The high stiffness of acrylic bone cements has been hypothesized to contribute to the increased number of fractures encountered after vertebroplasty, which has led to the development of low-modulus cements. However, there is no data available on the in vivo biocompatibility of any low-modulus cement. In this study, the in vitro cytotoxicity and in vivo biocompatibility of two types of low-modulus acrylic cements, one modified with castor oil and one with linoleic acid, were evaluated using human osteoblast-like cells and a rodent model, respectively. While the in vitro cytotoxicity appeared somewhat affected by the castor oil and linoleic acid additions, no difference could be found in the in vivo response to these cements in comparison to the base, commercially available cement, in terms of histology and flow cytometry analysis of the presence of immune cells. Furthermore, the in vivo radiopacity of the cements appeared unaltered. While these results are promising, the mechanical behavior of these cements in vivo remains to be investigated.
Poly(methyl methacrylate) (PMMA) is a synthetic thermosetting polymer that has been used as the base to produce bone cements for orthopedics since the 1960s, when Charnley first reported its use to anchor endoprostheses to bone [
In spite of their success in vertebroplasty—they provide pain relief and stability to the fracture site—acrylic bone cements present some issues. Their high stiffness in comparison to that of cancellous bone results in a property mismatch that has been hypothesized to be a contributing factor to adjacent vertebral fractures occurring shortly after vertebroplasty [
Unsaturated fatty acids and their glycerol esters are both natural compounds that can be used to modify the properties of bone cements. For instance, Vázquez et al. used aromatic amines as well as acrylic monomers, both derived from oleic acid, to optimize the properties of bone cements [
The present work hence aimed to evaluate castor oil- and linoleic acid-modified low-modulus acrylic bone cements both in vitro, using an osteoblastic-like cell model, and in vivo, using a subcutaneous rat model. Cell viability was evaluated on human osteoblast-like Saos-2 cells and the in vivo response was evaluated using histology and flow cytometry after implantation in Sprague-Dawley rats. The radiopacity of the modified cements was confirmed using in vivo microtomography.
OsteopalV (OP, Heraeus Medical GmbH, Hanau, Germany) radiopaque bone cement for vertebroplasty was used as the base cement. 12.3 wt% (of total cement weight) castor oil (CO, Sigma Aldrich, 259853, St Louis, MO, USA) was used, corresponding to 1.78 g CO for 10.0 g of OsteopalV powder and 2845
Disc-shaped cement samples (
The cytotoxicity of unmodified OsteopalV and of the low-modulus cements was evaluated by an indirect contact assay in which cells were cultured with cement extract (medium having been in contact with cement). Human osteoblast-like Saos-2 cells (HPACC) were used as the cell model. The cells were maintained in cell culture flasks in an incubator with a humidified atmosphere of 5% CO2 in air at 37°C. DME/F-12 medium (Thermo Scientific HyClone, reference number SH300023.01, Logan, UT, USA) supplemented with 1% penicillin/streptomycin (Sigma Aldrich, reference number P4333, St. Louis, Mo, USA) and 10% foetal bovine serum (Thermo Scientific HyClone, reference number SV30160.03, Logan, UT, USA) was used as culture medium. The medium was exchanged every second day. Upon confluence, cells were detached with a minimum amount of trypsin 0.25% in EDTA (Thermo Scientific HyClone, reference number SH30042.02, Logan, UT, USA) that was inactivated with supplemented medium after 10 min.
Cement extracts were prepared by immersing a cement disk (
6500 Saos-2 cells were seeded in a 96-well plate (2 × 104 cells/cm2) and were cultured for 24 h before starting the cytotoxicity assay. After 24 h, media were replaced by cement extract, which was added to the cells as obtained (100%), diluted 4-fold (25%) and diluted 10-fold (10%). Complete media were used as negative control (C−), media containing 0.1% Triton X-100 (Merck, reference number 1.08603.1000) were used as positive control (C+), and wells without cells were used as blank. Four replicates were included per sample.
Cells were incubated either for 1 day or 3 days, and cell viability was tested by AlamarBlue assay (Invitrogen, reference number DAL1100, Carlsbad, CA, USA). For this purpose, cells were washed once with PBS and afterwards 200
The animal study was approved by the local ethical committee (Approval number C208/12). In total, 18 male Sprague-Dawley rats, weighing 400–450 g (Taconic Farms Inc., Denmark), were used. The animals were randomly distributed into three groups (3 time points, 1, 4, and 12 weeks), and all individuals received implants of all three material compositions. Table
Design of the in vivo study. For each animal the end point as well as number and type of implants is specified. Total number of implants per formulation was 48, with 16 samples per end point. Six–ten samples were used for flow cytometry and the rest for histology.
