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Injectable Biodegradable Thermosensitive Hydrogel Composite for Orthopedic Tissue Engineering. 1. Preparation and Characterization of Nanohydroxyapatite/Poly(ethylene glycol)-Poly(ε-caprolactone)-Poly(ethylene glycol) Hydrogel Nanocomposites ShaoZhi Fu,†,§ Gang Guo,†,§ ChangYang Gong,† Shi Zeng,† Hang Liang,† Feng Luo,† XiaoNing Zhang,‡ Xia Zhao,† YuQuan Wei,† and ZhiYong Qian*,† State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan UniVersity, Chengdu, 610041, China, and School of Medicine, Tsinghua UniVersity, Beijing, 100084, China ReceiVed: August 14, 2009; ReVised Manuscript ReceiVed: October 25, 2009
In this study, we synthesized a biodegradable triblock copolymer poly(ethylene glycol)-poly(εcaprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) by ring-opening copolymerization, and nanohydroxyapatite (n-HA) powder was prepared by a hydrothermal precipitation method. The obtained n-HA was incorporated into the PECE matrix to prepare injectable thermosensitive hydrogel nanocomposites. 1H NMR, FT-IR, XRD, DSC, and TEM were used to investigate the properties of PECE copolymer and n-HA/ PECE nanocomposites. The rheological measurements for n-HA/PECE nanocomposites revealed that the gelation temperature was approximately 36 °C. The sol-gel-sol transition behavior and phase transition diagrams were recorded through a test tube inverting method. The results showed that n-HA/PECE nanocomposites still had thermoresponsivity like that of PECE thermosensitive hydrogel. The morphology of the nanocomposites was observed by SEM; the results showed that the nanocomposites had a 3D network structure. In addition, the effects of n-HA contents on the properties of n-HA/PECE nanocomposites are also discussed in the paper. From the results, n-HA/PECE hydrogel is believed to be promising for injectable orthopedic tissue engineering due to its good thermosensitivity and injectability. 1. Introduction In the biomedical field, hydrogel systems have gained much attention owing to their excellent physical-chemical properties and convenience in handling. Because they can provide eligible environments for controlled drug release, and suitable scaffolds for cell growth, they are promising biomaterials for drug delivery devices as well as for tissue engineering.1-4 In the past decade, many naturally based and synthetic hydrogels have been intensively studied and used in drug delivery systems and regeneration of cartilage or bone, such as chitosan-based hydrogels5-11 and polyester copolymer hydrogels.12-18 Among the synthetic materials, a lot of biodegradable multiblock polyesters have been explored and used as drug carriers or tissue engineering scaffolds. Poly(ε-caprolactone) (PCL), poly(D,L-lactide) (PDLLA), poly(D,L-lactic acid-coglycolic acid) (PLGA) and its block copolymers with poly(ethylene glycol) (PEG) or poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (trademark Pluronic or Poloxamer) have been intensively studied for use in the biomedical field. In these studies, the researchers prepared the thermoresponsive polymers and hydrogels and investigated the drug release behavior, thermoresponsivity, or thermoreversible gelation mechanisms of these copolymers. Thermoresponsive polymers and hydrogels have been largely studied due to their capability of imbibing large amounts of water and their sensitivity to environmental temperature, which enables them * To whom correspondence should be addressed. Telephone: +86-2885164063. Fax: +86-28-85164060. E-mail:
[email protected]. † West China Medical School. ‡ School of Medicine. § These authors contributed equally to this work.
