Evaluation of inherent toxicology and biocompatibility of magnesium phosphate bone cement

Colloids and Surfaces B: Biointerfaces 76 (2010) 496–504

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Evaluation of inherent toxicology and biocompatibility of magnesium phosphate bone cement Yonglin Yu a , Jing Wang b , Changsheng Liu b,∗ , Bingwen Zhang a , Honghong Chen c , Han Guo b , Gaoren Zhong d , Weidong Qu e , Songhui Jiang e , Huangyuan Huang a a

Department of Orthopaedics, Huashan Hospital, Fudan University, Shanghai 200040, China Key Laboratory for Ultrafine Materials of Ministry of Education, and Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, China c Institute of Radiological Medicine, Fudan University, Shanghai 200032, China d Department of Radiopharmacy, School of Pharmacy, Fudan University, Shanghai 200032, China e School of Health, Fudan University, Shanghai 200032, China b

a r t i c l e

i n f o

Article history: Received 10 August 2009 Received in revised form 27 November 2009 Accepted 15 December 2009 Available online 23 December 2009 Keywords: Magnesium phosphate cement Unscheduled DNA synthesis test Ames test Micronuclei test Toxicity

a b s t r a c t Magnesium phosphate cement (MPC) is a kind of novel biodegradable bone adhesive for its distinct performance. However, there is few research work concerning on the systemic biocompatibility and genetic toxicological evaluation of MPC. In this study, the investigation on the inherited toxicology of MPC including gene mutation assay (Ames test), chromosome aberration assay (micronucleus test), and DNA damage assay (unscheduled DNA synthesis test) were carried out. Fracture healing and degradation behavior were explored for the evaluation of the biocompatibility of MPC, using macroscopical histological, histomorphometrical, and scanning electron microscopical methods. The results of mutagenicity and potential carcinogenicity of MPC extracts were negative, and the animal implantation illustrated no toxicity and good resorption. The study suggested that bioresorbable MPC was safe for application and might have potential applications for physiological fracture fixation. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Unstable fracture, especially comminuted fracture, frequently occurs in clinic and is difficult to cure. Diaplasis and fixation of small fragments of bone are arduous and shift easily appears after fixation, always concomitant with bone defect. Therefore, therapy and regeneration of serious comminuted fracture and bone defect is one of the challenges for orthopedic surgeon [1]. Gluing is an attractive technique to fix small bone fragments [2,3]. Treatment of the unstable fracture (comminuted fracture) using bone adhesive is a longstanding and challenge problem for the orthopedist. At present, the methods of incision restoration with internal fixation have been widely adopted in clinics, followed by microplates screw fixation or intramedullary fixation, which is effective for the massive bone fracture. However, it is much difficult to the fixation of the small blocks of bone fracture. In spongy bone areas, moreover, the screw fixation method still has the disadvantage of weak strength, even for the massive bone fracture. The postoperative slippage occurs frequently, which affects the postoperative effects

∗ Corresponding author. E-mail address: [email protected] (C. Liu). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.12.010

and makes the secondary operation necessary [4]. Although the absorbable internal fixation materials have been developed to eliminate the secondary operation, the rapid decline in their mechanical strength limits their applications [5,6]. Therefore, it is desirable to develop novel kind of degradable and nontoxic bone adhesive with rapid-setting and high early strength. Recently, a novel kind of adhesive material, magnesium phosphate cement (MPC) has attracted much attention as potential biodegradable bone implant materials [7,8]. The main components of MPC are dead-burnt magnesia (MgO), acid ammonium phosphates, retardant, and setting liquid. Compared with the traditional calcium phosphate cements (CPC), Mg-substitution phosphate cement has the advantage of rapid setting, higher initial mechanical strength, as well as excellent in vivo degradability. Our previous studies on animal tests indicated that MPC had significant higher adhesive strength than CPCs, and could be used to directly conglutinate small fragments of no-loaded bone [7–10]. Furthermore, the results of evaluation on cytotoxicity, skin sensitization, subcuticular stimulation and acute toxicity have shown that MPC is nontoxic and could be applied on animal test safely. The aim of the present study was to investigate the inherited toxicology and biocompatibility of MPC-based adhesive. The unscheduled DNA synthesis (UDS) test and Ames mutagenic test

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was carried out to survey the effect of MPC on DNA damnification and repairing in human peripheral blood lymphocyte and the back mutation in murine typhoid salmonella. The mice’s marrow erythrocyte micronuclei rate analysis of MPC had been surveyed to evaluate the heredity toxin. Fracture healing and in vivo degradation pattern of the adhesive cement during implantation, especially osteointegration due to the cellular migration of osteoblasts, as well as inflammatory tissue reactions were studied. Furthermore, the blood biochemical parameters were determined in a rabbit model. The study would not only show the preliminary in vivo observation on biocompatibility and biodegradability characteristics of this novel material, but also supply the basic datum on the influence on genetic damage and immune function, which are essential aspects that should be taken into consideration for clinical use for bone regeneration.

