In Vitro and In Vivo Characterization of Premixed PMMA-CaP

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In vitro and in vivo characterization of premixed PMMA-CaP composite bone cements Shant Aghyarian, Elizabeth Bentley, Thao N. Hoang, Izabelle de Mello Gindri, Victor Kosmopoulos, Harry K. W. Kim, and Danieli C. Rodrigues ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00276 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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ACS Biomaterials Science & Engineering

In vitro and in vivo characterization of premixed PMMA-CaP composite bone cements Shant Aghyarian1, Elizabeth Bentley1, Thao N. Hoang1, Izabelle M. Gindri1, Victor Kosmopoulos2,3, Harry K. W. Kim4,5, Danieli C. Rodrigues* 1 1

Biomaterials for Osseointegration and Novel Engineering Laboratory (BONE Lab), Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080

2

Department of Orthopaedic Surgery, University of North Texas Health Science Center (UNTHSC), Fort Worth, TX

76107 3

Department of Materials Science and Engineering, University of North Texas, Denton, TX 76203

4

Center for Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children, 2222 Welborn Street, Dallas, TX

75219 5

Department of Orthopaedic Surgery, UT Southwestern Medical Center, Dallas, TX 75390

Corresponding Author: Dr. Danieli C. Rodrigues Email: [email protected] Tel: (972) 883-4703

Keywords: Pre-mixed Bone Cement, Composite, Calcium Phosphate, PMMA, Fracture Augmentation

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ABSTRACT

Acrylic bone cements although successful in the field of orthopedics suffer from a lack of bioactivity, not truly integrating with surrounding bone. Bioactive fixation is expected to enhance cement performance because of the natural interlocking and bonding with bone, which can improve the augmentative potential of the material in applications such as vertebroplasty (VP). In a recent study, two composite cements (PMMA-hydroxyapatite and PMMA-brushite) showed promising results demonstrating no deterioration in rheological and mechanical properties after CaP filler addition. In this study, the dynamic properties of the cements were investigated in vitro and in vivo. The hypothesis was that these composite cements will provide osseointegration around the implanted cement and increase new bone formation, thus decreasing the risk of bone structural failure. The effects of CaP elution were thus analyzed in vitro using these cements. Mass-loss, pore formation, and mechanical changes were tracked after cement immersion in Hank’s salt solution. PMMA-brushite was the only cement with a significant mass loss; however it showed low bulk porosity. Surface porosity increases were observed in both composite cements. Mechanical properties were maintained after cement immersion. In vitro culture studies tested pre-osteoblast cell viability and differentiation on the cement surface. Cell viability was demonstrated with MTT assay and confirmed on the cement surface. ALP assays showed no inhibition of osteoblast differentiation on the cement surface. In vivo experiments were performed using a rat tibiae model to demonstrate bone ingrowth around the cements implanted. Critical size defects were created and then filled with the cements. The animal studies showed no loss in mechanical strength after implantation and increased bone ingrowth around the composite cements. In summary, the composite cements provided bioactivity without sacrificing mechanical strength. 2 ACS Paragon Plus Environment

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Keywords: Composite Bone Cement, Vertebral Compression Fracture, Vertebroplasty, Osseointegration

INTRODUCTION

Osteoporosis is a degenerative disease that gradually affects bone over time. This age-related disease affects 50% of women and 25% of men above the age of 50 1,2. It is estimated that the size of the population aged over 50 years will increase about 200% between 1990 and 2025, correlating to a rise in fracture incidence 3. Bone mass is gradually reduced leaving behind weakened and brittle bones. The spine and hip, composed mostly of trabecular bone, experience continuous compressive loading and are subject to increased risk of failure. As osteoporotic fracture incidences increase with the aging population, so will the need for innovative orthopedic implants and bone augmentation solutions. Commercial bone cements are typically all acrylic and thus bioinert, being primarily composed of poly-methylmethacrylate (PMMA). These powder-liquid formulations are mainly used to provide fixation in orthopedic implants. This fixation is through mechanical locking which is susceptible to micromotion that can lead to implant loosening. Thus, a multitude of methods have been developed in order to decrease the risk of implant loosening through enhanced implant-bone interlocking 4–6. Techniques that provide surface porosity such as nanobrush coatings, porous resorbable metals, calcium phosphate coatings enhance biological fixation of surfaces by giving venues for cell anchorage and tissue growth within a porous matrix 4,5,7. Additionally, calcium phosphates when used as bulk material provide bioactivity because it involves crucial chemical interactions with the native tissue 4,5,7.Because of the lack of bioactive 3 ACS Paragon Plus Environment


