Appl Biochem Biotechnol DOI 10.1007/s12010-013-0696-y
Preparation, Characterization, and In Vitro Biological Evaluation of PLGA/Nano-Fluorohydroxyapatite (FHA) Microsphere-Sintered Scaffolds for Biomedical Applications Mohammadreza Tahriri & Fathollah Moztarzadeh
Received: 24 August 2013 / Accepted: 25 December 2013 # Springer Science+Business Media New York 2014
Abstract In this research, the novel three-dimensional (3D) porous scaffolds made of poly(lactic-co-glycolic acid) (PLGA)/nano-fluorohydroxyapatite (FHA) composite microspheres was prepared and characterize for potential bone repair applications. We employed a microsphere sintering method to produce 3D PLGA/nano-FHA scaffolds composite microspheres. The mechanical properties, pore size, and porosity of the composite scaffolds were controlled by varying parameters, such as sintering temperature, sintering time, and PLGA/nano-FHA ratio. The experimental results showed that the PLGA/nano-FHA (4:1) scaffold sintered at 90 °C for 2 h demonstrated the highest mechanical properties and an appropriate pore structure for bone tissue engineering applications. Furthermore, MTT assay and alkaline phosphatase activity (ALP activity) results ascertained that a general trend of increasing in cell viability was seen for PLGA/nano-FHA (4:1) scaffold sintered at 90 °C for 2 h by time with compared to control group. Eventually, obtained experimental results demonstrated PLGA/nano-FHA microsphere-sintered scaffold deserve attention utilizing for bone tissue engineering. Keywords PLGA . FHA . Microsphere . Sintering . Scaffold
Introduction Tissue engineering is a multidisciplinary field which combines the science of engineering, biology, and chemistry which this field has emerged as a well-promising alternative route to treat the loss or malfunction of a tissue or organ without the limitations of current therapies [1–7]. Tissue engineering involves the expansion of cells through a small biopsy from a patient, followed by the controlled culturing of the cells in temporary 3D scaffolds to form the new organ or tissue [8]. Therefore, one common approach is to implant biodegradable scaffolds for tissue ingrowth M. Tahriri : F. Moztarzadeh (*) Biomaterial Group, Faculty of Biomedical Engineering, Amirkabir University of Technology, P.O. Box: 15875-4413, Tehran, Iran e-mail: [email protected]
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directly in vivo to stimulate and direct tissue formation in situ [9, 10]. Using the patient’s own cells, the mentioned approach has the many advantages of autografts, but without the problems associated with sufficient supply. Recently, numerous biomaterials such as metals, ceramics, and polymers from biologic and synthetic origins have been employed as scaffolds for bone tissue engineering [11]. The bone is a polymer/ceramic hybrid composite, therefore it seems logical to look for a new generation of implantable materials with the same hybrid structure to combine the mechanical strength, and osteoconductivity of an inorganic phase with the formability, toughness, and resorbability of an organic phase [12, 13]. Poly(DL-lactide-co-glycolide) (PLGA) is a biodegradable polymer that has been evaluated widely since the 1960s and is commonly used as organic component of scaffold composites because of its good biocompatibility and tailored degradation rate [14, 15]. However, the chain of PLGA has no functional groups, and each lactic acid residue contains a pendant methyl group, giving a hydrophobic surface. One of the main routes in improving PLGA-based scaffold is to composite PLGA matrix with nanoceramic such as hydroxyapatite [16], tricalcium phosphate (TCP) [17], and bioactive glass [18]. Hydroxyapatite (HA; Ca5(PO4)3OH), the mineral component of bones and hard tissues in mammals, has been the subject of much research over the years, particularly in the field of biomaterials science. This is due to its importance in clinical applications involving medical devices and implants and more recently in the broad field of tissue engineering. Substitutions within the HA lattice are observed both for naturally occurring and synthetic HA. The most common are substitutions involving carbonate, fluoride, and chloride ions for hydroxyl ions [19]. However, bone mineral HA contains small amounts of fluoride ions, sodium, magnesium, and other trace components [20]. The fluoride ion has attracted attention due to its therapeutic ability of osteoporosis healing since the bone mass is increased by F− ion administration [21]. F− is known to stimulate osteoblast activity both in vitro and in vivo. Sodium fluoride directly increases the proliferation rate and the alkaline phosphatase activity of osteoblastic cells, thus enhance the new bone tissue formation [22]. F− may influence the proliferation and differentiation of orthoclastic cells which strongly offers the alteration of one or several G-protein-dependent tyrosine phosphorylation processes, activation of the extracellular signal-regulated kinase, and possibly other signaling pathways [23, 24]. Therefore, sodium fluoride therapy has been investigated as one of the main treatments for osteoporosis [25–27]. When F− substitutes for the hydroxyl (OH−) group, solubility of the bone mineral decreases due to partially F-substituted hydroxyapatite or fluorhydroxyapatite (FHA; Ca5(PO4)3(OH)1−xFx 0≤x≤1) and fluorapatite (FA; Ca5(PO4)3 F) are less soluble than pure hydroxyapatite at pH 5–7 [21]. The purpose of this project is to design and develop a porous composite with good mechanical and biological properties as potential scaffolds for bone tissue engineering applications. In the present work, we have prepared and characterized a PLGA/nanoFHA composite scaffold with different PLGA/FHA ratio based on a sintered microsphere method. The scaffolds were produced by mixing PLGA microspheres and heating them above the glass transition temperature of PLGA. The effects of PLGA/nano-FHA ratio and sintering conditions on the mechanical properties, pore structure, and porosity of the scaffolds were evaluated. Eventually, for in vitro evaluation, the osteoblast-like cells were seeded on the surface of composite scaffolds and the cell proliferation on them was investigated.
