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polypropylene carbonate biocomposite
Materials Science and Engineering C 63 (2016) 285–291
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Preparation, characterization and properties of nano-hydroxyapatite/ polypropylene carbonate biocomposite Jianguo Liao a,⁎, Yanqun Li a, Qin Zou b, Xingze Duan a, Zhengpeng Yang a, Yufen Xie a, Haohuai Liu c a b c
School of Materials Science and Engineering, Henan Polytechnic University, Jiaozuo 454000, China Analytical & Testing Center, Sichuan University, Chengdu 610064, China School of Chemistry and Chemical Engineering, Analytical and Testing Center, Guangzhou University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 21 January 2016 Accepted 19 February 2016 Available online 22 February 2016 Keywords: Nano-hydroxyapatite Polypropylene carbonate Bone substitute Biocompatibility Composite
a b s t r a c t The combination of nano-hydroxyapatite (n-HA) and polypropylene carbonate (PPC) was used to make a composite materials by a coprecipitation method. The physical and chemical properties of the composite were tested. Scanning electron microscope (SEM) observation indicated that the biomimetic n-HA crystals were uniformly distributed in the polymer matrix. As the n-HA content increased in the composite, the fracture mechanism of the composites changes from gliding fracture to gliding and brittle fracture. Furthermore, the chemical interaction between inorganic n-HA and polypropylene carbonate was also investigated and discussed in detail. The hyof n-HA crystal and the ester group (–COO–) of PPC. The drogen bonds might be formed between –OH/CO2− 3 tensile strength of n-HA/PPC (40/60) was similar to that of the cancellous bone, and reached ca 58 MPa. The osteoblasts were cultured for up to 7 days, and then the adhesion and proliferation of osteoblasts were measured by Methyl thiazolyl tetrazolium (MTT) colorimetry assay and SEM. The cells proliferated, grew normally in fusiform shape and well attached. The in vitro test confirmed that the n-HA/PPC composites were biocompatible and showed undetectable negative effect on osteoblasts. In vivo implantation of the composite in New Zealand white rabbits was performed. It can stimulate the growth of a new bone, and at the same time the material begins to degrade. These results suggested that the composite may be suitable for the reparation or replacement of bone defects and possessed the potential for clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (HA), the main inorganic component of human bone tissues, possesses good biocompatibility and high bioactivity [1, 2], and has been widely used to repair bone defects [3–5]. However, it is not suitable to be used for load-bearing applications for bone repair due to its brittleness, fatigue failure and slow degradation rate in vivo [6]. Since natural bone is a nano-hydroxyapatite/collagen composite at the microscopic scale, a polymer matrix composed of particulate and bioactive components provides a suitable material for substitute of the cortical bone [7,8]. The bioactivity of the composite, which is rendered by the bioactive component in the composite, can promote the ingrowth of the tissues adjacent to the implant which leads to the formation of a strong bond between bone tissues and the implant after implantation. Therefore, by simulating the composition of natural bones, n-HA/natural polymer [9–14] and n-HA/synthetic polymer [15–19]
⁎ Corresponding author. E-mail address: [email protected] (J. Liao).
http://dx.doi.org/10.1016/j.msec.2016.02.054 0928-4931/© 2016 Elsevier B.V. All rights reserved.
composites are widely studied for the applications in hard tissue repair materials. PPC, the copolymer of carbon dioxide (CO2) and propylene oxide (PO), is a class of biodegradable aliphatic polycarbonate polymers [20, 21], which has flexibility with elongation of 200–1000% at break. Studies have shown that PPC can be degraded in acid and alkali solution, and does not cause inflammation in vivo [22]. Low molecular weight PPC can degrade even faster compared to high molecular weight polymers [23]. Besides, PPC has shown desirable biocompatibility, which can be applied in biomedical area. Thus, it can make a great contribution to this novel inorganic/organic composite system. In this study, n-HA and polypropylene carbonate (PPC) were used to form a composite biomaterial by solution blending and microwave heating techniques. Similar composites have been studied previously, however, the conventional preparation methods for such composites have some drawbacks, such as i) the aggregation of the nanoparticles in the continuous polymeric phase leading to non-uniform dispersion and consequently decrease composite's properties, and ii) the preparation technology of in-situ synthesis is complex [24–26]. The process of coprecipitation can precipitate n-HA and PPC simultaneously and form
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n-HA/PPC composites in which the n-HA is homogeneously distributed in the PPC matrix. Microwave heating has the advantages of immediacy, integrity, efficiency utilization of energy, etc. Therefore, we choose coprecipitation method — microwave method. 2. Materials and methods 2.1. Materials The slurry of nano-hydroxyapatite (n-HA) used for composites was prepared by our laboratory according to Ref. [27]. Calcium nitrate and sodium phosphate were separately dissolved in deionized water. Sodium phosphate solution was dropped slowly into the calcium nitrate solution with stirring and was heated to 70 °C–80 °C. The pH value of the solution was kept between 10 and 12 by adding sodium hydrate aqueous solution. When the reaction ended, HA precipitate was obtained after aging 48 h, and after being fully washed with deionized water, the nanoscale needle-like HA crystal (n-HA) slurry was obtained. Polypropylene carbonate (PPC) was supplied by Henan Tianguan Group Co., Ltd., China. PEG6000 was from Chengdu Chemical Agent Co., Ltd., China. The ethanol was purchased from Chengdu Chemical Agent Co., Ltd., China, AR grade. 