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In situ synthesis and characterization of porous polymer-ceramic composites as scaffolds for gene delivery
Materials Science and Engineering C 27 (2007) 479 – 483 www.elsevier.com/locate/msec
In situ synthesis and characterization of porous polymer-ceramic composites as scaffolds for gene delivery Hsu-Feng Ko b , Charles Sfeir c , Prashant N. Kumta a,b,⁎ a
Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA, United States b Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, United States c Department of Oral Medicine, University of Pittsburgh, Pittsburgh, PA, United States Available online 5 July 2006
Abstract The use of three-dimensional scaffolds in gene delivery has emerged as a popular and necessary delivery vehicle for obtaining controlled gene delivery. In this report, techniques to synthesize composite scaffolds by combining natural polymers such as agarose and alginate with calcium phosphate (CaP) are described. The incorporation of CaP into the agarose or alginate hydrogels was performed in situ and the presence of CaP was confirmed by X-ray diffraction (XRD). The crystallite size of the CaP particles was determined to be 7.20 nm. Lyophilized, porous composites were examined under scanning electron microscopy (SEM) to estimate the size of the pores, an essential requirement for an ideal scaffold. The swelling properties of the composite samples were also investigated to study the effect of CaP incorporation on the behavior of the hydrogels. By incorporating CaP into the hydrogel, the aim is to synthesize a scaffold that is mechanically strong and chemically suitable for use as a gene delivery vehicle in tissue engineering. © 2006 Elsevier B.V. All rights reserved. Keywords: Agarose; Alginate; Calcium phosphate; Composite scaffold
1. Introduction The role of gene therapy in aiding wound healing and treating various diseases/defects has become increasingly important in the field of tissue engineering. As a result, there have been growing interests in the medical community to develop effective gene delivery strategies as well as study the gene delivery mechanisms to further enhance the potential of gene therapy. Gene therapy is the insertion of genetic material into a cell's genetic pool either to correct an underlying defect or to modify the characteristics of the cell via expression of the newly inserted gene [1]. Gene therapy can potentially be used to treat genetically inherited or acquired disorders or heal accidental or surgical wounds. Given the combinations of available genes and cell types, the application of gene therapy to attain these goals appears to be very promising. The procedure of inserting the genetic material into the cell is accomplished by the process of gene delivery. Gene delivery can be ⁎
Corresponding author. Department of Materials Science and Engineering, Department of Biomedical Engineering, Carnegie Mellon University, 4309 Wean Hall, 5000 Forbes Ave, Pittsburgh, PA 15213, United States. Tel.: +1 412 268 8739. E-mail address: [email protected] (P.N. Kumta). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.05.045
divided into two types: viral and non-viral gene delivery. Viral gene delivery has traditionally been used more frequently due to the inherent nature of viruses to penetrate and insert genetic material into target cells [1], resulting in very efficient transfection. Popular viral carriers include adenovirus, adeno-associated virus, and retrovirus. Drawbacks of viral gene delivery include restricted packaging capacity, insufficient expression, and immune responses triggered by the virus (and in some cases fatal responses) [2]. Nonviral gene delivery, on the other hand, is a method of delivering genetic material without the use of viruses. Some commonly known techniques in non-viral gene delivery include microinjection, electroporation, cationic lipid or polymer, and calcium phosphate (CaP) precipitation approaches [1]. Although non-viral delivery techniques have lower transfection efficiency, they are less expensive, less toxic, less immunogenic, and easier and safer to synthesize and manufacture. With non-viral delivery, the delivery technique also depends on carriers to complex with genetic materials as well as the need for a delivery system. One of the main delivery techniques utilizes a three-dimensional scaffold as a delivery system. The scaffold serves as a mechanical support and a temporary template that mimics the extracellular matrix (ECM) for cells. An ideal scaffold should be