Appl Biochem Biotechnol
Experimental Procedure Materials Methylene chloride (CH2Cl2) was purchased from Sigma-Aldrich Company and poly(vinyl alcohol) (PVA, MW =31,000–50,000 Da) was obtained from Merck Company. All materials were used as received without any further purification. It is worth mentioning that the used PLGA and FHA in this research were synthesized by bulk polymerization and pH-cycling method, respectively as described by Wang et al. and Eslami et al. [19, 28]. Preparation of PLGA/Nano-FHA Composite Microspheres PLGA/nano-FHA composite microspheres were prepared using a modification of the emulsion and solvent evaporation method. In brief, PLGA was dissolved in methylene chloride to make a 20 % (w/v) solution. To make PLAA/nano-FHA microspheres with certain PLGA to FHA ratio, a known amount of FHA powders were dispersed in this solution by agitating on a vortex machine for 2 h. The solution was then poured into a 1 % PVA solution stirring at 1,000 rpm. Stirring continued for 12 h to allow complete evaporation of the methylene chloride. The resultant microspheres washed with deionized water, and dried in a freezedrier system (Alpha 1-2 LD, Germany) for 10 h. Preparation of PLGA/Nano-FHA Scaffolds The 3D composite scaffolds were prepared by a sintered microsphere method. For this purpose, the PLGA microspheres were packed tightly in a stainless mold and sintered above the glass transition temperature of PLGA for a certain time. Different sintering temperatures (80 or 90 °C) and sintering times (2, 3 and 4 h) were evaluated. It is worth mentioning that a little shrinkage in the samples was observed. Characterization of PLGA/Nano-FHA Scaffolds X-ray Diffraction X-ray diffraction (XRD) of the samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) was carried out using a Siemens-Brucker D5000 diffractometer, with voltage and current setting of 40 kV and 40 mA, respectively, and uses Cu-Kα radiation (1.5406 Å). For qualitative analysis, XRD diagrams were recorded in the interval 10°≤2θ≤80° at a scan speed of 2°/min giving a step size 0.02° and the step time 1 s. Transform Infrared Spectroscopy The samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) were examined by Fourier transform infrared spectroscopy with a Bomem MB 100 spectrometer. For IR analysis, first 1 mg of the sample was carefully mixed with 300 mg of KBr (infrared grade) and palletized under vacuum. Then the pellet was analyzed in the range of 400 to 4,000 cm−1 at a scan speed of 23 scan/min with 4 cm−1 resolution.
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Thermal Analysis The thermal behavior of the samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) was studied by simultaneously thermal analysis (STA). A thermoanalyzer (STA; Polymer Laboratories PL-STA 1640) that starting from room temperature up to 1,000 °C with the heating rate of 10 °C/min was used to record the conventional thermoanalytical curves [differential thermal analysis (DTA) and thermogravimetric analysis (TGA)]. Scanning Electron Microscopy Microstructure and morphology of porous composite scaffold was evaluated using scanning electron microscopy (SEM). The scaffold sample were coated with a thin layer of Gold (Au) by sputtering (EMITECH K450X, England) and then the morphology of it was observed on a scanning electron microscope (SEM—Tescan Vega 2XMU) that operated at the acceleration voltage of 10 kV. Mechanical Properties The compressive strength and modulus of the composite scaffolds were measured using a mechanical testing machine (SMT-20, Santam, Iran). According to ASTM D 5024-95 standard, cylindrical samples with length-to-diameter ratio of 2:1 (10 mm in length and 5 mm in diameter) were prepared. The cross-head speed was set at 0.5 mm/min, and the load was applied until the specimen was compressed to approximately 30 % of its original length. The elastic modulus was determined as the slope of the initial linear portion of the stress–strain curve. The compressive strength was calculated as the maximum point of the stress–strain curve. Density and Porosity Measurement The porosity of the scaffold samples was calculated using liquid substitution method [29]. For this purpose, ethanol was used as the displacing liquid because it penetrated easily into the pores of the scaffold, but not into the composite itself. Briefly, a sample of measured weight W was immersed in a graduated cylinder containing a known volume (V1) of ethanol and kept for 5 min to allow the ethanol to penetrate into the pores of the scaffold samples. The total volume of the remaining ethanol and the ethanol-impregnated scaffolds was then recorded as V2 by simply reading the level in the cylinder. The volume difference (V 2 − V 1 ) represents the volume of the PLGA/nano-FHA scaffold sample. The ethanol-impregnated scaffolds were then removed from the graduated cylinder and the residual ethanol volume was recorded as V3. Hence, the total volume of the scaffolds was V=(V2 −V3) and the bulk density of the scaffold was expressed as ρ=W/(V2 −V3). By determining the initial and final weights Wi and Wf, respectively, of the scaffolds after soaking in ethanol (ρethanol = 0.789 g/cm3) for 24 h, the pore volume of scaffolds can be determined as (Wf −Wi)/ ρethanol and the porosity can be calculated using the following equation [29]: W f −W i =ρethanol Porosity ¼ V 2 −V 1
ð1Þ
Appl Biochem Biotechnol
In Vitro Biological Evaluation Cytotoxicity L929 mouse fibroblast cell line (ATCC) was employed for cytotoxicity investigation of mechanically optimum composite scaffold (PLGA/FHA=4:1). The cells were seeded in polystyrene plates enriched with Minimal Essential Medium supplemented with 10 % fetal bovine serum, 100 units/ml of penicillin and 100 μg/ml streptomycin, respectively, and incubated at 37 °C in humid atmosphere and 5 % CO2. When the cells attained confluency, the sterilized discs were placed in direct contact with the cells and incubated for 2 days under the same condition. It is noticeable that negative control sample (ultra high molecular weight poly ethylene) were used. After 2 days, the cells were observed under optical microscopy (Nicon E200). Cell Proliferation (MTT Assay) The cell proliferation on the nanocomposite scaffolds was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For the assay, cells were then seeded onto 96-well plates at a density of 2×103 cells/ well and were incubated under standard culturing conditions. The PLGA and PLGA/nanoFHA (4:1) scaffolds were placed on the cells after an overnight incubation at 37 °C with 5 % CO2 in a humidified atmosphere. Three wells in the absence of scaffolds were used as negative controls [tissue culture polystyrene (TCPS)]. For each sample, five wells of microliter plate were selected. The plates were incubated for 3, 7, and 14 days with half media changed every 2 days. Then, nanocomposite scaffolds were removed from the wells and 10 μL of a 5 mg/mL solution of MTT was added to each well followed by incubation for 5 h at 37 °C. Formed formazan crystals were dissolved by addition of 100 μL/well of isopropanol. Subsequently, the plates were incubated at 37 °C for 10 min and transferred to 48 °C for 15 min before absorbance measurements. Optical density (OD) was recorded on a multiwell microplate reader at a wavelength of 570 nm. ALP Activity The functional activity of the cells on the prepared scaffolds was evaluated by measuring the ALP activity. ALP activity was conducted using a commercial kinetics method. Briefly, the G-292 cell lines were transferred into 24-well microliter plates at 2×103 cells/well, separately. The PLGA and PLGA/nano-FHA (4:1) scaffolds were placed in the wells. Three wells in the absence of scaffolds were used as negative controls (TCPS). The plates were incubated for 3, 7, and 14 days at 37 °C in humidified air with 5 % CO2. Ten microliters of the supernatant was removed from each well at 3, 7, and 14 days and processed according to the guideline. It is noticeable that the color changing was measured by spectrophotometer at 405 nm. The standard p-nitrophenol curve was employed to convert the obtained absorbance data to the ALP content.