2.2. Preparation of n-HA/PPC composites The n-HA slurry (36%) and DMAC (N, N-dimethylacetamide, 400 mL) were mixed in a three-neck flask with continuous stirring at 120 °C. After water was completely evaporated, n-HA/DMAC slurry was obtained. PEG was used to modify n-HA crystals and improve their dispersion in the solution. At 140 °C, PEG (the ratio of PEG dose and n-HA dose was 8:100, in wt.%) and PPC (from 20 to 60 wt.% separately) were added into the above n-HA/DMAC solution and stirred for 3 h followed by full washing with anhydrous ethanol and dried in a vacuum oven at 40 °C for 48 h, thus n-HA/PPC composite powder was obtained. The n-HA: PPC ratios (wt.%) of the composites range from 10:90 to 60:40. Samples with standard shape, according to GB/T 14472005 standard, were made for mechanical testing by microwave heating at 40–50 °C. The dimensions of testing specimens were carefully machined to be 150 mm × 10 mm × 4 mm, and the gauge length is 50 mm. All tests were performed at an ambient temperature (25 °C), and five specimens were used in each test to obtain the average value. 2.3. Characterization and analysis of n-HA/PPC composites The samples for X-ray diffraction (XRD, D8 ADVANCE, Bruker, Germany), Fourier transform infrared absorption spectra (FT-IR, Netzsch, STA409PC/4/H-TENSOR27, Germany) and Raman spectra (RS, inVia, Renishaw, England) analysis were ground to fine powders and dried in a vacuum oven at 40 °C for 24 h before testing. XRD from a Cu X-ray tube was used to detect the phase composition and crystallinity. The 2θ of the measured samples ranged from 10° to 65°. FT-IR and RS were used to determine the bonding between inorganic phase and polymer phase. The morphology of the n-HA/PPC composites was observed by field emission gun scanning electron microscopy (FEG-SEM, FEI Quanta 250) and an X-Max 30 mm2 detector energy dispersive X-ray spectrometer (Bruker Quantax 200 Xflash 6|30 EDS). Mechanical properties were evaluated using a WDW-2C electronic universal testing machine (Jinan Kehui Test Equipment Co., Ltd., China). The average values of 5+ tests were reported. 2.4. Biocompatibility test 2.4.1. Cell culture Osteoblast MG-63 was isolated via a sequential collagenase digestion from neonatal rat calvaria according to an established protocol. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM,
Gibco) supplemented with 10% bovine serum (FBS, Gibco) under 5% CO2 atmosphere at 37 °C. The medium was changed every two days. After confluence, the monolayer cell was washed twice with phosphate buffered saline (PBS) and incubated with a trypsin–EDTA solution (0.25% trypsin, 1 mm EDTA, Gibco) to detach the cells. The effect of trypsin was then inhibited for the complete medium joining at room temperature. Thereafter, the cells were re-suspended in a complete medium for re-seeding and growing in new culture flasks. Osteoblasts at the third passage were used for cell experiments. The initial cell seeding density was 1 × 104 cells well−1 (24-well plate) in this study. 2.4.2. Cell viability Each specimen (Ø8.0 mm × 1.0 mm) was sterilized by ethylene oxide gas, immersed in a well with 2 mL of fresh medium (without cells), and extracted overnight in an incubator. MG63 cells cultured in media for 3 days were seeded on the top of pre-wetted specimens (1 × 104 cells/specimen). The specimens were then placed in the wells of plastic dishes (24-well cell culture plates, Corning, USA) and left undisturbed in an incubator for 3 h to allow the cells to attach and an additional 1 mL culture medium was added into each well. The cell/composite constructs were cultured in a humidified incubator at 37 °C with 95% air and 5% CO2 for 1, 4, and 7 day(s). The media were changed every 2 days. The morphology of MG63 cells cultured with the cement and plastic (as a control) was observed by an inverted phase contrast microscope (Nikon TE300, Japan). Cell proliferation was measured at 1, 4, and 7 day(s) using MTT assay. MTT reagent (3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) is enzymatically converted by living cells into a blue/purple formazan product. The intensity of the colored formazan is directly related to the number of viable cells and thus to their proliferation in vitro. MTT reagent was added to each sample and incubated at 37 °C for 4 h. The colored formazan product was solubilized into a solution with dimethyl sulfoxide (DMSO) and the solution of each sample was removed out for assay in a 96-well plate. The suspension was centrifuged and the absorbance of the supernatant was measured by a microplate reader (Bio-Rad 680) at the wavelength of 490 nm. Each treatment was performed five times and the average value was used as the final result. 2.4.3. Statistical analysis and SEM observation Statistical analysis was assessed using software SPSS (v10.0). The least significant difference method (assuming equal variances) was performed to measure the statistical significance between experimental groups. A value of **p b 0.01 was considered to be an outstanding statistical significance. The scanning electron microscope (JEOL, JEM-100CX, Japan) was used to observe the specimens. Cells cultured for 2 and 4 day(s) on cements were rinsed with PBS, fixed with 1 vol.% glutaraldehyde, subjected to graded alcohol dehydration, rinsed with isoamyl acetate, and sputter coated with gold. 2.4.4. Biocompatibility in vivo The n-HA/PPC (40/60) composite was implanted into eight healthy New Zealand white rabbits with the weight of about 2.0 kg each. The rabbits were anesthetized with pentobarbital sodium. A 3-cm parallel incision was made on the femur of the rabbit. The periosteum was retracted and the femur was exposed. A defect (Ø2 mm × 5 mm) was made by a drill bit. A composite construct (Ø2 mm × 5 mm) was inserted into the defect and the gap was sutured by suture line. Rabbits were sacrificed at 4 and 12 weeks after implantation. The composite was excised together with surrounding tissue fixed in 10% neutral buffered formalin, decalcified and embedded in paraffin. Tissue blocks were sectioned at 5 μm in thickness and stained with hematoxylin and eosin (H&E), then observed by an optical microscope (Olympus, Japan). Moreover, X-ray microradiography was employed to monitor the process of ectopic bone formation.
