Article pubs.acs.org/Langmuir
Rapid Biomimetic Mineralization of Hydroxyapatite‑g‑PDLLA Hybrid Microspheres Ke Du,†,§ Xudong Shi,† and Zhihua Gan*,†,‡ †
The CAS Key Laboratory of Engineering Plastics, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China ‡ The State Key Laboratory of Organic−Inorganic Composites, Beijing Laboratory of Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China § The Graduate University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: Hydroxyapatite-graf t-poly(D,L-lactide) (HA-gPDLLA) nanoparticles were synthesized here to fabricate hybrid microspheres with diameter in the range of 150−200 μm by emulsion solvent evaporation techniques. The asobtained microspheres were treated with alkaline solution in order to selectively degrade the PDLLA layer which covered on the surface of hybrid microspheres and instead to generate a dense coating of HA nanoparticles. The hybrid microspheres with enriched HA nanoparticles on the surface were further immersed in simulated body fluid (SBF) solution to evaluate the bone-forming ability of the bioactive hybrid microspheres via the in vitro biomimetic mineralization process. The resultant microspheres were analyzed by using X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA) to understand the nucleation and growth of bioactive calcium phosphate (Ca−P) crystals as a function of surface treatment. Results in this work clearly demonstrated that the existing HA nanoparticles on the surface of hybrid microspheres after alkaline treatment greatly affect the growth of the bone-like Ca−P crystals in SBF solutions. The biomimetic hybrid microspheres were found to be excellent candidates for use as injectable scaffolds for bone tissue engineering.
1. INTRODUCTION
may be preferred due to the minimal incision required during transplantation. A desirable artificial substitute for bone defect healing should be capable of mimicking the natural properties of bone and provide a temporary scaffold for tissue regeneration.9 Taken these requirements into consideration, a biocompatible and biodegradable scaffold with the same or similar constituents to bone is crucial to enhance bone regeneration.5 It has been known that natural bone is constituted of mineralized collagen fibrils with platelike hydroxyapatite (HA) crystals oriented preferentially with c-axes parallel to the longitudinal axis of the fibrils.10 With a general formula of Ca10(OH)2(PO4)6, HA constitutes the inorganic component of the natural bone, which implies the strong affinity to the bone-forming cells or the host bone.11 For these reasons, the HA has been used as the most popular graft substitute in bone tissue engineering.12−14 Combined with soft polymers when designing the scaffolds, which can provide similar structure and/or biological functions as the collagen template, is one of the promising research trends for bone regeneration purposes. Typically, the HA incorporated polymeric microspheres, which act as seeds for
The repair of bone defects is one of the challenging issues in clinical research.1−3 Autografting has been considered as an available method of repairing bone defect. However, the limited supply and the additional damage to the surgical site have restricted its wide application in the clinic. As another conventional bone-grafting approach, allografting, may cause the possible disease transmission and inconsistent bone healing.4 Consequently, bone tissue engineering has been developing rapidly because it has emerged as a promising approach to supply grafting substitutes as an alternative method to autografts and allografts.5 Biodegradable polymeric microspheres have been widely investigated as delivery systems for sustained release of bioactive compounds.6 Besides, as excellent cell-loading scaffolds, the microspheres are also used for bone defect repair purpose. Typically, the development of injectable scaffolds for bone tissue engineering boosts the studies on biodegradable polymeric microspheres as temporary cell-loading microcarriers. The cell-loading microspheres can localize and control delivery of cells with a high viability, which offers an attractive strategy to fill in bone defects.7 Moreover, since the artificial implants must be placed at the defect site for adjacent bone to bridge the gap caused by the defect,8 injectable microspheres © 2013 American Chemical Society
Received: June 1, 2013 Revised: November 16, 2013 Published: November 18, 2013 15293