Results and Discussion XRD Analysis Figure 1 shows the XRD patterns of the FHA, PLGA, and PLGA/FHA (4:1) composite samples. As it can be seen in the XRD pattern of FHA, no tricalcium phosphate was detected in FHA powders. It is important to point out that the XRD results suggest that FHA has a structure like HA. However, no structures for partially fluorine ion substituted apatites are recorded in the ICDD database. Also in this figure, the peaks that related to pure PLGA has been observed and indexed.
Appl Biochem Biotechnol
Fig. 1 The XRD patterns of FHA, PLGA, and PLGA/FHA (4:1) composite
FTIR Analysis Figure 2 shows the Fourier transform infrared spectroscopy (FTIR) spectra of the FHA, PLGA, and PLGA/FHA (4:1) composite samples. The characteristic bands exhibited in the sample spectra assigned here: (a) The band at 1,002 cm−1 arises from ν3 PO4, the bands at 514 cm−1 and 550 cm−1 arise from ν4 PO4, the bands at 916 cm−1 arises from ν1 PO4 and the bands at 437 cm−1 arise from ν2 PO4. (b) The bands at 868 cm−1 and about 1,434 cm−1 arise from CO3 which an indication of the presence of carbonate apatite [30, 31]. This might have
Fig. 2 The FTIR spectra of FHA, PLGA, and PLGA/FHA (4:1) composite
Appl Biochem Biotechnol
originated through the absorption of carbon dioxide from the atmosphere. (c) The bands at 3,436 cm−1 arises from OH….F bond that demonstrated some of the OH− groups in the crystalline network of HA were replaced by F−. The PLGA/FHA spectrum is similar to the pure PLGA alone. This is due to the high percent of PLGA in the composite. It is worth mentioning that the PLGA has covered the FHA particle and thus the scaffold composite (PLGA/FHA) did not sufficient transparent for the IR spectroscopy analysis, and therefore, FHA-related vibration bands did not appear very sharp and become very broad. Thermal Analysis The TG-DTA curves for the PLGA/nano-FHA (4:1) composite are illustrated in Fig. 3. As it can be seen in this curve, the composite has a thermal stability until about 260 °C. The weight loss of pure PLGA, attributed to thermal decomposition, occurs in two steps (390 and 470 °C). The gradual decrease in weight which is observed from 390 to 700 °C is because of the slow elimination of the carbonate groups linked to FHA which has been confirmed with the FTIR analysis discussed later. Also, as it can be observed from this figure, in the given temperature interval, FHA is thermally stable. Thus, the weight percentage at the end of the experiment (700 °C) is the percentage of FHA in the composite scaffolds. SEM Observations Figure 4 showed the SEM micrograph of PLGA/nano-FHA (4:1) microsphere scaffolds sintered at 90 °C for 2 h. The scaffolds were made by microspheres, and all types of PLGA-based microspheres maintained in spherical shape with visible rough surfaces because of great nano-FHA conglomeration enrichment on their surfaces. Mechanical Properties The preparation of 3D porous composite scaffolds is obtained through a microsphere sintering technique. The sintering temperature and sintering time are two processing parameters that can
Fig. 3 TG-DTA curve of the PLGA/FHA (4:1) composite
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Fig. 4 SEM micrograph of PLGA/nano-FHA (4:1) composite scaffolds sintered at 90 °C, for 2 h
be controlled the mechanical properties and porous structures of the sintered microsphere scaffolds. Figures 5 and 6 show the measured compressive modulus and strength of the composite scaffolds sintered under different conditions. As it can be seen in this figure, the compressive
Fig. 5 Effects of sintering temperature and sintering time on the compressive modulus of the PLGA/nano-FHA (4:1) composite scaffolds
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Fig. 6 Effects of sintering temperature and sintering time on the compressive strength of the PLGA/nano-FHA (4:1) composite scaffolds
modulus and compressive strength of the scaffolds sintered at 90 °C for 2 h are remarkably higher than the scaffolds prepared at other sintering conditions. Increasing either sintering temperature or sintering time resulted in better mechanical properties because of a greater bonding area. On the other hand, when the sintering time and sintering temperature was further increased, the PLGA microspheres in scaffolds is being collapsed resulting in scaffolds with inferior mechanical properties [32]. Also, the pore structures of the composite scaffolds are affected by sintering conditions. Jiang et al. [33] showed that increasing sintering time resulted in possible closure of pores, which this phenomenon caused to decrease scaffold porosity. This study ascertained that porosity decreased with sintering time (see Fig. 7). Scaffolds sintered at 90 °C for 2 h had porosity of 31 %.