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fixation with acrylic cements, calcium phosphate based cements have been attempted in different applications and investigational studies 8,15. Calcium phosphates (CaPs) have displayed desirable osseointegrative properties due to their compositional similarities with bone 8,12,13,15. Hydroxyapatite (HA) for example, is a calcium phosphate compound with near identical compositional similarities to the inorganic phase found in bone 6,7. Despite suitable biological properties, the application of calcium phosphate-based cements in load bearing applications may be challenging. A few previous studies have reported on the performance of CaP cements in vitro and in vivo. For example, the biocompatibility of the bioglass ceramic cement Cortoss® in comparison to a hydroxyapatite cement (Kyphos®) was investigated in vitro 12. The results showed that the polymer glass cement showed a reasonable cytotoxic effect with no signs of osteoblast cell function recovery within a period of 5 days in culture; whereas the calcium phosphate cement showed morphological signs of apatite formation 12. In a clinical study, the safety and efficacy of vertebroplasty using Cortoss was investigated 13. The study showed a 38% rate of asymptomatic cement leakage with 11.8% of patients experiencing significant complications. However, it was concluded that this material provides comparable efficacy and safety to PMMA augmentation 13. Other clinical studies have reported mixed results, with one particular case describing intra-cardiac Cortoss embolization 14, and another reporting effective augmentation using low volumes of the material 15. In a case study, Piazolla et. Al., reported observed that calcium phosphate cements used in vertebroplasty procedures were shown to result in future vertebral recollapse 9. PMMA-CaP composite cements combining the mechanical characteristics of acrylic cement with the bioactive properties of calcium phosphate could result in enhanced fixation and biological performance. Commercially available PMMA-CaP cement such as Vertecem V+, a highly filled


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cement containing 40% w/total w Zirconium dioxide and 15% w/total w hydroxyapatite, was investigated in vitro and in an animal model. Results showed cell-cement interaction in vitro with cell growth and differentiation followed by signs of osseointegration when implanted in an animal model 16. However, a limitation that could be expected from the addition of a calcium phosphate phase in an acrylic matrix is deterioration of the cement mechanical strength, handling and injectability, especially at high concentrations 10,11. The introduction of high CaP concentrations in traditional powder-liquid acrylic cements may lead to filler clumping due to inadequate mixing. The aggregates can create stress risers weakening the overall strength of the cement. Recent studies, however, have reported the successful development and characterization of pre-mixed bone cement system with the ability to completely incorporate high concentrations of CaP fillers in an acrylic PMMA matrix 17,18. The investigated pre-mixed cement system had its properties specifically tailored for use in minimally invasive spinal augmentation procedures such as vertebroplasty 17,18,19. In a vertebroplasty procedure, cement is directly injected into a collapsed vertebral body (VB), stabilizing it through stiffness restoration. The main complication with these procedures is cement extravasation out of the VB 20-22. Thus in order to decrease the risk of extravasation, a high cement viscosity is desirable. This however, decreases the ease of injection. The proposed composite cement system is advantageous in the sense that all components are pre-mixed and allowed to reach complete swelling resulting in a homogenous system. A previous study has demonstrated that these premixed cements can retain mechanical strength and pseudoplasticity, allowing for injection under pressure through the cannula while retaining a high viscosity upon reaching its target location in the VB 11. In summary, the pre-mixed composite cements investigated have shown superior handling properties and less extravasation risk compared to



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purely acrylic cements while successfully incorporating bioactive fillers. In addition to achieving properties suitable for vertebral compression fracture augmentation, the pre-mixed composite cements could incorporate CaP at greater concentrations compared to commercially available composite cements (e.g. 44.85 % wt/total wt) with no risk of phase separation due to the complete swelling achieved, and were able to achieve sufficient radiopacity with lower, 10%, radiopacifier concentration 17,18. In a previous study, the optical density of pre-mixed composite cements with 10% w/w ZrO2 radiopacifier was compared to a commercially available cement with 30% w/w BaSO4 radiopacifier, and demonstrated significantly higher optical density values 18

. In this study, two pre-mixed composite cement formulations, PMMA-HA and PMMA-

brushite, were chosen for investigation of osseointegrative potential using in vitro and in vivo experiments. The PMMA-HA cement was designed to provide a surface reactive phase to the otherwise bioinert PMMA structure. This is expected to induce osseointegration of the cement with the cancellous trabecular network, enhancing the augmentation 8,23,24,25. The PMMAbrushite formulation was designed to add a resorbability dynamic, allowing bone ingrowth through the cement scaffold. The ceramic phase of the cement can be released, leaving behind a porous network through the remaining acrylic scaffold. This cement dynamic can allow for bone regeneration and ingrowth to occur while maintaining mechanical strength in the cement implanted bone. The purpose of this study was to investigate the cell-cement interactions and performance post-implantation of the selected pre-mixed composite cement formulations to establish how their properties may change after injection into the VB. From mass-loss to cell and animal studies, the bioactivity and augmentative performance of these premixed composite cements was


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investigated. We hypothesized both the PMMA-HA and PMMA-brushite pre-mixed cements would maintain mechanical integrity in dynamic conditions, demonstrating biocompatibility, and enhanced bone ingrowth in vivo.

MATERIALS AND METHODS

All chemicals were used as received from manufacturers without any further modification. PMMA (Monomer-Polymer, Dajac Lab, Trevose, PA) (80,000 g mol) with particle size in the range of 100–200 mm in diameter was used as an aid to increase the viscosity of the cement mixture. Methyl methacrylate (MMA) (Fisher Scientific, Waltham, MA) was used as the monomer for the mixture. N,N-dimethyl p-toluidine (DMPT) (Sigma Aldrich, St. Louis, MO) and benzoyl peroxide (BPO) (Sigma Aldrich) were used as the activator and initiator, respectively, of the free radical polymerization reaction. Hydroxyapatite (HA) (Sigma Aldrich) and CaP dibasic dehydrate (Brushite) (Fisher) were used separately as the CaP filler additives in the cements. The Brushite and HA utilized have unreported particle size. The hygroscopic nature of these materials can lead to the formation of large clumps, which makes the characterization of individual particle size difficult. The manufacturers (Sigma Aldrich and Fisher) do not quantify the specific particle size for the materials due to the variability in water content, which can yield variations in particle size. Zirconium dioxide (ZrO2) (Fisher), with particle size in the range of 12–15 µm, was used as a radiopacifier.