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scintillating container, add 1 mL scintillators (2,5-diphenyloxazole (PPO), 0.1%, and 1,4-bis(5-phenyl-2-oxazolyl) benzene, (POPOP), 0.04%), and the radio counts per minute (CPM) were measured by Beckman LS 3801 liquid scintillation counter. Each test was performed in octuple. 2.4. Ames mutagenic test [13]

All the chemicals used were purchased from Sinopham Chemical Reagent Co. Ltd. MPC used in this study was composed of magnesium oxide (MgO) and ammonium dihydrogen phosphate (NH4 H2 PO4 ), and was fabricated according to the previous patent [8]. Briefly, the MgO was prepared by heating basic magnesium carbonate pentahydrate [(MgCO3 )4 • Mg(OH)2 • 5H2 O] in a furnace at 1500 ◦ C for 6 h. The resultant powder was first cooled to room temperature, and then ground in a planetary ball mill for 5 min, followed by sieving through 200 and 300 meshes, respectively. The grains in the range of 200 and 300 meshes were kept for further experiment. The obtained MgO and NH4 H2 PO4 powders with a molar ratio of 4:1 and retarder were well mixed to form the MPC powder. The cement was formed by mixing the powder with the cement liquid and storing the paste at 37 ◦ C in a 100% relative humidity box for 24 h.

The evaluation of mutagenic action of MPC was carried out on the basis of the reference Ames test. The test was prepared according to the procedure proposed by Maron and Ames. Briefly, the concentration of the original extract was 250 mg/mL, and it was diluted with physiological saline to following concentrations: 125 mg/mL, 62.5 mg/mL, 31.25 mg/mL, and 15.625 mg/mL. Salmonella typhimurium TA98, TA98, TA100 and TA102 strains were used to detect the mutagenicities of the extracts. The experimental samples were divided into activation group (addition of S9 solution) and non-activation group (no S9 solution). The mice hepatocyte cytomicrosome (S9), induced by polyammonia biphenyls, was homogenated and preserved at −80 ◦ C. Atebrin, 2-aminofluorene (2-AF), 2,7-diaminofluorene (2,7-AF), 1,8-dihydroxyanthraquinone, methyl methanesulfonate (MMS), and sodium azide (SA) were used as positive control, and physiological saline with same volume was negative control. Triplet parallels were carried out for each fungus. Different concentration groups of mixture of suspension and individual fungus were co-cultured by plate intermingle method. The test strains were observed after 48 h of incubation at 37 ◦ C. The average rate of test group (Rt) was calculated according to the average of 3 samples, and the ability of MPC to induce reverse mutation was evaluated according to the mutation rate value (calculated as follows). Mutation rate (MR) was the ratio of the number of reverse mutation of test (Rt) to that of negative control (Rc). The extracts of MPC and positive control are experimental group, and the physiological saline was negative control.

2.2. Preparation of extracts

2.5. Micronuclei test [14]

50 g of MPC was covered with tinfoil and extracted with 200 mL physiological saline in 37 ◦ C for 72 h. The extract was filtered through a 0.2 ␮m microporous membrane for sterilization and then used for test in 24 h. The concentration of the original extract was 250 mg/mL. The extract was then diluted with physiological saline to designated concentrations, such as 125 mg/mL, 31.25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, and 2.5 mg/mL for UDS test and 125 mg/mL, 62.5 mg/mL, 31.25 mg/mL, 15.625 mg/mL for Ames test, respectively.

Regarded as the best documentation on in vivo genotoxic effects (chromosome aberrations), the effect of MPC on inherited toxicology was investigated by examining the change of mice’s marrow red blood cell micronucleus rate. 50 healthy Kunming mice were prepared with the average weight of 18–20 g, and male and female in a half. All the mice were divided evenly into 5 groups. The MPC extracts with different dosage (125 mg/mL, 250 mg/mL and 500 mg/mL) were delivered via abdominal cavity injection, physiological saline served as a negative control while cyclophosphamide (CPA) with concentration of 100 mg/kg was used as positive control. All the injections were carried out in quartic at interval of 24 h. The mice were sacrificed at 6 h after the fourth injection and their thighbone marrow cells were removed, fixed with methanol, and then stained with Giemsa solution observed under a fluorescent microscope. At least 1000 polychromatic erythrocytes stained binucleated cells were scored for each sample for the micronuclei measurement. MN was formed during the metaphase/anaphase transition of mitosis (cell division).