Fig. 7 Effects of sintering temperature and sintering time on the porosity content of the PLGA/nano-FHA (4:1) composite scaffolds
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Density is another important parameter in the composite scaffold design. It is worth mentioning that the results showed that the sintering time and temperature did not largely affect scaffold density (see Fig. 8). Also, this figure demonstrated that all the sintering conditions resulted in composite scaffolds with density less than the media density (1 g/cm3). The optimal sintering condition was evaluated for different composite scaffolds with various PLAGA/nano-FHA ratios. The highest compressive modulus and strength were observed at the optimal sintering condition for all ratios (data not shown). Figures 9 and 10 show the effects of the FHA content in the composite scaffold on the compressive modulus and strength. Under the optimal sintering condition, the PLGA/nanoFHA (4:1) scaffolds ascertained that the highest mechanical properties, which were remarkably higher than other scaffolds (other PLGA/nano-FHA ratios). Additionally, the compressive modulus and strength of PLGA/nano-FHA (4:1) scaffolds were significantly higher than pure PLGA scaffolds, suggesting that the introducing of nano-FHA into PLGA scaffolds at specific ratio could boost the mechanical properties of the mentioned polymeric scaffolds. It is noticeable that, when a low amount of FHA was introduced into PLGA scaffold, approximately 100 % of the FHA particles were entranced into the microspheres. However, when the initial loading was increased, the efficiency of introducing was slightly decreased. Although the initial loading FHA is changed, the concentration of polymer solution (meaning that viscosity) is kept constant. Since the density of FHA is higher than PLGA solution and water, they tend to sediment at the tank bottom. Thus, the efficiency of FHA introducing decreases with an increase in initial loading. Borden et al. [34] showed that increasing either the time or temperature of sintering could result in an increase in compressive modulus and a decrease in porosity. In PLGA/nano-FHA (4:1) scaffold, a similar trend up to 90 °C/2 h was seen, while, further increasing sintering time caused to a decrease in compressive modulus. Hence, for each type of composite scaffold, there are optimal sintering conditions at which highest mechanical properties could be achieved. In order to study the effect of the PLGA/FHA ratio on the mechanical properties of the scaffolds, the optimal sintering conditions, as discussed earlier, was selected, and the mechanical characteristics of the various scaffold types were compared together. Since FHA is a high-
Fig. 8 Effects of sintering temperature and sintering time on the density of the PLGA/nano-FHA (4:1) composite scaffolds
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Fig. 9 Effects of scaffold composition on the compressive modulus of composite scaffolds (Scaffolds sintered at 90 °C, for 2 h)
mechanical strength bioceramic, hence, it seems that the scaffolds with higher compressive modulus and strength can be obtained by enhancing the amount of FHA in the composite scaffolds. In spite of that, the obtained results disclosed that the PLGA/nano-FHA (4:1) scaffold was the strongest among the four types of prepared scaffolds that mechanically analyzed. When packed microspheres were heated above the polymer’s glass transition temperature (Tg), sintering takes place because of the intertwining of polymer chains between adjacent microspheres, resulting in the formation of bonds [35]. Thus, the mechanical strength of the 3D microsphere-based scaffolds significantly depends on the fusion area between the microspheres.
Fig. 10 Effects of scaffold composition on the compressive strength of composite scaffolds (Scaffolds sintered at 90 °C, for 2 h)
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When greater FHA particles were introduced (such as PLGA/nano-FHA 1:1) in the microspheres, greater FHA particles existed on the microsphere surface which oppositely affect the PLGA sintering. Eventually, we concluded that the incorporation of appropriate amounts of FHA could boost the mechanical characteristics of 3D polymeric scaffolds prepared by a sintered microsphere method. In Vitro Biological Evaluation Cytotoxicity Based on number and form of cells covering the surface of the PLGA/nano-FHA (4:1) scaffold, the amount of cytotoxicity was evaluated by optical microscopy shown in Fig. 11. According to Fig. 10, it can be observed that a layer of fusiform cells of fibroblast has been covered on the surface of the PLGA/nano-FHA scaffold. The cytotoxicity of the scaffold is distinguished by development of layer of cells and also their fusiform morphology. The spherical cells are dead cells which are related to the lack of food but not the toxicity of scaffold sample. Therefore, the cytotoxic scale of the composite scaffold for L929 mouse fibroblast cells was measured as zero, which corresponds to non-cytotoxicity. MTT Assay The results obtained by MTT assay were compared with control group (polystyrene well) as shown in Fig. 12. According to the results, addition of FHA to polymeric scaffolds had no negative effect on the proliferation of G-292 or osteoblasts cells. Also, viabilities were better for nanocomposite scaffolds [PLGA/FHA(4:1)] using G-292 cell line; however, it resulted in a significant reduction in the biocompatibility of the nanocomposite samples in comparison with the control group just after 3 days incubation time. This phenomenon was completely revealed later on with longer incubation periods (7 or 14 days), which resulted in improved viabilities even comparing with control group. Fig. 11 Optical micrograph of L929 fibroblast cells in direct contact with PLGA/nano-FHA (4:1) composite scaffold
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Fig. 12 Cell proliferation of G-292 cells proliferated on the PLGA/nano-FHA (4:1) scaffolds along with negative control after incubation for 3, 7, and 14 days
Using G-292, viabilities were improved for nanocomposite scaffold [PLGA/FHA(4:1)] showed improved viabilities after 7 days. ALP Activity ALP is a well-known analysis for differentiation of osteoblasts on its expression during osteogenesis. ALP activity results for G-292 cells on the PLGA and PLGA/nano-FHA (4:1) scaffold is given for 3, 7, or 14 days of incubation period (Fig. 13). The obtained results ascertained that there is a general trend of increasing in ALP activity for PLGA/nano-FHA (4:1) scaffold by time.
Fig. 13 ALP activity test for G-292 cells proliferated on the PLGA/nano-FHA (4:1) scaffolds along with negative control after incubation for 3, 7, and 14 days
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Conclusions In conclusion, composite scaffolds containing nano-FHA and PLGA were successfully prepared by sintering method suitable for bone tissue engineering. It was found that the mechanical characteristics and pore structures to be modulated by sintering condition and PLGA/nano-FHA ratio. We chose the PLGA/nanoFHA (4:1) scaffold as it showed the greatest compressive modulus and strength and its pore structure was suitable for cell penetration. Also, the experimental results ascertained that the compressive modulus and compressive strength of the scaffolds sintered at 90 °C for 2 h are remarkably higher than the scaffolds prepared at other sintering conditions. Also, the obtained results exhibited that the sintering time and temperature did not significantly affect scaffold density. Also, all sintered composite scaffolds had a density less than the media density (1 g/cm3). In addition, MTT assay results showed that a general trend of increasing cell viability was observed for nanocomposite scaffolds [PLGA/FHA (4:1)] with time compared to control group. Eventually, it can be suggested that the newly developed PLGA/nano-FHA composite fulfills most of the requirements as suitable bone scaffolds for bone tissue engineering applications.