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highly porous with interconnected pores, exhibiting biocompatible and biodegradable characteristics. It should provide a suitable support for cell attachment, proliferation, and differentiation, while being mechanically strong [3]. It should also provide a matrix for carrying delivery agents such as genes, proteins, or other external factors. Traditionally, scaffolds are divided into two categories: synthetic polymers and natural polymers. Common synthetic polymers include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their co-polymer poly(lactic-co-glycolic acid) (PLGA). Several forms of synthetic polymers have been FDA approved and typically have a good degradation rate and sufficient mechanical strength [4]. However, biocompatibility could be adversely affected by the acidic degradation products they release, and the lack of chemically reactive chains renders the binding of external factor difficult [4,5]. Natural polymers include collagen, alginate, agarose, fibrin, chitosan, etc. They are known to be biocompatible, and they exhibit an environment that resembles the highly hydrated state of natural tissues [6]. However, the main issues with natural polymers are their weak mechanical properties, lack of cellular interactions, and uncontrolled degradation [4,5,7]. The focus of this report is to fabricate composite scaffolds in situ by combining either agarose or alginate with calcium phosphate (CaP). Agarose is a polysaccharide with alternating copolymers of 1,4-linked 3,6-anhydro-α-L-galactose and 1,3-linked βD-galactose [8]. Its gelling conditions are temperature dependent since it is soluble in water above 65 °C and gels upon cooling to temperatures between 17–40 °C, [3,8]. Most studies done up to date using agarose as a scaffold involve cartilage repair [3,9,10]. On the other hand, Alginate is a polysaccharide derived from brown algae and bacteria. It is a linear polysaccharide copolymer of (1-4)-linked β-D mannuronic acid (M) and α-L-guluronic acid (G) monomers [4]. Alginate gels in the presence of divalent ions such as Ca2+, Ba2+, and Sr2+ [11]. Alginate has been used extensively in various applications, including cell encapsulation/ seeding [12,13], gene delivery [14,15], and antibody or growth factor entrapment and release [16,17]. Knowing the aforementioned weaknesses regarding natural polymers, the goal of this study is to incorporate CaP into agarose or alginate hydrogels. As a ceramic material, CaP has high mechanical strength [18], which could offset natural polymers' weak mechanical properties. One of the main phases of CaP, hydroxyapatite (HA), is also known to be biocompatible. HA is also osteoconductive [19] and very similar to bone, thus could tremendously enhance the composite's properties in a bone repair setting. Moreover, it has been shown that in situ synthesis of CaP in the presence of polymers resulted in the formation of nano-sized CaP and that the composite helps to improve the mechanical properties and cell attachment [20]. This paper therefore describes the in situ synthesis protocols and discusses in detail techniques used to characterize the composites. 2. Materials and methods 2.1. Materials The chemicals used in this report were CaCl2, Na3PO4, agarose, and alginate. CaCl2·2H2O (C79-500) and Na3PO4·12H2O (S377-
500) were both purchased from Fisher Scientific (Pittsburgh, PA). Ultrapure™ agarose (15510-027) was purchased from Invitrogen Corporation (Carlsbad, CA). Alginic acid, sodium salt (177772500) was purchased from Acros Organics through Fisher Scientific. 2.2. Syntheses of agarose hydrogel and agarose–CaP composite The synthesis protocol for synthesizing the agarose hydrogel was adapted from protocols commonly known to fabricate agarose hydrogels for use in gel electrophoresis [21]. The protocol for agarose–CaP composite was adapted from one of our published work [22]. 1.2% (w/v) of agarose was prepared in doubly distilled, deionized water (ddH2O) with a conductivity of 18.3 Ω cm− 1. Plain agarose hydrogel was synthesized by heating 100 ml of 1.2% agarose solution for approximately 1 min in a conventional microwave oven. The agarose solution turned transparent as agarose dissolved in solution after microwave heating, and the hot solution was allowed to cool and solidify into a gel. Agarose– CaP composite was synthesized by first preparing a CaCl2 solution such that the weight ratio of agarose to CaCl2 is 2:1. Na3PO4 was dissolved in the 1.2% agarose solution and the mixture was heated in the microwave for 1 min. CaCl2 was then added to the mixture drop by drop. The Ca/P ratio was 1.67. The final volume of CaCl2 solution and (agarose + Na3PO4) solution was 100 ml. The solution was again allowed to cool and gel. Some of the agarose–CaP samples were immersed in ddH2O for 72 h to remove the NaCl salt byproduct and were then left to dry in an oven at approximately 60 °C prior to characterization of the gel. 2.3. Syntheses of alginate hydrogel and alginate–CaP composite The protocol for synthesizing alginate hydrogel and alginate– CaP composite was very similar to those reported in the literature [11,23,24]. 