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analytical grade) was obtained from Beijing Chemical Co. (China). Other chemical reagents used in this work were all of analytical grade and purchased from Beijing Chemical Co. (China). 2.2. Synthesis of HA-g-PDLLA Hybrid Nanocomposites via in Situ Grafting Polymerization. HA-g-PDLLA nanocomposites were synthesized via the following procedures. A suspension of HA (2.5 g) in dry toluene (20 mL) and D,L-lactide (DLLA) (7.5 g) monomer were added into 100 mL reaction tube under an argon atmosphere. Then, 30 mL of freshly dried toluene was added into the reaction tube. The catalyst stannous octoate (Sn(Oct)2) in dry toluene was added with a molar ratio of [Sn(Oct)2]/[DLLA] = 1/500. Polymerization was carried out at 120 °C under an argon atmosphere with stirring for 48 h and then quenched to room temperature. The resultant products were dissolved in dichloromethane and precipitated by cold methanol twice before use. 2.3. Fabrication and Alkaline Treatment of Microspheres. HA-g-PDLLA microspheres were fabricated by means of single emulsion29 and double emulsion30,31 solvent evaporation techniques. For the single emulsion method, HA-g-PDLLA (5% w/v) was dissolved in dichloromethane and added dropwise to an aqueous solution of PVA (0.1% w/v). After stirring at 400 rpm for 4 h at ambient temperature, the resultant microspheres were collected and washed with deionized water for three times to remove possible traces of organic solvent and PVA. Finally, the microspheres were freezedried overnight and stored at 4 °C. For the double emulsion method to fabricate porous HA-g-PDLLA microspheres, pore-foaming agent NH4HCO3 was dissolved in 0.5 mL of PVA aqueous solution (0.25% w/v). Then, HA-g-PDLLA solution in dichloromethane (5% w/v) was added in and emulsified for 3 min by using a sonicator (Grant ultrasonic bath XB3, 50−60 Hz, 60 W, UK) in an ice water bath. The obtained primary emulsion (W/O) was added dropwise into 200 mL of PVA aqueous solution (0.1% (w/v)). After gently stirring for 4 h at room temperature with a magnetic stirrer at 400 rpm, the final microspheres were collected and washed thrice with deionized water. The microspheres were then freeze-dried overnight and stored at 4 °C. The purpose of introducing single emulsion method is to well compare the surface morphologies among the microspheres with different alkaline treatment time. Because no pore forming agents were added during preparation process, almost no interconnected big opening-pores were found on the surface of microspheres. It is much easier and intuitionistic to utilize these simple microspheres as model to study the effect of alkaline treatment on the biomimetic mineralization process of the hybrid microspheres. However, the scaffold should possess an interconnected and spread porosity (usually exceeding 90%) with a highly porous surface and microstructure.32 Owning to the double emulsion method, we obtained the microspheres with aforementioned characters. For alkaline treatment, the HA-g-PDLLA hybrid microspheres were first separated by standard sieves to obtain the microspheres with diameters in the range of 150−250 μm. Then, a certain amount of HAg-PDLLA microspheres was taken and then immersed into a NaOH aqueous solution (0.2 M) for different times at 37 °C. Finally, the resultant microspheres were washed with distilled water thoroughly to remove the remained alkaline solution. 2.4. Biomimetic Coating of Ca−P Crystals. Simulated body fluid (SBF) solution was prepared according to Kokubo’s work.18 To accelerate the SBF-coating processes, the solution with the ionic concentration 1.5 times higher than SBF was used in this study.33−35 The SBF solution was prepared by adding the chemicals in sequence on the condition of complete dissolution of the former salt, in the following order: 12.053 g NaCl, 0.352 g of NaHCO3, 0.338 g KCl, 0.347 g K2HPO4·3H2O, 0.467 g MgCl2·6H2O, 58.5 mL 1.0 M HCl solution, 0.438 g CaCl2, and 0.108 g Na2SO4. Finally, the temperature was increased to 36.5 °C and the pH was adjusted to 7.4 by the addition of 9.177 g of (CH2OH)3CNH2 and little amount of 1.0 M HCl. Finally, distilled water was added up to 1 L to obtain the final SBF solution. It is worth to point out that SBF solution should be stored at 4 °C for no longer than a month during its use. The microspheres were soaked in SBF solution for different periods within