Animal | End point (weeks) | Number of OP implants | Number of OP + LA implants | Number of OP + CO implants |
---|---|---|---|---|
1 | 1 | 4 | 2 | 2 |
2 | 4 | 2 | 2 | 4 |
3 | 12 | 2 | 4 | 2 |
4 | 1 | 4 | 2 | 2 |
5 | 12 | 2 | 2 | 4 |
6 | 4 | 2 | 4 | 2 |
7 | 12 | 4 | 2 | 2 |
8 | 12 | 2 | 2 | 4 |
9 | 1 | 2 | 4 | 2 |
10 | 4 | 4 | 2 | 2 |
11 | 4 | 2 | 2 | 4 |
12 | 1 | 2 | 4 | 2 |
13 | 4 | 4 | 2 | 2 |
14 | 1 | 2 | 2 | 4 |
15 | 4 | 2 | 4 | 2 |
16 | 12 | 4 | 2 | 2 |
17 | 1 | 2 | 2 | 4 |
18 | 12 | 2 | 4 | 2 |
|
||||
|
48 | 48 | 48 |
The base material (unmodified OsteopalV) was used as the control. The rats were kept in pairs in Macron 4 cages, at the animal facility at Uppsala University Hospital, with daily monitoring by the animal facility personnel. The end points were chosen based on the three contact duration categories recommended for biomaterials and medical devices: (1) limited contact (<24 h), (2) prolonged contact (>24 h and <30 days), and (3) permanent contact (>30 days) [
The surgeries were performed under aseptic conditions. The rats were anaesthetized in an induction chamber with 5% isoflurane (Baxter, reference number KDG9623, Kista, Sweden), 0.3 L/min oxygen, and 0.7 L/min nitrous oxide for a few minutes and then transferred to an anesthesia mask (the anesthesia reduced to 1–2.5% isoflurane, 1.0 L/min oxygen, and 0.8 L/min nitrous oxide). One dose of 225 mg/kg antibiotics (Zinacef, GlaxoSmithKline AB, Sweden) was administered subcutaneously. The animals were placed on a heated pad (37°C) and the anterolateral back was shaved and disinfected with chlorhexidine (5 mg/mL; Fresenius Kabi, reference number 53 80 58, Uppsala, Sweden) and ethanol (70%). Eight cement discs (
The wound was closed intracutaneously with a resorbable 4.0 suture (Polysorb, reference number SL-691, Tyco Healthcare, Gosport, UK). Immediately after operation, the rat was given 1.0 mL physiological saline solution subcutaneously, to avoid dehydration. During the first two postoperative days, 0.05 mg/kg buprenorphine (Temgesic, reference number 08 61 88, Sheringer Plough, Brussel, Belgium) was administered subcutaneously for analgesia. The rats were allowed to move freely in the cages directly after surgery. At the end points the implantation sites were macroscopically assessed for presence of tissue reactions. The implants were then collected by careful dissection, along with 5 mm of surrounding tissue, by first separating the dermis and hypodermis from the underlying muscle and bone and then excising a circular piece of tissue with the cement disc in the center.
The cement disc and epidermis layer were gently removed using scalpels, and the remaining subcutaneous tissue was cut into small pieces and enzymatically digested at 37°C for 90 min, in an enzyme mixture containing 0.2% hyaluronidase (hyaluronidase from bovine testes, reference number H3506, Sigma) in PBS and 0.5% collagenase (crude collagenase from
Since acrylic bone cements are known as inert, permanent biomaterials a flow-cytometry-based method for evaluating only the surrounding tissue, and not the implant itself, was optimized from Ryhänen et al. [
The implant-tissue-complex was fixed in 4% phosphate buffered formaldehyde (reference number 02176, Histolab Products AB, Gothenburg, Sweden) at room temperature for 7–14 days. After fixation, the soft tissue at one end of the implant was cut and the cement disc was gently removed. The samples (soft tissue with implant removed) were mounted in paraffin, and several 7
The radiopacity of the modified materials was evaluated and compared to the base material (OP) to determine if the material modifications had an influence on this property. The samples were scanned with a microtomography (
Statistical analysis was performed in IBM SPSS Statistics version 21 (IBM, Chicago, IL, USA) using a one-way ANOVA at a significance level of
Figure
Viability of Saos-2 cultured for 1 and 3 days in 1, 6, 12, and 24 h extracts prepared with OP, OP + LA, and OP + CO. (a) Undiluted extracts; (b) 4-fold diluted extracts; (c) 10-fold diluted extracts. For each extract time,
In diluted 4-fold extracts (Figure
When 10-fold diluted extracts were used (Figure
All animals tolerated the surgery and the postoperative period well, and macroscopic assessment of the implant sites during the study period and at the end points showed no signs of tissue irritation or prolonged immune reactions, such as hematoma or edema.