1.5–2% (w/v) of alginate solution was prepared in ddH2O with a conductivity of 18.3 Ω cm− 1. Plain alginate hydrogel was synthesized by crosslinking alginate solution with a 0.03 M CaCl2 solution. CaCl2 was introduced to the alginate solution drop by drop while stirring intensely to ensure uniform crosslinking. The volume ratio of CaCl2 solution to that of alginate was 1:1. The hydrogel was immersed in CaCl2 overnight followed by immersion in ddH2O overnight. Excess solution was discarded. Alginate–CaP composite was synthesized by first dissolving Na3PO4 into the alginate solution. The crosslinker CaCl2 was then added drop by drop into the mixture while stirring. The Ca/P ratio was also 1.67. The volume ratio of CaCl2 solution to that of (alginate +Na3PO4 solution) was 1:1. The hydrogel was immersed in CaCl2 and then ddH2O in the same manner as the plain hydrogel to remove unreacted salt byproducts. The gel samples were left to dry in an oven at approximately 60 °C prior to characterization. An alternative method of synthesizing the alginate hydrogel was generating alginate in the shape of beads. The alginate solution was added drop by drop into CaCl2 solution, allowing the crosslinking of alginate to occur resulting in spherical beads. The same procedure was used to generate spherical beads of alginate–CaP composites. A solution containing alginate and Na3PO4 was added drop by drop into a CaCl2 solution. The
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observation under SEM. The SEM images were taken on a JEOL 9335 field emission gun SEM. The average pore size (d) was calculated by measuring the pore length (l) and p width ffiffiffiffiffiffiffiffiffiffi(w) ffi from SEM images taken, and using the relation d ¼ l w as previous reported in literature [23,24]. The average pore size for each scaffold was calculated from 3 selected areas of several SEM images, each area containing approximately 5 pores (∼ 15 pores total). 2.8. Swelling behavior study The swelling properties of the hydrogels and the composites were examined by immersing dried samples into distilled water for a period of 48 h. The diameters of the samples before and after the immersion were compared using the equation 1 DS ¼ d2d−d 100%, where DS is the degree of swelling, d1 is 1 the initial diameter, and d2 is the final diameter. The degree of swelling was averaged from 3 samples for each scaffold. Fig. 1. Digital images of agarose and alginate hydrogels. a: Left, plain agarose. Right, agarose–CaP. b: Left, plain alginate. Right, alginate–CaP.
3. Results and discussion
beads were immersed in CaCl2 solution then into ddH2O following similar approaches as the plain hydrogel. The concentrations of the solutions used to synthesize the beads were the same as those used to synthesize the hydrogel.
From the appearances of the hydrogels, plain hydrogels appear transparent or translucent while composites appear white
2.4. Characterization by X-ray Diffraction (XRD) Dried gel samples were collected and XRD patterns were obtained using Philips Analytical, Inc.'s Philips X'Pert PRO X-ray diffraction system using CuKα radiation (λ = 1.5418 Å). The diffractometer was operated at 45 kVand 40 mA over the 2θ range of 20–60°. The step size was 0.03 and the time per step was 50.17 s. 2.5. Calculation of crystallite size The crystallite size of the CaP particles formed within the hydrogel matrix was estimated based on the XRD pattern. The broadening of the peaks was deconvoluted using the Philips Profile Fit software, and the crystallite size was obtained from 0:9k the Debye–Scherrer's formula, t ¼ Bcosh , where t is the crysb tallite size, λ is the wavelength of the X-ray beam, B is one-half the difference between the two extreme angles around the peak at which the intensity is zero, and θb is one-half of the 2θ angle for the peak. The (310) peak (2θ = 40.430°) from three XRD scans was used to calculate the crystallite size. 2.6. Synthesis of porous scaffolds by lyophilization In order to create porosity in the hydrogels and composites, techniques previously described in literature [23,24] were followed. Samples were frozen at a −80 °C freezer and lyophilized for 72 h. 2.7. Characterization by Scanning Electron Microscopy (SEM) Hydrogel and composite samples that were frozen at − 80 °C and lyophilized for 72 h were cut to the appropriate size prior to
Fig. 2. XRD patterns showing CaP peaks in composites. a: As-prepared CaP. b: Agarose–CaP without rinsing off salt. c: Agarose–CaP after rinsing off salt. d: Alginate–CaP.