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Cement Preparation The cements investigated here were developed as described in previous studies 17,26. The novel premixed cement system allows for the addition of CaP fillers to acrylic cement without deterioration in handling and mechanical strength. These properties are achieved due to the complete swelling achieved after pre-mixing the cement components in the dual barrel system developed. The cement setting process begins on injection and mixing of both sides of the cartridge allowing ample time for injection. The composite cement compositions (PMMA-HA and PMMA-brushite) were compared with purely acrylic premixed cement (PMMA), and a commercially available cement, Kyphon HV-R (Medtronic, Minneapolis, MN) (PMMA-K). Briefly, the composite cements were prepared at powder-to-liquid ratios of 1.65:1, and had equal concentrations of PMMA and their respective CaP fillers (0.74 g/mL or 44.85 % w/total w). PMMA cement was prepared with a powder-to-liquid ratio of 0.9:1 with a 0.89 g/mL PMMA concentration (maximum allowed ratio to enable handling and injection of the cement through cannulas as shown in previous work) 17. The cements contained a fixed concentration of BPO, DMPT, and ZrO2, at 1.25% w/v, 0.7% v/v, and 10% w/w respectively (w/w or w/v referring to the percent amount of the compound in relation to the PMMA weight or MMA volume). The composite formulations were selected based on initial rheological and mechanical characterization 17. The 10% w/w ZrO2 radiopacifier concentration was chosen based on previously performed contrast studies performed in porcine VBs, which demonstrated increased opacity compared to commercially available cement 18. PMMA-K has a reported powder-toliquid ratio of 2:1 with a 1.36 g/mL PMMA concentration and utilizes Barium sulfate as radiopacifier at 30% w/w concentration.


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Mass Loss The PMMA-brushite, PMMA-HA, and PMMA cements were evaluated for any mass loss. Cements were molded into bars (70 x 13 x 4 mm) for testing. Cements were immersed in Hank’s salt solution (HSS), maintained at 37°C, and placed in a shaker. The cements were weighed every 2 days after overnight drying at 80°C. After weighing, the samples were placed in solution for another 2 days. This was repeated until a mass-loss plateau was observed. The tests were performed in triplicates (i.e., 3 bone cements including the control, tested 3 times each, thus resulting in a total of 9 tested samples).

Pore Formation Pore formation in the composite cements was tracked using two methods: (1) microscopy to track surface pores; and (2) micro-CT for bulk porosity. Samples of 3 molded bars (70 x 13 x 4 mm) per group, were immersed in HSS and kept in a shaker at 37°C for 2 weeks. Samples were polished before the start of the experiment to eliminate larger preformed surface pores. A digital microscope (Keyence Corporation of America, Itasca, IL) was used to image surface porosity. Micro-CT (Skyscan 1172, Bruker, Kontich, Belgium) scans were performed at 90W of power, at 4.7 µm resolution, with exposure time adjusted according to threshold values. A 0.5-mm Al filter was used during the scans.

Mechanical Characterization In a previous study, we reported favorable mechanical behavior (compressive strength and modulus; flexural strength and modulus) of the developed composite cements 17. In this study, 9 ACS Paragon Plus Environment


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we investigated whether the mechanical strength is maintained after immersion in HSS solution for PMMA-brushite, PMMA-HA, and PMMA cement. A set of samples, within 2 hours of sample preparation, were tested to failure without being immersed in solution to characterize the pre-immersion properties. Another set of samples were placed in HSS and kept in a shaker at 37°C for 2 weeks and then tested without drying, with the samples taken directly from solution. Compression, flexural, fracture toughness, and cyclic fatigue tests were employed using a materials testing system (Bionix Model 370, MTS, Eden Prairie, MN) according to ASTM standards to determine the mechanical properties 27–30. The compressive tests were performed on 6 samples per test group. The cements were molded into cylinders (6 x 12 mm) and loaded at a displacement controlled rate of 20 mm min-1, with force and displacement recorded simultaneously. Previously reported values were used for the pre-immersion cements 17. Flexural 3-point bend tests were performed on cements (5 per group) molded into bars (70 x 13 x 4 mm). The samples were deflected at a rate of 2.15 mm min-1. Flexural stress, strain, and modulus were calculated. Strain at failure was also noted. Previously reported values were used for the pre-immersion samples 17. Fracture toughness cement samples (3 per group) were molded into single-edge notch bars (70 x 13 x 4 mm) with a 6 mm crack created along the width. The samples were placed in the 3-point bend fixture with the crack facing down and loaded at a rate of 10 mm min-1. The cyclic fatigue tests were performed in an environmental chamber filled with 1X PBS maintained at 37ºC. The composite cements, tested in triplicates, were molded into dumbbells and subjected to a fully reversible stress of 9MPa at 5 Hz for 5,000,000 cycles 30.