the regeneration of destroyed bone, could be considered as potential scaffold candidates for bone tissue engineering. It is predicted that the presence of bioactive HA in scaffolds may help to stimulate cellular responses such as osteoblast proliferation, differentiation, and mineralization and finally promote the formation of new bone. Biological mineralization, or biomimetic mineralization, is the process of in situ formation of inorganic minerals.15 Simulated body fluid (SBF) is an electrolyte solution that mimics the inorganic composition of human blood plasma.16 Kokubo17 proposed that the formation of bonelike apatite on the surface of an artificial material when implanted in the living body is the essential requirement for evaluating its ability to bond to living bone. However, this in vivo formation of apatite can be reproduced in a simulated body fluid (SBF).18 Therefore, the ability of implants to induce bone-formation could be evaluated via the SBF treatment in vitro. Many attempts have been made to utilize SBF treatment as a route to create a biomimetic coating on biomaterials surface, which contributes significantly to the biological function of engineered scaffolds.19−21 Currently, most of the contributions focus on the surface modification by introducing functional groups to enhance the HA nucleation in SBF solutions.16,22 However, low crystallization efficiency and inhomogeneous distribution of apatite between exterior and interior of the bioinert scaffold are the major issues for biomimetic coating in SBF. Based on the above concerns, the focus of present research is to create bioactive microspheres for bone defect healing purpose. Specifically, in this study we developed new HA-gPDLLA hybrid microspheres with bioactive HA coatings and further evaluated the in vitro bone-forming ability by SBF treatment. Although many studies have reported the biomimetic mineralization of various scaffolds, however, so far many of them mainly focused on the scaffolds (1) without any chemical modification,23−25 (2) via physical pretreatment with Ca2+ containing solution to catalyze the mineralization,16,26 or (3) via functionalization with carboxyl groups by different means (i.e., selective oxidation reactions, exposure to plasma of oxidative gases, or adsorption of citric acid).27,28 Most reports concentrated on introducing new ions to the substrate in order to promote the biomimetic mineralization. Less concern has been paid on the alkaline treatment of HA incorporated polymer scaffolds and its effects on biomimetic mineralization behavior. So in our present work, rapid biomimetic mineralization in SBF was first found on the hybrid microspheres after alkaline treatment, which exhibited a different apatite deposition mechanism compared with the pristine polymeric microspheres. The rapid coating ability of bonelike apatite minerals on the surface of microspheres indicates the alkaline-treated HA-g-PDLLA microspheres could serve as an excellent bone substitute material to induce new bone formation in vivo. The work presented here thus becomes the first systematic study of utilizing alkaline treatment to modify the HA incorporated polymer scaffolds to make it accelerate during biomimetic mineralization, which is our contribution and novelty of this work.
2. EXPERIMENTAL SECTION 2.1. Materials. HA nanoparticles were purchased from Aldrich. DLLA monomer was purchased from Purac. HA-g-PDLLA hybrid nanocomposite was synthesized in our lab. Poly(vinyl alcohol) (PVA) (with a hydrolyzed degree of 87−89% and Mw 85 000−146 000) was purchased from Aldrich. Ammonium bicarbonate (NH4HCO3, 15294
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9 days. The specimens were taken out from the SBF and gently washed with pure water for several times and then freeze-dried for further characterization. 2.5. Analyses and Characterization. Morphologies of HA-gPDLLA microspheres before and after SBF treatment were observed by a JEOL JSM-6700F scanning electron microscope (SEM). The microspheres were freeze-dried, mounted on metal stubs with doublesided tape, and coated with platinum. X-ray diffraction (XRD) studies were performed on a Rigaku D/ Max 2500 diffractometer (40 kV, 200 mA, Cu Kα, k = 0.154 nm) at ambient temperature with a range from 10° to 70°; the scanning rate was 8°/min at a scanning step of 0.02°. X-ray photoelectron spectroscopy (XPS) experiments were performed on an ESCALab220i-XL electron spectrometer from VG Scientific using monochromatic Al Kα radiation (1486.7 eV) as the excitation source. All spectra were acquired at pass energy of 80 eV with the anode operated at 300 W. The base pressure was about 3 × 10−9 mbar. The binding energies were referenced to the C 1s line at 284.8 eV from adventitious carbon. Surface atomic percentages were calculated by normalizing the area of each peak with the total peak area of all atomic elements. The inorganic contents of HA-g-PDLLA microspheres during the apatite growth were measured on a thermogravimetric analyzer (TGA, PerkinElmer, Pyris 1). The samples were heated from 100 to 600 °C at a rate of 20 °C/min under a N2 atmosphere.
