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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility ac
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Qianqian Yao , Jun Ye , Qian Xu , Anchun Mo
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& Ping Gong
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State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, P.R. China b
Dental Implant Center, West China College of Stomatology, Sichuan University, Chengdu 610064, P.R. China
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c
Department of Stomatology, The Second Xiangya Hospital, Central-South University, Changsha 410011, P.R. China d
School of Stomatology, Kunming Medical University, Kunming 650031, P.R. China Published online: 02 Jan 2015.
To cite this article: Qianqian Yao, Jun Ye, Qian Xu, Anchun Mo & Ping Gong (2015): Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2014.994946 To link to this article: http://dx.doi.org/10.1080/09205063.2014.994946
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Journal of Biomaterials Science, Polymer Edition, 2014 http://dx.doi.org/10.1080/09205063.2014.994946
Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility Qianqian Yaoa,c, Jun Yea,b, Qian Xua,d, Anchun Moa,b* and Ping Gonga,b* a
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, P.R. China; Dental Implant Center, West China College of Stomatology, Sichuan University, Chengdu 610064, P.R. China; cDepartment of Stomatology, The Second Xiangya Hospital, Central-South University, Changsha 410011, P.R. China; dSchool of Stomatology, Kunming Medical University, Kunming 650031, P.R. China
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(Received 23 August 2014; accepted 28 November 2014) This study aims to evaluate in vitro degradability and osteoblast biocompatibility of dicalcium phosphate anhydrate/multi-(amino acid) (DCPA/MAA) composites prepared by in situ polymerization method. The results revealed that the composites could be slowly degraded in PBS solution, with weight loss of 9.5 ± 0.2 wt.% compared with 12.2 ± 0.2 wt.% of MAA copolymer after eight weeks, and the changes of pH value were in the range of 7.18–7.4 and stabilized at 7.24. In addition, the compressive strength of the composite decreased from 98 to 62 MPa while that of MAA copolymer from 117 to 86 MPa. Furthermore, with non-toxicity demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay, the addition of DCPA to the MAA copolymer evidenced an enhancement of osteoblast differentiation and attachment compared with pure MAA materials regarding to alkaline phosphatase activity as well as initial cell adhesion. The results indicated that the DCPA/MAA scaffolds with good osteoblast biocompatibility, degradability, and sufficient strength had promising potential application in bone tissue engineering. Keywords: composite; dicalcium phosphate anhydrate; multi-(amino acid) copolymer; degradation; osteoblast biocompatibility
1. Introduction Biomaterials play a critical role in bone tissue engineering. Developing ideal biomaterials, including excellent biocompatibility, proper mechanical strength, and consistent mechanical performance during degradation, remains a main task in biomaterial research. Degradable polymer, a versatile class of functional materials, has been extensively investigated on account of its sufficient mechanical property, outstanding biocompatibility, and controllable degradability.[1,2] In absence of bioactivity, however, is the major drawback narrowed application of degradable polymers.[3,4] Calcium phosphate (CaP) biomaterials, which are similar to the mineral part of bone, have excellent biocompatibility and bioactivity.[5,6] It was found that CaP could provide a large number of sites for heterogeneous nucleation and crystallization, formed by a biologically reactive apatite layer equivalent to the inorganic mineral phase of bone and bonding to bone tissue in vivo.[7,8] Brittleness of CaP scaffolds, however, *Corresponding authors. Email: [email protected] (A. Mo); [email protected] (P. Gong) © 2014 Taylor & Francis
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prevents its further application in load-bearing area.[9] For the polymer phase brings toughness while inorganic particles provide with bioactivity, many researches were focused on bioactive scaffolds of inorganic/organic composites.[4,10] In addition to the mechanical strength of composites, biological properties and cell behaviors of polymer can be affected by the incorporation of inorganic bioactive materials.[9,11] Furthermore, the choice of degradable polymers with different molecular weight and the content ratio of inorganic/organic material would influence its structural stability and biological performance.[10,12] Multi-(amino acid) (MAA) copolymer, composed of ε-aminohexanoic acid, γ-aminobutanoic acid, L-proline, and L-lysine, exhibited clinical-controlled chemical breakdown and resorption, which was hydrolytically decomposing into CO2 and H2O without discernible difference between the implant site and the host tissue ultimately.[13] In previous studies, results indicated that MAA was effective in enhancing the biomechanical and biocompatibility, but ineffective in producing bioactive components.[13] Aimed to improve its bioactivity, bioactive materials, including various compositions of bioactive glasses, ceramics and glass–ceramics, have been researched for the effect of inorganic doping in MAA on its degradability and mineralization in clinical applications.[14,15] Recently, great interest was raised on dicalcium phosphate (DCP) for its significant role in the basic process of mineralization and fast osteotransduction.[10,16–18] Depending on various implanted sites or physiological conditions, they would dissolve and/or hydrolyze to carbonated hydroxyapatite. Moreover, with the resorption rate faster than that of carbonated apatite and beta-tricalcium phosphate, DCPs are one of the fastest resorbable CaP materials.[19] Hence, a bioactive dicalcium phosphate anhydrate/multi(amino acid) (DCPA/MAA) composite was fabricated to fit these characteristics. In this study, we introduced DCPA into MAA to synthesize DCPA/MAA composite in situ polymerization. However, little is known with respect to the influence of bioactive DCPA incorporated to MAA on their microstructures and mechanical properties. In addition, we further discussed DCPA/MAA scaffolds regarding their biodegradability, biomechanical property, and osteoblast biocompatibility in vitro. To characterize the effects of the composite on cell behaviors, rat primary osteoblasts were seeded in samples to evaluate cell adhesion, proliferation, and differentiation. The study aimed to develop new scaffolds with controllable biodegradability and bioactivity, which would provide a kind of material for potential clinical application. 2. Materials and methods 2.1. Preparation and characterization of samples ε-Aminohexanoic acid, γ-aminobutanoic acid, L-proline, and L-lysine were purchased from Shanghai Experimental Reagent Co., Ltd, and DCPA from Shanghai Aladdin Reagent Co., Ltd. The DCPA/MAA composite was synthesized in situ polymerization method: in brief, 98.25 g ε-aminohexanoic acid, 22.0 g γ-aminobutanoic acid, 5.0 g L-proline, 3.0 g L-lysine, and 0.5 mL phosphorous acid as catalyst were added into the reaction flask, kept at 200 °C until water was evaporated, followed with melting at 210 °C.[13] Then, 73.5 g DCPA (2–20 μm amorphous white powders) was added into the melt mixture with stirring (500 rpm) for 4 h at 220 °C. After cooling down to room temperature, the target composite with 40.0 ± 2.0 wt.% DCPA was obtained (data not showed). To avoid undesirable oxidation reactions, the system was protected by a continuous flow of nitrogen gas. The MAA material was prepared in the same condition