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Cell Culture Studies Cytotoxicity and cell differentiation staining procedures were performed on pre-osteoblast cells (MC3T3-E1 cells, ATCC, Manassas, VA) in contact with the cement formulations, according to ISO 10993-5 31. The cells were cultured in alpha minimum essential media supplemented with 10% fetal bovine serum that was replenished every 2 days and incubated at 37°C in a humidified atmosphere. For both cytotoxicity and differentiation assays three PMMA-HA samples, three PMMA-brushite samples, and three PMMA-samples were placed in each 24-well plate, with three empty wells per plate used as a positive control. Cement samples with surface area equal to 1% of the well area, (1 cm x 1 cm x 2 mm) were used to perform the experiments. Cells were seeded at a density of 50,000 cells/cm2 in each well. For the cytotoxicity tests, cement samples were placed in the culture plates after cells reached confluence. After 24 hours, the cells were stained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and then read at a wavelength of 570 nm (Plate reader, Biotek, Winooski, VT). For the alkaline phosphatase (ALP) cell differentiation assay, the cells were plated in cement-containing wells, as well as in empty wells (positive control) and allowed to grow for 7 days in non-differentiation media. An ALP assay kit (Abcam, Cambridge, MA) was used to stain the ALP which was detected at 405 nm wavelength. The MTT assay was performed at 7 days as well to normalize the ALP results at a cellular level. Cells were imaged using optical microscopy (Keyence, Itasca, IL) after ALP staining (Sigma).

In Vivo Experimental Model The animal studies were approved by the University of Texas at Dallas IACUC (15-02). Seventeen 9-11 weeks old male Lewis rats (Taconic, Hudson, NY) were used. The animals were 11 ACS Paragon Plus Environment


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housed in the Animal Facility at The University of Texas at Dallas, and cared for according to established protocols. The rats were exposed to a 12:12 hour light-to-dark cycle with free access to water and food. The testing groups were divided between the two composite cements, PMMAHA and PMMA-brushite, with the commercially available Kyphon HV-R (denoted as PMMAK) as the acrylic control. Positive and negative controls were also used, and were comprised of unoperated rats and an operated-nontreated rat, respectively. The rats were randomly organized into the following test groups: I [Positive Control, 3 rats], II [PMMA-K, 4 rats], III [PMMA-HA, 4 rats], IV [PMMA-brushite, 5 rats], and V [Negative control, 1 rat].

Surgical Procedure Surgery was performed according to IACUC approved protocols. The rats were anesthetized by injection of ketamine (25 µg/kg IM) and inhalation of 4% isoflurane. After shaving and disinfecting, the tibia was exposed by a 15 mm longitudinal incision below the knee. The tissue was reflected and the metaphysis of the tibia exposed. A full-thickness critical size defect ranging between 3-4 mm in diameter (according to tibia size) was created 10 mm below the knee using a drill and metal burr. This procedure was performed on both tibiae of the animal. The critical size defects were filled with cement according to each test group, as visualized in Figure 1. PMMA-K was mixed using the provided Kyphon kit and allowed to reach working phase prior to use. The composite cements were injected using static mixing nozzles. The three groups required additional packing of the cement in the defect with a spatula. The cements were allowed to set for 1 minute prior to suturing with non-resorbable sutures. For the negative control the created defects were left empty. After surgery the rats were allowed to move freely and received injections of sustained release buprenorphine for pain relief and baytril as prophylactic antibiotic. 12 ACS Paragon Plus Environment

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Biomechanical Testing After two months of surgical placement of cements, the test groups (I-IV) were euthanized (3 rats each for biomechanical) with Beuthanasia overdose and the tibiae were excised and prepared for testing by removing any remaining tissue. The tibiae were imaged using a Keyence VHX HDR microscope (Keyence Corporation of America, Itasca, IL) prior to testing to visualize the cement-bone contact. Three-point bend flexural tests were performed using a materials testing system (Model 370, MTS Systems Corporation, Eden Prairie, MN) with force and displacement recorded simultaneously in order to analyze strength and stiffness restoration. The tibiae (6 per group) were placed in the testing fixture with a 35 mm span length and subjected to a 0.5 mm/min deflection rate. The maximum strength (kN) and flexural stiffness (kN/mm) were compared between the test groups.

Histological Analysis The rats were euthanized with Beuthanasia injection after one month (1 PMMA-brushite rat and 1 negative control), and two months (1 rat per test group II-IV) of healing. Profusion was performed with 100 mL phosphate buffered saline (PBS) followed by 100 mL 4% paraformaldehyde prior to tibia excision. The tibiae were then fixed in 50% ethanol, dehydrated, and embedded in methylmethacrylate. The embedded tibiae were sectioned transversally (SM 2500, Leica, Buffalo Grove, IL) at 5 µm thickness and stained with Weigert’s iron hematoxylin and Gio’s trichrome (0.5 % phosphomolybdic acid, 1 % Orange g, 1 % light green, 2 % acid fuchsin, 1 % ponceau xylidine; dissolved in 1% acetic acid) 32. The expected color effects from 13 ACS Paragon Plus Environment


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the staining procedure were as follows: nuclei, dark blue; bone matrix, dark red; osteoid border, blood red; connective tissue, pink; synthetic bone substitute, bright green to turquoise 32. The stained tibiae were visualized using light microscopy (DP73, Olympus, Tokyo, Japan) and bone growth was evaluated around and within the created critical size defect.

