Oleic acid surfactant in polycaprolactone/hydroxyapatite-composites for bone tissue engineering Guinea B. C. Cardoso,1 Devid Maniglio,2 Fabio Z. Volpato,2 Abhishek Tondon,3 Claudio Migliaresi,2 Roland R. Kaunas,3 Cecilia A. C. Zavaglia1 1

University of Campinas, Materials Engineering Department, Faculty of Mechanical Engineering, Campinas, Brazil University of Trento, Department of Industrial Engineering, BIOtech Research Center, Trento 38123, Italy 3 Texas A&M University, Department of Biomedical Engineering, College Station, Texas 2

Received 20 February 2015; revised 30 April 2015; accepted 13 May 2015 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33457 Abstract: Bone substitutes are required to repair osseous defects caused by a number of factors, such as traumas, degenerative diseases, and cancer. Autologous bone grafting is typically used to bridge bone defects, but suffers from chronic pain at the donor-site and limited availability of graft material. Tissue engineering approaches are being investigated as viable alternatives, which ideal scaffold should be biocompatible, biodegradable, and promote cellular interactions and tissue development, need to present proper mechanical and physical properties. In this study, poly(e-caprolactone) (PCL), oleic acid (OA) and hydroxyapatite (HAp) were used to obtain films whose properties were investigated by contact angle, scanning electron microscopy, atomic force microscopy, tensile mechanical tests, and in vitro tests with U2OS human osteosarcoma cells by direct contact. Our results indicate that by using OA as surfactant/dispersant, it was possible to obtain a homogenous film

with HAp. The PCL/OA/Hap sample had twice the roughness of the control (PCL) and a lower contact angle, indicating increased hydrophilicity of the film. Furthermore, mechanical testing showed that the addition of HAp decreased the load at yield point and tensile strength and increased tensile modulus, indicating a more brittle composition vs. PCL matrix. Preliminary cell culture experiments carried out with the films demonstrated that U2OS cells adhered and proliferated on all surfaces. The data demonstrate the improved dispersion of HAp using OA and the important consequences of this addition on the composite, unveiling the potentially of this composition for bone C 2015 Wiley Periodicals, Inc. J Biomed Mater Res growth support. V Part B: Appl Biomater 00B: 000–000, 2015.

Key Words: engineering

composite/hard

tissue,

bone

graft,

tissue

How to cite this article: Cardoso GBC, Maniglio D, Volpato FZ, Tondon A, Migliaresi C, Kaunas RR, Zavaglia CAC. 2015. Oleic acid surfactant in polycaprolactone/hydroxyapatite-composites for bone tissue engineering. J Biomed Mater Res Part B 2015:00B:000–000.

INTRODUCTION

Nonunion defects of bone are a major challenge in orthopedics. Of the 13 million yearly fractures that occur in the United States, about 10% fail to repair.1,2 In many cases, synthetic implants can temporarily stabilize such injuries, but inadequate cellular responses can delay healing, and poor bone quality can cause rapid loss of fixation. Failed implants are notoriously difficult to revise and bones that fail to heal exact a high cost on the medical system in general and on the health of the patient in particular.3 Autologous bone grafting is typically used to bridge bone defects, but the available graft material is limited and involves additional surgery causing chronic donor-site pain in many patients.4 Consequently, various synthetic biomaterials are being investigated as viable alternatives to autografts. The choice of the biomaterials (usually biodegradable polymers) play a crucial role in the fabrication of the 3-dimensional scaffolds designed to support and guide cell growth and proliferation. The surface design of these bioma-

terials has attracted much attention in tissue engineering technology, since the surface can influence the protein adsorption and cell behavior, and thus influence the biological performance and integration of the scaffolds.5 e-Polycaprolactone (PCL) is a popular polymer for tissue engineering applications because it is biodegradable, biocompatible, and relatively easy to process via a wide range of fabrication techniques. Recently, poly(e-caprolactone) (PCL) composite scaffolds incorporating bioactive components have been studied in effort to improve osteoconductivity.6 Calcium phosphates, for example, hydroxyapatite (HAp), are regularly applied as bioactive factors due to their chemical similarities with the natural bone. Despite its inherent osteoconductivity, HAp bears the disadvantage of brittleness, which makes it unsuitable for handling and processing. However, when used as whiskers, the mechanical property disadvantage can be overcome, since there is greater resistance to crack propagation due load transfer along the length of whiskers.7 It follows that load transfer is