to maintain certain shapes and fulfill drug controlled release or tissue repair, especially in the normal body temperature range.12,13,19 Hydroxyapatite (HA) is an important component of natural bone and teeth. It has been widely used as bone implant material in clinical settings due to its excellent bioactivity and biocompatibility. It can provide a favorable environment for osteoconduction, protein adhesion, and osteoblast proliferation.20-22 However, its brittleness has limited its application as implants. In order to overcome this shortcoming and widen the application scope of HA, addition of HA into a polymer matrix for preparation of HA/polymer composites is a common approach. It was reported that these kinds of composite materials have improved bioactivity and mechanical properties, and induce cells to attach or proliferate on them.20 In previous works of our group,13,23 Gong et al. successfully prepared the poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) triblock copolymer, and investigated the sol-gel-sol phase transition behavior in vitro, gel formation and degradation in vivo, and drug release behavior. Therefore, in this study, we added n-HA into PECE copolymer for preparing HA/ PECE hydrogel nanocomposite, which is a kind of injectable and thermosensitive material, and is expected to be used in tissue engineering. 2. Materials and Methods 2.1. Materials. ε-Caprolactone (ε-CL), poly(ethylene glycol) methyl ether (MPEG, Mn ) 550), and stannous octoate (Sn(Oct)2) were obtained from Aldrich, USA. Hexamethylene diisocyanate (HMDI) was purchased from Sigma, USA. Calcium chloride dihydrate (CaCl2 · 2H2O) and sodium phosphate
10.1021/jp907974d 2009 American Chemical Society Published on Web 11/30/2009
Tissue Engineering n-HA/PECE Hydrogel Composites (Na3PO4) were purchased from Kelong Chemicals, Chengdu, China. All materials used in this work were of analytical reagent (AR) grade and were used directly without further purification. 2.2. Synthesis and Purification of PECE Popolymer. The triblock PECE copolymer was synthesized in two steps. First, MPEG-PCL diblock copolymer was synthesized from ε-CL initiated by MPEG using Sn(Oct)2 as a catalyst by ring-opening copolymerization.24-26 Second, the obtained diblock copolymer was cross-linked by HMDI to prepare PECE triblock copolymer according to the protocol reported previously.13 In this study, PECE (Mn ) 3300) triblock copolymer was prepared as follows: In the first step, 22 g (0.02 mol) of ε-CL, 11 g (0.02 mol) of MPEG (Mn ) 550), and 0.16 g of Sn(Oct)2 (0.5 wt % of total reactants) as catalyst were mixed and reacted for 6 h at 130 °C in a three-necked glass flask. In the second step, the obtained MPEG-PCL copolymer was linked by HMDI at a molar ratio of 1.1/1(linker/copolymer); the reaction was kept at 80 °C for 6 h under vigorous stirring. The purified product was dried in a vacuum and stored in an airtight bag before use. 2.3. Preparation of n-HA Powder. It has been reported that the Ca(NO3)2-(NH4)2HPO4 and Ca(OH)2-H3PO4 systems are common approaches for preparing n-HA. However, Choi et al. developed a novel aqueous approach for synthesizing nanostructured calcium phosphates using CaCl2 and Na3PO4 as starting materials.27-29 In this paper, we synthesized pure n-HA powder with minimum impurities by a reaction of CaCl2 with Na3PO4 in a solution according to the following reaction:
10CaCl2 · 2H2O + 6Na3PO4 + 2NaOH ) Ca10(PO4)6(OH)2 + 20NaCl + 20H2O A 200 mL, 0.1 mol/L Na3PO4 solution was dropped into an equal volume of 0.167 mol/L CaCl2 solution under stirring in a homeothermal water bath at 60 °C, and the pH of the solution was maintained at 10.5 by adding 0.1 mol/L sodium hydroxide (NaOH) solution. After the titration was finished, the mixture was aged at 60 °C for 20 h and autoclaved for another 20 h at 160 °C under a vapor pressure of 0.5 MPa; then the so-obtained precipitate was washed with deionized water and anhydrous ethanol four times to remove the byproduct of the reaction. The obtained n-HA slurry was dried in a vacuum at 80 °C for 24 h and sintered at 1000 °C for 2 h, and then ground to powder. 