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Preparation, Characterization, and In Vitro Biological Evaluation of PLGA/Nano-Fluorohydroxyapatite (FHA) Microsphere-Sintered Scaffolds for Biomedical Applications Mohammadreza Tahriri & Fathollah Moztarzadeh
Received: 24 August 2013 / Accepted: 25 December 2013 # Springer Science+Business Media New York 2014
Abstract In this research, the novel three-dimensional (3D) porous scaffolds made of poly(lactic-co-glycolic acid) (PLGA)/nano-fluorohydroxyapatite (FHA) composite microspheres was prepared and characterize for potential bone repair applications. We employed a microsphere sintering method to produce 3D PLGA/nano-FHA scaffolds composite microspheres. The mechanical properties, pore size, and porosity of the composite scaffolds were controlled by varying parameters, such as sintering temperature, sintering time, and PLGA/nano-FHA ratio. The experimental results showed that the PLGA/nano-FHA (4:1) scaffold sintered at 90 °C for 2 h demonstrated the highest mechanical properties and an appropriate pore structure for bone tissue engineering applications. Furthermore, MTT assay and alkaline phosphatase activity (ALP activity) results ascertained that a general trend of increasing in cell viability was seen for PLGA/nano-FHA (4:1) scaffold sintered at 90 °C for 2 h by time with compared to control group. Eventually, obtained experimental results demonstrated PLGA/nano-FHA microsphere-sintered scaffold deserve attention utilizing for bone tissue engineering. Keywords PLGA . FHA . Microsphere . Sintering . Scaffold
Introduction Tissue engineering is a multidisciplinary field which combines the science of engineering, biology, and chemistry which this field has emerged as a well-promising alternative route to treat the loss or malfunction of a tissue or organ without the limitations of current therapies [1–7]. Tissue engineering involves the expansion of cells through a small biopsy from a patient, followed by the controlled culturing of the cells in temporary 3D scaffolds to form the new organ or tissue [8]. Therefore, one common approach is to implant biodegradable scaffolds for tissue ingrowth M. Tahriri : F. Moztarzadeh (*) Biomaterial Group, Faculty of Biomedical Engineering, Amirkabir University of Technology, P.O. Box: 15875-4413, Tehran, Iran e-mail: [email protected]
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directly in vivo to stimulate and direct tissue formation in situ [9, 10]. Using the patient’s own cells, the mentioned approach has the many advantages of autografts, but without the problems associated with sufficient supply. Recently, numerous biomaterials such as metals, ceramics, and polymers from biologic and synthetic origins have been employed as scaffolds for bone tissue engineering [11]. The bone is a polymer/ceramic hybrid composite, therefore it seems logical to look for a new generation of implantable materials with the same hybrid structure to combine the mechanical strength, and osteoconductivity of an inorganic phase with the formability, toughness, and resorbability of an organic phase [12, 13]. Poly(DL-lactide-co-glycolide) (PLGA) is a biodegradable polymer that has been evaluated widely since the 1960s and is commonly used as organic component of scaffold composites because of its good biocompatibility and tailored degradation rate [14, 15]. However, the chain of PLGA has no functional groups, and each lactic acid residue contains a pendant methyl group, giving a hydrophobic surface. One of the main routes in improving PLGA-based scaffold is to composite PLGA matrix with nanoceramic such as hydroxyapatite [16], tricalcium phosphate (TCP) [17], and bioactive glass [18]. Hydroxyapatite (HA; Ca5(PO4)3OH), the mineral component of bones and hard tissues in mammals, has been the subject of much research over the years, particularly in the field of biomaterials science. This is due to its importance in clinical applications involving medical devices and implants and more recently in the broad field of tissue engineering. Substitutions within the HA lattice are observed both for naturally occurring and synthetic HA. The most common are substitutions involving carbonate, fluoride, and chloride ions for hydroxyl ions [19]. However, bone mineral HA contains small amounts of fluoride ions, sodium, magnesium, and other trace components [20]. The fluoride ion has attracted attention due to its therapeutic ability of osteoporosis healing since the bone mass is increased by F− ion administration [21]. F− is known to stimulate osteoblast activity both in vitro and in vivo. Sodium fluoride directly increases the proliferation rate and the alkaline phosphatase activity of osteoblastic cells, thus enhance the new bone tissue formation [22]. F− may influence the proliferation and differentiation of orthoclastic cells which strongly offers the alteration of one or several G-protein-dependent tyrosine phosphorylation processes, activation of the extracellular signal-regulated kinase, and possibly other signaling pathways [23, 24]. Therefore, sodium fluoride therapy has been investigated as one of the main treatments for osteoporosis [25–27]. When F− substitutes for the hydroxyl (OH−) group, solubility of the bone mineral decreases due to partially F-substituted hydroxyapatite or fluorhydroxyapatite (FHA; Ca5(PO4)3(OH)1−xFx 0≤x≤1) and fluorapatite (FA; Ca5(PO4)3 F) are less soluble than pure hydroxyapatite at pH 5–7 [21]. The purpose of this project is to design and develop a porous composite with good mechanical and biological properties as potential scaffolds for bone tissue engineering applications. In the present work, we have prepared and characterized a PLGA/nanoFHA composite scaffold with different PLGA/FHA ratio based on a sintered microsphere method. The scaffolds were produced by mixing PLGA microspheres and heating them above the glass transition temperature of PLGA. The effects of PLGA/nano-FHA ratio and sintering conditions on the mechanical properties, pore structure, and porosity of the scaffolds were evaluated. Eventually, for in vitro evaluation, the osteoblast-like cells were seeded on the surface of composite scaffolds and the cell proliferation on them was investigated.