Correspondence to: G. B. C. Cardoso; e-mail: [email protected]

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enhanced with greater surface interaction between the crystals and the polymeric matrix, which is not easily achieved. To improve ceramic dispersion into hydrophobic polymer matrices, one simple strategy is to use amphiphilic molecules, for example, oleic acid (OA), which acts as a surfactant.8–11 In this study, we investigated the potential of using OA to improve PCL/HAp integration for composites intended for use in bone tissue engineering. Pure PCL, PCL/OA, and PCL/OA/HAp samples were prepared and characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (static and Wilhelmy methods), and tensile mechanical testing. Cell adhesion was characterized with human osteosarcoma cell line (U2OS) expressing GFP-actin. MATERIALS AND METHODS

Materials The commercials reagents used were PCL [Aldrich (Mw 5 80 K)] and OA (Carbosynth). The HAp whiskers were synthesized as previously described.12 Films were cast by dissolving OA in chloroform (10:1 v/w) for 30 min, followed by addition of HAp (15% v/v) and stirring at room temperature for 2 h. PCL was then added and stirred for another 24 h. Preparation of PCL with different reagents All the different films were prepared using the casting method, with chloroform as the solvent. The concentration ratio of the polymer/solvent was 12.5% w/v. The preparation of the three sample types is given in detail below. PCL film. The solution was stirred for 6 h, then the solution was cast into a glass Petri dish (diameter 90 mm), which was covered with a lid (diameter 100 mm) and kept for 48 h in a closed ambient container for the slow chloroform evaporation. The dried film was then collected and vacuum dried for 48 h. PCL/OA. The initial solution was produced using chloroform and the dispersant (OA) in a concentration of 10:1%w/w, and stirred for 30 min before adding the polymer. The obtained solution was used for film casting, following the method described above (PCL film). PCL/HAp/OA. The initial solution was mixed with the HAp whiskers for 30 min using tip sonication (Heilscher UP400S). Then the polymer was added and processed as described above (PCL films). CHARACTERIZATION

Scanning electron microscopy Surface morphologies of various films were characterized using SEM (Cambridge Stereoscan 200) using samples that were gold coated using a sputter coater (SEM Coating Unit PS3, Assing S.p.A, Rome, Italy) with conditions of 20 mA, 5 3 1027 Pa for 30 s. The SEM images of cross-sections were collected using a JEOL JCM 5000 (Texas A&M, College Station).