2.4. Preparation of n-HA/PECE Hydrogel Nanocomposites. A 0.4 g sample of PECE copolymer and 0.1 g of n-HA powder were added into a glass tube with an inner diameter of 10 mm and a length of 100 mm. Then pure water was added to the mixture and the made PECE copolymer dissolved completely; the volume of the suspension was kept at 1 mL by adding water. After the mixture was incubated in a water bath at 60 °C for 2 min and cooled in ice-water bath at 0 °C for 10 min under ultrasonication, the injectable thermosensitive n-HA/ PECE hydrogel composite was prepared. In this paper, the percentages of n-HA particles which were contained in n-HA/PECE hydrogel nanocomposites were expressed as the mass ratio of n-HA weight (WHA) to the total mass of the solid state (WHA + WPECE). For example, the aboveobtained hydrogel composite contained 20 wt % n-HA. Changing the mass of n-HA powder, we prepared n-HA/PECE hydrogel composites with 10, 20, and 30 wt % HA, and pure PECE hydrogel without n-HA was prepared through methods similar to that described above. 2.5. Characterization of PECE Copolymer, n-HA, and n-HA/PECE Hydrogel Nanocomposites. 2.5.1. Nuclear Magnetic Resonance Analysis (1H NMR). 1H NMR spectroscopy was performed on a Varian 400 instrument (Varian, USA) at
J. Phys. Chem. B, Vol. 113, No. 52, 2009 16519 400 MHz to determine the chemical composition and molecular structure of the PECE copolymer. The copolymer was dissolved in deuterated chloroform (CDCl3) at room temperature, and tetramethylsilane (TMS) was used as an internal reference standard. 2.5.2. Fourier Transform Infrared Spectroscopy (FT-IR). The pure HA powder was mixed with KBr powder and pressed into a pellet. In order to obtain the spectra of PECE alone, the copolymer was dissolved in chloroform and cast onto a KBr plate to form a thin film. In the case of n-HA/PECE composite hydrogel analysis, the composite material was rolled in a manually operated rolling machine until the sample was presumed to be thin enough to be considered optically transparent. FT-IR spectra were recorded on a NICOLET 200SXV infrared spectrophotometer (Nicolet, USA) in the range 4004000 cm-1. 2.5.3. X-ray Diffraction (XRD). The samples for XRD measurements were as-synthesized n-HA powder, PECE copolymer, and PECE/n-HA composites. XRD was performed on an X’Pert Pro MPD DY1291 (PHILIPS, Netherlands) diffractometer using graphite monochromatized Cu K radiation (λ ) 0.1542 nm; 40 kV; 40 mA). The samples were measured in the 2θ range from 10° to 60° at a scanning rate of 4°/min. 2.5.4. Transmission Electron Microscopy (TEM). The particle size of n-HA powder was characterized on a HITACHI H-600 electron microscope operating at 100 kV. A 2 mg sample of n-HA powder was dispersed in ethanol to form a suspension by ultrasonication for 60 min; a drop of the resulting suspension was then directly dripped onto a copper TEM grid coated with a thin Formvar-carbon support film and dried on a filter. 2.5.5. Differential Scanning Calorimetry (DSC). The thermal properties of dried pure PECE copolymer and n-HA/PECE hydrogel composites were characterized by using a differential scanning calorimeter (NETSCZ 204, NETSCZ, Germany). The samples were first heated from -40 to 90 °C under nitrogen atmosphere at a heating rate of 10 °C/min, then cooled to -40 °C, and then reheated to 90 °C at the same rate. 2.5.6. Rheological Study of n-HA/PECE Hydrogel Nanocomposites. Rheological measurements of the aqueous PECE copolymer solution and n-HA/PECE hydrogel nanocomposites were carried out by using an AR2000 ex rheometer (TA Instruments, USA). The samples were placed between parallel plates of 40 mm diameter and with a gap of 1 mm. The heating rate was 2 °C/min in the range 10-60 °C. The storage modulus (G′), loss modulus (G′′), and complex viscosity (n*) were measured as functions of temperature. A frequency of 1 Hz and a strain of 1% were applied in order to maintain a linear viscoelastic region. 2.5.7. Scanning Electron Microscopy (SEM). The morphology of freeze-dried PECE hydrogel and n-HA/PECE hydrogel nanocomposite samples were observed by scanning electron microscopy (SEM). For SEM analysis, the samples were dried in a vacuum for 2 days; then were cross sectioned and sputtercoated a thin layer of gold with a KYKY SBC-12 Sputter Coater System. The surface morphologies of samples were observed on a JEOL SEM (JSM-5900LV, JEOL, Japan) and operated at 20 kV accelerating voltage. 2.5.8. Sol-Gel-Sol Phase Transition BehaWior Study. For this study, samples with different n-HA contents were prepared by dissolving a known amount of PECE copolymer in deionized water at a designated temperature, and then n-HA powder was added to aqueous PECE solution to form a suspension under ultrasonication. The volume of the suspension was kept at 1 mL in total regardless of the concentration. After being
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SCHEME 1: Scheme of the Synthesis Pathway of the PECE Triblock Copolymer
Figure 1. 1H NMR spectrum of PECE (MPEG550-PCL2200-MPEG550) triblock copolymer.