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3. Results and discussion 3.1. XRD analysis The XRD patterns of PPC, n-HA/PPC composite, n-HA crystal and modified n-HA are shown in Fig. 1. The characteristic peaks at 2θ = 25.9°, 31.8°, 32.9° and 39.9° showed that the n-HA was poorly crystallized. The hydroxyapatite in the bone was also found to be poorly crystallized [28]. This means that the n-HA crystal, from the point of crystallinity, is similar to hydroxyapatite in the bone. Compared with previous HA, modified n-HA showed weaker crystallization peaks due to the attachment of PEG6000 on the surface. According to literature [29], the two peaks at 2θ = 13.1° and 19.8° could be assigned to the characteristic peak and the amorphous peak of PPC, respectively. The n-HA/PPC composite powder still showed weak peaks of the n-HA and PPC. As shown in Fig. 1c, the crystallinity of PPC phase was decreased in composite, indicating that the crystal structure of PPC was changed after composite formation with n-HA crystal. The hydrogen bonds in PPC contribute to its crystallinity. In view of the formation of composite, the interface binding between n-HA and PPC might result in the decline in the number of hydrogen bonds, thus lessens PPC crystallinity. 3.2. FT-IR analysis Fig. 2 shows the FT-IR spectra of the n-HA, modified n-HA, n-HA/PC composite and PPC. In n-HA (Fig. 2a), the peaks at 3572 cm−1 and 630 cm−1 belong to the stretching vibration of hydroxyl (–OH) [18], while the peaks at 1039 cm−1, 1100 cm−1, 957 cm−1, and 603 cm−1, [30,31]. belong to PO3− 4 In the modified n-HA (Fig. 2b), there are antisymmetric stretching vibration peak (2930 cm−1) and symmetric stretching vibration peak (2860 cm−1) of –CH2–, which are the characteristic peaks of PEG6000. In PPC (Fig. 2d), the peak at 2992 cm−1 belongs to the antisymmetric stretching vibration of CH3, which moves to 2980 cm−1 in the composite. 1749 cm−1 belongs to the symmetric stretching vibration of C_O, 785 cm−1, 857 cm−1 is the out-of-plane bending vibration of _CH–, and 1074 cm−1 belongs to symmetric stretching vibration of C–O. It can be seen that in the n-HA/PPC composite (Fig. 2c), characteristic peaks of n-HA and PPC have been slightly changed. Fig. 2c shows C–O–C stretching vibration peak (1074 cm−1), however, there is no peak of nHA/PPC (40/60) composite. In the composite (Fig. 2c), the peak of hydroxyl (–OH) at 630 cm−1, the peak of PO34 − at 1090 cm− 1 and the peak at 870 cm− 1 of n-HA
Fig. 1. XRD patterns of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
Fig. 2. IR spectra of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
crystal that can be attributed to CO23 − disappear in the IR spectra of the composite [30,31]. The C_O peak of the PPC at 1749 cm−1 was moved to 1747 cm−1 in the composite. Overall it can be said that, the disappearance of the peaks of –OH and CO23 − at 630 cm− 1 and 870 cm− 1 confirms the possibility that hydrogen bonds might be formed between –OH/CO23 − of n-HA crystal and the ester group (–COO–) of PPC. 3.3. RS analysis The Raman spectra in Fig. 3 represent the n-HA (a), modified n-HA (b), n-HA/PPC composite (c) and PPC (d) samples. The similarities and differences of Raman spectra among the samples of n-HA and modified n-HA of apatite are summarized in Fig. 3a and b, which shows individual plots of measurements of the three most important Raman spectral features. P–O–P symmetric stretching vibration is at ca 961 cm−1,
Fig. 3. Raman spectra of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
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antisymmetric bending vibration at ca 428 cm−1, bending vibration at ca 598 cm−1, and asymmetric stretching mode at 1051 cm− 1 (Fig. 3a). Fig. 3a and b are similar, however the peak of Fig. 3a is weaker because of the PEG6000 attaching to the n-HA surface. In Fig. 3c, the peaks at 1452 cm−1, 1746 cm−1, 2882 cm−1 and 2936 cm−1 belong to PPC, and the other peaks belong to n-HA. Fig. 3 indicates that the chemistry component of n-HA and PPC is unchanged after composite formation. 3.4. SEM observation The SEM photographs of the n-HA/PPC composite with different weight rations are shown in Fig. 4. In Fig. 4a, n-HA without modification was unevenly distributed in the PPC matrix, and poor bonding caused the most fractured surface along the interface between the PPC matrix and n-HA. The fracture was brittle, and some holes existed at the surface of the fracture. Fig. 4b, c and d shows fracture surface of modified n-HA/ PPC composite. Good interconnections between the PPC matrix and modified n-HA were observed, modified n-HA was homogeneously
distributed in the PPC matrix, the interface of the PPC matrix and modified n-HA was fuzzy and the fracture was gliding. It was noted that the bonding force at the interface of PPC and n-HA was increased for PEG6000-modified n-HA, thus the composite exhibited a very good combination effect, that is to say that the properties of the composite will be improved. Fig. 4d shows EDS spectrum of position I in Fig. 4(c) and the sample contains calcium, phosphorous, carbon, oxygen and hydrogen in certain contents. Carbon elements mainly belong to the PPC and calcium and phosphorous belong to n-HA. As the n-HA content was increased in composite, as shown in Fig.4, the fracture mechanism of the composite changes from gliding fracture to the coexistence of gliding and brittle. 3.5. Mechanical properties As seen in Fig. 5, the tensile strength and elongation at break of nHA/PPC composite were investigated as the content of n-HA was varied from 10 to 60 wt.%. With the increase of the content of n-HA, the tensile strength was first increased and then decreased. If the content of n-HA
Fig. 4. SEM images of n-HA/PPC composites with different weight rations (a) n-HA/PPC (40/60, without modified n-HA), (b) n-HA/PPC (30/70, modified n-HA), (c) n-HA/PPC (40/60, modified n-HA), (d) EDS spectrum of position I in panel (c), (e) n-HA/PPC (50/50, modified n-HA).
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For the control group, osteoblasts were cultured under the same condition without materials (Fig. 6). In various cell culture periods, the cell number was increased with the culture time on test groups and control groups. Statistical analysis indicated that the proliferation of osteoblast cells cultured on n-HA/PPC composite was higher than that of the control groups for 1 and 7 day incubation. However, there was no significant difference in the cell number between osteoblasts cultured on nHA/PPC composites and control groups for 4 and 7 day incubation. This similar ascendant tendency of the cell population demonstrated that n-HA/PPC composite imposed little influence on cell growth. Fig. 7 shows the representative SEM photographs of cell attachment onto the n-HA/PPC composite after 2 and 4 day cell culture. From the micrographs, it can be seen that after 2 day culture, the cells proliferated, grew normally in fusiform shape, and well attached. Obviously, the osteoblasts/composite constructs show no negative effect on the cell morphology, viability and proliferation. Fig. 5. Tensile strength and elongation at break of n-HA/PPC composite with n-HA content.