2. Materials and methods 2.1. Preparation of MPC

2.3. Unscheduled DNA synthesis (UDS) test [11,12] RPMI 1640 culture medium containing 5% newborn bovine serum, hydroxyurea 20 mmol/L and 3 H-TdR 20 ␮Ci/mL was added into fresh human peripheral blood and mixed homogeneous, then transferred into 0.5 mL cuvettes. Physiological saline (200 ␮L) served as negative control and nickel sulfate with the concentrations of 0.01 mmol/L, 0.1 mmol/L and 1 mmol/L individually served as positive control. 2 ␮L, 5 ␮L, 10 ␮L, 25 ␮L, 100 ␮L, 200 ␮L extract were added to each experimental group, respectively, and was complemented with physiological saline to uniform 200 ␮L. The specimens were incubated in constant water bath at 37 ◦ C and shaken for 4 h until 2 mL cold physiological saline was added to stop reaction. The reactant was filtrated in vacuum using 49-type glass fiber filter paper, washed twice with 6 mL distilled water to remove the free 3 H-TdR, and then fixed with 3 mL trichloroacetic acid (TDA), dehydrated and decolored with 3 mL 75% alcohol and absolute alcohol gradually. After drying at 37 ◦ C, the filter film was kept flat in

2.6. Surgical procedures In the study, 27 healthy animals were used. Both ectopic subcutaneous and femur condylus implantation were carried out under aseptic conditions. The experiment was approved by the Animal Experimentation Ethics Committee of Medical College at Fudan University. For subcutaneous implantation, twelve healthy New Zealand white rabbits with mature skeleton and an average weight of

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3.0 kg were divided into three groups for time intervals of 1, 2, and 3 months. Each respective group was consisting of 4 animals, thereinto three samples were taken out for mechanical test and the rest was for degradation observation. General anesthesia was induced using intramuscular injections of 3% pentobarbital (30 mg/kg). After shaving and disinfecting, small skin incisions were made in the proximal part of each limb and in the lumbar area. The cylindrical MPC samples with the size of Ø3.2 × 10 mm3 were implanted into the left dorsal muscle pouches (one implant per animal). Similar process was carried out for femur condyle implantation. Approval by an institutional review board was obtained prior to surgery. Fifteen healthy New Zealand white rabbits with mature skeleton and an average body weight of 3.0 kg were divided into five groups with preset sampling time of 0.5, 1, 2, 3, and 6 months for the current study. The operations were performed in an operating room under aseptic conditions. Under general anesthesia (3% pentobarbital) and sterile conditions, the left femur of each rabbit was exposed and one defect (Ø3.2 mm) was drilled in the distal condylus part of the femur. The bone cavities were carefully washed to eliminate bone dregs and dried with gauze. Cylindrical preset samples of MPC with the size of Ø3.2 × 10 mm3 were implanted into the defects in the rabbit femora and periosteum was layered sutured. Postoperatively, X-rays were taken and the animals were allowed to recover in a well-temperated wake-up area without pelvic limb activity restriction. 2.7. Follow-up and sacrifices In all cases the animals were permitted to walk immediately after surgery. For subcutaneous pocket implantation, at 1, 2 and 3 months after surgery, rabbits were sacrificed and the specimens were taken out. Degradation pattern were observed via light microscopy. For those femur condyle implantations, three animals of each group were sacrificed by an overdose abdominal injection of pentobarbital sodium after 0.5, 1, 2, 3 and 6 months. The bone specimens were harvested immediately after sacrifice. The distal femur was dissected and separated from all adjacent soft tissue, and the condyle was harvested for histological analyses. 2.8. Biological mechanics analysis and degradation test in vivo At time intervals of 1, 2, and 3 months, four animals of each group were sacrificed and the implants and their surrounding tissues were dissected. After taken out from the subcutaneous pocket site, compressive strength of the MPC specimen was measured at a loading rate of 1 mm min−1 using a universal testing machine (AG-2000A, Shimadzu Autograph, Shimadzu Co. Ltd., Japan). Three replicates were carried out for each group, and the results were expressed as means ± standard deviation (means ± SD). The remanent of each group was used for observation of degradation behavior via light microscopy and scanning electron microscopy (SEM). For SEM observation, specimens were washed with physiological saline, and then fixed with glutaraldehyde (2.5%) and osmic acid (1%) at 4 ◦ C for 2 h. After eluted with 0.1 M phosphate buffer solution (PBS) for 3 times, the samples were gradient dehydrated in ethanol series (50%, 70%, 90%, and 100%) and dried. Specimens were glued onto copper stubs and sputter-coated with gold palladium prior to observation (JSM-6360LV/Falcon, JEOL/EDAX, Japan).