incubated in an ice-water bath at 0 °C for 10 min, the hydrated samples were slowly heated at a rate of 0.5 °C/min from 0 to 60 °C. Sol-gel-sol phase transition diagrams of PECE hydrogel and n-HA/PECE hydrogel nanocomposites were recorded using a test tube inverting method with a 10 mL tightly screw-capped vial with an inner diameter of 13 mm. The sol-gel-sol transition was visually observed by inverting the vials, and conditions of sol and gel were defined as “flow liquid sol” and “no flow solid gel” in 1 min, respectively.13 3. Results 3.1. Synthesis and Characterization of PECE Copolymer. The PECE triblock copolymer was synthesized through a twostep method, as depicted in Scheme 1. First, MPEG-PCL diblock copolymer was prepared by ring-opening copolymerization of ε-caprolactone initiated by poly(ethylene glycol) methyl ether using Sn(Oct)2 as catalyst. Second, the obtained MPEG-PCL was linked by the coupling agent HMDI.23 The chemical structure of PECE copolymer was characterized by 1 H NMR. The 1H NMR spectrum of PECE copolymer was recorded and is shown in Figure 1. The characteristic absorption peaks are also presented in the figure. In the 1H NMR spectrum, the sharp peak at 3.60 ppm was attributed to the ethylene peak of the ethylene glycol (-CH2CH2O-) unit, the peak at 3.45 ppm belonged to methyl protons of a CH3O- unit in MPEG, and two weak peaks at 4.22 and 3.70 ppm belonged to methylene protons of -O-CH2-CH2- in the MPEG end unit which linked with PCL blocks. Two strong peaks at 2.32 and 4.07 ppm were attributed to methylene protons of -OCCH2and -CH2OOC- in PCL blocks, respectively. Some weak peaks at 3.12 and 1.51 ppm belonged to methylene protons of linker HMDI. The number-average molecular weight (Mn) of PECE copolymer could be determined by using the integral intensities of characteristic absorption peaks at 3.60 and 4.07 ppm. The Mn value for as-obtained PECE copolymer was 3150 according to the 1H NMR spectrum. Figure 2 presents the typical FT-IR spectra of n-HA (a), PECE (b), and PECE/n-HA composite (c). In Figure 2a, the peaks at 3568.4 and 634.9 cm-1 were assigned to the hydroxyl group
Figure 2. FT-IR spectra for (a, top) n-HA, (b, middle) PECE copolymer, and (c, bottom) n-HA/PECE composite.
(-OH) stretching and bending modes in the crystal lattice of n-HA, respectively. A broad peak at 3434.1 cm-1 was related to the absorbed water. The peaks at 1093.6 and 1045.9 cm-1 were attributed to ν3(P-O) vibrations of phosphate groups (PO43-) and the absorption peaks at 602.4 and 569.8 cm-1 corresponded to ν4(P-O) vibrations of PO43-; besides the described absorption peaks, there were two weak peaks at 952.7 and 470.3 cm-1, which were attributed to ν1 and ν2 vibrations of PO43- ions, respectively. These absorption bands were characteristic functional groups of hydroxyapatite lattices; therefore, the FT-IR results confirmed that the as-prepared powder was pure phase HA. In Figure 2b, a strong CdO stretching band appearing at 1734 cm-1 was attributed to the ester bond of PECE copolymer. There was no absorption at 2250-2270 cm-1, which indicated that the -NCO groups of hexamethylene diisocyanate (HMDI) disappeared completely due to the coupling reaction of -NCO with the -OH groups. The absorption bands at 1525 cm-1 were due to the N-H bending vibrations, which confirmed the formation of PECE
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Figure 4. TEM micrograph of the hydroxyapatite powder as obtained in this study.
Figure 3. XRD patterns of (a, top) n-HA, (b, middle) n-HA/PECE composite, and (c, bottom) PECE copolymer.