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due to the presence of CaP. Fig. 1a shows the digital images of plain agarose and agarose–CaP composite. The hydrogels originally covered the entire area of the Petri dishes. However, during drying, the hydrogels started shrinking uniformly toward the center of the Petri dishes as water evaporated. Fig. 1b shows the digital images of beads of plain alginate and alginate–CaP composite.
peaks can be seen and appear amorphous (not shown). Fig. 2d shows the XRD patterns for alginate–CaP composite. Similar to the agarose–CaP composite, CaP peaks are also seen in the XRD patterns, while the pattern for alginate also appears amorphous (not shown), demonstrating CaP's presence in the composites for both hydrogels. 3.2. Calculation of crystallite size
3.1. Characterization by X-ray diffraction XRD was used to confirm the successful in situ synthesis of CaP and the incorporation of CaP into the hydrogels. Fig. 2a shows the XRD patterns for the as-prepared CaP. The broad peaks are characteristic of a lack of crystallinity in the asprepared CaP. The major peaks in the XRD pattern appear to match well with HA. The broad nature of the peaks, however, does not preclude the presence of other CaP phases. Hence, we refer to phase present in the XRD pattern as CaP. Fig. 2b shows the XRD pattern for agarose–CaP composite. Without removing the NaCl byproduct formed during the in situ synthesis of CaP, NaCl was trapped within the hydrogel and resulted in the formation of NaCl crystals upon drying. This was evident in Fig. 2b, where major NaCl peaks centered at 2θ = 31.70° and 45.45° [(200) and (220) planes, respectively] overlap the much lower CaP peaks. The agarose–CaP composite samples were then immersed in ddH2O for 72 h, replacing the water multiple times, and then dried again for XRD analysis. Fig. 2c shows the XRD patterns for agarose–CaP composite after rinsing off the NaCl residue. NaCl peaks are no longer present, and broad, overlapping CaP peaks centered around 2θ = 32–34° [(112), (300), (202) planes] are seen. Agarose is not crystalline, thus no
The crystallite size was determined from the agarose–CaP XRD pattern shown in Fig. 2c. The (310) peak (2θ =40.430°) from the XRD pattern was used since it contains the least overlap of the broadened peaks. The calculation of the crystallite size from 3 sample runs yielded an approximate crystallite size of 7.20 (±0.34) nm. The size calculation demonstrated and confirmed the nano-structured nature of the CaP within the hydrogel matrix. 3.3. Characterization by Scanning Electron Microscopy SEM images were taken to determine the structure of the pores and to ascertain the approximate size of the pores created after the hydrogels were lyophilized for 72 h. Fig. 3a shows the SEM images of agarose–CaP composite taken at two different magnifications (250× and 350×). An interconnected, porous structure could be seen from the SEM images. The average pore size for each scaffold was calculated from 3 selected areas of several SEM images, each area containing approximately 5 pores (∼15 pores total). The average pore size for the agarose–CaP composite was calculated to be 41.4 (±7.3) μm. Fig. 3b shows the SEM images of alginate–CaP composite taken at two different magnifications (95× and 150×). A similar porous structure to that
Fig. 3. SEM images of agarose and alginate–CaP composites. a: Agarose–CaP composite. b: Alginate–CaP composite.