Table 1 The UDS assay of MPC extracts, negative control and positive control. Sample

CPM

NiSO4 solution (0.01 mmol/L) NiSO4 solution (0.1 mmol/L) NiSO4 solution (1 mmol/L) MPC extract (2.5 mg/mL) MPC extract (6.25 mg/mL) MPC extract (12.5 mg/mL) MPC extract (31.25 mg/mL) MPC extract (125 mg/mL) MPC extract (250 mg/mL) Negative control

289.50 461.52 495.13 24.63 30.38 34.63 41.38 43.13 41.75 14.75

± ± ± ± ± ± ± ± ± ±

145.41 160.08 127.89 5.78 8.93 7.95 13.28 12.26 10.31 3.11

were excised, fixed in 10% neutral buffered formalin, fixed with formaldehyde, and dehydrated with acetone. Then the samples were embedded in polymethyl methacrylate (PMMA) solution and dried. Tissue blocks were sectioned at 5 ␮m in thickness and stained with hematoxylin and eosin (H&E), then observed by light microscope (TE2000U, Nikon Corp., Japan). 2.10. Blood biochemical test Blood biochemistry fluctuate of the femur condyle osteotomized animals were evaluated. The blood biochemical values were collected through 2 mL blood drawn from marginal ear vessels of the rabbit pre- and post-operation, respectively. After centrifugalizing (15 min, 3000 r/min) for 2 times, the supernatant solution was collected. The blood calcium, serum phosphate concentrations were analyzed using auto biochemical analyzer (Backman CX-3, Backman-Coulter, USA), while serum magnesium level was obtained via Atomic Absorption Spectrophotometer (VIDEO-22, Huayang Analyse Instruments (HAI) Co., China). 2.11. Statistical analysis Statistical analyses were performed on the means of the data obtained from three independent experiments. All of the results were illustrated as the mean ± standard deviations. Statistical difference was analyzed using one-way analysis of variance (ANOVA). A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. Inherent toxicology evaluation

2.9. Macroscopic and histological evaluation

3.1.1. Unscheduled DNA synthesis (UDS) test The data of UDS test were listed in Table 1. The average CPM value of MPC extract, negative control and positive control was 35.98, 14.75 and 415.38, respectively. Compared with the blank of negative group, the CPM value of MPC extracts was enhanced along with incipient increased of concentration. Nevertheless, it seemed that further increase of MPC concentration would not make the CPM sustained growth, which was dissimilar to the feature of continuous improvement in positive controlled group. Moreover, the results showed that the significance difference between MPC in various concentrations and the negative control was negligible (p < 0.01), but existed between MPC and the positive control (p > 0.05). This indicated that the MPC extracts had no carcinogenicity and mutagenicity, which coincided with the results of Ames test thereafter.

To evaluate the interfacial incorporation, new bone formation and adjacent tissue reactions of MPC cement, the macroscopic appearance of the femur condyle defects was assessed. For histological evaluation, the samples together with surrounding tissues

3.1.2. Ames mutagenic test The Ames test, named for its developer, Bruce Ames, is a method to test chemicals for their cancer-causing properties [15]. In this study, mutagenic activities of MPC extracts with various concen-

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– 1006.0 ± 45.1* 1004.0 ± 16.1* –

– –

1027.7 ± 68.5 *

3.6 2.0 2.1 2.0 2.6 3.2 41.0 40.0 43.7 41.0 38.0 34.7

–

5.0 2.5 1.5 1.5 2.1 2.1 ± ± ± ± ± ± ± ± ± ± ± ±

33.0 31.3 35.7 35.3 26.7 32.7

TA98 − S9 TA98 + S9

4.4 4.6 7.1 3.5 4.0 3.0

– –

– 32.6*

1038.3 ± 77.0*

± ± ± ± ± ± 146.0 141.0 145.7 142.3 138.7 154.0

51.5* 59.2*

*P < 0.05 (vs. negative controlled group).

TA97 − S9

9.5 7.4 10.4 4.9 7.8 4.2 ± ± ± ± ± ±

TA97 + S9

157.0 154.7 163.0 160.7 153.3 175.7 6.6 9.3 6.0 4.2 2.5 8.3

TA102 − S9

264.0 ± 273.3 ± 274.7 ± 276.3 ± 279.7 ± 258.7 ± – – 1019.7 ± – 10.4 5.6 7.6 5.0 9.9 1.5

Sample

Number of micronuclei

MPC extract (125 mg/mL) MPC extract (250 mg/mL) MPC extract (500 mg/mL) Negative control Positive control

2.9 2.7 2.9 3.2 46

± ± ± ± ±

0.9 0.7 1.0 0.6 3.1

trations were investigated through Ames test. Four Salmonella typhimurium strains (TA97, TA98, TA100, and TA102) were used in the experiments, and the results were listed in Tables 2 and 3. The strains differed with respect to the type of mutation in the histidine gene, which results in different susceptibility of these strains to the action of chemical mutagenesis and enables the determination of a compound mutagenic action evaluated as an induction of specific bacterial DNA changes. The results indicated that none of the tested samples caused the increase in his+ revertants number in any of the four studied bacterial strains, both in experiments with and without mice hepatocyte cytomicrosome (S9) and no matter which concentration. The mutagenesis rates of murine typhoid salmonella in the experimental groups of all dosage levers were less than 2 while the MRs of the positive control groups were more than 2, and the MPC would not cause an increase of the back mutation in murine typhoid salmonella, which strongly suggested that MPC do not exhibit any mutagenic activity.