The presence of leukocytes and the leukocyte subpopulations macrophages and granulocytes around the implantation sites was evaluated by flow cytometry and is presented in Figure
Evaluation by flow cytometry at 1, 4, and 12 weeks after implantation showed no statistical difference in the cell populations present in the tissue surrounding the modified material compared to the base materials. Immune cell populations are shown as mean percentage of entire cell population in each tissue sample. The error bars represent the standard deviations of the mean, with 6–10 replicates per group.
Assessment of histological sections stained with hematoxylin and eosin confirmed the macroscopic evaluation results. None of the material compositions caused any toxic reactions in the tissue surrounding the implantation sites. Also, no difference in tissue response between the base cement and the modified cements was visible, keeping in mind that the tissue surrounding the implants differs somewhat in composition (distribution of, e.g., fat and muscle tissue) between the implant locations, as the location in the body differs. Furthermore, for the assessed time points, no abnormal tissue organization could be seen at the implantation sites, apart from the necessary wound healing. At the later time points, the formation of a fibrous capsule had started around all implants. Representative histological sections are shown in Figure
Representative histological sections of tissue explants from 1, 4, and 12 weeks, cut horizontally and stained with hematoxylin and eosin. The implant space within each section is marked by an asterisk. Arrows indicate fibrous capsule. Scale bar applies to all sections.
Radiopacity and in vivo visibility of the modified materials were evaluated by
Representative scans of all material compositions in vivo (large picture) and ex vivo (small pictures). The modified materials have the same radiopacity as the base material and are equally visible in vivo. Five implants out of eight are observed in this image.
We have previously shown that fatty acids and triglyceride oils are able to substantially improve the mechanical properties of acrylic bone cements in terms of lowering their elastic modulus [
In the in vitro study, cells were incubated in the presence of cement extract. The extracts were evaluated undiluted as well as diluted 4- and 10-fold to more closely simulate the in vivo conditions, in which physiological fluid flows through the porous structure of cancellous bone within the vertebra [
The cytotoxic potential of PMMA cements has been known for a long time [
In this study, the response pattern observed was quite comparable to the earlier findings for subcutaneous models [
In summary, the low-modulus cements caused a decrease in in vitro cell viability in comparison to the nonmodified cement when using nondiluted cement extracts. However, for the worst-case, 6 h extracts, by diluting them only 4-fold, the cell growth showed no differences between samples and neither in comparison to the fresh media. In the in vivo study, the flow cytometry analysis and the histology results showed no significant differences between unmodified cement and the low-modulus cement samples. This is the first time low-modulus acrylic bone cements have been evaluated in an in vivo model. While these results are promising, the mechanical functionality of these types of cements remains to be evaluated in vivo.
In this study, we showed that two types of low-modulus PMMA-based bone cements have comparable in vitro cytocompatibility to commercially available conventional PMMA cement after only a small degree of extract dilution. Moreover, under in vivo conditions, all materials showed a similar biocompatibility and inflammatory response to conventional PMMA cement. The radiopacity of the cement also appeared unaffected by the modifications.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Funding from VINNOVA (VINNMER Project 2010-02073) is gratefully acknowledged. Gemma Mestres acknowledges VINNOVA (Project Grant no. 2013-01260) and Lars Hiertas Minne Foundation (Project no. FO2013-0337). Sune Larsson acknowledges funding from Inga Britt and Arne Lundbergs Forskningsstiftelse. Part of this work was performed at the BioMat Facility/Science for Life Laboratory at Uppsala University.