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without addition of DCPA as a control group. Before cell culture, samples press formed as a cylinder of 8 mm diameter and 1 mm thickness, and sterilized at 121 °C for 5 min. DCPA powder, MAA copolymer, and the DCPA/MAA composite were analyzed by X-ray diffraction (XRD) experiments performed on the X-ray diffractometer (X’Pert Pro-MPD) with monochromated Cu Kα radiation (Philips, Netherlands). Surface microstructures of MAA copolymer and the composite were characterized by scanning electron microscopy (SEM, JSM-5900LV, JEOL Techniques, Tokyo, Japan). 2.2. In vitro degradation test The degradation test for MAA copolymer and the composites was performed in phosphate-buffered saline (PBS, pH 7.4) for eight weeks at 37 ± 0.5 °C in vitro, with weight loss rate, pH value, and compress test investigated. Samples were incubated in PBS with a weight-to-volume ratio of 1/30 g/cm3 in sealed polyethene vials for 0 (W0), 3, 7, 14, 21, 28, 35, 42, 49, and 56 days, respectively. At each time, the scaffolds were rinsed in distilled water and dried in vacuum at 80 °C for 4 h before accurately weighting (Wt). The percentage of weight loss was calculated by the following equation: Weight loss ¼
ðW0 Wt Þ 100% W0
(1)
In addition, the change of the pH value was measured using an electrolyte-type pH meter (pHS-2C, Jingke Leici Co., Shanghai, China) at the preset soaking time. Furthermore, the compressive strength was determined by mechanical testing machine (REGER 30-50, Shenzhen Reger Co. Ltd, China) after soaking into PBS for 0, 2, 4, and 8 weeks. The cross-head speed was 1 mm/min, and the load was conducted by mechanical testing machine (REGER30-50, Shenzhen Reger Co., Ltd, China) with 50 kN load cells. It was recorded until these specimens were compressed to about 50% in height. After eight weeks degradation, the surface morphology of samples was characterized by SEM. Three samples were tested at each time point, and the results were expressed as mean ± SD. 2.3. Cell culture Primary osteoblasts, harvested from newborn SD rats according to the guideline of Animal Ethics Committee of Sichuan University, were testified by ALP and alizarin red S histochemical staining. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal calf serum (FCS, Gibco, USA) and penicillin–streptomycin (100 U/ml) in a 37 °C −5% CO2 incubator. Media were replenished every three day. In order to characterize, morphologically, cell behavior on the material surface, subcultured osteoblasts of passage 3–6 were seeded in samples directly and performed on osteoblast biocompatibility experiments. The non-cytotoxic HDPE (highdensity polyethylene, Hebei Bangji Co., China) was used as a negative control, while the cytotoxic phenol dilution (5 g/L, Tianjin Bodi Co. Ltd, China) as a positive and culture medium as a blank. All cultures were analyzed in triplicate. 2.4. MTT assay MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma, USA) assay was used to evaluate cell metabolic activity. Primary osteoblasts were seeded and
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incubated with samples at a density of 2 × 104 cells/well in 24-well tissue culture plates (Costar, USA) described before. After 1, 3, 5, and 7 days, changes in morphology were observed by an inverse phase contrast microscope (IPCM, IX70-S8F2, OLYMPUS, Japan) and the culture medium was removed before adding 20 μL of MTT (5 g/L) into each well. The supernatant was taken out after incubated for 4 h. Dimethyl sulfoxide (Sinopharm, Shanghai, China) was added (200 μL/well) to stop the reaction and dissolve the purple formazan. Then, the supernatant solution (100 μL/well) was transferred to another 96-well plate and optical densities (OD) were measured at 37 °C with a microplate reader (Thermo Scientific Varioskan Flash, Finland) setting the wavelength at 570 nm. 2.5. Alkaline phosphatase activity Osteoblasts seeded samples at a density of 2 × 105 cells/well in 6-well plates for 1, 3, 5, and 7 days after removing culture medium, which were washed with PBS, respectively. Nonidet P-40 (200 μL/well 1%) was added for 1 h incubation to obtain cell lysate. Then p-nitrophenylphosphate (Sangon, Shanghai, China) substrate solution (50 μL/well, 1 mg/mL, pH 9) composed of 0.1 mol/L glycine and 1 mmol/L MgCl2·6H2O was added and incubated for 30 min. By adding 100 μL/well of 0.1 N NaOH to quench the reaction, OD value was quantified by a microplate reader (Thermo Scientific Varioskan Flash, Finland) at the wavelength of 405 nm. According to the Pierce protein assay kit (Pierce Biotechnology Inc, Rockford, IL), total protein content of the cell lysate was examined by bicinchoninic acid method on the same condition, read at 560 nm and calculated with bovine serum albumin as the standard. Consequently, the ALP activity was expressed as OD value per total protein (mU/mg). 2.6. Cell morphology In order to evaluate the preliminary cell–material interaction, osteoblasts at a density of 2 × 104 cells/well attached on DCPA/MAA composites and MAA copolymers directly in 24-well tissue culture plates were harvested at 4, 8, and 24 h for SEM observation. Fixed in 2.5% glutaraldehyde in 0.1 M sodium-PBS for 30 min, samples went through alcohol gradient dehydration for 15 min, respectively. Specimens were immersed in 50% alcohol-hexamethyldisilazane solution (v/v) for 10 min and subsequently pure hexamethyldisilazane for another 10 min, vacuum dried at 37 °C overnight. Samples which were gold coated by ion sputtering were investigated by SEM. 2.7. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA), with statistically significant at p < 0.05. Results were expressed as mean ± standard deviation (SD). 3. Results 3.1. XRD analysis XRD patterns of the MAA copolymer, DCPA, and DCPA/MAA composite were obtained in this study (Figure 1). It was seen that the XRD pattern of the synthesis composite consisted of DCPA phase and MAA copolymer phase that without any new
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Figure 1.
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XRD patterns of the MAA (a), DCPA (b), and the DCPA/MAA composite (c).
peaks. As compared with the MAA copolymer, however, the intensity of two diffraction peaks in the composite dramatically declined, which is confirmed from observed characteristic peaks at diffracted angles (2θ) 19.75 and 23.57, respectively. 3.2. In vitro degradability 3.2.1. Weight loss and PH change in PBS After soaking in PBS for various time periods, the weight loss ratio of both MAA copolymer and the composite increased fast at the initial two weeks, then gradually decreased in the following time (Figure 2). In addition, the weight loss ratio of MAA
Figure 2. Weight loss of DCPA/MAA composite scaffolds and MAA copolymers after soaking in PBS over time. Error bars represented as mean ± SD. n = 3.
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copolymer was significantly higher than that of the composite, for the former was 12.2 ± 0.2 wt.% after eight weeks while the latter was 9.5 ± 0.2 wt.%. Although volumes of samples reduced gradually, no collapse degradation existed in observation time. The changes of pH value of the PBS solution were acquired with samples immersion (Figure 3). A significant decrease in pH was observed during the first 7 days, then no obvious changes in following soaking time. It was noticed that changes of pH value of MAA copolymer and the composite were in the range of 7.18–7.4 during immersion and stabilized at 7.36 and 7.24, respectively. 3.2.2. Compressive strength and microstructure analysis It was found that the compressive strength of scaffolds gradually decreased with a prolonged time (Figure 4). Before immersing in PBS, the initial compressive strength of MAA copolymer and the DCPA/MAA composite was 117 and 98 MPa, respectively. After eight weeks of soaking, however, the compressive strength in the former was 86 MPa and the latter was 62 MPa. Figure 5 shows surface morphology of the scaffolds with an interconnected solid profile before and after immersing into PBS solution for eight weeks. It was seen that obvious amorphous porous area existing in the composite, while the surface morphology of MAA was dense and smooth (Figures 5(A) and (B)). After eight week immersion, clearly, the surface of the MAA copolymer was eroded and many debris formed (Figure 5(C)), but some deep cracks were found on the surface of DCPA/MAA composite (Figure 5(D)). 3.3. In vitro osteoblast biocompatibility 3.3.1. Cytotoxicity in vitro As OD values showed, in general, DCPA/MAA composites had no toxic effect on cell growth, similar to the negative and blank control (p > 0.05) (Figure 6). In addition, the percentage of cell viability from the composites oscillated between 91.40 and 99.65%, with the highest at day three, compared with that of MAA copolymer varied between 92.28 and 98.19% (Figure 6). In contrast, cell shrinkage and lysis exposed to phenol indicated an obvious toxicity and significant decrease in viability (p < 0.05).