286.3 ± 285.0 ± 285.7 ± 263.3 ± 269.3 ± 282.7 ± – – – 1044.0 ± 126.3 ± 125.0 ± 121.3 ± 129.7 ± 129.7 ± 125.7 ± 1061.3 ± – – –

TA102 + S9

Table 3 Results of micronuclei assay.

2.5 4.6 4.5 5.0 5.0 6.4 38.8*

TA100 − S9

129.7 ± 132.0 ± 129.3 ± 133.7 ± 133.7 ± 135.7 ± – 1057.7 ± – – MPC extract (250 mg/mL) MPC extract (125 mg/mL) MPC extract (62.5 mg/mL) MPC extract (31 mg/mL) MPC extract (15.625 mg/mL) Physiological saline SA– 2-AF MMS 1,8-Dihydroxyanthraquinone Atebrin 2,7-AF

1.5 2.6 2.1 6.7 6.7 4.5

TA100 + S9 Sample

Table 2 The influence of MPC on the number of revertants his+ of bacterial strains Salmonella typhimurium.

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3.1.3. Micronuclei test As the amount of micronuclei is directly proportional to the mutagen dose, frequency of micronuclei occurrence is always utilized to evaluate the genetic damage degree. Scoring of micronuclei can be performed relatively easily and on different cell types relevant for human biomonitoring: lymphocytes, fibroblasts and exfoliated epithelial cells, without extra in vitro cultivation step [16–18]. In this work, polychromatic erythrocytes (PCE, usually named reticulocytes) in mouse marrow with Gimsa were utilized to evaluate the genetic damage of MPC. MN observed in exfoliated cells was not induced when the cells were at the epithelial surface, but when they were in the basal layer. Cellular chromosome breaks can be embodied by a high micronuclei ratio which predict poor biocompatibility. Table 3 showed the micronucleus numbers of polychromatic erythrocytes stained thighbone marrow cells. The mice micronucleus in all the exposed groups was similar to the negative control while cyclophosphamide group serving as the positive control was markedly different from the physiological saline (p < 0.01). These results were coincident with the microscopically observation (Fig. 1). If a chromosome break or loss occurs, a micronucleus (i.e., a ‘small’ nucleus) is formed and DNA damage will results in more micronuclei in the daughter cells. From the photomicrograph Fig. 1a, there were no statistically significant differences in the micronucleated polychromatic erythrocytes when compared with the negative control. In micronuclei in the positive group were identified as DNA-containing structures in the cytoplasm, clearly separated from the main nucleus. In addition, no mutagenic in a dose-dependent manner in MPC group was observed, which suggested that the extracts of MPC would not result in an increase of micronucleus frequency in mice bone marrow eosinophile cells. 3.2. Evaluation of biocompatibility and osteogenesis in vivo 3.2.1. In vivo degradation behavior The muscle implantations were taken out at designed intervals to investigate the degradation patterns. The morphologies of the MPC specimens were exhibited in Fig. 2. Compared with the

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Fig. 1. Microscopically observation of micronuclei formation after treatment with MPC and controlled groups. Each dosage is uniformed as 100 mg/kg, and magnification times is 10 × 1000. (a) MPC extracts; (b) negative control of physiological saline; (c) positive control of cyclophosphamide. The arrow showed the representative micronucleus of mice bone marrow polychromatic erythrocytes in positive control.

Fig. 2. Light microscopy and scanning electron microscopy (SEM) photograph of MPC samples which were taken out at different intervals. (a) Standard sample preset, (b) 2 months, (c) 3 months (at 10× magnification) and (d) SEM of external surface of 3 months’ implantation. The red vector indicated the forming pores via degradation process. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

smooth and neat surface of the standard pre-implantation sample (Fig. 2a), surface turned rough after implanted for 2 months (Fig. 2b). Great amount of scallops had been appeared in the specimen of 3 months (Fig. 2c), indicating the degradation happened in vivo. Further proof came from the SEM of the interface between material and tissue (Fig. 2d), faster degradation was observed at the interfacial lamella compared to the interior part, and a great deal of pores about 200 ␮m had been generated at the interface, which would benefit for the new bone ingrowths.