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Experimental Procedure Materials Methylene chloride (CH2Cl2) was purchased from Sigma-Aldrich Company and poly(vinyl alcohol) (PVA, MW =31,000–50,000 Da) was obtained from Merck Company. All materials were used as received without any further purification. It is worth mentioning that the used PLGA and FHA in this research were synthesized by bulk polymerization and pH-cycling method, respectively as described by Wang et al. and Eslami et al. [19, 28]. Preparation of PLGA/Nano-FHA Composite Microspheres PLGA/nano-FHA composite microspheres were prepared using a modification of the emulsion and solvent evaporation method. In brief, PLGA was dissolved in methylene chloride to make a 20 % (w/v) solution. To make PLAA/nano-FHA microspheres with certain PLGA to FHA ratio, a known amount of FHA powders were dispersed in this solution by agitating on a vortex machine for 2 h. The solution was then poured into a 1 % PVA solution stirring at 1,000 rpm. Stirring continued for 12 h to allow complete evaporation of the methylene chloride. The resultant microspheres washed with deionized water, and dried in a freezedrier system (Alpha 1-2 LD, Germany) for 10 h. Preparation of PLGA/Nano-FHA Scaffolds The 3D composite scaffolds were prepared by a sintered microsphere method. For this purpose, the PLGA microspheres were packed tightly in a stainless mold and sintered above the glass transition temperature of PLGA for a certain time. Different sintering temperatures (80 or 90 °C) and sintering times (2, 3 and 4 h) were evaluated. It is worth mentioning that a little shrinkage in the samples was observed. Characterization of PLGA/Nano-FHA Scaffolds X-ray Diffraction X-ray diffraction (XRD) of the samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) was carried out using a Siemens-Brucker D5000 diffractometer, with voltage and current setting of 40 kV and 40 mA, respectively, and uses Cu-Kα radiation (1.5406 Å). For qualitative analysis, XRD diagrams were recorded in the interval 10°≤2θ≤80° at a scan speed of 2°/min giving a step size 0.02° and the step time 1 s. Transform Infrared Spectroscopy The samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) were examined by Fourier transform infrared spectroscopy with a Bomem MB 100 spectrometer. For IR analysis, first 1 mg of the sample was carefully mixed with 300 mg of KBr (infrared grade) and palletized under vacuum. Then the pellet was analyzed in the range of 400 to 4,000 cm−1 at a scan speed of 23 scan/min with 4 cm−1 resolution.
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Thermal Analysis The thermal behavior of the samples (composite scaffold, as well as on chemically synthesized FHA powder and PLGA) was studied by simultaneously thermal analysis (STA). A thermoanalyzer (STA; Polymer Laboratories PL-STA 1640) that starting from room temperature up to 1,000 °C with the heating rate of 10 °C/min was used to record the conventional thermoanalytical curves [differential thermal analysis (DTA) and thermogravimetric analysis (TGA)]. Scanning Electron Microscopy Microstructure and morphology of porous composite scaffold was evaluated using scanning electron microscopy (SEM). The scaffold sample were coated with a thin layer of Gold (Au) by sputtering (EMITECH K450X, England) and then the morphology of it was observed on a scanning electron microscope (SEM—Tescan Vega 2XMU) that operated at the acceleration voltage of 10 kV. Mechanical Properties The compressive strength and modulus of the composite scaffolds were measured using a mechanical testing machine (SMT-20, Santam, Iran). According to ASTM D 5024-95 standard, cylindrical samples with length-to-diameter ratio of 2:1 (10 mm in length and 5 mm in diameter) were prepared. The cross-head speed was set at 0.5 mm/min, and the load was applied until the specimen was compressed to approximately 30 % of its original length. The elastic modulus was determined as the slope of the initial linear portion of the stress–strain curve. The compressive strength was calculated as the maximum point of the stress–strain curve. Density and Porosity Measurement The porosity of the scaffold samples was calculated using liquid substitution method [29]. For this purpose, ethanol was used as the displacing liquid because it penetrated easily into the pores of the scaffold, but not into the composite itself. Briefly, a sample of measured weight W was immersed in a graduated cylinder containing a known volume (V1) of ethanol and kept for 5 min to allow the ethanol to penetrate into the pores of the scaffold samples. The total volume of the remaining ethanol and the ethanol-impregnated scaffolds was then recorded as V2 by simply reading the level in the cylinder. The volume difference (V 2 − V 1 ) represents the volume of the PLGA/nano-FHA scaffold sample. The ethanol-impregnated scaffolds were then removed from the graduated cylinder and the residual ethanol volume was recorded as V3. Hence, the total volume of the scaffolds was V=(V2 −V3) and the bulk density of the scaffold was expressed as ρ=W/(V2 −V3). By determining the initial and final weights Wi and Wf, respectively, of the scaffolds after soaking in ethanol (ρethanol = 0.789 g/cm3) for 24 h, the pore volume of scaffolds can be determined as (Wf −Wi)/ ρethanol and the porosity can be calculated using the following equation [29]: W f −W i =ρethanol Porosity ¼ V 2 −V 1
ð1Þ
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In Vitro Biological Evaluation Cytotoxicity L929 mouse fibroblast cell line (ATCC) was employed for cytotoxicity investigation of mechanically optimum composite scaffold (PLGA/FHA=4:1). The cells were seeded in polystyrene plates enriched with Minimal Essential Medium supplemented with 10 % fetal bovine serum, 100 units/ml of penicillin and 100 μg/ml streptomycin, respectively, and incubated at 37 °C in humid atmosphere and 5 % CO2. When the cells attained confluency, the sterilized discs were placed in direct contact with the cells and incubated for 2 days under the same condition. It is noticeable that negative control sample (ultra high molecular weight poly ethylene) were used. After 2 days, the cells were observed under optical microscopy (Nicon E200). Cell Proliferation (MTT Assay) The cell proliferation on the nanocomposite scaffolds was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For the assay, cells were then seeded onto 96-well plates at a density of 2×103 cells/ well and were incubated under standard culturing conditions. The PLGA and PLGA/nanoFHA (4:1) scaffolds were placed on the cells after an overnight incubation at 37 °C with 5 % CO2 in a humidified atmosphere. Three wells in the absence of scaffolds were used as negative controls [tissue culture polystyrene (TCPS)]. For each sample, five wells of microliter plate were selected. The plates were incubated for 3, 7, and 14 days with half media changed every 2 days. Then, nanocomposite scaffolds were removed from the wells and 10 μL of a 5 mg/mL solution of MTT was added to each well followed by incubation for 5 h at 37 °C. Formed formazan crystals were dissolved by addition of 100 μL/well of isopropanol. Subsequently, the plates were incubated at 37 °C for 10 min and transferred to 48 °C for 15 min before absorbance measurements. Optical density (OD) was recorded on a multiwell microplate reader at a wavelength of 570 nm. ALP Activity The functional activity of the cells on the prepared scaffolds was evaluated by measuring the ALP activity. ALP activity was conducted using a commercial kinetics method. Briefly, the G-292 cell lines were transferred into 24-well microliter plates at 2×103 cells/well, separately. The PLGA and PLGA/nano-FHA (4:1) scaffolds were placed in the wells. Three wells in the absence of scaffolds were used as negative controls (TCPS). The plates were incubated for 3, 7, and 14 days at 37 °C in humidified air with 5 % CO2. Ten microliters of the supernatant was removed from each well at 3, 7, and 14 days and processed according to the guideline. It is noticeable that the color changing was measured by spectrophotometer at 405 nm. The standard p-nitrophenol curve was employed to convert the obtained absorbance data to the ALP content.
