In our experiments we included TheraCal LC ® as an internal positive control for monomer cytotoxicity. TheraCal LC ® contains hydrophilic monomers such as HEMA and PEGDMA. These hydrophilic monomers have been reported to reduce cell viability and to induce cell cycle perturbation in peripheral blood mononuclear cells obtained from healthy and HEMA-sensitized patients, and in murine RAW cells in a dose-dependent manner (Chang et al., 2005;Orimoto et al., 2013). Our results showing that TheraCal LC ® increased LDH activity and decreased metabolic activity and DNA content in hASCs and hOBs at day 1 and 7 agree with these studies. The use of TheraCal LC ® as a positive control for cytotoxicity strengthens our findings that PMMA is not cytotoxic for MSCs and hOBs, as it shows that our assays are sensitive enough to measure adverse effects of monomers.
Polymerization temperature and time considerably affect the residual monomer content of polymers. Increasing the polymerization temperature in denture base resins from 30 to 60 C has been shown to decrease residual monomer of the polymer (Vallittu et al., 1998). In addition, when heat-cured denture base resins polymerized at 70 C are allowed to polymerize for an additional period at 100 C, then the concentration of residual monomers of the polymer are significantly reduced when compared with a resin polymerized at 70 C only (Vallittu et al., 1998) (Ayre, Denyer, & Evans, 2014). PMMA polymerized at room temperature enhanced the expression of RANKL as well as OPG in hASCs at day 1. The increased expression of RANKL and OPG did not result in differences in RANKL/OPG ratio, which indicates that production of osteoclasts factors which promote osteoclast activation are not enhanced by PMMA. PMMA polymerized at 100 C decreased OPG expression in hOB only at day 1, which did not result in differences in RANKL/OPG ratio. PMMA polymerized at room temperature enhanced OPG gene expression, and similar to PMMA polymerized at 60 C, it decreased the RANKL/OPG ratio in hOB, indicating that osteoclast activation potential may be decreased. Thus, PMMA does not appear to induce osteoclast activation potential by hOBs and hASCs, and therefore PMMA is unlikely to enhance bone resorption at the implant site, although further in vivo studies using PMMA should be performed to unequivocally demonstrate that PMMA do not induce bone resorption.
The present study has some limitations. The measurement of the osteogenic differentiation markers was performed at the level of mRNA expression rather than protein. However, the results of gene expression of osteogenic markers were in agreement with the results of ALP activity measurement and bone nodule formation, strengthening the conclusion that PMMA does not affect osteogenic differentiation of hOBs or hASCs. Although we did not perform osteoclast culture and bone resorption assays in our experiments, we analyzed the expression of RANKL and OPG in hASCs and hOBs. RANKL and OPG are widely acknowledged as key regulators of osteoclastogenesis (Kobayashi, Udagawa, & Takahashi, 2009). The study of the effect of the exothermic reaction during PMMA bone cement is highly relevant and therefore future studies should address this issue. The study of the exothermic reaction during PMMA polymerization was out of the scope in our study.
In conclusion, our results show that PMMA is not cytotoxic, and does not interfere with the osteogenic differentiation potential of hASCs and hOBs, even when polymerization of the material occurs at distinct temperatures. In addition, PMMA does not enhance production of osteoclast regulatory markers by hASCs and hOBs in vitro.
Thus, in contrast to our hypothesis, our data suggests that PMMA, polymerized at distinct temperatures, does not inhibit bone formation.
Therefore, these data support the notion that PMMA bone cement is suitable for the treatment of critical size cranial defects.
F I G U R E 8 Effect of polymethyl methacrylate (PMMA) on RANKL and OPG gene expression in human osteoblasts (hOBs). hOBs were cultured with PMMA polymerized at room temperature, at 60 C, or at 100 C. (a) RANKL gene expression at day 1 and 7. (b) OPG gene expression of at day 1 and 7. (c) RANKL/OPG ratio at day 1 and 7. Values are mean ± SEM of duplicate cultures from three independent experiments (n = 3) using bone cells obtained from two donors. *Significant effect of PMMA, p < .05