3. RESULTS AND DISCUSSION 3.1. Alkaline Treatment on Microspheres. Figure 1 shows the typical morphologies of HA-g-PDLLA hybrid microspheres after alkaline treatment for different times. It was found that the hybrid microspheres preserved their structural integrity and maitained their spherical shape even treatment for 30 min. Importantly, a regular change in the surface morphology was observed with increasing alkaline treatment time. The surface of the untreated microspheres was relatively smooth, with slightly visible granular bulges due to the uniformly distributed HA nanoparticles under the PDLLA layer. With further hydrolysis to erode the organic PDLLA component, the inorganic HA nanoparticles became more visible and were exposed as a thick layer on the surface of the microspheres. Meanwhile, more pores with irregular shapes appeared on the surface. The tendency of morphological change during alkaline treatment suggested that the controlled hydrolysis in alkaline solution is an efficient method to prepare microspheres with a dense and bioactive HA nanoparticle coatings on the surface. It was also found that the average diameter of the hybrid microspheres decreased with increasing the alkaline treatment time. Figure 2 shows the statistic results from SEM images. The mean diameter of untreated microspheres is about 111 μm. The mean diameter was reduced from 111 μm to 107, 106, 83, 79, and 67 μm with a gradual narrower distribution for the hybrid microspheres after alkaline treatment for 5, 10, 20, 30, and 40 min, respectively. As revealed by the SEM and statistic results, it could be speculated that the gradual reduction of microsphere diameter was caused by the surface corrosion of PDLLA component in the alkaline environment. Figure 3 shows the thermogravimetric analysis of hybrid microspheres before and after alkaline treatments. It was found that all the traces showed a rapid weight loss around 300 °C, and only one thermal decomposition stage was observed. Evidently, the decomposition stage was attributed to the thermal degradation of PDLLA component in the hybrid microspheres. As shown for the untreated hybrid microspheres, the residue weight percentage was 22.5%, which was in
Figure 1. SEM images of HA-g-PDLLA hybrid microspheres before (a) and after alkaline treatment for 5 (b), 20 (c), and 30 min (d).
accordance with the composition ratio of HA and PDLLA component in microspheres. After alkaline treatment for 5 min, the residue weight percentage of hybrid microspheres reached up to 27.2%, which confirmed the selective degradation of PDLLA component. Moreover, the residues further increased to 32.1% when the hybrid microspheres were treated in NaOH solution for 20 min. This data would be further used to calculate the increase of apatite contents after biomimetic mineralization in SBF solution. 3.2. Biomimetic Mineralization of the AlkalineTreated Hybrid Microspheres. The alkaline-treated hybrid microspheres were further treated by SBF solution to investigate the capability of microspheres for nucleation and crystallization of apatites. Figure 4 shows the SEM images of microspheres via biomimetic mineralization process in SBF for 3 days. It is evident that the pristine PDLLA microsphere does 15295
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Figure 2. SEM images and size distribution of hybrid microspheres before and after alkaline treatment for different time length. Figure 4. SEM images of microspheres after immersion in SBF for 3 days: (a) untreated PDLLA microspheres alone; (b) untreated HA-gPDLLA hybrid microspheres; (c) hybrid microspheres after alkaline treatment for 5 min; (d) hybrid microspheres after alkaline treatment for 20 min.