3.7. Biocompatibility in vivo
accounted for 40%, the tensile strength was ca 58 MPa, which was similar to the tensile strength of cancellous bone. In addition, compared to pure PPC with the same molecular weight, the fracture elongation ratio of n-HA/PPC composite was decreased from 300% to b10%, which meant an enhancement of the fracture strength. The difference of mechanical properties between n-HA/PPC was mostly caused by the appropriate content, dispersion state and binding force. Small diameter resulted in higher surface energy and surface activity, even particles distribution, which, in turn, resulted in a strong bonding between n-HA and PPC. Thus the property of the composite was improved.
After the operation, animal's eating, moving, etc., were normal. Moreover, no bone resorption, osteomyelitis, rejection phenomenon and bone fracture in the operation area were observed. And no material break was found in the observation period. During the whole period, neither bone fracture nor material broken was observed. After 4 weeks (Fig. 8a), the gap between the material and the bone was completely healed, but the appearance and density of the material had not been changed. After 12 weeks (Fig. 8b), the new bone grew toward the implanted material, while no mature bones were formed on the ambient of material, and at the same time the material began to degrade and the fringe became irregular. The absence of an intervening connective layer suggested that this nano-biocomposite had an excellent bioactivity and was expected to be an analogous structure to the natural bone.
3.6. Biocompatibility in vitro The proliferation of osteoblasts co-cultured with PPC and n-HA/PPC composites was evaluated using MTT (Fig. 6). In various cell culture periods, the cell number was increased with the culture time on test groups and control groups. It was easy to find that, compared to the control group, the PPC group had lower cell viability (**p b 0.01) than other groups. However, with the increase of HA in composite, n-HA/PPC could enhance osteoblasts viability. On the first day, no statistical difference was found between the control group and n-HA/PPC (40% and 50%). However, after 4 and 7 days, n-HA/PPC (40% and 50%) showed higher cell proliferation than the control group (**p b 0.01). The statistical difference was not found between 40% n-HA/PPC and 50% n-HA/PPC, indicating that HA in n-HA/PPC composite could improve the cell viability. If the amount of HA was about 40%, the n-HA/PPC composite had the best cell viability.
4. Discussion The inorganic component of n-HA is similar to the human hard tissue. It has a good biocompatibility and can bond with the bone tissue in the body. The outstanding biological performance of n-HA and n-HA composite biomaterial has been widely proved in the form of bone filler, and as scaffolds for cell carrier in bone tissue engineering [32]. In this study, a novel method, co-solution, co-precipitation and dehydrated ethanol treatment under normal atmospheric pressure, was employed for n-HA/PPC composite biomaterial. From the SEM images, n-HA was distributed evenly in the PPC matrix, and a good interface bonding between n-HA and PPC was observed.
Fig. 6. MTT assay of the control group (a: without any materials), osteoblasts with PPC (b) and osteoblasts with n-HA/PPC composites (c: 20%, d: 40%, and e: 50%) at different periods, 4 and 7 days, respectively. Error bars represent means ± SD for n = 5, **p b 0.01.
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Fig. 7. SEM micrographs of the induced osteoblasts cultured on the n-HA/PPC (40/60) composite, after 2 day cell culture (a), after 4 days (b).
To achieve the desired bioactivity, it is necessary to improve n-HA content in the composite. A conventional melting method by mechanically mixing n-HA powders with polymer to prepare n-HA/polymer composite is difficult to ensure a high n-HA content in composite with good homogeneity. The new method of co-solution, co-precipitation and dehydrated ethanol treatment used in this experiment is more helpful to make a n-HA/polymer composite than mechanically mixing with clogged powders under high temperature or/and high pressure. In order to make a n-HA/polymer composite with good mechanical properties, the interface between the inorganic mineral and organic polymer should be first optimized to create proper chemical bonding between the two phases. Polypropylene carbonate is a polymer with nonpolar groups in its molecular chain. The nonpolar polymer has relatively low affinity to polar fillers (e.g. n-HA). Therefore, the PEG6000 was used to modify nHA surface. IR analysis shows that some adsorption peaks of the n-HA peaks of the n-HA and PPC have variations in the composite: the PO3− 4 at 1090 cm− 1, –OH at 630 cm− 1, CO23 − at 870 cm− 1, –CH3 peaks at 2992 cm− 1 move to 2980 cm−1, and –CH2– vibration peaks at 2890 cm−1 move to 2870 cm−1. These results indicate that some molecule interactions may be present among the n-HA, PEG600 and PPC in the composite. The results of in vitro cell culture show that n-HA/PPC composite has no cell toxicity and has good biocompatibility with osteoblast cells. The results of in vivo tests indicate that the composite exhibits no toxicity, no irritation, and possesses no negative tissue responses after being
implanted into the rabbit bone. The histological observation shows that the specimens are gradually biodegradable with time. However, after 4 weeks, new bone was formed around the defects of the rabbit femur where the n-HA/PPC composite was implanted, and gradually matured with the implantation time. The results demonstrate that the n-HA/PPC composite has good biocompatibility and bioactivity, and has a potential to be used for the reparation and substitution of bone defects. 5. Conclusion Improved properties of n-HA/PCC composites have been achieved in the current study based on a biomimetic strategy and a new preparation method. The combination of n-HA and PPC causes the improvement in mechanic properties. Direct use of n-HA slurry and PPC solution by the co-solution method to prepare composite contributes to the uniform dispersion of n-HA crystals in PPC matrix and prevents the aggregation of n-HA nanoparticles within the polymer matrix. The use of nano-scale HA allows high n-HA content within the composite, better homogeneity and mechanical properties. Based on in vitro and in vivo experimental results, the n-HA/PPC composite exhibits undetectable negative effect on the growth and proliferation of osteoblasts, and has shown proper compatibility. This composite can stimulate the growth of a new bone, indicating that it is an osteoconductive material. Therefore, this class of composites shows a great potential for the reparation or replacement of bone defects.