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of agarose–CaP could also be seen in the SEM. The size of the pores appears to be larger than that of the agarose–CaP composite, and the average pore size calculation yields 103 (±20) μm. With the porous structure containing pores large enough for cells to infiltrate, and the potential to manipulate the pore size by adjusting the freezing temperature, freezing medium, duration, etc, these composites could become suitable candidates as scaffolds in numerous tissue engineering applications. 3.4. Swelling behavior study The swelling properties of agarose hydrogel, alginate hydrogel, and the composites were examined by comparing the change in diameter before and after immersion in water. The degree of swelling, calculated from 3 samples for each scaffold, shows average diameter increases of 4.78 (±0.51) %, 10.64 (±0.42) %, 9.38 (±1.1) %, and 11.98 (±0.65) % for plain agarose, CaP– agarose, plain alginate, and CaP–alginate, respectively. It should be noted that the composites underwent a larger reduction in diameter than the hydrogels during drying. Upon rehydration, the degree of swelling for the composites was much greater compared to that of the plain hydrogels. These results demonstrate that both agarose and alginate have limited swelling behavior. It was also observed that upon incorporation of CaP, the hydrogels exhibit a larger shrinkage and correspondingly, a larger swelling upon rehydration. 4. Conclusion In this report, methods to fabricate agarose–CaP and alginate– CaP in situ have been described. CaP incorporation into the hydrogel matrix was verified by XRD patterns showing peaks characteristic of CaP. By lyophilizing the composites, porous structures were observed by SEM, creating an environment with sufficient pore size for cells to infiltrate. The average pore sizes for agarose– CaP and alginate–CaP were 41.4 (±7.3) μm and 103 (±20) μm, respectively. Both of the composites also exhibited swelling properties, showing average diameter increases of 10.64 (±0.42) % and 11.98 (±0.65) % for CaP–agarose and CaP–alginate, respectively. Agarose–CaP and alginate–CaP composites are thus a promising approach to enhance the integrity of plain hydrogels. CaP's other characteristics such as osteoconductivity and ability to degrade at physiological pH can also render the composite to be one of the more superior scaffolds for use in tissue engineering.
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Acknowledgement This work was supported by the Defense University Research Initiative on Nanotechnology (DURINT) program administered by the Office of Naval Research under Grant N00014-0110715. This work was also supported by the Carnegie Mellon University, the Pittsburgh Tissue Engineering Initiative (PTEI), NSF-NIRT (CTS0210238), NIH-NIBIB (1R01EB002706-01), NIHNIDC (1R03DE015905-01) and the National Science Foundation (grants CTS-0000563 and DMR-0073586). References [1] R. Valani, Queen's Health Sci. J. 1 (1998) 1. [2] K. Lundstorm, Trends Biotech. 21 (2003) 117. [3] D.W. Hutmacher, J.C.H. Goh, S.H. Teoh, Ann. Acad. Med. Singap. 30 (2001) 183. [4] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337. [5] S. Yang, K.F. Leong, Z. Du, C.K. Chua, Tissue Eng. 7 (2001) 679. [6] B.D. Ratner, S.J. Bryant, Annu. Rev. Biomed. Eng. 6 (2004) 41. [7] X. Liu, P.X. Ma, Ann. Biomed. Eng. 32 (2004) 477. [8] A.P. Balgude, X. Yu, A. Szymanski, R.V. Bellamkonda, Biomaterials 22 (2001) 1077. [9] J.C. Hu, K.A. Athanasiou, Biomaterials 26 (2005) 2001. [10] A.C. Aufderheide, K.A. Athanasiou, Tissue Eng. 11 (2005) 1095. [11] H.R. Lin, Y.J. Yeh, J. Biomed. Mater. Res. B 71 (2004) 52. [12] R. Glicklis, L. Shapiro, R. Agbaria, J.C. Merchuk, S. Cohen, Biotechnol. Bioeng. 67 (2000) 344. [13] S. Gerecht-Nir, S. Cohen, A. Ziskind, J. Itskovitz-Eldor, Biotechnol. Bioeng. 88 (2004) 313. [14] T. Sone, E. Nagamori, T. Ikeuchi, A. Mizukami, Y. Takakura, S. Kajiyama, E. Fukusaki, S. Harashima, A. Kobayashi, K. Fukui, J. Biosci. Bioeng. 94 (2002) 87. [15] T. Higashi, E. Nagamori, T. Sone, S. Matsunaga, K. Fukui, J. Biosci. Bioeng. 97 (2004) 191. [16] M. Sivakumar, K.P. Rao, J. Biomed. Mater. Res. A 65 (2003) 222. [17] A. Perets, Y. Baruch, F. Weisbuch, G. Shaoshany, G. Neufeld, S. Cohen, J. Biomed. Mater. Res. A 65 (2003) 489. [18] K. Burg, S. Porter, J. Kellam, Biomaterials 21 (2000) 2347. [19] R.Z. LeGeros, Clin. Orthop. Relat. Res. 395 (2002) 81. [20] K.G. Marra, J.W. Szem, P.N. Kumta, P.A. DiMilla, L.E. Weiss, J. Biomed. Mater. Res. 47 (1999) 324. [21] A. Nussinovitch, N. Jaffe, M. Gillilov, Food Hydrocoll. 18 (2004) 825. [22] D. Choi, P.N. Kumta, J. Amer. Cer. Soc. 89 (2006) 444. [23] L. Shapiro, S. Cohen, Biomaterials 18 (1997) 583. [24] S. Zmora, R. Glicklis, S. Cohen, Biomaterials 23 (2002) 4087.