triblock copolymer. From Figure 2c, it can be seen that the absorption bands of n-HA and PECE copolymer were overlapped, but the main characteristic absorption bands still appeared and changed slightly. 3.2. Preparation of n-HA Powder. In this work, neat n-HA powder was prepared by the hydrothermal precipitation method. XRD analysis was used to detect the phase composition and crystallinity of the n-HA powder and the n-HA/PECE nanocomposite powder which contains 20 wt % n-HA. The relevant XRD patterns are displayed in Figure 3. From Figure 3a, three strong characteristic diffraction peaks for HA at 2θ ) 25.8° (002), 31.7° (211), and 32.9° (300) can be seen.30 Two crystalline peaks at 25.8° and 39.8° were used to calculate the n-HA crystal sizes, according to the Debye-Scherrer formula:
D ) Kλ/(β1/2 cos θ)
(1)
where D is the average crystallite size in the chosen direction, K is a constant (here chosen as 0.89) which depends on the crystal habit, λ is the wavelength of the Cu KR radiation (1.542 Å), β1/2 is the full width at half-maximum (fwhm) of the XRD peak, and θ is the Bragg angle. The crystal size along (002) and (310) corresponds to the length and width of hydroxyapatite. Figure 3b,c shows XRD patterns for n-HA/PECE composite which contains 20 wt % n-HA (b) and pure PECE copolymer (c). From Figure 3c, two strong diffraction peaks at 21.3° and 23.6° were attributed to PECE copolymer. All characterization peaks of n-HA and PECE copolymer could be observed in the pattern of n-HA/PECE nanocomposites as shown in Figure 3b. However, the intensity of these peaks decreased slightly, the reason for which might be that the interactions of HA particles and PECE matrix weaken the crystallization ability of each other. A TEM photograph of as-prepared n-HA particles is shown in Figure 4. The size of needle-like n-HA crystals was about 20-30 nm in diameter and 90-110 nm in length. 3.3. Thermal Property Analysis. Thermal properties, such as the melting temperature (Tm) and crystallization temperature (Tc), of the pure PECE copolymer and n-HA/PECE hydrogel nanocomposites with different HA loadings were measured through DSC. The DSC curves are shown in Figure 5; the values
Figure 5. DSC curves of pure PECE and n-HA/PECE hydrogel nanocomposites with 10, 20, and 30 wt % HA for (A) heating process and (B) cooling process. The rates of heating and cooling were 10 °C/ min.
TABLE 1: Melting Temperature (Tm) and Crystallization Temperature (Tc) of n-HA/PECE Hydrogel Nanocomposites Containing 0, 10, 20, and 30 wt % HA Tm (°C) n-HA (wt %)
peak 1
peak 2
Tc (°C)
0 10 20 30
38.4 38.8 39.6 39.8
46.5 46.8 48.1 48.7
11.6 12.0 11.5 11.0
of temperature for melting and crystallization were obtained from DSC curves and are listed in Table 1. From Figure 5A, it can be seen that all samples displayed two melting peaks at 35-40 °C and 45-50 °C in the heating process; the two endothermic peaks were attributed to the melting of PEG and PCL segments, respectively. The temperature of peak 1 (lower temperature) increased from 38.4 to 39.8 °C as the n-HA content increased from 0 to 30 wt %, and peak 2 (higher temperature) increased from 46.5 to 48.7 °C in the same trend. The result
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Figure 6. Rheological results showing changes of storage modulus (G′), loss modulus (G′′), and complex viscosity (n*) of samples upon heating process: (a) HA, 0 wt %; (b) HA, 10 wt %; (c) HA, 20 wt %; (d) HA, 30 wt %.
revealed that adding n-HA into PECE matrix can improve the melting temperature of nanocomposites. Figure 5B shows the cooling process of all samples; there was only one exothermic peak that could be seen in every cooling curve. This indicates that the crystallization temperatures of the samples were at 8-15 °C and the crystallization temperature decreased as the n-HA content increased, the reason for which might be that addition of n-HA particles into PECE matrix weakens the crystallinity of PECE chains. 3.4. Rheological Analysis. The mechanical properties of the PECE copolymer solution and aqueous n-HA/PECE hydrogel nanocomposites were examined by oscillatory rheological tests as a function of temperature. The storage modulus (G′), loss modulus (G′′), and complex viscosity (n*) were measured to characterize the kinetics of hydrogel formation. Figure 6 shows changes in the G′, G′′, and complex viscosity for PECE solution and n-HA/PECE nanocomposites with different HA contents. When the temperature was below 32 °C, the values of G′ and G′′ were about 1 Pa for all samples, which indicated that the samples all remained in the liquid state and could flow freely. When the temperature increased above 32 °C, the G′, G′′, and complex viscosity values increased rapidly. The change of aqueous samples from a liquid-like to an elastic gel-like state occurred at the crossover point of G′ and G′′, which was observed at about 36 °C. When the temperature increased above 38 °C, the curves of storage modulus, loss modulus, and complex viscosity leveled off, indicating that the samples were at a stable gel state. At temperatures above 43 °C, the G′, G′′, and complex viscosity decreased quickly, revealing that the