Fig. 8. Hematoxylin/eosin-stained sections of n-HA/PPC (40/60) composite materials. In the photos, M denotes n-HA/PPC (40/60) composite materials, B denotes bone, while NB denotes newly formed bone tissue.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, characterization and properties of nano-hydroxyapatite/ polypropylene carbonate biocomposite Jianguo Liao a,⁎, Yanqun Li a, Qin Zou b, Xingze Duan a, Zhengpeng Yang a, Yufen Xie a, Haohuai Liu c a b c
School of Materials Science and Engineering, Henan Polytechnic University, Jiaozuo 454000, China Analytical & Testing Center, Sichuan University, Chengdu 610064, China School of Chemistry and Chemical Engineering, Analytical and Testing Center, Guangzhou University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 21 January 2016 Accepted 19 February 2016 Available online 22 February 2016 Keywords: Nano-hydroxyapatite Polypropylene carbonate Bone substitute Biocompatibility Composite
a b s t r a c t The combination of nano-hydroxyapatite (n-HA) and polypropylene carbonate (PPC) was used to make a composite materials by a coprecipitation method. The physical and chemical properties of the composite were tested. Scanning electron microscope (SEM) observation indicated that the biomimetic n-HA crystals were uniformly distributed in the polymer matrix. As the n-HA content increased in the composite, the fracture mechanism of the composites changes from gliding fracture to gliding and brittle fracture. Furthermore, the chemical interaction between inorganic n-HA and polypropylene carbonate was also investigated and discussed in detail. The hyof n-HA crystal and the ester group (–COO–) of PPC. The drogen bonds might be formed between –OH/CO2− 3 tensile strength of n-HA/PPC (40/60) was similar to that of the cancellous bone, and reached ca 58 MPa. The osteoblasts were cultured for up to 7 days, and then the adhesion and proliferation of osteoblasts were measured by Methyl thiazolyl tetrazolium (MTT) colorimetry assay and SEM. The cells proliferated, grew normally in fusiform shape and well attached. The in vitro test confirmed that the n-HA/PPC composites were biocompatible and showed undetectable negative effect on osteoblasts. In vivo implantation of the composite in New Zealand white rabbits was performed. It can stimulate the growth of a new bone, and at the same time the material begins to degrade. These results suggested that the composite may be suitable for the reparation or replacement of bone defects and possessed the potential for clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (HA), the main inorganic component of human bone tissues, possesses good biocompatibility and high bioactivity [1, 2], and has been widely used to repair bone defects [3–5]. However, it is not suitable to be used for load-bearing applications for bone repair due to its brittleness, fatigue failure and slow degradation rate in vivo [6]. Since natural bone is a nano-hydroxyapatite/collagen composite at the microscopic scale, a polymer matrix composed of particulate and bioactive components provides a suitable material for substitute of the cortical bone [7,8]. The bioactivity of the composite, which is rendered by the bioactive component in the composite, can promote the ingrowth of the tissues adjacent to the implant which leads to the formation of a strong bond between bone tissues and the implant after implantation. Therefore, by simulating the composition of natural bones, n-HA/natural polymer [9–14] and n-HA/synthetic polymer [15–19]
⁎ Corresponding author. E-mail address: [email protected] (J. Liao).
http://dx.doi.org/10.1016/j.msec.2016.02.054 0928-4931/© 2016 Elsevier B.V. All rights reserved.
composites are widely studied for the applications in hard tissue repair materials. PPC, the copolymer of carbon dioxide (CO2) and propylene oxide (PO), is a class of biodegradable aliphatic polycarbonate polymers [20, 21], which has flexibility with elongation of 200–1000% at break. Studies have shown that PPC can be degraded in acid and alkali solution, and does not cause inflammation in vivo [22]. Low molecular weight PPC can degrade even faster compared to high molecular weight polymers [23]. Besides, PPC has shown desirable biocompatibility, which can be applied in biomedical area. Thus, it can make a great contribution to this novel inorganic/organic composite system. In this study, n-HA and polypropylene carbonate (PPC) were used to form a composite biomaterial by solution blending and microwave heating techniques. Similar composites have been studied previously, however, the conventional preparation methods for such composites have some drawbacks, such as i) the aggregation of the nanoparticles in the continuous polymeric phase leading to non-uniform dispersion and consequently decrease composite's properties, and ii) the preparation technology of in-situ synthesis is complex [24–26]. The process of coprecipitation can precipitate n-HA and PPC simultaneously and form
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n-HA/PPC composites in which the n-HA is homogeneously distributed in the PPC matrix. Microwave heating has the advantages of immediacy, integrity, efficiency utilization of energy, etc. Therefore, we choose coprecipitation method — microwave method. 2. Materials and methods 2.1. Materials The slurry of nano-hydroxyapatite (n-HA) used for composites was prepared by our laboratory according to Ref. [27]. Calcium nitrate and sodium phosphate were separately dissolved in deionized water. Sodium phosphate solution was dropped slowly into the calcium nitrate solution with stirring and was heated to 70 °C–80 °C. The pH value of the solution was kept between 10 and 12 by adding sodium hydrate aqueous solution. When the reaction ended, HA precipitate was obtained after aging 48 h, and after being fully washed with deionized water, the nanoscale needle-like HA crystal (n-HA) slurry was obtained. Polypropylene carbonate (PPC) was supplied by Henan Tianguan Group Co., Ltd., China. PEG6000 was from Chengdu Chemical Agent Co., Ltd., China. The ethanol was purchased from Chengdu Chemical Agent Co., Ltd., China, AR grade. 