not induce mineralization after incubation with SBF solution. However, in the situation of HA-g-PDLLA hybrid microspheres without alkaline treatment, mineralized clusters with diameter sizes ranging from 100 to 400 nm were sparsely and randomly dispersed on the surface of microspheres. For the hybrid microspheres after alkaline treatment, mineralized clusters could be seen obviously on the surface. As shown in Figures 4c1 and 4c2, it was observed that the whole surface almost completely covered with the apatite clusters after 5 min alkaline treatment; only a few bare spaces were observed between the
adjacent mineralized clusters. Moreover, a very dense layer of mineralized clusters was formed on the surface of HA-g-PDLLA microspheres pretreated in alkaline solution for 20 min. It can be clearly seen from the magnified SEM images that the biomineral cluster was composed of a stack of small platelike
Figure 3. Thermogravimetric analysis of HA-g-PDLLA microspheres before and after alkaline treatment for different time length: (a) weight loss curves; (b) dependence of HA contents on time. 15296
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biominerals, which were reported to be the dominant morphology of nature bone.36,37 It may be concluded that the significant difference of the mineralization ability of HA-gPDLLA microspheres before and after alkaline treatment indicated the crucial role of HA nanoparticles in promoting the nucleation and growth of apatite crystals. In order to confirm the crystalline structure of the platelike minerals formed in SBF solutions, XRD characterization was performed on the microspheres after immersing in SBF for 3 days, and the results are shown in Figure 5. Appearance of the
Figure 6. Thermogravimetric analysis of HA-g-PDLLA microspheres after alkaline pretreatment for different time length and then immersion in SBF for 3 days.
and 5 and 20 min for alkaline-treated hybrid microspheres. This quantitative result agrees well to the SEM images of Figure 4. Figure 7 shows the XPS results of the hybrid microspheres with different alkaline treatment history and after immersion in
Figure 5. X-ray diffraction patterns of microspheres after biomineralizated for 3 days: (1) PDLLA microspheres, (2) untreated HA-gPDLLA hybrid microspheres, (3) hybrid microspheres after alkaline treatment for 5 min, (4) hybrid microspheres after alkaline treatment for 20 min, and (5) commercial HA nanoparticles (Sigma-Aldrich).
broad peak at 10°−40° was attributed to the amorphous nature of PDLLA. For the hybrid microspheres after alkaline treatment for 5 min, the characteristic peaks at 25.9°, 27.8°, 29.0°, 31.8°, 32.2°, 32.9°, 34.3°, 39.8°, 46.7°, 48.1°, and 49.4° which correspond to reflection of (002), (102), (210), (211), (112), (300), (202), (310), (222), (312), and (213) of apatite crystals,25 are clearly observed. For the purpose of quantitative comparison, the diffraction peak at 25.9° which corresponds to the (002) reflection was recommended due to its well resolution and no interferences.38 It was found that that the diffraction peak at 25.9° began to emerge in untreated HA-gPDLLA microspheres and subsequently increased with increasing the alkaline treatment time. Evidently, the peak intensity of microspheres of 20 min alkaline treatment is bigger than other peaks, indicating the amount of newly formed apatite was more than others. In addition, it was evident that the newly formed diffraction peaks of hybrid microspheres after alkaline treatment were extremely coincident with those peaks of pristine HA, indicating that the bonelike crystals were successfully formed on the alkaline-treated hybrid microspheres by biomimetic mineralization in SBF solution. Figure 6 shows the thermogravimetric curves of hybrid microspheres with different alkaline treatment history and after biomimetic mineralization in SBF for 3 days. It was found that the PDLLA microspheres nearly lost all the weight with the temperature increasing up to around 340 °C, while the hybrid microspheres showed an obvious increase in residual weight. Comparing to the results from Figure 3, the residual weights of hybrid microspheres after incubation in SBF were higher than that of hybrid microspheres before biomimetic mineralization. The increment of newly formed apatite via SBF treatment was 5.7%, 11.9%, and 12.1% for the untreated hybrid microspheres
Figure 7. X-ray photoelectron spectroscopy of microspheres after biomineralizaed for 3 days: (1) PDLLA microspheres, (2) untreated HA-g-PDLLA hybrid microspheres, (3) hybrid microspheres after alkaline treatment for 5 min, and (4) hybrid microspheres after alkaline treatment for 20 min.