samples changed from gel state to sol state except for the sample containing 30 wt % HA. 3.5. Morphology. SEM analysis was conducted in order to investigate the microstructure and the dispersion homogeneity of n-HA particles in the PECE matrix. Figure 7 presents the morphologies of the porous structure of the PECE hydrogel (a) and n-HA/PECE hydrogel nanocomposites (b-d) which contain n-HA from 10 to 30 wt %. It can be seen that the inner pores of the PECE hydrogel were interconnected with irregular shapes and pore sizes ranging from 5 to 20 µm as shown in Figure 7a. The micrographs of Figure 7b-d display the effect of n-HA contents on the microstructure of n-HA/PECE hydrogel nanocomposites: with the increase of n-HA from 10 to 30 wt %, the morphologies of n-HA/PECE hydrogel nanocomposites changed slightly, the interconnected pores became less and less, the pores changed in integrity, and the aggregation of n-HA particles became serious as n-HA loadings increased. Some aggregated HA particles even could be observed in the walls of the PECE copolymer matrix. On the other hand, the polymeric matrix became fragile with the increase of n-HA. This might be because the incorporation of n-HA particles into the PECE matrix disturbed the arrangement of PECE chains. 3.6. Temperature-Dependent Sol-Gel-Sol Transition of n-HA/PECE Hydrogel Nanocomposites. Figure 8 presents photographs of aqueous PECE solution and n-HA/PECE nanocomposites with 20 wt % n-HA at room temperature and body temperature of about 37 °C, which revealed the temperaturedependent sol-gel transition behavior of PECE copolymer and n-HA/PECE nanocomposites. At lower temperature, the sample
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Figure 7. SEM photographs showing cross-sectional morphologies of (a, top left) pure PECE hydrogel and n-HA/PECE hydrogel nanocomposites containing (b, top right) 10, (c, bottom left) 20, and (d, bottom right) 30 wt % HA.
Figure 9. Sol-gel-sol transition phase diagram of PECE hydrogel and n-HA/PECE hydrogel nanocomposites containing 10, 20, and 30 wt % HA at different PECE concentrations. Figure 8. Thermosensitive hydrogel formation of pure PECE and n-HA/PECE nanocomposites with 20 wt % HA at room temperature (R.T.) and 37 °C.
could flow freely, but it became opaque and lost its flowability as the temperature rose to about body temperature (37 °C). Figure 9 displays the sol-gel-sol transition phase diagram of aqueous PECE copolymer solution and n-HA/PECE hydrogel nanocomposites with different loadings of n-HA. All aqueous samples had sol-gel transition and gel-sol transition: when the organic phase concentrations of PECE solution and n-HA/ PECE nanocomposites were above the critical gelation concentration (CGC), the samples changed from “sol” phase to “gel” phase following with the increase in temperature. With further increase in temperature, the samples changed from “gel” phase to “sol” phase. On the other hand, the “sol-gel” transition temperature of aqueous n-HA/PECE hydrogel nanocomposites decreased as the HA content increased, and the “gel-sol” transition temperature shifted to higher temperature at the same time.
4. Discussion Over the past decades, much attention focused on bone substitute materials for the repair of bone defects. Among the implant materials, the demand for in situ gellable hydrogel composites is rising, because they can be applied in surgery via minimally invasive techniques such as injection, with fastsetting properties to form a scaffold at any shape of defect. In particular, thermosensitive hydrogels have received much attention in orthopedic tissue engineering owing to the fact that they can be injected in vivo as a liquid with in situ gel formation at physiological temperature.31 In previous studies of our group, Gong et al. and Liu et al. have studied the thermosensitive hydrogels consisting of PCL and PEG.23,32 However, their studies mainly focused on the use of hydrogels as a drug delivery system; thus in this study, we combined thermosensitive PECE hydrogel with inorganic filler n-HA for preparing the injectable and thermosensitive n-HA/PECE hydrogel nanocomposites which were expected to be applied in the tissue engineering field.