Figure 3. Chang of pH value of the solution after DCPA/MAA composite and MAA immersing into PBS with time. Error bars represented as mean ± SD. n = 3.
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Figure 4. Chang of compressive strength of the DCPA/MAA composite and MAA after immersing into PBS with time. Error bars represented as mean ± SD. n = 3.
Figure 5. Surface morphology of microstructure of the MAA copolymer (A, C) and DCPA/ MAA composite (B, D) observed by SEM, before (A, B) and after (C, D) soaking in PBS solution for eight weeks. (Scale bar represents 20 μm.)
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Figure 6. OD value of MTT assay on the DCPA/MAA composite, MAA copolymer, negative (HDPE), blank (culture medium) or positive (phenol) control at 1, 3, 5, and 7 days. Error bars represented as mean ± SD. n = 3. The symbol * represents a statistically difference between phenol and other groups (p < 0.05).
3.3.2. Osteoblast differentiation ALP activity of DCPA/MAA composite increased with prolonged time (Figure 7), and exhibited higher than that of negative and blank control, yet the difference was not statistically significant (p > 0.05). After three days in culture, however, the value of ALP activity of the composite was significantly higher than that of MAA copolymer (p < 0.05). When phenol group was considered, ALP activity decreased clearly and the difference between it and the composite was statistically significant (p < 0.05). 3.3.3. Osteoblast adhesion Cell adhesion, one of the crucial parameters influencing cell–biomaterial interactions as well as cell viabilities, was examined by SEM observation (Figure 8), since it was possible to demonstrate the initial cell–material cross-talk on the surface of samples. The adherent osteoblasts exhibited round morphology and dispersed in few cell protrusions
Figure 7. ALP activity of osteoblast cells cultured on the DCPA/MAA composite, MAA copolymer, negative (HDPE), blank (culture medium) or positive (phenol) control at 1, 3, 5, and 7 days. Error bars represented as mean ± SD. n = 3. The symbol * represents a statistically difference between phenol and other groups (p < 0.05). ** Represents an obvious statistically difference between positive and other groups (p < 0.01).
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Figure 8. Osteoblasts attached to the surface of MAA copolymer (A, C, and E) and DCPA/ MAA composite (B, D, and F) after 24 h incubation, which demonstrated the initial cell–material cross-talk. A, B: 4 h; C, D: 8 h; E, F: 24 h. (Scale bar represents 20 μm for A–D and 50 μm for E, F.)
on MAA material surface (Figures 8(A) and (C)). On the other hand, osteoblasts firmly attached and spread more processes on coarse surface of the composite, exhibiting a flattened appearance even at the preliminary 4 h (Figure 8(B)). After 8 h incubation, guided filopodia protrusions (Figure 8(D)) were formed probably due to enhanced cell adhesion. About 24 h later, an affluent layer of osteoblasts on DCPA/MAA material
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was almost formed, connecting with each other through digitations (Figure 8(F)). However, there were still fewer adherent cells on the surface of MAA with irregular morphology (Figure 8(E)).
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4. Discussion Bone can be regarded as a composite made up of inorganic mineral and organic collagen. In accordance to biomimetic principle, DCP anhydrate and multi-amino acid copolymer composed of the DCPA/MAA composite, expecting to obtain novel scaffolds for tissue engineering. In this study, the results revealed that the DCPA/MAA composite scaffolds showed appropriate biodegradability and enhanced cell–material interaction in vitro, indicating that the addition of DCPA into MAA could improve biodegradability property and osteoblast biocompatibility of MAA copolymer. Because degradability is crucial for bone induction, conduction, metabolism, and longevity when implanted into body; proper degradation in a physiological environment is one of the most important characteristics for a degradable biomaterial.[20,21] Chemical factors, especially, composition play an important role in the degradation behavior of MAA copolymer in vitro, which ultimately hydrolytically decomposing into CO2 and H2O. Some degradable scaffolds, such as poly(lactic acid) and poly(D, L-lactic acid-coglycolic acid), showed a bulk degradation manner and collapsed after implantation in vivo, and hence could not provide enough support.[22] For DCPA/MAA composites, apart from chemical constitute, the degradation behavior in vitro may also be the result of physical abrasion, such as porosity, crystallinity and grain size, and different content ratio of inorganic/organic material.[10,23,24] In the present study, although the composite had weight loss of 9.5 ± 0.2 wt.% after soaking for eight weeks, no collapse degradation manner was observed, with maintained bulk structure, which indicated the incorporation of DCPA enhanced the degradation property of MAA copolymer with slow degradable rate. In addition, the compressive strength of the composite and MAA material decreased from 98 to 62 MPa and 117 to 86 MPa, respectively, closed to the range of 50–150 MPa of natural cortical bone.[25] Therefore, the composite could maintain sufficient mechanical properties, indicating potential in clinical applications for fundamental support during bone regeneration. The pH value caused by the degradable products was one of the important properties of the degradable biomaterial in local environment in vivo, which would have great influence on cell response to implants. Acidic byproducts decomposed by some biomaterials (such as PLA and its copolymer) decrease the pH value in the ambient solution, resulted in clinically undesirable inflammatory reaction.[20,26] The results showed that the changes of pH value of PBS solution with the composite soaking were in the range of 7.4–7.18, and stabilized at 7.24, approaching to physiological fluid, which suggested innoxious to tissue in vivo.[27] Cell behavior on biomaterials can be affected by both the material structure and the soluble monomers that may leach out. In addition, primary osteoblast cell was chosen to mimic the chief cell-type in vivo physiological environment with which DCPA/MAA composites will interact as bone tissue engineering materials. The substituted DCPA/MAA composite was designed to serve as bone scaffold guiding the migration, adhesion, and proliferation of osteoblast, and new bone formation induced by scaffold was identified as the extracellular matrix production of differentiated osteoblast.[28] ALP activity, an early stage important marker of differentiation, plays a vital role for the synthesis of CaP crystal during the calcification process in bone healing.
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Reflected by enhancement in ALP activity, the DCPA/MAA composite could stimulate cell proliferation and differentiation on account of a bioactive apatite element which increased concentration of CaP,[29,30] contributing to bone healing in vivo.[31] Furthermore, calcium and phosphate ions released during the resorption can be used to mineralize new bone in the bone remodeling process.[32,33] Therefore, the enhanced differentiation of osteoblasts observed in our study may be explained by the presence of DCPA bioactive particles that play an important role in stimulatory processes. Various factors, such as surface patterning to hydrophobic, hydrophilic, or electrostatic, could influence the initial interactions between cells and synthetic polymers surface.[34] Of particular interest, in this study, were the different responses of osteoblast adhesion and differentiation to DCPA/MAA composites and MAA copolymer scaffolds. Results showed that the composite not only stimulated osteoblast differentiation, but also promoted osteoblast adhesion at initial 24 h, which was possibly caused by the addition of bioactive DCPA facilitating an apatite layer and improving the cell behavior directly. Moreover, several other reports have suggested that divalent cations, such as Ca2+, play a significant role in cellular adhesion to biomaterial surface, mediated mainly by proteins of the integrin type.[31,32] Compared with these methods, the addition of DCPA to MAA increasing inorganic concentration to control cell adhesion seems to be a simple, reproducible, and high cost-effective procedure. This study, furthermore, should need further experiments to explore the exact cell–material interaction mechanism and future work will investigate the long-term application properties of the DCPA/MAA composite in order to better control degradability and osteoblast biocompatibility. 5. Conclusions In this work, a degradable bone tissue engineering biomaterial DCPA/MAA composite was prepared by adding DCPA to MAA copolymer in situ polymerization. The composite was biodegraded slowly, without significant changes of the pH value caused by degradation products, confirmed by weight loss after immersion in PBS. Besides, the DCPA incorporation had effects on the degradation behavior and mechanical property of MAA copolymer, and exhibited low cytotoxicity. Furthermore, the bioactive particles DCPA enhanced osteoblast adhesion, spreading, and osteogenic differentiation. According to the histological evaluation results, the DCPA/MAA scaffolds showed good osteoblast biocompatibility and degradability, with promising potential application in bone implantation. Acknowledgments The authors are grateful for the financial support from National Key Technology R&D Program in the 11th Five year Plan of China (No. 2007BAE13B03). They would also like to acknowledge Ms. Li Xiaoyu at the State Key Laboratory of Oral Diseases (West China College of Stomatology) for her assistance in cell culture.