3.2.2. In vivo biological mechanics The compressive strengths of the MPC samples after implanted in muscle were shown in Fig. 3. MPC had higher original strength of more than 80 MPa. After implanting for 2 months, the cement began to degradation, along with the obvious loss of compress strength. In spite of this, complete bony restoration at the osteotomy site with excellent reorganized and remodelled vital trabecular bone was occurred during the 2–3 months. Thus the decrease of mechanical strength had little negative effect on the callusogenesis of bone

Fig. 3. Compressive strength of implanted MPC as a function of time.

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Fig. 4. Macroscopic evaluation of MPC implanted into bone defects of rabbits for 1 month (a), 2 months (b), 3 months (c), 6 months (d), and X-ray radiography for 6 months (e).

defect. Appropriate degradation and resorption were expected for clinical application which would expedient for new bones formation. 3.2.3. Macroscopic evaluation Fig. 4 illustrated the macroscopic evaluation of MPC samples that were implanted in the bone cavities of rabbit femora for different times. All animals were survived with compact integration between the MPC and contiguous bone tissues. For all implants, neither inflammation response nor gangrene was

observed throughout the observation periods. After 1-month implantation (Fig. 4a), the cement sample was integrated well and did not display any obvious inflammatory response in the adjacent tissue. The bone plug was effectively bonded to the interior surfaces of the bone tunnel by the MPC. From Fig. 4b and c, it could be seen that, after 2 and 3 months’ implantation, the bounds of MPC cement remnants were vague, indicating the degradation occurred during the process, and the newly formed bone grew simultaneously from the periphery inwards, which was indistinguishable from the original bone. As shown in Fig. 4d, the defect area was completely filled

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Fig. 5. Hematoxylin/eosin-stained sections of MPC samples, which were harvested at (a) 0.5, (b) 1, (c) 3 and (d) 6 months after implantation (at 10× magnification). Red arrow denoted osteoblasts and white arrow denoted bone ingrowth at the interface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

with newly formed bone after 6 months’ implantation. X-ray radiography of 6 months was coincident with abovementioned (Fig. 4e). None shift had been seen, which implying the locked fixation, and the vague profile at interface showed the obvious degradation. 3.2.4. Histological evaluation The results of the histological evaluation of the MPC specimens implanted in the bone defects of rabbit femora are shown in Fig. 5. In 0.5-month groups (Fig. 5a), the MPC implant was incorporated tightly in the center of the osteotomy gap, the surrounding fracture hematoma and trabecular fragments was visible. No fibroid film was found, but the band of material was covered with a thin layer of osteoblast at the interface. After 1-month’s implantation (Fig. 5b), a good resorption of the fracture hematoma and of trabecular fragments with a high increase of osteoblasts and woven bone formation without inflammatory tissue reactions was observed. The MPC implant began to degrade from the edge of the implant and the interface between MPC implant and the host bone was clearly visible. After 3 months, as shown in Fig. 5c, the boundary between the cement and the host bone was unclear due to the formation of mature bone tissue, which had grown into the pores of the cement and bonded tightly with the material. The direct contact between the new bone and the cement increased with time, which indicated the MPC did not act as barrier for cellular migration and did not interfere with bone ingrowth or osteogenesis. MPC resorption at the bone–cement interface was prominent and the new bone was in direct contact with the surface of the implant. The woven bone was partly transformed to new lamellar bone with vital osteocytes (indicated as red arrow). After 6 months (Fig. 5d), multi-location of the original MPC implant was replaced with the new bone and the interface between the cement and the host bone was hardly detectable and a close union was formed without a gap. The woven bone was partly transformed to new lamellar bone with vital osteocytes. 3.2.5. Blood biochemical analysis The versatility of blood calcium, serum phosphate and serum magnesium concentrations were exhibited in Fig. 6. The hema-

Fig. 6. Concentration variation of serum calcium, phosphate and magnesium as a function of time.

tological examination showed that the blood calcium, serum phosphate, and serum magnesium level were maintained at normal physiological level, no obvious variations of ions concentrations have been observed except a little fluctuation in blood phosphate level. The results indicated that the implant of MPC would not cause the significant change of in vivo metabolic level, and the metabolism would balance the concentration of calcium, phosphate, and magnesium in serum. 4. Discussion Regarding as a kind of novel implantation material, the interactions between MPC and the ambient tissue and cells and the inherited toxicology have not been clarified. It is necessary to evaluate the toxicology, including the potential genotoxicity, carcinogenicity, and reproductive toxicity [19]. However, it is difficult to carry out the genotoxicity tests for carcinogenicity,