2.2. Preparation of n-HA/PPC composites The n-HA slurry (36%) and DMAC (N, N-dimethylacetamide, 400 mL) were mixed in a three-neck flask with continuous stirring at 120 °C. After water was completely evaporated, n-HA/DMAC slurry was obtained. PEG was used to modify n-HA crystals and improve their dispersion in the solution. At 140 °C, PEG (the ratio of PEG dose and n-HA dose was 8:100, in wt.%) and PPC (from 20 to 60 wt.% separately) were added into the above n-HA/DMAC solution and stirred for 3 h followed by full washing with anhydrous ethanol and dried in a vacuum oven at 40 °C for 48 h, thus n-HA/PPC composite powder was obtained. The n-HA: PPC ratios (wt.%) of the composites range from 10:90 to 60:40. Samples with standard shape, according to GB/T 14472005 standard, were made for mechanical testing by microwave heating at 40–50 °C. The dimensions of testing specimens were carefully machined to be 150 mm × 10 mm × 4 mm, and the gauge length is 50 mm. All tests were performed at an ambient temperature (25 °C), and five specimens were used in each test to obtain the average value. 2.3. Characterization and analysis of n-HA/PPC composites The samples for X-ray diffraction (XRD, D8 ADVANCE, Bruker, Germany), Fourier transform infrared absorption spectra (FT-IR, Netzsch, STA409PC/4/H-TENSOR27, Germany) and Raman spectra (RS, inVia, Renishaw, England) analysis were ground to fine powders and dried in a vacuum oven at 40 °C for 24 h before testing. XRD from a Cu X-ray tube was used to detect the phase composition and crystallinity. The 2θ of the measured samples ranged from 10° to 65°. FT-IR and RS were used to determine the bonding between inorganic phase and polymer phase. The morphology of the n-HA/PPC composites was observed by field emission gun scanning electron microscopy (FEG-SEM, FEI Quanta 250) and an X-Max 30 mm2 detector energy dispersive X-ray spectrometer (Bruker Quantax 200 Xflash 6|30 EDS). Mechanical properties were evaluated using a WDW-2C electronic universal testing machine (Jinan Kehui Test Equipment Co., Ltd., China). The average values of 5+ tests were reported. 2.4. Biocompatibility test 2.4.1. Cell culture Osteoblast MG-63 was isolated via a sequential collagenase digestion from neonatal rat calvaria according to an established protocol. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM,
Gibco) supplemented with 10% bovine serum (FBS, Gibco) under 5% CO2 atmosphere at 37 °C. The medium was changed every two days. After confluence, the monolayer cell was washed twice with phosphate buffered saline (PBS) and incubated with a trypsin–EDTA solution (0.25% trypsin, 1 mm EDTA, Gibco) to detach the cells. The effect of trypsin was then inhibited for the complete medium joining at room temperature. Thereafter, the cells were re-suspended in a complete medium for re-seeding and growing in new culture flasks. Osteoblasts at the third passage were used for cell experiments. The initial cell seeding density was 1 × 104 cells well−1 (24-well plate) in this study. 2.4.2. Cell viability Each specimen (Ø8.0 mm × 1.0 mm) was sterilized by ethylene oxide gas, immersed in a well with 2 mL of fresh medium (without cells), and extracted overnight in an incubator. MG63 cells cultured in media for 3 days were seeded on the top of pre-wetted specimens (1 × 104 cells/specimen). The specimens were then placed in the wells of plastic dishes (24-well cell culture plates, Corning, USA) and left undisturbed in an incubator for 3 h to allow the cells to attach and an additional 1 mL culture medium was added into each well. The cell/composite constructs were cultured in a humidified incubator at 37 °C with 95% air and 5% CO2 for 1, 4, and 7 day(s). The media were changed every 2 days. The morphology of MG63 cells cultured with the cement and plastic (as a control) was observed by an inverted phase contrast microscope (Nikon TE300, Japan). Cell proliferation was measured at 1, 4, and 7 day(s) using MTT assay. MTT reagent (3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) is enzymatically converted by living cells into a blue/purple formazan product. The intensity of the colored formazan is directly related to the number of viable cells and thus to their proliferation in vitro. MTT reagent was added to each sample and incubated at 37 °C for 4 h. The colored formazan product was solubilized into a solution with dimethyl sulfoxide (DMSO) and the solution of each sample was removed out for assay in a 96-well plate. The suspension was centrifuged and the absorbance of the supernatant was measured by a microplate reader (Bio-Rad 680) at the wavelength of 490 nm. Each treatment was performed five times and the average value was used as the final result. 2.4.3. Statistical analysis and SEM observation Statistical analysis was assessed using software SPSS (v10.0). The least significant difference method (assuming equal variances) was performed to measure the statistical significance between experimental groups. A value of **p b 0.01 was considered to be an outstanding statistical significance. The scanning electron microscope (JEOL, JEM-100CX, Japan) was used to observe the specimens. Cells cultured for 2 and 4 day(s) on cements were rinsed with PBS, fixed with 1 vol.% glutaraldehyde, subjected to graded alcohol dehydration, rinsed with isoamyl acetate, and sputter coated with gold. 2.4.4. Biocompatibility in vivo The n-HA/PPC (40/60) composite was implanted into eight healthy New Zealand white rabbits with the weight of about 2.0 kg each. The rabbits were anesthetized with pentobarbital sodium. A 3-cm parallel incision was made on the femur of the rabbit. The periosteum was retracted and the femur was exposed. A defect (Ø2 mm × 5 mm) was made by a drill bit. A composite construct (Ø2 mm × 5 mm) was inserted into the defect and the gap was sutured by suture line. Rabbits were sacrificed at 4 and 12 weeks after implantation. The composite was excised together with surrounding tissue fixed in 10% neutral buffered formalin, decalcified and embedded in paraffin. Tissue blocks were sectioned at 5 μm in thickness and stained with hematoxylin and eosin (H&E), then observed by an optical microscope (Olympus, Japan). Moreover, X-ray microradiography was employed to monitor the process of ectopic bone formation.