References [1] Cortizo MS, Molinuevo MS, Cortizo AM. Biocompatibility and biodegradation of polyester and polyfumarate based-scaffolds for bone tissue engineering. J. Tissue Eng. Regen. Med. 2008;2:33–42.
Downloaded by [Florida Atlantic University] at 05:28 18 January 2015
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[2] Lombello CB, Santos AR Jr., Malmonge SM, Barbanti SH, Wada ML, Duek EA. Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials. J. Mater. Sci. - Mater. Med. 2002;13:867–874. [3] Hench LL, Polak JM. Third-generation biomedical materials. Science (New York, NY). 2002;295:1014–1017. [4] Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006;27:3413–3431. [5] Jyoti MA, Thai VV, Min YK, Lee B-T, Song HY. In vitro bioactivity and biocompatibility of calcium phosphate cements using hydroxy-propyl-methyl-Cellulose (HPMC). Appl. Surf. Sci. 2010;257:1533–1539. [6] Shor L, Güçeri S, Wen X, Gandhi M, Sun W. Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials. 2007;28:5291–5297. [7] Al-Munajjed AA, O’Brien FJ. Influence of a novel calcium-phosphate coating on the mechanical properties of highly porous collagen scaffolds for bone repair. J. Mech. Behav. Biomed. Mater. 2009;2:138–146. [8] LeGeros RZ. Properties of osteoconductive biomaterials: calcium phosphates. Clin. Orthop. Relat. Res. 2002;395:81–98. [9] Wagoner JAJ, Herschler BA. A review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair. Acta Biomater. 2011;7:16–30. [10] Chae T, Yang H, Ko F, Troczynski T. Bio-inspired dicalcium phosphate anhydrate/poly(lactic acid) nanocomposite fibrous scaffolds for hard tissue regeneration: in situ synthesis and electrospinning. J. Biomed. Mater. Res. A. 2014;102:514–522. [11] Lao L, Wang Y, Zhu Y, Zhang Y, Gao C. Poly(lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering. J. Mater. Sci. Mater. Med. 2011;22:1873–1884. [12] Garg T, Singh O, Arora S, Murthy R. Scaffold: a novel carrier for cell and drug delivery. Crit. Rev. Ther. Drug Carrier Syst. 2012;29:1–63. [13] Li H, Yan Y, Wei J, Jian M, Min G, Xiaoman L, Yunfei Z. Bone substitute biomedical material of multi-(amino acid) copolymer: in vitro degradation and biocompatibility. J. Mater. Sci. - Mater. Med. 2011;22:2555–2563. [14] Li H, Yang L, Dong X, Gu Y, Lv G, Yan Y. Composite scaffolds of nano calcium deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration. J. Mater. Sci. Mater. Med. 2014;25:1257–1265. [15] Qi X, Li H, Qiao B, Li W, Hao X, Wu J, Su B, Jiang D. Development and characterization of an injectable cement of nano calcium-deficient hydroxyapatite/multi(amino acid) copolymer/calcium sulfate hemihydrate for bone repair. Int. J. Nanomed. 2013;8:4441–4452. [16] Tamimi F, Le ND, Eimar H, Sheikh Z, Komarova S, Barralet J. The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: brushite vs. monetite. Acta Biomater. 2012;8:3161–3169. [17] Galea L, Bohner M, Thuering J, Doebelin N, Aneziris CG, Graule T. Control of the size, shape and composition of highly uniform, non-agglomerated, sub-micrometer β-tricalcium phosphate and dicalcium phosphate platelets. Biomaterials. 2013;34:6388–6401. [18] Habibovic P, Gbureck U, Doillon CJ, Bassett DC, van Blitterswijk CA, Barralet JE. Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants. Biomaterials. 2008;29:944–953. [19] Tang R, Hass M, Wu W, Gulde S, Nancollas GH. Constant composition dissolution of mixed phases. J. Colloid Interface Sci. 2003;260:379–384. [20] Ambrose CG, Clanton TO. Bioabsorbable implants: review of clinical experience in orthopedic surgery. Ann. Biomed. Eng. 2004;32:171–177. [21] Barbanti SH, Santos AR Jr., Zavaglia CA, Duek EA. Poly(ε-caprolactone) and poly(d,l-lactic acid-co-glycolic acid) scaffolds used in bone tissue engineering prepared by melt compression–particulate leaching method. J. Mater. Sci. - Mater. Med. 2011;22:2377–2385. [22] Barbanti SH, Santos AR Jr., Zavaglia CA, Duek EA. Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture. J. Mater. Sci. - Mater. Med. 2004;15:1315–1321.
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[23] Dias AG, Lopes MA, Gibson IR, Santos JD. In vitro degradation studies of calcium phosphate glass ceramics prepared by controlled crystallization. J. Non-Cryst. Solids. 2003;330:81–89. [24] Qiu K, Wang CX, Zhang Q, Chen X, Su H. In vitro degradation studies of calcium polyphosphate ceramics prepared by controlled degree of polymerization and crystallization. Adv. Biomater. VI 2005;288–289:553–556. [25] Jie W, Yubao L. Tissue engineering scaffold material of nano-apatite crystals and polyamide composite. Eur. Polym. J. 2004;40:509–515. [26] Griffith LG. Polymeric biomaterials. Acta Mater. 2000;48:263–277. [27] Chaudhry MA, Bowen BD, Piret JM. Culture pH and osmolality influence proliferation and embryoid body yields of murine embryonic stem cells. Biochem. Eng. J. 2009;45:126–135. [28] LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem. Rev. 2008;108:4742–4753. [29] Chen QZ, Efthymiou A, Salih V, Boccaccini AR. Bioglass®-derived glass–ceramic scaffolds: study of cell proliferation and scaffold degradation in vitro. J. Biomed. Mater. Res. A. 2008;84A:1049–1060. [30] Yang L, Perez-Amodio S, Barrère-de Groot FY, Everts V, van Blitterswijk CA, Habibovic P. The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts. Biomaterials. 2010;31:2976–2989. [31] Habibovic P, Barralet JE. Bioinorganics and biomaterials: bone repair. Acta Biomater. 2011;7:3013–3026. [32] Tsang EJ, Arakawa CK, Zuk PA, Wu BM. Osteoblast interactions within a biomimetic apatite microenvironment. Ann. Biomed. Eng. 2011;39:1186–1200. [33] Wang J, de Boer J, de Groot K. Proliferation and differentiation of osteoblast-like MC3T3E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings. J. Biomed. Mater. Res. A. 2009;90A:664–670. [34] Teramura Y, Kaneda Y, Totani T, Iwata H. Behavior of synthetic polymers immobilized on a cell membrane. Biomaterials. 2008;29:1345–1355.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility ac
ab
ad
ab
Qianqian Yao , Jun Ye , Qian Xu , Anchun Mo
ab
& Ping Gong
a
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, P.R. China b
Dental Implant Center, West China College of Stomatology, Sichuan University, Chengdu 610064, P.R. China
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c
Department of Stomatology, The Second Xiangya Hospital, Central-South University, Changsha 410011, P.R. China d
School of Stomatology, Kunming Medical University, Kunming 650031, P.R. China Published online: 02 Jan 2015.