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Atomic force microscopy The topography and roughness of the surfaces of PCL, PCL/ OA, and PCL/OA/HAp films under dry conditions were characterized using AFM (NT-MDT Solver AFM). Contact angle Contact angles were measured by the tensiometry method using Wilhelmy microbalance. This method is based on the measurement of the tension along the wetting perimeter of a regularly shaped object (typically a plate or cylinder) as it is immersed into or emerged out from a liquid. It takes advantage of the accessibility of the contact angles by force measurements. The detected hysteresis force loops were used to calculate advancing and receding contact angles. Measurements where performed using a CAHN DCA322 Wilhelmy microbalance. The static contact angle (h) was measured with a KSV-CAM 200 goniometer (KSV Instruments) equipped with an auto dispenser, video camera, and drop-shape analysis software. The static measurements were acquired from a 5 lL sessile water droplet at 15 and 120 s (short and long time responses), following deposition. The values reported are an average of three measurements taken on different areas of the same specimen at the air surface: PCL, PCL/OA, and PCL/OA/HAp. Tensile mechanical tests Tensile tests of samples were performed at room temperature on a tensile tester (Instron 3345). Rectangular strips (10 3 3.2 3 1.0 mm3) were subjected to tension at a constant strain rate (50 mm/min), until breaking. From the stress-strain curves, tensile modulus (E), and tensile strength (TS) were determined. The reported values are an average of five measurements. In vitro tests The samples 11 mm in diameter were sterilized by immersion in 70% ethanol for 2 h. The samples were then attached to a petri dish with the air contact side (during evaporation) up using medical grade tape and kept under UV light for 25 min for further sterilization. U20S osteosarcoma cells stably expressing GFP-actin (Marin Pharma GmbH) between passages 2 and 6 were cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1 mM penicillin/streptomycin in a humidified incubator kept at 378 and 5% CO2, as described previously.13 After periods of 1, 3, and 7 days, the PCL, PCL/OA, and PCL/OA/HAp samples were rinsed with PBS and fixed with 4% paraformaldehyde for 10 min. Images were capture using the Nikon C1 laser scanning confocal head with a 603 water-dipping objective illuminated with a 40-mW Argon ion laser (Melles Griot).14 GFP-actin expression in U2OS cells localizes to stress fibers, but also results in some diffuse fluorescence throughout the cytoplasm. Cell outlines were determined from images of GFP fluorescence in the U2OS cells by image thresholding at a grayscale level 5 units greater than the noise in the background surrounding the cell. Cell spreading and shape factor were then calculated

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FIGURE 1. Images of the samples: (A) PCL/HAp and (B) PCL/AO/HAp. Arrows show the HAp agglomerations. Scale bar: 20 mm.

using the Analyze Particles macro in ImageJ (NIH). Shape factor is defined as 4p 3 area/perimeter2. A shape factor approaching unity corresponds to a cell shape approaching a circle.13 Statistical analyses All data are expressed as mean 6 ST. Comparisons between the groups were made using ANOVA, followed by StudentNewman-Keuls correction factor for multiple comparisons as a post hoc. RESULTS

Cross section samples The morphology of the cross-sections showed that the sample without OA dispersant exhibits HAp aggregation at the area in contact with the glass [Figure 1(A)]. Addition of OA resulted in detection of HAp on the opposite surface in contact with air [Figure 1(B)]. Morphology and roughness characterization of the membranes AFM showed the effects of the surfactants and HAp addition on the surface roughness (Table I) and the effect on morphology (Figure 2) of the films were demonstrated by the SEM images. As expected, all surfaces displayed higher roughness when in contact with air during solvent evaporation than when in contact with glass. Comparing the air contact sur-

face, the roughest sample was the PCL/OA/HAp that presented the value 200 nm and the smoothest surface was the PCL sample. Consistent with the AFM results, the morphology of the membrane imaged by SEM displayed very different characteristics on the air vs. glass surfaces. The side of the PCL sample in contact with air [Figure 2-(1A)] showed a slightly rough surface characterized by discrete spherule-like aggregation occupying the entire surface of the film. In contrast, the side in contact with glass [Figure 2-(1B)] was smooth. Higher magnification [Figure 2-(1C)] reveled spherules structures as well. The PCL/OA and PCL/OA/HAp (Figures 222 and 223) samples had similar surface morphologies and exhibited a rough surface on both sides, though more on the side in contact with air. Contact angle Table II summarizes the contact angles measured from the PCL, PCL/OA, and PCL/OA/Hap films. The polymer matrix without any modification (PCL) had advancing and receding angles of 888 and 598, respectively, resulting in hysteresis of 288. When OA was present, the values were advancing angle of 1088 and receding angle of 548, resulting in hysteresis angle of 838. On addition of HAp, the values were 95.38, 378, and 588 as advancing, receding, and hysteresis angles, respectively. ANOVA and multiple comparisons indicated significant differences in hysteresis angles. Static contact angle measurements were 103.68 (6 6.9) for the PCL, 99.28 (6 2.7) for PCL/OA, and 63.38 (6 4.7) with HAp addition.