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3. Results and discussion 3.1. XRD analysis The XRD patterns of PPC, n-HA/PPC composite, n-HA crystal and modified n-HA are shown in Fig. 1. The characteristic peaks at 2θ = 25.9°, 31.8°, 32.9° and 39.9° showed that the n-HA was poorly crystallized. The hydroxyapatite in the bone was also found to be poorly crystallized [28]. This means that the n-HA crystal, from the point of crystallinity, is similar to hydroxyapatite in the bone. Compared with previous HA, modified n-HA showed weaker crystallization peaks due to the attachment of PEG6000 on the surface. According to literature [29], the two peaks at 2θ = 13.1° and 19.8° could be assigned to the characteristic peak and the amorphous peak of PPC, respectively. The n-HA/PPC composite powder still showed weak peaks of the n-HA and PPC. As shown in Fig. 1c, the crystallinity of PPC phase was decreased in composite, indicating that the crystal structure of PPC was changed after composite formation with n-HA crystal. The hydrogen bonds in PPC contribute to its crystallinity. In view of the formation of composite, the interface binding between n-HA and PPC might result in the decline in the number of hydrogen bonds, thus lessens PPC crystallinity. 3.2. FT-IR analysis Fig. 2 shows the FT-IR spectra of the n-HA, modified n-HA, n-HA/PC composite and PPC. In n-HA (Fig. 2a), the peaks at 3572 cm−1 and 630 cm−1 belong to the stretching vibration of hydroxyl (–OH) [18], while the peaks at 1039 cm−1, 1100 cm−1, 957 cm−1, and 603 cm−1, [30,31]. belong to PO3− 4 In the modified n-HA (Fig. 2b), there are antisymmetric stretching vibration peak (2930 cm−1) and symmetric stretching vibration peak (2860 cm−1) of –CH2–, which are the characteristic peaks of PEG6000. In PPC (Fig. 2d), the peak at 2992 cm−1 belongs to the antisymmetric stretching vibration of CH3, which moves to 2980 cm−1 in the composite. 1749 cm−1 belongs to the symmetric stretching vibration of C_O, 785 cm−1, 857 cm−1 is the out-of-plane bending vibration of _CH–, and 1074 cm−1 belongs to symmetric stretching vibration of C–O. It can be seen that in the n-HA/PPC composite (Fig. 2c), characteristic peaks of n-HA and PPC have been slightly changed. Fig. 2c shows C–O–C stretching vibration peak (1074 cm−1), however, there is no peak of nHA/PPC (40/60) composite. In the composite (Fig. 2c), the peak of hydroxyl (–OH) at 630 cm−1, the peak of PO34 − at 1090 cm− 1 and the peak at 870 cm− 1 of n-HA
Fig. 1. XRD patterns of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
Fig. 2. IR spectra of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
crystal that can be attributed to CO23 − disappear in the IR spectra of the composite [30,31]. The C_O peak of the PPC at 1749 cm−1 was moved to 1747 cm−1 in the composite. Overall it can be said that, the disappearance of the peaks of –OH and CO23 − at 630 cm− 1 and 870 cm− 1 confirms the possibility that hydrogen bonds might be formed between –OH/CO23 − of n-HA crystal and the ester group (–COO–) of PPC. 3.3. RS analysis The Raman spectra in Fig. 3 represent the n-HA (a), modified n-HA (b), n-HA/PPC composite (c) and PPC (d) samples. The similarities and differences of Raman spectra among the samples of n-HA and modified n-HA of apatite are summarized in Fig. 3a and b, which shows individual plots of measurements of the three most important Raman spectral features. P–O–P symmetric stretching vibration is at ca 961 cm−1,
Fig. 3. Raman spectra of n-HA (a), modified n-HA (b), n-HA/PPC (40/60) composite (c) and PPC (d).
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antisymmetric bending vibration at ca 428 cm−1, bending vibration at ca 598 cm−1, and asymmetric stretching mode at 1051 cm− 1 (Fig. 3a). Fig. 3a and b are similar, however the peak of Fig. 3a is weaker because of the PEG6000 attaching to the n-HA surface. In Fig. 3c, the peaks at 1452 cm−1, 1746 cm−1, 2882 cm−1 and 2936 cm−1 belong to PPC, and the other peaks belong to n-HA. Fig. 3 indicates that the chemistry component of n-HA and PPC is unchanged after composite formation. 3.4. SEM observation The SEM photographs of the n-HA/PPC composite with different weight rations are shown in Fig. 4. In Fig. 4a, n-HA without modification was unevenly distributed in the PPC matrix, and poor bonding caused the most fractured surface along the interface between the PPC matrix and n-HA. The fracture was brittle, and some holes existed at the surface of the fracture. Fig. 4b, c and d shows fracture surface of modified n-HA/ PPC composite. Good interconnections between the PPC matrix and modified n-HA were observed, modified n-HA was homogeneously
distributed in the PPC matrix, the interface of the PPC matrix and modified n-HA was fuzzy and the fracture was gliding. It was noted that the bonding force at the interface of PPC and n-HA was increased for PEG6000-modified n-HA, thus the composite exhibited a very good combination effect, that is to say that the properties of the composite will be improved. Fig. 4d shows EDS spectrum of position I in Fig. 4(c) and the sample contains calcium, phosphorous, carbon, oxygen and hydrogen in certain contents. Carbon elements mainly belong to the PPC and calcium and phosphorous belong to n-HA. As the n-HA content was increased in composite, as shown in Fig.4, the fracture mechanism of the composite changes from gliding fracture to the coexistence of gliding and brittle. 3.5. Mechanical properties As seen in Fig. 5, the tensile strength and elongation at break of nHA/PPC composite were investigated as the content of n-HA was varied from 10 to 60 wt.%. With the increase of the content of n-HA, the tensile strength was first increased and then decreased. If the content of n-HA
Fig. 4. SEM images of n-HA/PPC composites with different weight rations (a) n-HA/PPC (40/60, without modified n-HA), (b) n-HA/PPC (30/70, modified n-HA), (c) n-HA/PPC (40/60, modified n-HA), (d) EDS spectrum of position I in panel (c), (e) n-HA/PPC (50/50, modified n-HA).
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For the control group, osteoblasts were cultured under the same condition without materials (Fig. 6). In various cell culture periods, the cell number was increased with the culture time on test groups and control groups. Statistical analysis indicated that the proliferation of osteoblast cells cultured on n-HA/PPC composite was higher than that of the control groups for 1 and 7 day incubation. However, there was no significant difference in the cell number between osteoblasts cultured on nHA/PPC composites and control groups for 4 and 7 day incubation. This similar ascendant tendency of the cell population demonstrated that n-HA/PPC composite imposed little influence on cell growth. Fig. 7 shows the representative SEM photographs of cell attachment onto the n-HA/PPC composite after 2 and 4 day cell culture. From the micrographs, it can be seen that after 2 day culture, the cells proliferated, grew normally in fusiform shape, and well attached. Obviously, the osteoblasts/composite constructs show no negative effect on the cell morphology, viability and proliferation. Fig. 5. Tensile strength and elongation at break of n-HA/PPC composite with n-HA content.