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mutagemicity and teratogenicity which are enormous time and cost consuming [20,21]. Short-termed genotoxicity tests are recommended as the original screening, and conduct the carcinogenic test for those positive groups only. In the light of this, UDS test and Ames mutagenic test have been carried out in this study to survey the effect on DNA damnification and repairing in human peripheral blood lymphocyte and the back mutation in murine typhoid salmonella [22,23]. The analysis of the rate of mice’s marrow erythrocyte micronuclei affected by MPC has been determined to evaluate the heredity toxin [24,25]. Data obtained from the inherent toxicological tests demonstrated that the MPC would neither cause the increase of the unscheduled DNA synthesis of human peripheral blood lymphocyte, the back mutation in murine typhoid salmonella, nor the back mutation in murine typhoid salmonella. Though the CPM value of MPC was a little higher than the negative group, there was no significant difference (p > 0.05) between the experimental MPC group and the negative controlled group, whereas the significant difference existed as compared with the positive controlled group. As a result, it was reasonable to suggest that MPC was safety for it would not induce DNA damage, and gene mutations. Despite the preferable correlation between micronuclei frequencies and carcinogenicity could be obtained, the mutation is not the sufficient condition for cancerogenicity. Multiplicate inheritance toxicology tests, such as stable chromosome aberration (balanced chromosomal translocation), should be incorporated carrying out to evaluate the cancerogenicity [26,27]. As to the biocompatibility of MPC, due to its characteristics of quick setting, high early strength, and gradually biodegradable, magnesium phosphate cement was expected as a novel bone adhesive. The results from animal (rabbit) tests showed that adhesive strength of MPC was distinct higher than the traditional calcium phosphate cement, and the tiny fracture fragment at nonloading region could be adhered directly. The safety of MPC has been studied in our previous studies [28,29]; including the cytotoxicity, skin sensitization, intracutaneous test, and acute systemic toxicity test according to ISO 10993-1998 standards. The results indicated that all the tests are qualified and the material proves to be nontoxic and would be safe when used into the animal experiment, which laid a solid foundation for the application of this kind of inorganic bone cement. The focus of the current work was to evaluate degradation behavior and mechanical variation in vivo, as well as the osteogenesis and blood biochemical index in the bone defect of rabbits. Although there is variation existed in the animal tests, especially the data of the mechanical strength after in vivo degradation and hematological examination, the general tendency of is not being offset. Each implanted specimen exhibited erosion surface with obvious scallops. In the femur condyle implantation, only one animal in 3-month implantation was found nonunion. This might probably due to the individual difference. As a whole, the tested group showed well synosteosis. From the point of view of compositions, the main components of MPC are dead-burnt magnesia (MgO) and acid ammonium phosphates, particularly ammonium dihydrogen phosphate (NH4 H2 PO4 ). Magnesia was a kind of oral antiacid medicine of 400–840 mg dosage; ammonium dihydrogen phosphate could be used as raising agent and yeast buffer. After hydration, MPC is conversed into ammonium magnesium phosphate in vivo, which was the common biomineral existed in body. Therefore, considering whatever the main compositions and the final products, MPC should be safety and nontoxicity. It is well documented that Mg-doped bone substitute can decrease stress concentrations at the interface between the implant and bone, reduce healing time for bone fracture, and eliminate surgery for implant removal. Good degradation behavior by

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chemical dissolution and cell-mediated resorption of magnesium phosphate-based adhesive cement has already been reported in our previous study. In the present study, the in vivo degradation behavior of MPC through the 6-month implantation in rabbit bone defects demonstrated that resorption of the implant occurred gradually accompanied with the regeneration of new bone and subsequent replaced by autologous tissue. The dominated degradation mechanism is presumed as gradually dissolving of Mg ion. Along with the creeping substitution, marrow cavity and intrabony Haversian canal extend to the interface between implant and bone, and the MPC is marinated into the marrow liquid, which makes for the dissolution of MPC, as well as being conveyed to distant defect. Thus, following with growing of osteoblast cells and formation of newly bone, the dissolving and metabolic process of MPC occur simultaneously and gradually. This has been confirmed in our regeneration study (the photo has not been shown in this article) that MPC particles can be observed penetrating into bone canaliculus or emerging beyond the adhering interface. In other words, though not as well-blooded microenvironment as in muscle, MPC still can be degraded via the cooperation of gradually bone formation, which supplying the adequate intramedullary fluid for MPC dissolving. Though the dissolving of Mg might not as fast as in muscle, the gradual solvability is feasible for bone regeneration process. It has been reported that MPC has higher mechanical properties than CPC [31]. However, the mechanical strength is measured in vitro conditions. To the best of our knowledge, the actual strength fluctuate in vivo has not been reported. Considering the tight integrating between MPC and bone, subcutaneous implantation is selected for observations for its easier separating. The biomechanics results showed that even though the mechanical strength was loss during the degradation process, the early mechanics was enough higher, while the micropores would facilitate the migration of cells. Furthermore, once new bone has grown into the microporous layer generated via surface degradation of MPC and penetrated into the implant, the bonding would be increased to compensate the strength loss. Macroscopic evaluation results and histological studies exhibited good in vivo biocompatibility without impairment of physiological fracture healing. Application of MPC did not lead to prolonged inflammation or infection complications in any animal. Histological evaluation revealed that the cement contacted with the bone intimate and direct, exhibiting better osteogenesis at the interfacial areas and enhanced osteointegration in the defect area. The MPC did not act as a barrier for bridging of the osteotomy gap by osteoblasts and after 6 months the initial woven bone was completely transformed into vital lamellar bone. The need for bone regeneration is continuously expanding due to the improvement of life quality and the consequent increase in life expectancy [30]. The results combining the biocompatibility and inherent toxicology indicated that MPC adhesive cement could be developed as a kind of potential product for bone gluing and regeneration in clinical applications. Despite these promising results, further investigations are necessary to confirm long-term biocompatibility and to elucidate degradation properties. Further research is ongoing.