TABLE I. Roughness Values by AFM Samples PCL air PCL glass PCL/OA air PCL/OAglass PCL/OA/HAp air PCL/OA/HAp glass

Ra 89 nm 61 nm 117 nm 49 nm 200 nm 154 nm

Mechanical properties To observe the mechanical properties of the samples, a tensile load was applied to each sample with dimension of 10 3 3.2 3 1.0 mm3. The mechanical properties of the samples are summarized in Table III. For the PCL films, the average tensile modulus was 181 6 8 MPa, with a peak load at yield of 37.3 6 7.6 N. Addition of OA showed similar value with the PCL (160 6 4 MPa and 48.2 6 7.4 N).

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FIGURE 2. SEM images of the films surfaces: (1) Sample PCL; (2) Sample PCL/OA; (3) Sample PCL/OA/HAp; (A-D-G) Samples with air contact during evaporation, scale bar: 100 mm; (B-E-H) Samples with glass contact during evaporation, scale bar: 100 mm; (C-F-I) Samples with glass contact during evaporation, scale bar: 30 mm (C) and 20 mm (F-I).

Addition of HAp increased elastic modulus (244 6 8 MPa) and had lower load at yield peak (21.2 6 4.8). In vitro tests To better understand the influence of OA and HAp on cell growth, morphology and behavior, U2OS cells were cultured on the films and stained with Alexa 488-phalloidin. Figure 3 shows the cell morphology after 1, 3, and 7 days of culture. Cell proliferation studies showed that, in general, the

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number of adherent cells on all the films increased with culture time. The day 3 images indicate that PCL/OA was most conductive to US02 cells adhesion. Whereas, PCL/OA/HAp was the least, due the presence of cells clumps. At day 7, in all the samples multiple cell layers were observed thus preventing the counting of relative cell area. Figure 4 shows the relative cells area on PCL, PCL/OA, and PCL/OA/HAp samples. No significant difference was observed in cell shapes between the samples.

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TABLE II. Contact Angles Results: Advancing, Receding, Hysteresis Angles, and Static Angle Samples

Advancing angle ha (standard deviation) [8]

Receding angle hr (standard deviation) [8]

Hysteresis angle (ha 2 hr) [ ]

Static angle – air contact (standard deviation)

88.1 (3.4) 108.9 (4.2) 95.3 (2.1)

59.5 (3.2) 25.7 (1.2) 37.2 (0.2)

28.6 83.3 58.0

103.6 (6.9) 99.2 (2.7) 63.3 (4.7)

PCL PCL/OA PCL/OA/HAp

DISCUSSIONS

PCL is a biodegradable and biocompatible polymer often used for bone tissue-engineering applications.9,12,15 However, the hydrophobic character of the material is one of the main obstacles for cell attachment.16,17 The addition of HAp whiskers to the PCL matrix is supposed to generate composites able to overcome this issue, as well improve mechanical stiffness.18 However, when the composition does not present a uniform surface this improvement is not achieved. Amphiphilic molecules, for example, OA, have been shown to have a significant impact on the distribution of particles (cf. Figure 1). The film containing OA results in a homogeneous distribution of HAp, while films lacking OA were characterized by HAp clustering isolated to the glass surface. This homogeneous composition has an effect on others properties such as morphology, roughness, and surface energy and can interfere with the regulation of bone formation.19,20 The nature of the solvent is known to induce morphological differences in terms of aggregates: acetone and ethyl acetate solvents yields filamentous PCL structures, while chloroform and tetrahydrofuran produces particulate structure.21 Indeed, the samples with OA and HAp resulted in films with filamentous aggregation. This morphology may be attributed to the reorganization of the PCL molecules after the dissolution and subsequent separation, since OA and HAp components affect the nucleation of the PCL, interfering with intramolecular aggregation. The addition of OA and HAp also alters the surface roughness and the contact angle (cf. Tables I and II). Wettability characterization is necessary to identify the hydrophobic or hydrophilic properties of the material. Hydrophilic surfaces promote protein adsorption, which is expected to lead to higher cell affinity. Addition of HAp increased the wettability of the film as evidenced by the decrease in static contact angle compared with the pure PCL formulation. It is well known that surface roughness and chemical heterogeneity can modify both the advancing and the receding contact angles.8 The addition of OA addition resulted in a large change in the receding angle (25.78 6 1.2 vs. 59.586 3.2 for the PCL control), which is responsible for the high