3.7. Biocompatibility in vivo
accounted for 40%, the tensile strength was ca 58 MPa, which was similar to the tensile strength of cancellous bone. In addition, compared to pure PPC with the same molecular weight, the fracture elongation ratio of n-HA/PPC composite was decreased from 300% to b10%, which meant an enhancement of the fracture strength. The difference of mechanical properties between n-HA/PPC was mostly caused by the appropriate content, dispersion state and binding force. Small diameter resulted in higher surface energy and surface activity, even particles distribution, which, in turn, resulted in a strong bonding between n-HA and PPC. Thus the property of the composite was improved.
After the operation, animal's eating, moving, etc., were normal. Moreover, no bone resorption, osteomyelitis, rejection phenomenon and bone fracture in the operation area were observed. And no material break was found in the observation period. During the whole period, neither bone fracture nor material broken was observed. After 4 weeks (Fig. 8a), the gap between the material and the bone was completely healed, but the appearance and density of the material had not been changed. After 12 weeks (Fig. 8b), the new bone grew toward the implanted material, while no mature bones were formed on the ambient of material, and at the same time the material began to degrade and the fringe became irregular. The absence of an intervening connective layer suggested that this nano-biocomposite had an excellent bioactivity and was expected to be an analogous structure to the natural bone.
3.6. Biocompatibility in vitro The proliferation of osteoblasts co-cultured with PPC and n-HA/PPC composites was evaluated using MTT (Fig. 6). In various cell culture periods, the cell number was increased with the culture time on test groups and control groups. It was easy to find that, compared to the control group, the PPC group had lower cell viability (**p b 0.01) than other groups. However, with the increase of HA in composite, n-HA/PPC could enhance osteoblasts viability. On the first day, no statistical difference was found between the control group and n-HA/PPC (40% and 50%). However, after 4 and 7 days, n-HA/PPC (40% and 50%) showed higher cell proliferation than the control group (**p b 0.01). The statistical difference was not found between 40% n-HA/PPC and 50% n-HA/PPC, indicating that HA in n-HA/PPC composite could improve the cell viability. If the amount of HA was about 40%, the n-HA/PPC composite had the best cell viability.
4. Discussion The inorganic component of n-HA is similar to the human hard tissue. It has a good biocompatibility and can bond with the bone tissue in the body. The outstanding biological performance of n-HA and n-HA composite biomaterial has been widely proved in the form of bone filler, and as scaffolds for cell carrier in bone tissue engineering [32]. In this study, a novel method, co-solution, co-precipitation and dehydrated ethanol treatment under normal atmospheric pressure, was employed for n-HA/PPC composite biomaterial. From the SEM images, n-HA was distributed evenly in the PPC matrix, and a good interface bonding between n-HA and PPC was observed.
Fig. 6. MTT assay of the control group (a: without any materials), osteoblasts with PPC (b) and osteoblasts with n-HA/PPC composites (c: 20%, d: 40%, and e: 50%) at different periods, 4 and 7 days, respectively. Error bars represent means ± SD for n = 5, **p b 0.01.
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Fig. 7. SEM micrographs of the induced osteoblasts cultured on the n-HA/PPC (40/60) composite, after 2 day cell culture (a), after 4 days (b).
To achieve the desired bioactivity, it is necessary to improve n-HA content in the composite. A conventional melting method by mechanically mixing n-HA powders with polymer to prepare n-HA/polymer composite is difficult to ensure a high n-HA content in composite with good homogeneity. The new method of co-solution, co-precipitation and dehydrated ethanol treatment used in this experiment is more helpful to make a n-HA/polymer composite than mechanically mixing with clogged powders under high temperature or/and high pressure. In order to make a n-HA/polymer composite with good mechanical properties, the interface between the inorganic mineral and organic polymer should be first optimized to create proper chemical bonding between the two phases. Polypropylene carbonate is a polymer with nonpolar groups in its molecular chain. The nonpolar polymer has relatively low affinity to polar fillers (e.g. n-HA). Therefore, the PEG6000 was used to modify nHA surface. IR analysis shows that some adsorption peaks of the n-HA peaks of the n-HA and PPC have variations in the composite: the PO3− 4 at 1090 cm− 1, –OH at 630 cm− 1, CO23 − at 870 cm− 1, –CH3 peaks at 2992 cm− 1 move to 2980 cm−1, and –CH2– vibration peaks at 2890 cm−1 move to 2870 cm−1. These results indicate that some molecule interactions may be present among the n-HA, PEG600 and PPC in the composite. The results of in vitro cell culture show that n-HA/PPC composite has no cell toxicity and has good biocompatibility with osteoblast cells. The results of in vivo tests indicate that the composite exhibits no toxicity, no irritation, and possesses no negative tissue responses after being
implanted into the rabbit bone. The histological observation shows that the specimens are gradually biodegradable with time. However, after 4 weeks, new bone was formed around the defects of the rabbit femur where the n-HA/PPC composite was implanted, and gradually matured with the implantation time. The results demonstrate that the n-HA/PPC composite has good biocompatibility and bioactivity, and has a potential to be used for the reparation and substitution of bone defects. 5. Conclusion Improved properties of n-HA/PCC composites have been achieved in the current study based on a biomimetic strategy and a new preparation method. The combination of n-HA and PPC causes the improvement in mechanic properties. Direct use of n-HA slurry and PPC solution by the co-solution method to prepare composite contributes to the uniform dispersion of n-HA crystals in PPC matrix and prevents the aggregation of n-HA nanoparticles within the polymer matrix. The use of nano-scale HA allows high n-HA content within the composite, better homogeneity and mechanical properties. Based on in vitro and in vivo experimental results, the n-HA/PPC composite exhibits undetectable negative effect on the growth and proliferation of osteoblasts, and has shown proper compatibility. This composite can stimulate the growth of a new bone, indicating that it is an osteoconductive material. Therefore, this class of composites shows a great potential for the reparation or replacement of bone defects.
Fig. 8. Hematoxylin/eosin-stained sections of n-HA/PPC (40/60) composite materials. In the photos, M denotes n-HA/PPC (40/60) composite materials, B denotes bone, while NB denotes newly formed bone tissue.
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