Statistical Analysis One-way Analysis of Variance (ANOVA), with multiple comparison tests across values was performed to determine differences between groups at a confidence level of 95% using MATLAB (R2012a, Natick, MA).

RESULTS

Mass Loss The PMMA-HA, PMMA-brushite, and PMMA cements were studied in HSS for mass-loss in a 37°C shaker bath. PMMA-brushite, PMMA-HA, and PMMA showed average decreases in mass (g) of 6.62 ± 0.05 %, 0.07 ± 0.09 %, and 1.05 ± 0.56 % after 12 days, respectively (n=3). PMMA-brushite had a significantly higher mass-loss (p 0.05) effect on osteoblast differentiation compared to PMMA and the control (no cement). Osteoblast growth on the cement is clearly observed in Figure 3 (top) after ALP staining.

Biomechanical Testing The tibial defects were stabilized after 2 months for all test groups. Figure 4 depicts the proper contact between the cement and bone and the slight decrease in defect size. The augmented tibiae 15 ACS Paragon Plus Environment


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demonstrated comparable flexural strength and stiffness restoration to the control, as seen in Figure 5. The tibiae treated with the PMMA-HA and PMMA-brushite cements had average strength and stiffness values of 63.92 ± 11.98 kN and 93.88 ± 6.34 kN, and 67.06 ± 8.44 kN/mm and 90.59 ± 6.60 kN/mm, respectively (n=3). There was no significant difference (p>0.05) between any of the test groups. The post-testing tibiae are shown in Figure 6. The cracks through the bone were found to be close to the cement bone interface for all cements.

Histological Analysis After a one month healing period, only 1 tibia from the negative control test group were stabilized, with the other tibia having fractured at the defect site. The histology results for one month are shown in Figure 7 (top). The defect area was still observed in the stabilized negative control with no new bone around the area. For the PMMA-brushite cement, dark green lamellar bone was present around the defect. At a higher magnification (20x), a cutting cone was observed into the lamellar bone (area highlighted by a square), indicating bone remodeling (Figure 7). Figure 7 (bottom) shows the histology after two months of healing for the PMMA-K, PMMA-HA, and PMMA-brushite cements. Bone formation around the defect was observed for the PMMA-HA and PMMA-brushite composite cements but not PMMA-K cement. Bone growth through the cement body into the defect area was not observed.


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DISCUSSION

Understanding the dynamic interaction of newly developed cements with surrounding bone is important in predicting whether osseointegration, leading to stable fixation, will occur. A stepwise approach was taken in characterizing the multiple factors that can affect this process. Two composite cements with osseointegrative potential, PMMA-HA and PMMA-brushite, were tested (0.74 g/mL PMMA and 0.74 g/mL CaP concentration). Brushite, with its lower calcium to phosphate ratio is more resorbable than HA. Hence, PMMA-brushite was expected to provide venues for bone ingrowth into the cement matrix. The pore formation, however, may also impact the mechanical integrity of the cements, and was therefore tested after HSS immersion. The remaining acrylic matrix, after any CaP resorption, must be able to withstand the various loads despite any pore formation. We hypothesized in this study both the PMMA-HA and PMMAbrushite pre-mixed cements would maintain mechanical integrity in dynamic conditions, demonstrating biocompatibility, and enhanced bone ingrowth in vivo. This pre-mixed composite cement system is advantageous given the potential to incorporate high concentrations of CaP filler (up to 44.85% wt/total wt) without detrimental effects on its rheological and mechanical properties. This is an important characteristics given commercial composite formulations are more limited in terms of incorporating high amounts of CaP because they also need to contain high concentrations of radiopacifier (typically 40%). PMMA-brushite formulation was demonstrated filler resorption in the mass loss experiment. The PMMA-brushite decreased in mass by an average 6.62%, which was significantly higher than the negligible loss of the acrylic control and PMMA-HA cements. PMMA-brushite showed an initial burst of mass loss which finally plateaued at 12 days (data not shown). It is important to note that this experiment analyzes mass-loss from filler elution and did 17 ACS Paragon Plus Environment


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not account for potential cellular interactions with the cement when injected into a VB. Osteoclast attachment on the cement can lead to the acidic degradation of the CaP phase in both PMMA-HA and PMMA-brushite, which can further accelerate degradation 8,23,33. Factors that may have influenced the results include any ZrO2 radiopacifier elution increasing mass loss and any salt deposition from HSS onto the cement decreasing mass loss. The mass loss experiment demonstrated early filler elution dynamics, which can help in understanding the effects on cement porosity and mechanical properties. Formation of cement porosity was investigated after immersion in HSS. Initial pore formation was observed on the cement surface upon molding, and these pores were polished out prior to immersion. Characterization of both surface and bulk pore formation was necessary, as these pores can have varying effects on cement osseointegration and mechanical properties. Optical microscopy was used to observe surface porosity in both composite cements, revealing pores sizes up to 300 µm in diameter. This increases the cement surface area, which enhances the PMMA-HA surface reactivity. HA resorption is much slower than brushite, therefore a surface interaction will be the main factor in establishing osseointegration. Bulk pores play a crucial role in scaffold formation, allowing cells to infiltrate and deposit bone inside the cement matrix. PMMA-brushite can create a regenerative scaffold through the resorption of the brushite phase. The remaining acrylic scaffold can provide mechanical support, with the resorbed brushite providing calcium and phosphate ions for new bone deposition. Bulk porosity was visualized using micro-CT imaging which showed an average of 3.3% increase after immersion. The pores were homogenously distributed throughout the cement, with larger pores located closer to the cement surface as depicted in Figure 2. This bulk porosity increase may not be enough for bone growth to infiltrate the cement. The results reported here are for a two week immersion period