To cite this article: Qianqian Yao, Jun Ye, Qian Xu, Anchun Mo & Ping Gong (2015): Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2014.994946 To link to this article: http://dx.doi.org/10.1080/09205063.2014.994946
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Journal of Biomaterials Science, Polymer Edition, 2014 http://dx.doi.org/10.1080/09205063.2014.994946
Composite scaffolds of dicalcium phosphate anhydrate /multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility Qianqian Yaoa,c, Jun Yea,b, Qian Xua,d, Anchun Moa,b* and Ping Gonga,b* a
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, P.R. China; Dental Implant Center, West China College of Stomatology, Sichuan University, Chengdu 610064, P.R. China; cDepartment of Stomatology, The Second Xiangya Hospital, Central-South University, Changsha 410011, P.R. China; dSchool of Stomatology, Kunming Medical University, Kunming 650031, P.R. China
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b
(Received 23 August 2014; accepted 28 November 2014) This study aims to evaluate in vitro degradability and osteoblast biocompatibility of dicalcium phosphate anhydrate/multi-(amino acid) (DCPA/MAA) composites prepared by in situ polymerization method. The results revealed that the composites could be slowly degraded in PBS solution, with weight loss of 9.5 ± 0.2 wt.% compared with 12.2 ± 0.2 wt.% of MAA copolymer after eight weeks, and the changes of pH value were in the range of 7.18–7.4 and stabilized at 7.24. In addition, the compressive strength of the composite decreased from 98 to 62 MPa while that of MAA copolymer from 117 to 86 MPa. Furthermore, with non-toxicity demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay, the addition of DCPA to the MAA copolymer evidenced an enhancement of osteoblast differentiation and attachment compared with pure MAA materials regarding to alkaline phosphatase activity as well as initial cell adhesion. The results indicated that the DCPA/MAA scaffolds with good osteoblast biocompatibility, degradability, and sufficient strength had promising potential application in bone tissue engineering. Keywords: composite; dicalcium phosphate anhydrate; multi-(amino acid) copolymer; degradation; osteoblast biocompatibility
1. Introduction Biomaterials play a critical role in bone tissue engineering. Developing ideal biomaterials, including excellent biocompatibility, proper mechanical strength, and consistent mechanical performance during degradation, remains a main task in biomaterial research. Degradable polymer, a versatile class of functional materials, has been extensively investigated on account of its sufficient mechanical property, outstanding biocompatibility, and controllable degradability.[1,2] In absence of bioactivity, however, is the major drawback narrowed application of degradable polymers.[3,4] Calcium phosphate (CaP) biomaterials, which are similar to the mineral part of bone, have excellent biocompatibility and bioactivity.[5,6] It was found that CaP could provide a large number of sites for heterogeneous nucleation and crystallization, formed by a biologically reactive apatite layer equivalent to the inorganic mineral phase of bone and bonding to bone tissue in vivo.[7,8] Brittleness of CaP scaffolds, however, *Corresponding authors. Email: [email protected] (A. Mo); [email protected] (P. Gong) © 2014 Taylor & Francis
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prevents its further application in load-bearing area.[9] For the polymer phase brings toughness while inorganic particles provide with bioactivity, many researches were focused on bioactive scaffolds of inorganic/organic composites.[4,10] In addition to the mechanical strength of composites, biological properties and cell behaviors of polymer can be affected by the incorporation of inorganic bioactive materials.[9,11] Furthermore, the choice of degradable polymers with different molecular weight and the content ratio of inorganic/organic material would influence its structural stability and biological performance.[10,12] Multi-(amino acid) (MAA) copolymer, composed of ε-aminohexanoic acid, γ-aminobutanoic acid, L-proline, and L-lysine, exhibited clinical-controlled chemical breakdown and resorption, which was hydrolytically decomposing into CO2 and H2O without discernible difference between the implant site and the host tissue ultimately.[13] In previous studies, results indicated that MAA was effective in enhancing the biomechanical and biocompatibility, but ineffective in producing bioactive components.[13] Aimed to improve its bioactivity, bioactive materials, including various compositions of bioactive glasses, ceramics and glass–ceramics, have been researched for the effect of inorganic doping in MAA on its degradability and mineralization in clinical applications.[14,15] Recently, great interest was raised on dicalcium phosphate (DCP) for its significant role in the basic process of mineralization and fast osteotransduction.[10,16–18] Depending on various implanted sites or physiological conditions, they would dissolve and/or hydrolyze to carbonated hydroxyapatite. Moreover, with the resorption rate faster than that of carbonated apatite and beta-tricalcium phosphate, DCPs are one of the fastest resorbable CaP materials.[19] Hence, a bioactive dicalcium phosphate anhydrate/multi(amino acid) (DCPA/MAA) composite was fabricated to fit these characteristics. In this study, we introduced DCPA into MAA to synthesize DCPA/MAA composite in situ polymerization. However, little is known with respect to the influence of bioactive DCPA incorporated to MAA on their microstructures and mechanical properties. In addition, we further discussed DCPA/MAA scaffolds regarding their biodegradability, biomechanical property, and osteoblast biocompatibility in vitro. To characterize the effects of the composite on cell behaviors, rat primary osteoblasts were seeded in samples to evaluate cell adhesion, proliferation, and differentiation. The study aimed to develop new scaffolds with controllable biodegradability and bioactivity, which would provide a kind of material for potential clinical application. 2. Materials and methods 2.1. Preparation and characterization of samples ε-Aminohexanoic acid, γ-aminobutanoic acid, L-proline, and L-lysine were purchased from Shanghai Experimental Reagent Co., Ltd, and DCPA from Shanghai Aladdin Reagent Co., Ltd. The DCPA/MAA composite was synthesized in situ polymerization method: in brief, 98.25 g ε-aminohexanoic acid, 22.0 g γ-aminobutanoic acid, 5.0 g L-proline, 3.0 g L-lysine, and 0.5 mL phosphorous acid as catalyst were added into the reaction flask, kept at 200 °C until water was evaporated, followed with melting at 210 °C.[13] Then, 73.5 g DCPA (2–20 μm amorphous white powders) was added into the melt mixture with stirring (500 rpm) for 4 h at 220 °C. After cooling down to room temperature, the target composite with 40.0 ± 2.0 wt.% DCPA was obtained (data not showed). To avoid undesirable oxidation reactions, the system was protected by a continuous flow of nitrogen gas. The MAA material was prepared in the same condition
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without addition of DCPA as a control group. Before cell culture, samples press formed as a cylinder of 8 mm diameter and 1 mm thickness, and sterilized at 121 °C for 5 min. DCPA powder, MAA copolymer, and the DCPA/MAA composite were analyzed by X-ray diffraction (XRD) experiments performed on the X-ray diffractometer (X’Pert Pro-MPD) with monochromated Cu Kα radiation (Philips, Netherlands). Surface microstructures of MAA copolymer and the composite were characterized by scanning electron microscopy (SEM, JSM-5900LV, JEOL Techniques, Tokyo, Japan). 2.2. In vitro degradation test The degradation test for MAA copolymer and the composites was performed in phosphate-buffered saline (PBS, pH 7.4) for eight weeks at 37 ± 0.5 °C in vitro, with weight loss rate, pH value, and compress test investigated. Samples were incubated in PBS with a weight-to-volume ratio of 1/30 g/cm3 in sealed polyethene vials for 0 (W0), 3, 7, 14, 21, 28, 35, 42, 49, and 56 days, respectively. At each time, the scaffolds were rinsed in distilled water and dried in vacuum at 80 °C for 4 h before accurately weighting (Wt). The percentage of weight loss was calculated by the following equation: Weight loss ¼