TABLE III. Data of Tensile Mechanical Test: PCL, PCL/OA, and PCL/OA/HAp

Samples

Maxim load (N)

Tensile strength 2 TS (MPa)

PCL 42.4 (4.3) 22.81 (2.66) PCL/OA 54.2 (3.4) 26.72 (1.61) PCL/OA/HAp 29.3 (2.6) 13.14 (1.01)

Tensile Peak load modulus 2 at yield (N) E (MPa) 37.3 (7.6) 48.2 (7.4) 21.2 (4.8)

181 (8) 160 (4) 244 (8)

hysteresis angle of 83.38. This could be attributed to the mobility of the OA molecule. The chemical structure of OA contains a long non-polar hydrocarbon chain (tail) and a polar functional group at the other end of the chain (head). Thus, when the OA molecules come in contact with water, it moves “up” so the hydrophilic end is in contact with the water and the tail stay within the structure, which forces attractions between hydrophobic molecules and lowers the receding angle. Mechanical properties are generally improved with the addition of HAp. The shape of the filler is crucial for act as reinforcement agent. A needle-like shape increases the compression modulus relative to that for rod and spherical shapes.15 We investigated the effect of needle-shaped filler on the mechanical properties of PCL composites using a tensile mechanical test (cf. Table III). HAp addition decreased the extent of elastic deformation under a given applied load and increased the amount of load that structure could withstand before permanent deformation. This occurs because the PCL/OA/HAp structure became more brittle rather than ductile than in PCL and PCL/OA formulations. Therefore, the PCL/OA/HAp samples showed less elongation before failure, but considerable higher elastic modulus. The organization of cells and the corresponding tissue properties is known to be highly dependent on the structure of the extracellular matrix (ECM). The ECM provides a complex spatial and temporal structure that spans several orders of magnitude. Protein adsorption is dependent on surface properties for example, roughness, charge, chemistry, and wettability, which in turns influences cell attachment.22 Lee et al. observed that cell adhesion and proliferation are inversely related to micropore size of polycarbonate membrane surfaces.23 Likewise, Van Kooten et al. reported that fibroblast proliferate better on grooves that were 2 and 5 mm in width than on wider 10 mm grooves.24 Thus our results (cf. Figure 3) are consistent with the previous studies demonstrating that fibroblast prefers smoother surface to proliferate, for example, PCL and PCL/OA.21 In addition, the chemistry of the films can influence the cell adhesion and proliferation. Samples with soluble bioceramics, like b-TCP-a-TCP-CDHA, can lead to an alteration at the media concentration of inorganic phosphate (Pi) and calcium ion (Ca12).25 Cell proliferation and differentiation decrease significantly with the Pi concentration higher than 0.09 mM and the amount of Ca12 do not change cell proliferation, but inhibited cell differentiation and also enhance cell mineralization.26,27 Further investigation was conducted by culturing adipose stem cells (ADSC) on PCL, PCL/OA, and PCL/OA/HAp samples for 14 and 21 days. These samples were analyzed using Alizarin red assay to check for mineralization. It was demonstrated that samples with HAp did not showed