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however, and do not account for the in vivo enzymatic and cellular interactions that may further break down the brushite phase. Negligible changes in PMMA-HA bulk porosity were recorded correlating to the low mass-loss values recorded, but surface porosity was still observed. Therefore bulk porosity seems to be the main factor contributing to cement mass-loss. The resolution of the employed techniques must be noted as a limitation since most of the porosity of HA and brushite is smaller than what can be detected using these methods 34. Despite this, the goal of this experiment was to highlight pores of 100 microns or larger diameter that would allow for possible cell migration into the cement scaffold. Mechanical stability is necessary for the success of composite cements 4,35-37. In previous studies, the composite cements used here demonstrated desirable mechanical properties, comparable to a commercially available acrylic control 17-19. The introduction of a CaP phase did not deteriorate the mechanical strength as it was completely incorporated in the acrylic matrix upon swelling. Despite this, the previous studies did not account for any changes the CaP phase may create over time. In pure CaP cements, dynamic changes in the structure can have a drastic effect on mechanical stability. The high porosity of pure CaP cements may contribute to their brittle nature. A 3% increase in ceramics-based materials porosity can account for a 10 times decrease in mechanical strength and stiffness 7. Therefore, it was necessary to study the mechanical strength after HSS immersion of the composite cements. Pore formation, both surface and bulk, can create stress risers in the cement structure which decrease the mechanical strength. Deterioration of compressive strength was not observed with the composite cements as seen in Table 1. On the other hand, the flexural properties of the composite cements decreased. Calcium phosphates are notorious for their brittle nature. It is this, factor that has held back their adoption into fracture augmentation procedures. Using a pre-mixed cement, mitigation of the



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loss in ductility in a composite cement was hypothesized and shown in previous studies 17. However, in this study the effects on cement properties were tested after immersion. The PMMA-HA and PMMA-brushite both significantly increased in compressive properties and decreased in flexural properties, specifically flexural modulus. A possible rationale may be the accentuation of minute CaP pockets on the cement surface, creating stress risers. This can be correlated to the surface pore formation (Figure 2) that was observed, indicating that the surface accessible CaP of the composite cement creates a dynamic interaction with the material’s environment. Interestingly, the purely acrylic pre-mixed cement (PMMA) differed in that the flexural modulus was shown to increase. Taking all factors into consideration, the affect of possible salt deposition from the HSS on the cement surface cannot be ruled out. An increase in flexural modulus in PMMA after immersion was observed, however, the samples did not fail before immersion and did fail after the immersion failure as shown by the strain to failure. Despite this, fracture toughness tests demonstrated the composite cements’ resistance to crack propagation with no significant changes detected after the immersion period. This is important to note since pore formation, surface and bulk (Figure 2), tends to deteriorate fracture toughness in cements. The lack of fracture toughness decrease, despite the pore formation observed in the composite cements, can be attributed to the attenuation of detrimental effects of CaP addition and pore formation by the supportive acrylic matrix. The acrylic phase of the pre-mixed cement is allowed to maintain its mechanical properties due to the homogenous distribution of CaP within the cement. This acrylic scaffolding may also explain the insignificant difference between post-immersion fracture toughness values of the composite cements compared to that of the purely acrylic cement. The composite cements’ strength was further demonstrated with the cyclic fatigue tests performed at the ASTM defined maximum stress level of 9MPa for 5,000,000


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cycles 30. The complete incorporation of the CaP filler within the acrylic phase in the composite cements creates a homogenous distribution of PMMA and CaP 17. The lack of clumping enables the acrylic phase to thoroughly support the composite cements without the generation of any potential stress rising areas as the CaP phase is resorbed. Bioactive fixation requires an interaction with surrounding cells. In order to introduce osseointegrative characteristics, composite cements were developed with a bioactive CaP phase 12,13,16

. CaP bioceramics have shown the ability to chemically bond with bone through physico-

chemical and enzymatic interactions 12,16,23,24. Diffusion and ion exchange are the main mechanisms through which the CaP phase can start initial interactions with the biological environment 24,33. Depending on their calcium to phosphate ratios, CaP bioceramics can vary in their solubility 6,33. In this study we have chosen HA and Brushite, which are the least soluble and most soluble forms of CaP cements, respectively. Protein interactions with the surface can also add to the dissolution of the CaP phase 38. The release of calcium and phosphate ions has shown to decrease osteoclast activity and promote bone deposition by osteoblasts 33,38. Cellular interactions with the CaP phase include osteoblast activity, osteoclast attachment, and osteoprogenitor cell recruitment. Osteoclasts and osteoblasts are involved in a continuous remodeling of bone. It is believed this remodeling process accounts for a 10% turnover of bone per year in adults 38,39. Rapid dissolution of CaP, as with brushite, can activate osteoblasts to deposit bone, whereas a slower dissolution, as with HA, can allow for osteoclast attachment on the cement leading to acidulation and a gradual release of ions 39. During fracture healing, new bone formation has been shown to involved mobile osteoprogenitor cells which possess the ability to differentiate into osteoblasts. The osteoblast differentiation is divided into three stages: cellular proliferation, maturation, and matrix mineralization. It is during the cell maturation stage