SBF for 3 days. It was found that the atomic concentrations of both calcium and phosphorus increased with increasing the time of alkaline treatment. Evidently, the alkaline treatment induced more Ca−P crystallization on hybrid microspheres, which was in good agreement with XRD and SEM results. According to the XPS results, the Ca/P ratio is calculated as 1.56, 1.63, and 1.63 for the untreated hybrid microspheres, the hybrid microspheres after alkaline treatment for 5 and 20 min. It was reported that the Ca/P ratio of 1.56 in the case of untreated microspheres was the indicative of calcium-deficient HA,39 while the Ca/P ratio value reached up to 1.63 as for the alkaline-treated hybrid microspheres, which was very close to the Ca/P ration of natural bone tissue (1.67), indicating the successful growth of a bonelike apatite minerals for the alkalinetreated microspheres in SBF solution. Based on the discussion above, it is evident that the presence of the HA nanoparticles coating on the surface of microspheres may play a crucial role in promoting the nucleation and growth of Ca−P crystals. The possible mechanism of this rapid biomimetic mineralization behavior on alkaline-pretreated HAg-PDLLA microspheres is proposed in Figure 8. For the alkaline-pretreated microspheres, the HA nanoparticles on the 15297
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Figure 8. Schematic depiction of rapid mineralization process of alkaline-pretreated hybrid microspheres.
Figure 9. Time-lapse SEM images of untreated hybrid microspheres after biomimetic mineralization in SBF for (a) 3, (b) 5, (c) 7, and (d) 9 days. Each column from top to bottom shows the gradual magnified images.
surface released a certain amount of Ca2+ and PO43− ions in SBF solution. These local enriched Ca2+ and PO43− ions caused the increase of ion concentration and enhanced the nucleation of new apatite in the surrounding simulative body fluid. Meanwhile, the hydrolysis of the PDLLA component in NaOH solution generated numbers of COOH groups on the surface layer of microspheres, which provided a preferred nucleation sites for apatite. By continuously consuming the calcium and phosphate ions40 from the surrounding environment, numerous apatite nuclei formed on the favorable sites and grew spontaneously. It is possible to conclude, however, that the presence of HA nanoparticles will promote newly formed apatite deposition in SBF. This is in line with earlier observations which reported that the incorporated HA particles into PLGA nanofibrous composite scaffolds could function as nucleation sites to accelerate the mineral growth.25
3.3. Biomimetic Mineralization with Time. Figure 9 shows the time-lapse SEM analyses of untreated hybrid microspheres after incubating with SBF for 3, 5, 7, and 9 days. Based on the SEM time-lapse images, it is feasible to get a better understanding about the mineral growth process on the hybrid microspheres. As shown in Figures 9a1−a3, scattered platelike biominerals began to emerge and uniformly dispersed on the surfaces of hybrid microspheres at day 3. This is an obvious nucleation process of the crystals at the initial stage. After incubating with SBF for 5 days, the spherical mineral deposits grew rapidly and appeared like “blossoms” on the surfaces of the untreated hybrid microspheres. At day 7, the diameter of the spherical mineral deposits further increased, and adjacent minerals began to become mutually integrated. Gradually, after 9 days of incubation, the minerals integrated into a dense coating covering the entire surface. 15298
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Figure 10. Time-lapse SEM images of 5 min alkaline-pretreated hybrid microspheres after biomimetic mineralization in SBF for (a) 3, (b) 5, (c) 7, and (d) 9 days. Each column from top to bottom shows the gradual magnified images.
Figure 11. TGA traces of 20 min alkaline-pretreated hybrid microspheres after immersion in SBF for 1, 3, 5, and 9 days: (a) weight loss curves; (b) dependence of HA contents on biomimetic mineralization time.