In situ synthesis and characterization of porous polymer-ceramic composites as scaffolds for gene delivery Hsu-Feng Ko b , Charles Sfeir c , Prashant N. Kumta a,b,⁎ a
Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA, United States b Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, United States c Department of Oral Medicine, University of Pittsburgh, Pittsburgh, PA, United States Available online 5 July 2006
Abstract The use of three-dimensional scaffolds in gene delivery has emerged as a popular and necessary delivery vehicle for obtaining controlled gene delivery. In this report, techniques to synthesize composite scaffolds by combining natural polymers such as agarose and alginate with calcium phosphate (CaP) are described. The incorporation of CaP into the agarose or alginate hydrogels was performed in situ and the presence of CaP was confirmed by X-ray diffraction (XRD). The crystallite size of the CaP particles was determined to be 7.20 nm. Lyophilized, porous composites were examined under scanning electron microscopy (SEM) to estimate the size of the pores, an essential requirement for an ideal scaffold. The swelling properties of the composite samples were also investigated to study the effect of CaP incorporation on the behavior of the hydrogels. By incorporating CaP into the hydrogel, the aim is to synthesize a scaffold that is mechanically strong and chemically suitable for use as a gene delivery vehicle in tissue engineering. © 2006 Elsevier B.V. All rights reserved. Keywords: Agarose; Alginate; Calcium phosphate; Composite scaffold
1. Introduction The role of gene therapy in aiding wound healing and treating various diseases/defects has become increasingly important in the field of tissue engineering. As a result, there have been growing interests in the medical community to develop effective gene delivery strategies as well as study the gene delivery mechanisms to further enhance the potential of gene therapy. Gene therapy is the insertion of genetic material into a cell's genetic pool either to correct an underlying defect or to modify the characteristics of the cell via expression of the newly inserted gene [1]. Gene therapy can potentially be used to treat genetically inherited or acquired disorders or heal accidental or surgical wounds. Given the combinations of available genes and cell types, the application of gene therapy to attain these goals appears to be very promising. The procedure of inserting the genetic material into the cell is accomplished by the process of gene delivery. Gene delivery can be ⁎
Corresponding author. Department of Materials Science and Engineering, Department of Biomedical Engineering, Carnegie Mellon University, 4309 Wean Hall, 5000 Forbes Ave, Pittsburgh, PA 15213, United States. Tel.: +1 412 268 8739. E-mail address: [email protected] (P.N. Kumta). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.05.045
divided into two types: viral and non-viral gene delivery. Viral gene delivery has traditionally been used more frequently due to the inherent nature of viruses to penetrate and insert genetic material into target cells [1], resulting in very efficient transfection. Popular viral carriers include adenovirus, adeno-associated virus, and retrovirus. Drawbacks of viral gene delivery include restricted packaging capacity, insufficient expression, and immune responses triggered by the virus (and in some cases fatal responses) [2]. Nonviral gene delivery, on the other hand, is a method of delivering genetic material without the use of viruses. Some commonly known techniques in non-viral gene delivery include microinjection, electroporation, cationic lipid or polymer, and calcium phosphate (CaP) precipitation approaches [1]. Although non-viral delivery techniques have lower transfection efficiency, they are less expensive, less toxic, less immunogenic, and easier and safer to synthesize and manufacture. With non-viral delivery, the delivery technique also depends on carriers to complex with genetic materials as well as the need for a delivery system. One of the main delivery techniques utilizes a three-dimensional scaffold as a delivery system. The scaffold serves as a mechanical support and a temporary template that mimics the extracellular matrix (ECM) for cells. An ideal scaffold should be