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that ALP is expressed, and therefore is a well-established indicator of osteoblastic differentiation 38,40,41

. The presence of phosphate ions, however, may decrease ALP production without

deterring the mineralization process during healing 40. This limitation was not a concern, as it was crucial to show that this key differentiation process was not hindered due to the presence of cement. The composite cement formulations demonstrated increased cell viability using the MTT assay (Figure 3). The cell viability of the composite cements met the ISO 10993-5 standard of 70% 31. The cements were injected in the wells with cells and put in contact within 1 hour of preparation. This immediate contact could be why a low viability was detected with the acrylic control (Figure 3); however it represents a worst-case scenario for analysis. Also, the premixed acrylic control used in this study has a higher monomer concentration compared to commercially available powder-liquid formulations, as it was the standard two-solution bone cement used in previous studies 17. It was crucial to demonstrate cell viability as the pre-mixed cements lack the pre-administration steps of a “mixing time” and “swelling time” such as powder-liquid cement formulations. These times allow for the polymerization reaction to get a head start before cement application/injection. Combine the head start with evaporating methyl-methacrylate (MMA) and toxic monomer concentration is further decreased translating to increased cell viability. Of course there are drawbacks to these steps involved with powder-liquid cements which form the motivation behind pre-mixed cement development. Such drawbacks are difficult handling due to viscosity increase, and poor performance upon filler addition. Pre-mixed cements remove the “mixing time” and “swelling time”. Being pre-mixed cements, the cement components are combined in such a way, keeping the free-radical initiator and accelerator separate, that prevents the polymerization reaction from causing the cement to set. This traps the toxic monomer in the sealed container and decreases the amount that can evaporate out. Upon application, the pre-


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mixed cements are injected directly into the fracture site only using a static mixing nozzle to combine the aforementioned initiator and accelerator. This direct application eliminates a “swelling time”, removing another venue for monomer concentration reduction. As mentioned in this study, the swelling takes place in the cartridge and is the key to successful filler incorporation. Using this mechanism, pre-mixed cements are susceptible to having increased monomer concentrations leading to increased toxicity. This was demonstrated by the purely acrylic pre-mixed PMMA control, however it was not observed with the pre-mixed composite cements studied. The composite cements showed no significant enhancement in osteoblast differentiation; however the process was shown to be unhampered when in contact with the cement. Both PMMA-HA and PMMA-brushite reached differentiation values similar to the acrylic and the control well. The elution of CaP from PMMA-brushite was thought to increase cell differentiation, but this was not observed. Osteoblast attachment on the cement surface was visualized with ALP staining (Figure 3). Both composite cements showed ALP positive cells in contact with the cement surfaces. Since cell attachment is crucial for proper osseointegration, in vivo studies are needed to further investigate and confirm cement-cell interaction and new bone formation. It is important to note the limitation of our in-vitro studies of cement dynamics that do not factor in cellular interactions. Cellular interaction can translate to increased mass-loss, pore formation, and possibly greater effects on cement mechanical properties. The dynamic remodeling process between osteoclasts and osteoblasts can only be represented using an in vivo study 41,42. A rat tibia defect model was readily available and could expose injected cements to loads during the healing process. The various loads could exploit any weaknesses in the



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composite cements due to the CaP phase addition. Such models have been used in previous studies, testing the healing ability of a material within a critical size defect ranging from 3-6 mm in diameter 23,42,43. Defects with that size do not allow for spontaneous healing to take place and therefore can accurately assess the healing properties of the implanted material. In order to achieve critical size defects of the correct diameter, a tibia model was chosen instead of a rat tail model. Animal tail models have been used in such studies; however larger animals were not available for this study. Biomechanical testing was performed using 3-point bending flexural tests. CaP ceramics are known to have sufficient compressive strength, but are very poor in tension. Flexural tests subject the augmented tibiae to both compressive and tensile forces which exploit this weakness and act as a worst-case scenario 43. Figure 5 depicts the flexural data from the rat tibiae after two months of healing. The injected cements provided strength and stiffness restoration to positive control values. The commercially available PMMA-K and composite cements (PMMA-HA, PMMA-Brushite) showed no significant difference in strength and stiffness compared to the positive control. Despite the addition of a brittle CaP phase, the composite cements still withstood the various loads experienced in the rat tibiae without failure. This translates to efficient load transfer between the bone and the cement, which is achieved through direct interlocking of the cement-bone interface. This interlocking can be seen in Figure 4 for all cement groups. In this experiment the cement was over-packed into the created defect which can aid in the interlocking; however injecting cement through cannulas in VP makes achieving such contact less probable. Therefore, osseointegration is required in order to achieve optimum load transfer and stabilization of the defect 4.