ðW0 Wt Þ 100% W0
(1)
In addition, the change of the pH value was measured using an electrolyte-type pH meter (pHS-2C, Jingke Leici Co., Shanghai, China) at the preset soaking time. Furthermore, the compressive strength was determined by mechanical testing machine (REGER 30-50, Shenzhen Reger Co. Ltd, China) after soaking into PBS for 0, 2, 4, and 8 weeks. The cross-head speed was 1 mm/min, and the load was conducted by mechanical testing machine (REGER30-50, Shenzhen Reger Co., Ltd, China) with 50 kN load cells. It was recorded until these specimens were compressed to about 50% in height. After eight weeks degradation, the surface morphology of samples was characterized by SEM. Three samples were tested at each time point, and the results were expressed as mean ± SD. 2.3. Cell culture Primary osteoblasts, harvested from newborn SD rats according to the guideline of Animal Ethics Committee of Sichuan University, were testified by ALP and alizarin red S histochemical staining. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal calf serum (FCS, Gibco, USA) and penicillin–streptomycin (100 U/ml) in a 37 °C −5% CO2 incubator. Media were replenished every three day. In order to characterize, morphologically, cell behavior on the material surface, subcultured osteoblasts of passage 3–6 were seeded in samples directly and performed on osteoblast biocompatibility experiments. The non-cytotoxic HDPE (highdensity polyethylene, Hebei Bangji Co., China) was used as a negative control, while the cytotoxic phenol dilution (5 g/L, Tianjin Bodi Co. Ltd, China) as a positive and culture medium as a blank. All cultures were analyzed in triplicate. 2.4. MTT assay MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma, USA) assay was used to evaluate cell metabolic activity. Primary osteoblasts were seeded and
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incubated with samples at a density of 2 × 104 cells/well in 24-well tissue culture plates (Costar, USA) described before. After 1, 3, 5, and 7 days, changes in morphology were observed by an inverse phase contrast microscope (IPCM, IX70-S8F2, OLYMPUS, Japan) and the culture medium was removed before adding 20 μL of MTT (5 g/L) into each well. The supernatant was taken out after incubated for 4 h. Dimethyl sulfoxide (Sinopharm, Shanghai, China) was added (200 μL/well) to stop the reaction and dissolve the purple formazan. Then, the supernatant solution (100 μL/well) was transferred to another 96-well plate and optical densities (OD) were measured at 37 °C with a microplate reader (Thermo Scientific Varioskan Flash, Finland) setting the wavelength at 570 nm. 2.5. Alkaline phosphatase activity Osteoblasts seeded samples at a density of 2 × 105 cells/well in 6-well plates for 1, 3, 5, and 7 days after removing culture medium, which were washed with PBS, respectively. Nonidet P-40 (200 μL/well 1%) was added for 1 h incubation to obtain cell lysate. Then p-nitrophenylphosphate (Sangon, Shanghai, China) substrate solution (50 μL/well, 1 mg/mL, pH 9) composed of 0.1 mol/L glycine and 1 mmol/L MgCl2·6H2O was added and incubated for 30 min. By adding 100 μL/well of 0.1 N NaOH to quench the reaction, OD value was quantified by a microplate reader (Thermo Scientific Varioskan Flash, Finland) at the wavelength of 405 nm. According to the Pierce protein assay kit (Pierce Biotechnology Inc, Rockford, IL), total protein content of the cell lysate was examined by bicinchoninic acid method on the same condition, read at 560 nm and calculated with bovine serum albumin as the standard. Consequently, the ALP activity was expressed as OD value per total protein (mU/mg). 2.6. Cell morphology In order to evaluate the preliminary cell–material interaction, osteoblasts at a density of 2 × 104 cells/well attached on DCPA/MAA composites and MAA copolymers directly in 24-well tissue culture plates were harvested at 4, 8, and 24 h for SEM observation. Fixed in 2.5% glutaraldehyde in 0.1 M sodium-PBS for 30 min, samples went through alcohol gradient dehydration for 15 min, respectively. Specimens were immersed in 50% alcohol-hexamethyldisilazane solution (v/v) for 10 min and subsequently pure hexamethyldisilazane for another 10 min, vacuum dried at 37 °C overnight. Samples which were gold coated by ion sputtering were investigated by SEM. 2.7. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA), with statistically significant at p < 0.05. Results were expressed as mean ± standard deviation (SD). 3. Results 3.1. XRD analysis XRD patterns of the MAA copolymer, DCPA, and DCPA/MAA composite were obtained in this study (Figure 1). It was seen that the XRD pattern of the synthesis composite consisted of DCPA phase and MAA copolymer phase that without any new
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Figure 1.
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XRD patterns of the MAA (a), DCPA (b), and the DCPA/MAA composite (c).
peaks. As compared with the MAA copolymer, however, the intensity of two diffraction peaks in the composite dramatically declined, which is confirmed from observed characteristic peaks at diffracted angles (2θ) 19.75 and 23.57, respectively. 3.2. In vitro degradability 3.2.1. Weight loss and PH change in PBS After soaking in PBS for various time periods, the weight loss ratio of both MAA copolymer and the composite increased fast at the initial two weeks, then gradually decreased in the following time (Figure 2). In addition, the weight loss ratio of MAA
Figure 2. Weight loss of DCPA/MAA composite scaffolds and MAA copolymers after soaking in PBS over time. Error bars represented as mean ± SD. n = 3.
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copolymer was significantly higher than that of the composite, for the former was 12.2 ± 0.2 wt.% after eight weeks while the latter was 9.5 ± 0.2 wt.%. Although volumes of samples reduced gradually, no collapse degradation existed in observation time. The changes of pH value of the PBS solution were acquired with samples immersion (Figure 3). A significant decrease in pH was observed during the first 7 days, then no obvious changes in following soaking time. It was noticed that changes of pH value of MAA copolymer and the composite were in the range of 7.18–7.4 during immersion and stabilized at 7.36 and 7.24, respectively. 3.2.2. Compressive strength and microstructure analysis It was found that the compressive strength of scaffolds gradually decreased with a prolonged time (Figure 4). Before immersing in PBS, the initial compressive strength of MAA copolymer and the DCPA/MAA composite was 117 and 98 MPa, respectively. After eight weeks of soaking, however, the compressive strength in the former was 86 MPa and the latter was 62 MPa. Figure 5 shows surface morphology of the scaffolds with an interconnected solid profile before and after immersing into PBS solution for eight weeks. It was seen that obvious amorphous porous area existing in the composite, while the surface morphology of MAA was dense and smooth (Figures 5(A) and (B)). After eight week immersion, clearly, the surface of the MAA copolymer was eroded and many debris formed (Figure 5(C)), but some deep cracks were found on the surface of DCPA/MAA composite (Figure 5(D)). 3.3. In vitro osteoblast biocompatibility 3.3.1. Cytotoxicity in vitro As OD values showed, in general, DCPA/MAA composites had no toxic effect on cell growth, similar to the negative and blank control (p > 0.05) (Figure 6). In addition, the percentage of cell viability from the composites oscillated between 91.40 and 99.65%, with the highest at day three, compared with that of MAA copolymer varied between 92.28 and 98.19% (Figure 6). In contrast, cell shrinkage and lysis exposed to phenol indicated an obvious toxicity and significant decrease in viability (p < 0.05).