16524 J. Phys. Chem. B, Vol. 113, No. 52, 2009 In this paper, we synthesized PECE triblock copolymer by ring-opening copolymerization of ε-caprolactone on MPEG and coupled with HMDI. 1H NMR and FT-IR results were in agreement with those from Gong et al.’s works, indicating that PECE copolymer was prepared successfully. Nanohydroxyapatite (n-HA) is an important component of bone, which can promote the formation of bone-like apatite on its surface and has been widely used in various biomedical fields.33 We have prepared pure n-HA powder by using a hydrothermal precipitation method. The approach used NaOH for adjusting the pH value of solution which differed from the common Ca(NO3)2-(NH4)2HPO4 system using ammonium hydroxide, thus benefitting the environment and the health of the operator. FT-IR, XRD, and TEM were applied to characterize the asobtained n-HA; the results present that HA is a nanosize needlelike crystal and its shape and components were similar to the HA crystal contained in human bone. We introduced n-HA in thermoreversible PECE hydrogel for preparing injectable n-HA/PECE hydrogel nanocomposites through an in situ combination method. FT-IR results of n-HA/ PECE nanocomposites showed that the main characteristic absorption peaks changed slightly compared to those of pure PECE copolymer and n-HA. It may be that the hydrogen bonds formed between hydroxyl groups (-OH) of n-HA and carboxyl groups (sCdO) of PECE chains.34,35 XRD was used to determine the crystallinity of pure PECE, n-HA, and n-HA/ PECE nanocomposites as shown in Figure 3. Pure PECE copolymer has two strong characteristic peaks at 21.3° and 23.6°, indicating that the PECE copolymer is partly crystalline. The two peaks can be observed in the pattern of n-HA/PECE nanocomposites, but the intensity of peaks decreased obviously. It may be that the interactions of n-HA particles and PECE macromolecular chains thus weaken the crystallinity of PECE. To investigate the thermal properties of n-HA/PECE hydrogel nanocomposites, DSC analysis was performed. According to Figure 5, the heating process presented two melting peaks at 35-40 and 45-50 °C. Moreover, the two melting temperatures of n-HA/PECE hydrogel nanocomposites were higher than those of pure PECE copolymer and the temperatures improved with the increase of n-HA content. It might be that the interactions between n-HA particles and PECE matrix after incorporation of n-HA into PECE copolymer formed the hydrogen bonds between the n-HA particles and PECE chains as described above, which blocked the thermal motion of PECE chains. Therefore, the melting temperatures of n-HA/PECE hydrogel nanocomposites were higher than that of pure PECE copolymer. Hydrogen bonds increased with the increase of n-HA contents, so the melting peaks were shifted to higher temperature followed by increased n-HA loadings. In the cooling process, one exothermic peak at 8-15 °C can be observed. The crystallization temperature decreased slightly from 11.6 to 11.0 °C with the increase of HA content from 0 to 30 wt % except for n-HA/ PECE containing 10 wt % at 12.0 °C; the reason for this may be that loading of 10 wt % n-HA dispersed into PECE matrix more homogeneously than higher n-HA contents such as 20 and 30 wt %. The homogeneous distribution of HA nanoparticles makes them act as the center of nucleation and induce the crystallization of PECE domains. The mechanical properties of the PECE hydrogel and n-HA/ PECE hydrogel nanocomposites were studied by oscillatory rheology experiments. The rheological measurements confirmed the thermal sensitivity of n-HA/PECE hydrogel nanocomposites as presented in Figure 6. The change of G′ modulus, G′′ modulus, and complex viscosity of the samples containing 0
Fu et al. wt % (control), 10, 20, and 30 wt % HA upon the heating process can be seen in Figure 6. Before the samples were heated to 32 °C, the G′ and G′′ values were at approximately 1 Pa, indicating that the samples were at the sol state. Then the G′ modulus, G′′ modulus, and complex viscosity of all samples increased quickly followed by the increase of temperature because of gelation. From Figure 6a-c, we can see that the curves G′ and G′′ intersected at about 36 °C except for the sample containing 30 wt % HA at 40 °C. The gelation points were defined as the temperature at which both G′ and G′′ cross over.10,36 The gelation temperatures of n-HA/PECE nanocomposites were higher than that of pure PECE hydrogel; moreover, the gelation temperature increased with the increase of n-HA content. A possible explanation is that HA nanoparticles may influence the specific hydrophobic interactions between PCL block and PEG block of PECE copolymer. The formation of hydrogen bonds between HA and PECE chains or the aggregation of HA nanoparticles may facilitate the gelation event. Scanning electron microscopic (SEM) observations revealed porous structures within PECE hydrogel and n-HA/PECE hydrogel nanocomposites as shown in Figure 7. The