Results and Discussion XRD Analysis Figure 1 shows the XRD patterns of the FHA, PLGA, and PLGA/FHA (4:1) composite samples. As it can be seen in the XRD pattern of FHA, no tricalcium phosphate was detected in FHA powders. It is important to point out that the XRD results suggest that FHA has a structure like HA. However, no structures for partially fluorine ion substituted apatites are recorded in the ICDD database. Also in this figure, the peaks that related to pure PLGA has been observed and indexed.
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Fig. 1 The XRD patterns of FHA, PLGA, and PLGA/FHA (4:1) composite
FTIR Analysis Figure 2 shows the Fourier transform infrared spectroscopy (FTIR) spectra of the FHA, PLGA, and PLGA/FHA (4:1) composite samples. The characteristic bands exhibited in the sample spectra assigned here: (a) The band at 1,002 cm−1 arises from ν3 PO4, the bands at 514 cm−1 and 550 cm−1 arise from ν4 PO4, the bands at 916 cm−1 arises from ν1 PO4 and the bands at 437 cm−1 arise from ν2 PO4. (b) The bands at 868 cm−1 and about 1,434 cm−1 arise from CO3 which an indication of the presence of carbonate apatite [30, 31]. This might have
Fig. 2 The FTIR spectra of FHA, PLGA, and PLGA/FHA (4:1) composite
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originated through the absorption of carbon dioxide from the atmosphere. (c) The bands at 3,436 cm−1 arises from OH….F bond that demonstrated some of the OH− groups in the crystalline network of HA were replaced by F−. The PLGA/FHA spectrum is similar to the pure PLGA alone. This is due to the high percent of PLGA in the composite. It is worth mentioning that the PLGA has covered the FHA particle and thus the scaffold composite (PLGA/FHA) did not sufficient transparent for the IR spectroscopy analysis, and therefore, FHA-related vibration bands did not appear very sharp and become very broad. Thermal Analysis The TG-DTA curves for the PLGA/nano-FHA (4:1) composite are illustrated in Fig. 3. As it can be seen in this curve, the composite has a thermal stability until about 260 °C. The weight loss of pure PLGA, attributed to thermal decomposition, occurs in two steps (390 and 470 °C). The gradual decrease in weight which is observed from 390 to 700 °C is because of the slow elimination of the carbonate groups linked to FHA which has been confirmed with the FTIR analysis discussed later. Also, as it can be observed from this figure, in the given temperature interval, FHA is thermally stable. Thus, the weight percentage at the end of the experiment (700 °C) is the percentage of FHA in the composite scaffolds. SEM Observations Figure 4 showed the SEM micrograph of PLGA/nano-FHA (4:1) microsphere scaffolds sintered at 90 °C for 2 h. The scaffolds were made by microspheres, and all types of PLGA-based microspheres maintained in spherical shape with visible rough surfaces because of great nano-FHA conglomeration enrichment on their surfaces. Mechanical Properties The preparation of 3D porous composite scaffolds is obtained through a microsphere sintering technique. The sintering temperature and sintering time are two processing parameters that can
Fig. 3 TG-DTA curve of the PLGA/FHA (4:1) composite
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Fig. 4 SEM micrograph of PLGA/nano-FHA (4:1) composite scaffolds sintered at 90 °C, for 2 h
be controlled the mechanical properties and porous structures of the sintered microsphere scaffolds. Figures 5 and 6 show the measured compressive modulus and strength of the composite scaffolds sintered under different conditions. As it can be seen in this figure, the compressive
Fig. 5 Effects of sintering temperature and sintering time on the compressive modulus of the PLGA/nano-FHA (4:1) composite scaffolds
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Fig. 6 Effects of sintering temperature and sintering time on the compressive strength of the PLGA/nano-FHA (4:1) composite scaffolds
modulus and compressive strength of the scaffolds sintered at 90 °C for 2 h are remarkably higher than the scaffolds prepared at other sintering conditions. Increasing either sintering temperature or sintering time resulted in better mechanical properties because of a greater bonding area. On the other hand, when the sintering time and sintering temperature was further increased, the PLGA microspheres in scaffolds is being collapsed resulting in scaffolds with inferior mechanical properties [32]. Also, the pore structures of the composite scaffolds are affected by sintering conditions. Jiang et al. [33] showed that increasing sintering time resulted in possible closure of pores, which this phenomenon caused to decrease scaffold porosity. This study ascertained that porosity decreased with sintering time (see Fig. 7). Scaffolds sintered at 90 °C for 2 h had porosity of 31 %.