5. Conclusions The inherited toxicology and biocompatibility of MPC were investigated through UDS test, Ames test, micronuclei test, and animal implantation. The genetic results showed that MPC would neither cause the increase of the unscheduled DNA synthesis of human peripheral blood lymphocyte, the back mutation in murine typhoid salmonella, nor the back mutation in murine typhoid

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salmonella. This suggested that MPC was not toxic and safe since it would not induce DNA damage, and gene mutations. As regard implantation tests, no foreign body reaction, inflammation and necrosis were found in vivo from the macroscopic observation. Histological evaluation confirmed that MPC would form direct bonding with the host bone. The periodical implanted samples exhibited good degradation pattern in vivo. All results obtained would provide fundamentals for further animal test and clinic application. Acknowledgements The authors appreciate financial support from the National Natural Science Foundation of China (No. 50732002), and the Key Project of Basic Research Foundation of Shanghai Science and Technique Committee (No. 07JC14008). References [1] P. Habibovic, K. Groot, Osteoinductive biomaterials—properties and relevance in bone repair, Journal of Tissue Engineering and Regenerative Medicine 1 (2007) 25–32. [2] C. Heiss, N. Hahn, et al., The tissue response to an alkaline bis(dilactoyl)methacrylate bone adhesive, Biomaterials 26 (2005) 1389–1396. [3] C. Heiss, R. Kraus, D. Schluckebier, A.C. Stiller, S. Wenisch, R. Schnettler, Bone adhesives in trauma and orthopedic surgery, European Journal of Trauma 32 (2006) 141–148. [4] P. Weninger, M. Schueller, M. Jamek, S.T. Stefanie, H. Redl, E.K. Tschegg, Factors influencing interlocking screw failure in unreamed small diameter nails—a biomechanical study using a distal tibia fracture model, Clinical Biomechanics 24 (4) (2009) 379–384. [5] A.A. Zadpoor, J. Sinke, R. Benedictus, The mechanical behavior of adhesively bonded tailor-made blanks, International Journal of Adhesion and Adhesives 29 (5) (2009) 558–571. [6] A.M. Young, J.E. Barralet, et al., Chemical characterization of a degradable polymeric bone adhesive containing hydrolysable fillers and interpretation of anomalous mechanical properties, Acta Biomaterialia 5 (6) (2009) 2072–2083. [7] M. Waselau, V.F. Samii, S.E. Weisbrode, et al., Effects of a magnesium adhesive cement on bone stability and healing following a metatarsal osteotomy in horses, American Journal of Veterinary Research 68 (4) (2007) 370–378. [8] C.S. Liu, Inorganic;bone adhesion agent and its use in human hard tissue repair. US patent 2006; 7,094,286. [9] F. Wu, J. Wei, H. Guo, F.P. Chen, H. Hong, C.S. Liu, Self-setting bioactive calcium–magnesium phosphate cement with high strength and degradability for bone regeneration, Acta Biomaterialia 4 (2008) 1873–1884. [10] S. Pina, S.M. Olhero, S. Gheduzzi, A.W. Miles, J.M.F. Ferreira, Influence of setting liquid composition and liquid-to-powder ratio on properties of a Mg-substituted calcium phosphate cement, Acta Biomaterialia 5 (4) (2009) 1233–1240. [11] I. Brasnjevic, P.R. Hof, H.W.M. Steinbusch, C. Schmitz, Accumulation of nuclear DNA damage or neuron loss: molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases, DNA Repair 7 (2008) 1087–1097.

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