Figure 3. Chang of pH value of the solution after DCPA/MAA composite and MAA immersing into PBS with time. Error bars represented as mean ± SD. n = 3.
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Figure 4. Chang of compressive strength of the DCPA/MAA composite and MAA after immersing into PBS with time. Error bars represented as mean ± SD. n = 3.
Figure 5. Surface morphology of microstructure of the MAA copolymer (A, C) and DCPA/ MAA composite (B, D) observed by SEM, before (A, B) and after (C, D) soaking in PBS solution for eight weeks. (Scale bar represents 20 μm.)
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Figure 6. OD value of MTT assay on the DCPA/MAA composite, MAA copolymer, negative (HDPE), blank (culture medium) or positive (phenol) control at 1, 3, 5, and 7 days. Error bars represented as mean ± SD. n = 3. The symbol * represents a statistically difference between phenol and other groups (p < 0.05).
3.3.2. Osteoblast differentiation ALP activity of DCPA/MAA composite increased with prolonged time (Figure 7), and exhibited higher than that of negative and blank control, yet the difference was not statistically significant (p > 0.05). After three days in culture, however, the value of ALP activity of the composite was significantly higher than that of MAA copolymer (p < 0.05). When phenol group was considered, ALP activity decreased clearly and the difference between it and the composite was statistically significant (p < 0.05). 3.3.3. Osteoblast adhesion Cell adhesion, one of the crucial parameters influencing cell–biomaterial interactions as well as cell viabilities, was examined by SEM observation (Figure 8), since it was possible to demonstrate the initial cell–material cross-talk on the surface of samples. The adherent osteoblasts exhibited round morphology and dispersed in few cell protrusions
Figure 7. ALP activity of osteoblast cells cultured on the DCPA/MAA composite, MAA copolymer, negative (HDPE), blank (culture medium) or positive (phenol) control at 1, 3, 5, and 7 days. Error bars represented as mean ± SD. n = 3. The symbol * represents a statistically difference between phenol and other groups (p < 0.05). ** Represents an obvious statistically difference between positive and other groups (p < 0.01).
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Figure 8. Osteoblasts attached to the surface of MAA copolymer (A, C, and E) and DCPA/ MAA composite (B, D, and F) after 24 h incubation, which demonstrated the initial cell–material cross-talk. A, B: 4 h; C, D: 8 h; E, F: 24 h. (Scale bar represents 20 μm for A–D and 50 μm for E, F.)
on MAA material surface (Figures 8(A) and (C)). On the other hand, osteoblasts firmly attached and spread more processes on coarse surface of the composite, exhibiting a flattened appearance even at the preliminary 4 h (Figure 8(B)). After 8 h incubation, guided filopodia protrusions (Figure 8(D)) were formed probably due to enhanced cell adhesion. About 24 h later, an affluent layer of osteoblasts on DCPA/MAA material
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was almost formed, connecting with each other through digitations (Figure 8(F)). However, there were still fewer adherent cells on the surface of MAA with irregular morphology (Figure 8(E)).
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4. Discussion Bone can be regarded as a composite made up of inorganic mineral and organic collagen. In accordance to biomimetic principle, DCP anhydrate and multi-amino acid copolymer composed of the DCPA/MAA composite, expecting to obtain novel scaffolds for tissue engineering. In this study, the results revealed that the DCPA/MAA composite scaffolds showed appropriate biodegradability and enhanced cell–material interaction in vitro, indicating that the addition of DCPA into MAA could improve biodegradability property and osteoblast biocompatibility of MAA copolymer. Because degradability is crucial for bone induction, conduction, metabolism, and longevity when implanted into body; proper degradation in a physiological environment is one of the most important characteristics for a degradable biomaterial.[20,21] Chemical factors, especially, composition play an important role in the degradation behavior of MAA copolymer in vitro, which ultimately hydrolytically decomposing into CO2 and H2O. Some degradable scaffolds, such as poly(lactic acid) and poly(D, L-lactic acid-coglycolic acid), showed a bulk degradation manner and collapsed after implantation in vivo, and hence could not provide enough support.[22] For DCPA/MAA composites, apart from chemical constitute, the degradation behavior in vitro may also be the result of physical abrasion, such as porosity, crystallinity and grain size, and different content ratio of inorganic/organic material.[10,23,24] In the present study, although the composite had weight loss of 9.5 ± 0.2 wt.% after soaking for eight weeks, no collapse degradation manner was observed, with maintained bulk structure, which indicated the incorporation of DCPA enhanced the degradation property of MAA copolymer with slow degradable rate. In addition, the compressive strength of the composite and MAA material decreased from 98 to 62 MPa and 117 to 86 MPa, respectively, closed to the range of 50–150 MPa of natural cortical bone.[25] Therefore, the composite could maintain sufficient mechanical properties, indicating potential in clinical applications for fundamental support during bone regeneration. The pH value caused by the degradable products was one of the important properties of the degradable biomaterial in local environment in vivo, which would have great influence on cell response to implants. Acidic byproducts decomposed by some biomaterials (such as PLA and its copolymer) decrease the pH value in the ambient solution, resulted in clinically undesirable inflammatory reaction.[20,26] The results showed that the changes of pH value of PBS solution with the composite soaking were in the range of 7.4–7.18, and stabilized at 7.24, approaching to physiological fluid, which suggested innoxious to tissue in vivo.[27] Cell behavior on biomaterials can be affected by both the material structure and the soluble monomers that may leach out. In addition, primary osteoblast cell was chosen to mimic the chief cell-type in vivo physiological environment with which DCPA/MAA composites will interact as bone tissue engineering materials. The substituted DCPA/MAA composite was designed to serve as bone scaffold guiding the migration, adhesion, and proliferation of osteoblast, and new bone formation induced by scaffold was identified as the extracellular matrix production of differentiated osteoblast.[28] ALP activity, an early stage important marker of differentiation, plays a vital role for the synthesis of CaP crystal during the calcification process in bone healing.
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Reflected by enhancement in ALP activity, the DCPA/MAA composite could stimulate cell proliferation and differentiation on account of a bioactive apatite element which increased concentration of CaP,[29,30] contributing to bone healing in vivo.[31] Furthermore, calcium and phosphate ions released during the resorption can be used to mineralize new bone in the bone remodeling process.[32,33] Therefore, the enhanced differentiation of osteoblasts observed in our study may be explained by the presence of DCPA bioactive particles that play an important role in stimulatory processes. Various factors, such as surface patterning to hydrophobic, hydrophilic, or electrostatic, could influence the initial interactions between cells and synthetic polymers surface.[34] Of particular interest, in this study, were the different responses of osteoblast adhesion and differentiation to DCPA/MAA composites and MAA copolymer scaffolds. Results showed that the composite not only stimulated osteoblast differentiation, but also promoted osteoblast adhesion at initial 24 h, which was possibly caused by the addition of bioactive DCPA facilitating an apatite layer and improving the cell behavior directly. Moreover, several other reports have suggested that divalent cations, such as Ca2+, play a significant role in cellular adhesion to biomaterial surface, mediated mainly by proteins of the integrin type.[31,32] Compared with these methods, the addition of DCPA to MAA increasing inorganic concentration to control cell adhesion seems to be a simple, reproducible, and high cost-effective procedure. This study, furthermore, should need further experiments to explore the exact cell–material interaction mechanism and future work will investigate the long-term application properties of the DCPA/MAA composite in order to better control degradability and osteoblast biocompatibility. 5. Conclusions In this work, a degradable bone tissue engineering biomaterial DCPA/MAA composite was prepared by adding DCPA to MAA copolymer in situ polymerization. The composite was biodegraded slowly, without significant changes of the pH value caused by degradation products, confirmed by weight loss after immersion in PBS. Besides, the DCPA incorporation had effects on the degradation behavior and mechanical property of MAA copolymer, and exhibited low cytotoxicity. Furthermore, the bioactive particles DCPA enhanced osteoblast adhesion, spreading, and osteogenic differentiation. According to the histological evaluation results, the DCPA/MAA scaffolds showed good osteoblast biocompatibility and degradability, with promising potential application in bone implantation. Acknowledgments The authors are grateful for the financial support from National Key Technology R&D Program in the 11th Five year Plan of China (No. 2007BAE13B03). They would also like to acknowledge Ms. Li Xiaoyu at the State Key Laboratory of Oral Diseases (West China College of Stomatology) for her assistance in cell culture.