Figure 10 reveals the time-lapse SEM images of biomimetic mineralization process of hybrid microspheres after alkaline treatment for 5 min. Compared with untreated microspheres, it is evident that the exposed HA nanoparticles on the surface of microspheres can greatly accelerate the mineral growth at the given time intervals. The mineral nucleation was much more extensively and the Ca−P crystals almost occupied the whole surface within only 3 days of incubation. After then, the minerals integrated into a dense coating on the entire surface and the morphology remained same as time elapsed. Similar results were observed in the case of hybrid microspheres alkaline treated for 20 min, in which the mineral deposits became into integrated within 3 days and maintained the morphology over the remaining observation periods (data not shown). To better understand the minerals deposition process in SBF, the newly formed apatite content on the hybrid microspheres after alkaline pretreatment for 20 min was evaluated by thermogravimetric analysis, and the results are
shown in Figure 11. It was found that the weight percentage of residue increased with increasing the SBF incubation time. The residual content increased from 32.1% into 37.8%, 44.2%, 53.8%, and 75.3% after immersing in SBF for 1, 3, 5, and 9 days, respectively. Accordingly, the increments of newly formed apatite via SBF incubation were 5.7%, 12.1%, 21.7%, and 43.2%, respectively. These results clearly confirmed the gradual growth of apatite crystals after nucleation on the exposed HA nanoparticles of microsphere surface. On one hand, the cluster size of biominerals increased until the gap of the microsphere surface was filled, which is similar to the vivid process of blossoming. On the other hand, the SBF solution penetrated into the inner space of the microspheres through the porous structure and thus also led to the Ca−P crystal growth inside microspheres. Consequently, these combined factors resulted in the rapid and significant increase of biomineral contents after incubation in SBF. 3.4. Biomimetic Mineralization of Porous Microspheres. The microspheres with interconnected pores are 15299
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Figure 12. SEM images of porous HA-g-PDLLA microspheres via alkaline treatment for 5 min (a1−a3) and then incubated with SBF for 3 days (b1−b3).
desirable scaffolds that could serve as microcarriers for cells, growth factors, and/or other bioactive molecules.41 Particularly, owning to the interconnected porous structure, the porous microspheres are of great potential for tissue engineering and cell delivery purposes.42 In the present study, at beginning we employed single emulsion method to prepare microspheres with relative smooth surface. It is utilized as models to study the effect of alkaline treatment on the biomimetic mineralization process of the hybrid microspheres, by which we could get more intuitionistic and distinct results. Then in the following we used double emulsion solvent evaporation technique (i.e., w/o/w)43 to prepare hybrid microspheres with a highly interconnected porous structure. We wonder if these porous microspheres will show the same tendency after alkaline treatment and biomimetic mineralization in SBF, just as the previous models. Figure 12 reveals the SEM images of porous HA-g-PDLLA microspheres via alkaline treatment for 5 min (a1−a3) and then incubated with SBF for 3 days (b1−b3). It was found that the porous microsphere preserved its structural integrity after alkaline treatment. The magnified SEM image revealed that the porous surface of hybrid microspheres was covered with a layer of HA nanoparticles (a3). After these porous microspheres were incubated with SBF for 3 days, as shown in Figures 12b1− b3, both the outer surface and inner walls of porous microspheres were covered by the homogeneously distributed laminated minerals. Results indicated that the alkaline treatment did not destroy the porous structure and further modified the surface of the hybrid microspheres. Owning to the tailored interconnected structure and the homogeneous distribution of bioactive HA nanoparticles on the surface, the alkaline-treated porous HA-g-PDLLA microspheres have promising potentials as cell microcarriers for bone tissue engineering applications.
the nucleation and growth of the bonelike calcium phosphate (Ca−P) crystals in SBF solutions. The newly deposited Ca−P mineral showed a similar crystal structure to that of hydroxyapatite component found in bone. Our results implied the great potentials of bone-inducing formation of the alkalinetreated microspheres in vivo. This work suggested that the biomimetic HA-g-PDLLA hybrid microspheres treated with alkaline pretreatment are potentially excellent injectable scaffolds in bone tissue engineering for defect filling and regeneration.
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AUTHOR INFORMATION
Corresponding Author
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[email protected] (Z.G.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the Natural Science Foundation of China (Grant 51025314) and the “Strategic Priority Research Program” of the Chinese Academy of Sciences (Grant XDA01030301).
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REFERENCES
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4. CONCLUSIONS In summary, HA-g-PDLLA hybrid microspheres were successfully prepared by emulsion solvent evaporation techniques. Pretreatment in alkaline solution has been proved to be an efficient and simple method to modify the surface of microspheres to expose a layer of dense HA nanoparticles coating. These exposed HA nanoparticles can greatly accelerate 15300
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dx.doi.org/10.1021/la404209u | Langmuir 2013, 29, 15293−15301