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FIGURE 3. Confocal images of the films surfaces: Samples PCL, PCL/OA, and PCL/OA/HAp with 1, 3, and 7 days of culture. Scale bar: 25 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 4. Relative cells area onto the solution cast films: Samples PCL, PCL/OA, and PCL/OA/HAp, period 1 and 3 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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enhanced value of mineralization, which can be attributed to the amount of inorganic phosphate, since the media used was with b-glycerophosphate.28 The literature demonstrates that the amount of Pi supplied by the osteogenic differentiation stimulator (b-glycerophosphate) is sufficient resource for the cell mineralization and higher concentration can lead to cell apoptosis.25 Overall, our results are consistent with the literature, due the cytotoxicity of the films (PCL, PCL/OA, and PCL/OA/HAp) demonstrated biocompatible (cf. Figure 4).9,11 CONCLUSION

The improvement at tissue engineering research is increase every year. Researchers not only quest the response of cells to the biomaterial in question, but also the responses of the surface modifications and their properties. This research focused on the effect of OA and HAp addition to the polymeric matrix, with the special attention of morphology, roughness, wettability, and mechanical properties, listing all the changes ahead cellular response. The results indicated that with the dispersant used was able to obtain a homogenous film (PCL/OA/HAp). In addition, the HAp leads to a reduction of the contact angle, increasing the hydrophilic of the film. Furthermore, according to the mechanical test the addition of ceramics had a decrease the load at yield point, demonstrating less elongation before failure, while showing a relevant increase of elastic modulus, which we attribute to the ability of OA to disperse HAp inside the composite. In the present in vitro study, the films PCL, PCL/OA, and PCL/OA/HAp encouraged cell attachment, with no negative effects on the tissue formation. The preliminary investigations indicate that these strategies have great potential to improve current bone biomaterials and in the development of new scaffolds for bone tissue engineering. Although preliminary investigations seem to support the impact of HAp use in bone tissue engineering, significant advancements are necessary to realize their full potential in clinical use. ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial support from FAPESP (grant number 2013/19472-0 and number 2014/08625-2). REFERENCES 1. Marsh D. Concepts of fracture union, delayed union, and nounion. Clin Orthop Relat Res 1998;355:S22–S30. 2. United States Bone and Joint Decade: The Burden of Musculoskeletal Diseases and Musculoskeletal Injuries. Rosemont, IL: American Academy of Orthopedic Surgeons; 2008. 3. Hak DJ, McElvany M. Removal of broken hardware. J Am Acad Orthop Surg 2008;16:113–120. 4. Kao ST, Scott DD. A review of bone substitutes. Oral Maxillofac Surg Clin North Am 2007;19:513–521. 5. Lee JW, Kim YH, Park KD, Jee KS, Shin JW, Hahn SB. Importance of integrin b1-mediated cell adhesion on biodegradable polymers under serum depletion in mesenchymal stem cells and chondrocytes. Biomaterials 2004;25:1901–1909. 6. Scaglione S, Lazzarini E, Ilengo C, Quarto R. A composite material model for improved bone formation. J Tissue Eng Regen Med 2010;4:505–513.