Fig. 7 Effects of sintering temperature and sintering time on the porosity content of the PLGA/nano-FHA (4:1) composite scaffolds
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Density is another important parameter in the composite scaffold design. It is worth mentioning that the results showed that the sintering time and temperature did not largely affect scaffold density (see Fig. 8). Also, this figure demonstrated that all the sintering conditions resulted in composite scaffolds with density less than the media density (1 g/cm3). The optimal sintering condition was evaluated for different composite scaffolds with various PLAGA/nano-FHA ratios. The highest compressive modulus and strength were observed at the optimal sintering condition for all ratios (data not shown). Figures 9 and 10 show the effects of the FHA content in the composite scaffold on the compressive modulus and strength. Under the optimal sintering condition, the PLGA/nanoFHA (4:1) scaffolds ascertained that the highest mechanical properties, which were remarkably higher than other scaffolds (other PLGA/nano-FHA ratios). Additionally, the compressive modulus and strength of PLGA/nano-FHA (4:1) scaffolds were significantly higher than pure PLGA scaffolds, suggesting that the introducing of nano-FHA into PLGA scaffolds at specific ratio could boost the mechanical properties of the mentioned polymeric scaffolds. It is noticeable that, when a low amount of FHA was introduced into PLGA scaffold, approximately 100 % of the FHA particles were entranced into the microspheres. However, when the initial loading was increased, the efficiency of introducing was slightly decreased. Although the initial loading FHA is changed, the concentration of polymer solution (meaning that viscosity) is kept constant. Since the density of FHA is higher than PLGA solution and water, they tend to sediment at the tank bottom. Thus, the efficiency of FHA introducing decreases with an increase in initial loading. Borden et al. [34] showed that increasing either the time or temperature of sintering could result in an increase in compressive modulus and a decrease in porosity. In PLGA/nano-FHA (4:1) scaffold, a similar trend up to 90 °C/2 h was seen, while, further increasing sintering time caused to a decrease in compressive modulus. Hence, for each type of composite scaffold, there are optimal sintering conditions at which highest mechanical properties could be achieved. In order to study the effect of the PLGA/FHA ratio on the mechanical properties of the scaffolds, the optimal sintering conditions, as discussed earlier, was selected, and the mechanical characteristics of the various scaffold types were compared together. Since FHA is a high-
Fig. 8 Effects of sintering temperature and sintering time on the density of the PLGA/nano-FHA (4:1) composite scaffolds
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Fig. 9 Effects of scaffold composition on the compressive modulus of composite scaffolds (Scaffolds sintered at 90 °C, for 2 h)
mechanical strength bioceramic, hence, it seems that the scaffolds with higher compressive modulus and strength can be obtained by enhancing the amount of FHA in the composite scaffolds. In spite of that, the obtained results disclosed that the PLGA/nano-FHA (4:1) scaffold was the strongest among the four types of prepared scaffolds that mechanically analyzed. When packed microspheres were heated above the polymer’s glass transition temperature (Tg), sintering takes place because of the intertwining of polymer chains between adjacent microspheres, resulting in the formation of bonds [35]. Thus, the mechanical strength of the 3D microsphere-based scaffolds significantly depends on the fusion area between the microspheres.
Fig. 10 Effects of scaffold composition on the compressive strength of composite scaffolds (Scaffolds sintered at 90 °C, for 2 h)
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When greater FHA particles were introduced (such as PLGA/nano-FHA 1:1) in the microspheres, greater FHA particles existed on the microsphere surface which oppositely affect the PLGA sintering. Eventually, we concluded that the incorporation of appropriate amounts of FHA could boost the mechanical characteristics of 3D polymeric scaffolds prepared by a sintered microsphere method. In Vitro Biological Evaluation Cytotoxicity Based on number and form of cells covering the surface of the PLGA/nano-FHA (4:1) scaffold, the amount of cytotoxicity was evaluated by optical microscopy shown in Fig. 11. According to Fig. 10, it can be observed that a layer of fusiform cells of fibroblast has been covered on the surface of the PLGA/nano-FHA scaffold. The cytotoxicity of the scaffold is distinguished by development of layer of cells and also their fusiform morphology. The spherical cells are dead cells which are related to the lack of food but not the toxicity of scaffold sample. Therefore, the cytotoxic scale of the composite scaffold for L929 mouse fibroblast cells was measured as zero, which corresponds to non-cytotoxicity. MTT Assay The results obtained by MTT assay were compared with control group (polystyrene well) as shown in Fig. 12. According to the results, addition of FHA to polymeric scaffolds had no negative effect on the proliferation of G-292 or osteoblasts cells. Also, viabilities were better for nanocomposite scaffolds [PLGA/FHA(4:1)] using G-292 cell line; however, it resulted in a significant reduction in the biocompatibility of the nanocomposite samples in comparison with the control group just after 3 days incubation time. This phenomenon was completely revealed later on with longer incubation periods (7 or 14 days), which resulted in improved viabilities even comparing with control group. Fig. 11 Optical micrograph of L929 fibroblast cells in direct contact with PLGA/nano-FHA (4:1) composite scaffold
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Fig. 12 Cell proliferation of G-292 cells proliferated on the PLGA/nano-FHA (4:1) scaffolds along with negative control after incubation for 3, 7, and 14 days
Using G-292, viabilities were improved for nanocomposite scaffold [PLGA/FHA(4:1)] showed improved viabilities after 7 days. ALP Activity ALP is a well-known analysis for differentiation of osteoblasts on its expression during osteogenesis. ALP activity results for G-292 cells on the PLGA and PLGA/nano-FHA (4:1) scaffold is given for 3, 7, or 14 days of incubation period (Fig. 13). The obtained results ascertained that there is a general trend of increasing in ALP activity for PLGA/nano-FHA (4:1) scaffold by time.
Fig. 13 ALP activity test for G-292 cells proliferated on the PLGA/nano-FHA (4:1) scaffolds along with negative control after incubation for 3, 7, and 14 days
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Conclusions In conclusion, composite scaffolds containing nano-FHA and PLGA were successfully prepared by sintering method suitable for bone tissue engineering. It was found that the mechanical characteristics and pore structures to be modulated by sintering condition and PLGA/nano-FHA ratio. We chose the PLGA/nanoFHA (4:1) scaffold as it showed the greatest compressive modulus and strength and its pore structure was suitable for cell penetration. Also, the experimental results ascertained that the compressive modulus and compressive strength of the scaffolds sintered at 90 °C for 2 h are remarkably higher than the scaffolds prepared at other sintering conditions. Also, the obtained results exhibited that the sintering time and temperature did not significantly affect scaffold density. Also, all sintered composite scaffolds had a density less than the media density (1 g/cm3). In addition, MTT assay results showed that a general trend of increasing cell viability was observed for nanocomposite scaffolds [PLGA/FHA (4:1)] with time compared to control group. Eventually, it can be suggested that the newly developed PLGA/nano-FHA composite fulfills most of the requirements as suitable bone scaffolds for bone tissue engineering applications.
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