References [1] Cortizo MS, Molinuevo MS, Cortizo AM. Biocompatibility and biodegradation of polyester and polyfumarate based-scaffolds for bone tissue engineering. J. Tissue Eng. Regen. Med. 2008;2:33–42.
Downloaded by [Florida Atlantic University] at 05:28 18 January 2015
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Q. Yao et al.
[2] Lombello CB, Santos AR Jr., Malmonge SM, Barbanti SH, Wada ML, Duek EA. Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials. J. Mater. Sci. - Mater. Med. 2002;13:867–874. [3] Hench LL, Polak JM. Third-generation biomedical materials. Science (New York, NY). 2002;295:1014–1017. [4] Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006;27:3413–3431. [5] Jyoti MA, Thai VV, Min YK, Lee B-T, Song HY. In vitro bioactivity and biocompatibility of calcium phosphate cements using hydroxy-propyl-methyl-Cellulose (HPMC). Appl. Surf. Sci. 2010;257:1533–1539. [6] Shor L, Güçeri S, Wen X, Gandhi M, Sun W. Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials. 2007;28:5291–5297. [7] Al-Munajjed AA, O’Brien FJ. Influence of a novel calcium-phosphate coating on the mechanical properties of highly porous collagen scaffolds for bone repair. J. Mech. Behav. Biomed. Mater. 2009;2:138–146. [8] LeGeros RZ. Properties of osteoconductive biomaterials: calcium phosphates. Clin. Orthop. Relat. Res. 2002;395:81–98. [9] Wagoner JAJ, Herschler BA. A review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair. Acta Biomater. 2011;7:16–30. [10] Chae T, Yang H, Ko F, Troczynski T. Bio-inspired dicalcium phosphate anhydrate/poly(lactic acid) nanocomposite fibrous scaffolds for hard tissue regeneration: in situ synthesis and electrospinning. J. Biomed. Mater. Res. A. 2014;102:514–522. [11] Lao L, Wang Y, Zhu Y, Zhang Y, Gao C. Poly(lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering. J. Mater. Sci. Mater. Med. 2011;22:1873–1884. [12] Garg T, Singh O, Arora S, Murthy R. Scaffold: a novel carrier for cell and drug delivery. Crit. Rev. Ther. Drug Carrier Syst. 2012;29:1–63. [13] Li H, Yan Y, Wei J, Jian M, Min G, Xiaoman L, Yunfei Z. Bone substitute biomedical material of multi-(amino acid) copolymer: in vitro degradation and biocompatibility. J. Mater. Sci. - Mater. Med. 2011;22:2555–2563. [14] Li H, Yang L, Dong X, Gu Y, Lv G, Yan Y. Composite scaffolds of nano calcium deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration. J. Mater. Sci. Mater. Med. 2014;25:1257–1265. [15] Qi X, Li H, Qiao B, Li W, Hao X, Wu J, Su B, Jiang D. Development and characterization of an injectable cement of nano calcium-deficient hydroxyapatite/multi(amino acid) copolymer/calcium sulfate hemihydrate for bone repair. Int. J. Nanomed. 2013;8:4441–4452. [16] Tamimi F, Le ND, Eimar H, Sheikh Z, Komarova S, Barralet J. The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: brushite vs. monetite. Acta Biomater. 2012;8:3161–3169. [17] Galea L, Bohner M, Thuering J, Doebelin N, Aneziris CG, Graule T. Control of the size, shape and composition of highly uniform, non-agglomerated, sub-micrometer β-tricalcium phosphate and dicalcium phosphate platelets. Biomaterials. 2013;34:6388–6401. [18] Habibovic P, Gbureck U, Doillon CJ, Bassett DC, van Blitterswijk CA, Barralet JE. Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants. Biomaterials. 2008;29:944–953. [19] Tang R, Hass M, Wu W, Gulde S, Nancollas GH. Constant composition dissolution of mixed phases. J. Colloid Interface Sci. 2003;260:379–384. [20] Ambrose CG, Clanton TO. Bioabsorbable implants: review of clinical experience in orthopedic surgery. Ann. Biomed. Eng. 2004;32:171–177. [21] Barbanti SH, Santos AR Jr., Zavaglia CA, Duek EA. Poly(ε-caprolactone) and poly(d,l-lactic acid-co-glycolic acid) scaffolds used in bone tissue engineering prepared by melt compression–particulate leaching method. J. Mater. Sci. - Mater. Med. 2011;22:2377–2385. [22] Barbanti SH, Santos AR Jr., Zavaglia CA, Duek EA. Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture. J. Mater. Sci. - Mater. Med. 2004;15:1315–1321.
Downloaded by [Florida Atlantic University] at 05:28 18 January 2015
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[23] Dias AG, Lopes MA, Gibson IR, Santos JD. In vitro degradation studies of calcium phosphate glass ceramics prepared by controlled crystallization. J. Non-Cryst. Solids. 2003;330:81–89. [24] Qiu K, Wang CX, Zhang Q, Chen X, Su H. In vitro degradation studies of calcium polyphosphate ceramics prepared by controlled degree of polymerization and crystallization. Adv. Biomater. VI 2005;288–289:553–556. [25] Jie W, Yubao L. Tissue engineering scaffold material of nano-apatite crystals and polyamide composite. Eur. Polym. J. 2004;40:509–515. [26] Griffith LG. Polymeric biomaterials. Acta Mater. 2000;48:263–277. [27] Chaudhry MA, Bowen BD, Piret JM. Culture pH and osmolality influence proliferation and embryoid body yields of murine embryonic stem cells. Biochem. Eng. J. 2009;45:126–135. [28] LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem. Rev. 2008;108:4742–4753. [29] Chen QZ, Efthymiou A, Salih V, Boccaccini AR. Bioglass®-derived glass–ceramic scaffolds: study of cell proliferation and scaffold degradation in vitro. J. Biomed. Mater. Res. A. 2008;84A:1049–1060. [30] Yang L, Perez-Amodio S, Barrère-de Groot FY, Everts V, van Blitterswijk CA, Habibovic P. The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts. Biomaterials. 2010;31:2976–2989. [31] Habibovic P, Barralet JE. Bioinorganics and biomaterials: bone repair. Acta Biomater. 2011;7:3013–3026. [32] Tsang EJ, Arakawa CK, Zuk PA, Wu BM. Osteoblast interactions within a biomimetic apatite microenvironment. Ann. Biomed. Eng. 2011;39:1186–1200. [33] Wang J, de Boer J, de Groot K. Proliferation and differentiation of osteoblast-like MC3T3E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings. J. Biomed. Mater. Res. A. 2009;90A:664–670. [34] Teramura Y, Kaneda Y, Totani T, Iwata H. Behavior of synthetic polymers immobilized on a cell membrane. Biomaterials. 2008;29:1345–1355.