7. Kane RJ, Converse GL, Roeder RK. Effects of the reinforcement morphology on the fatigue properties of hydroxyapatite reinforced polymers. J Mech Behav Biomed Mater 2008;1:261–268.  re  D. Wetting and Roughness. Annu Rev Mater Res 2008;38: 8. Que 71–99. 9. Choi D, Marra KG, Kumta PN. Chemical synthesis of hydroxyapatite/ poly(e-caprolactone) composites. Mater Res Bull 2004;39:417–432. 10. Hae-Won K. Biomedical nanocomposites of hydroxyapatite/polycaprolactone obtained by surfactant mediation. J Biomed Mater Res A 2007;83A:169–177. 11. Liu J, Ye X, Wang H, Zhu M, Wang B, Yan H. The influence of pH and temperature on the morphology of hydroxyapatite synthesized by hydrothermal method. Ceram Int 2003;29:629–633. 12. Cardoso GBC, Ramos SLF, Rodas ACD, Higa OZ, Zavaglia CAC, Arruda ACF. Scaffolds of poly (e-caprolactone) with whiskers of hydroxyapatite. J Mater Sci 2010;45:4990–4993. 13. Hsu H-J, Lee C-F, Locke A, Vanderzyl SQ, Kaunas R. Stretchinduced stress fiber remodeling and the activations of JNK and ERK depend on mechanical strain rate, but not FAK. PloS One 2010;5:e12470. 14. Tondon A, Hsu H-J, Kaunas R. Dependence of cyclic stretchinduced stress fiber reorientation on stretch waveform. J Biomech 2012;45:728–735. 15. Roohani-Esfahani S-I, Nouri-Khorasani S, Lu Z, Appleyard R, Zreiqat H. The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites. Biomaterials 2010; 31:5498–5509. 16. Chen G, Zhou P, Mei NA, Chen XIN, Shao Z. Silk fibroin modified porous poly (e -caprolactone) scaffold for human fibroblast culture in vitro. J Mater Sci Med 2004;5:671–677. 17. Oyane A, Uchida M, Yokoyama Y, Choong C, Triffitt J, Ito A. Simple surface modification of poly(epsilon-caprolactone) to induce its apatite-forming ability. J Biomed Mater Res A 2005;75:138–145. € c¸eri S, Wen X, Gandhi M, Sun W. Fabrication of three18. Shor L, Gu dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials 2007;28: 5291–5297. 19. Schwartz Z, Kieswetter K, Dean DD, Boyan BD. Underlying mechanisms at the bone-surface interface during regeneration. J Periodontal Res 1997;32:166–171. 20. Junker R, Dimakis A, Thoneick M, Jansen JA. Effects of implant surface coatings and composition on bone integration: A systematic review. Clin Oral Implants Res 2009;20:185–206. 21. Tang ZG, Black RA, Curran JM, Hunt JA, Rhodes NP, Williams DF. Surface properties and biocompatibility of solvent-cast poly[-caprolactone] films. Biomaterials 2004;25:4741–4748. 22. Parker MC, Wilson MS, Menzies D, Sunderland G, Clark DN, Knight AD, Crowe AM. The SCAR-3 study: 5-year adhesion-related readmission risk following lower abdominal surgical procedures. Colorectal Dis 2005;7:551–558. 23. Khang G. Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity. J Biomater Sci Polym Ed 1999;10:283–294. 24. Kooten TG Van Whitesides JF, Von Recum AF. Influence of silicone (PDMS) surface texture on human skin fibroblast proliferation as determined by cell cycle analysis. J Biomed Mater Res 1998;43:1–14. 25. Liu YK, Lu QZ, Pei R, Ji HJ, Zhou GS, Zhao XL, Tang RK, Zhang M. The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: Implication for bone tissue engineering. Biomed Mater (Bristol, England) 2009;4:025004. 26. Lin T-M, Tsai J-L, Lin S-D, Lai C-S, Chang C-C. Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants. Stem Cells Dev 2005;14:92–102. 27. Meleti Z, Shapiro IM, Adams CS. Inorganic phosphate induces apoptosis of osteoblast-like cells in culture. Bone 2000;27:359– 366. 28. Cardoso GBC. Development of three-dimensional matrix composite of poly (e-caprolactone) and bioactive ceramic for use in tissue engineering. University of Campinas, Mechanical Engineering; Thesis 2013. p. 125.

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hydroxyapatite-composites for bone tissue engineering.

Bone substitutes are required to repair osseous defects caused by a number of factors, such as traumas, degenerative diseases, and cancer. Autologous ...
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