micrographs presented an irregular pore distribution throughout the PECE matrix and with pore sizes ranging from 5 to 20 µm. The threedimensional (3D) network structure of all samples formed owing to the hydrophobic interaction of the PCL segments in PECE chains. In addition, the interconnected porous structures suggested that n-HA/PECE hydrogel nanocomposites were potentially useful as an injectable scaffold for cell growth and migration in polymer matrix. Moreover, the pore sizes in all samples suitable for cell culture, such as the chondrocytes, are approximately 10 µm.37 In previous studies of our group, Gong et al. investigated the thermal sensitivity of PECE hydrogel by using a test tube inverting method. In this paper, we studied the thermosensitive behavior of n-HA/PECE hydrogel nanocomposites in the same way and show the results in Figure 8. The results display the nanocomposites had a thermoresponsivity similar to that of PECE hydrogel. At the lower temperature, the n-HA/PECE could remain in the liquid state and flowed freely, but under the higher temperature of about 37 °C, they became more viscose and even lost their mobility. Figure 9 records the sol-gel-sol transition phase diagram of all samples; the results reveal that the temperature ranges of which the samples remained in the gel state became wider followed by the increased n-HA contents. The reason may be the interactions of HA nanoparticles and PECE polymer matrix just as described above. Figure 9d is different from the other plots because the hydrogel nanocomposites containing 30 wt % HA have lost their flowability after gelation when the PECE concentrations were above 20%, so their gel-sol transition behaviors could not be observed by inverting the vials. The as-obtained n-HA/PECE hydrogel nanocomposites have thermal sensitivity as described above. They were injectable fluids and could form a gel in the desired tissue, organ, or body cavity in a minimally invasive manner. Thus, they could serve as a new biomaterial for orthopedic tissue engineering. 5. Conclusions In this paper, we successfully synthesized the PECE triblock copolymer and pure n-HA powder. PECE copolymer was combined with HA nanoparticles in order to prepare novel thermosensitive hydrogel nanocomposites. The HA particles were nanosized and acicular in shape, which guaranteed the HA particles could be dispersed homogeneously into the PECE
Tissue Engineering n-HA/PECE Hydrogel Composites matrix. The rheological measurements revealed that the gelation points of the n-HA/PECE hydrogel nanocomposites were adequate for intracorporeal injection. The nanocomposite system could remain in the sol state when they were at room temperature; subsequently, as the temperature increased, they grew stable in the gel state at a higher temperature of about 37 °C. The gelation temperature could be adjusted by changing the PECE concentration or HA contents. The 3D structure of n-HA/PECE hydrogel nanocomposites displayed that they could be used as scaffold material for cell proliferation. Although the concept of injectable and thermoresponsive hydrogel nanocomposites was introduced in this study, more research is need to validate the application of such systems in bone regeneration, possibly by investigating them with osteoblasts and growth factors in the future. Therefore, the n-HA/PECE hydrogel nanocomposite system is a prospective candidate as an injectable biomaterial for orthopedic tissue engineering. Acknowledgment. This work was financially supported by the National 863 project (2007AA021902), Specialized Research Fund for the Doctoral Program of Higher Education (200806100065), China Key Basis Research Program (529906), and New Century Excellent Talents in University (NCET-08-0371). References and Notes (1) Temenoff, J. S.; Mikos, A. G. Biomaterials 2000, 21, 2405–2412. (2) Schmaljohann, D. e-Polymers 2005, Article 021, pp 1-17. (3) Gutowska, A.; Jeong, B.; Jasionowski, M. Anat. Rec. 2001, 263, 342–349. (4) Payne, R. G.; Yaszemski, M. J.; Yasko, A. W.; Mikos, A. G. Biomaterials 2002, 23, 4359–4371. (5) Griffon, D. J.; Sedighi, M. R.; Schaeffer, D. V.; Eurell, J. A.; Johnson, A. L. Acta Biomater. 2006, 2, 313–320. (6) Bhattarai, N.; Ramay, H. R.; Gunn, J.; Matsen, F. A.; Zhang, M. Q. J. Controlled Release 2005, 103, 609–624. (7) Li, X. Y.; Zheng, X. L.; Wei, X. W.; Guo, G.; Gou, M. L.; Gong, C. Y.; Wang, X. H.; Dai, M.; Chen, L. J.; Wei, Y. Q.; Qian, Z. Y. J. Nanosci. Nanotechnol. 2009, 9, 4586–4592. (8) Park, K. M.; Joung, Y. K.; Na, J. S.; Lee, M. C.; Park, K. D. Acta Biomater. 2009, 5, 1956–1965. (9) Kim, I. Y.; Seo, S. J.; Moon, H. S.; Yoo, M. K.; Park, I. Y.; Kim, B. C.; Cho, C. S. Biotechnol. AdV. 2008, 26, 1–21. (10) Couto, D. S.; Hong, Z. K.; Mano, J. F. Acta Biomater. 2009, 5, 115–123. (11) Moreau, J. L.; Xu, H. K. Biomaterials 2009, 30, 2675–2682. (12) Brink, K. S.; Yang, P. J.; Temenoff, J. S. Acta Biomater. 2009, 5, 570–579.
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