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Histology was performed to assess whether bone formation had occurred around and within the cement. Analysis was performed after a 2 month healing period for all the cement groups. In addition, a time point after 1 month of healing was used for the PMMA-brushite cement and the negative control. Due to the resorbable nature of brushite, it was interesting to observe whether initial pore formation within the PMMA-brushite cement would promote bone ingrowth through the material. The negative control was used to confirm non-healing of the critical size defect. One negative control tibia did show a partial healing and the defect was still visible with histology (Figure 7). The partial healing could be attributed to the young age of the rats. In order to evaluate the osseointegrative effectiveness of the composite formulations, a comparison with the healing ability of an acrylic control (PMMA-K) was performed. After a two month healing period, tibiae from all three cement groups (II-IV) were analyzed with histology. Figure 7 depicts the tibial defects histology from the PMMA-K, PMMA-HA, and PMMAbrushite cements. PMMA-K clearly shows the outline of the defect area with no bone formation around it. The composite cements clearly illustrate large bone formation around the defect area. In order to substantiate the observed bone growth, a preliminary histomorphometric analysis was performed. Using Image J (NIH, Bethesda, MA) software the histological slides were cropped at an outer diameter of 50 pixels. This was done in order to focus on bone growth around the defect which enhances cement fixation. Using the software, thresholding was used to isolate the green color which indicates bone formation. The isolated green area was then calculated and converted to a percentage of the total area around the defect. The results found were 49.74% bone growth for PMMA-HA and 57.22% for PMMA-brushite. Both the negative control and the commercially available PMMA-K showed no bone growth around the defect. The effect of CaP



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phase addition is exhibited with these histology results. The bone formation may be a result of a bone remodeling cascade initiated by osteoclastic attachment on the cement. Osteoclasts can break down the CaP phase into calcium and phosphate ions, which can trigger osteoprogenitor cell recruitment and subsequent osteoblast differentiation. These osteoblasts can then deposit new bone at the cement-bone interface 38. Bone ingrowth through the cement body into the defect area was still not observed in the PMMA-brushite cement, suggesting that two month duration may not be sufficient to permit the formation of bulk porous venues within the cement. The in vivo study successfully evaluated biomechanical stabilization and osseointegration of the cement formulations. Despite this, some inherent limitations can be noted. It is important to point out that a small animal model was chosen for this study because this was the first time this cement is tested in vivo, thus data collected with this model will provide important data for the design of a future large animal model. The pre-mixed composite cements were developed for use in vertebroplasty and were compared to PMMA-K accordingly as it is indicated for use in vertebral compression fracture augmentation and is popular among many clinicians, however PMMA-K is an acrylic cement and it would have been beneficial to have a commercially available composite cement for comparison 12-16. However, PMMA-K has a larger number of published studies and clinical data compared to composite cements, and was used as control in previously published studies on the pre-mixed cement system 17-19. The rat tibia defect model provides a suitable load bearing environment during the healing process, however does not mimic cement injection into a complex trabecular structure like human vertebral body (VB). The cement-bone interface will show proper interdigitation due to over-packing which may not be the case during cement injection into a VB. Another limitation can be attributed to the staining procedure which was expected to allow for distinguishing between newly formed bone and the


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CaP filler (brushite or HA). This did not impede the work performed, as osseointegration and new bone formation was still visible with histology. Future studies are required for in depth study of the cellular processes at the cement-bone interface to track the effects of the CaP phase in the composite cements.

CONCLUSION

The composite cements demonstrated negligible effects on mechanical strength with CaP elution. Cell viability was increased when placed in contact with the cement with no hindrance of osteoblast differentiation. Finally, the PMMA-HA and PMMA-brushite composite cement formulations provided strength and stiffness restoration to control values in a rat tibia defect model. The composite formulations also induced new bone formation around the created defects.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Hayenga (The Vascular Mechanobiology Lab, UTD) and Dr. Rennaker (Director of Texas Biomedical Device Center, Head of Bioengineering Department) for their support and the UT Transform award system for providing partial funding and startup funds (D.C.R).



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Table 1. The mechanical properties of the cements before and after immersion.

Cement Immersion

PMMA-brushite Before After

PMMA-HA Before After

PMMA Before After

Compressive Strength σc

75.6 ± 3.0

101.1 ± 4.5*

103.5 ± 3.0

107.5 ± 6.7

80.9 ± 2.3

92.8 ± 5.5*

1.8 ± 0.3

2.6 ± 0.2*

1.8 ± 0.1

2.6 ± 0.1*

1.6 ± 0.7

2.1 ± 0.1*

63.6 ± 2.2

45.3 ± 3.4*

64.0 ± 3.1

51.2 ± 3.8*

48.0 ± 8.5

72.3 ± 10.6*

3.7 ± 0.1

2.1 ± 0.1*

4.2 ± 0.1

2.5 ± 0.2*

1.7 ± 0.3

2.6 ± 0.1*

3.1 ± 0.9

2.4 ± 0.3*

3.2 ± 1.0

2.3 ± 0.3*

N/A

3.2 ± 0.8*

1.4 ± 0.1

1.3 ± 0.1

1.4 ± 0.2

1.5 ± 0.1

(MPa) (n=6) Compressive Modulus Ec (GPa) (n=6) Flexural Strength σf (MPa) (n=5) Flexural Modulus Ef (GPa) (n=5) Strain to Failure εf (%) (n=5) Fracture toughness KIC

1.3 ± 0.1 1.1 ± 0.1

0.5

(MPa•m ) (n=3) *Significance (p

In Vitro and In Vivo Characterization of Premixed PMMA-CaP

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