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highly porous with interconnected pores, exhibiting biocompatible and biodegradable characteristics. It should provide a suitable support for cell attachment, proliferation, and differentiation, while being mechanically strong [3]. It should also provide a matrix for carrying delivery agents such as genes, proteins, or other external factors. Traditionally, scaffolds are divided into two categories: synthetic polymers and natural polymers. Common synthetic polymers include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their co-polymer poly(lactic-co-glycolic acid) (PLGA). Several forms of synthetic polymers have been FDA approved and typically have a good degradation rate and sufficient mechanical strength [4]. However, biocompatibility could be adversely affected by the acidic degradation products they release, and the lack of chemically reactive chains renders the binding of external factor difficult [4,5]. Natural polymers include collagen, alginate, agarose, fibrin, chitosan, etc. They are known to be biocompatible, and they exhibit an environment that resembles the highly hydrated state of natural tissues [6]. However, the main issues with natural polymers are their weak mechanical properties, lack of cellular interactions, and uncontrolled degradation [4,5,7]. The focus of this report is to fabricate composite scaffolds in situ by combining either agarose or alginate with calcium phosphate (CaP). Agarose is a polysaccharide with alternating copolymers of 1,4-linked 3,6-anhydro-α-L-galactose and 1,3-linked βD-galactose [8]. Its gelling conditions are temperature dependent since it is soluble in water above 65 °C and gels upon cooling to temperatures between 17–40 °C, [3,8]. Most studies done up to date using agarose as a scaffold involve cartilage repair [3,9,10]. On the other hand, Alginate is a polysaccharide derived from brown algae and bacteria. It is a linear polysaccharide copolymer of (1-4)-linked β-D mannuronic acid (M) and α-L-guluronic acid (G) monomers [4]. Alginate gels in the presence of divalent ions such as Ca2+, Ba2+, and Sr2+ [11]. Alginate has been used extensively in various applications, including cell encapsulation/ seeding [12,13], gene delivery [14,15], and antibody or growth factor entrapment and release [16,17]. Knowing the aforementioned weaknesses regarding natural polymers, the goal of this study is to incorporate CaP into agarose or alginate hydrogels. As a ceramic material, CaP has high mechanical strength [18], which could offset natural polymers' weak mechanical properties. One of the main phases of CaP, hydroxyapatite (HA), is also known to be biocompatible. HA is also osteoconductive [19] and very similar to bone, thus could tremendously enhance the composite's properties in a bone repair setting. Moreover, it has been shown that in situ synthesis of CaP in the presence of polymers resulted in the formation of nano-sized CaP and that the composite helps to improve the mechanical properties and cell attachment [20]. This paper therefore describes the in situ synthesis protocols and discusses in detail techniques used to characterize the composites. 2. Materials and methods 2.1. Materials The chemicals used in this report were CaCl2, Na3PO4, agarose, and alginate. CaCl2·2H2O (C79-500) and Na3PO4·12H2O (S377-
500) were both purchased from Fisher Scientific (Pittsburgh, PA). Ultrapure™ agarose (15510-027) was purchased from Invitrogen Corporation (Carlsbad, CA). Alginic acid, sodium salt (177772500) was purchased from Acros Organics through Fisher Scientific. 2.2. Syntheses of agarose hydrogel and agarose–CaP composite The synthesis protocol for synthesizing the agarose hydrogel was adapted from protocols commonly known to fabricate agarose hydrogels for use in gel electrophoresis [21]. The protocol for agarose–CaP composite was adapted from one of our published work [22]. 1.2% (w/v) of agarose was prepared in doubly distilled, deionized water (ddH2O) with a conductivity of 18.3 Ω cm− 1. Plain agarose hydrogel was synthesized by heating 100 ml of 1.2% agarose solution for approximately 1 min in a conventional microwave oven. The agarose solution turned transparent as agarose dissolved in solution after microwave heating, and the hot solution was allowed to cool and solidify into a gel. Agarose– CaP composite was synthesized by first preparing a CaCl2 solution such that the weight ratio of agarose to CaCl2 is 2:1. Na3PO4 was dissolved in the 1.2% agarose solution and the mixture was heated in the microwave for 1 min. CaCl2 was then added to the mixture drop by drop. The Ca/P ratio was 1.67. The final volume of CaCl2 solution and (agarose + Na3PO4) solution was 100 ml. The solution was again allowed to cool and gel. Some of the agarose–CaP samples were immersed in ddH2O for 72 h to remove the NaCl salt byproduct and were then left to dry in an oven at approximately 60 °C prior to characterization of the gel. 2.3. Syntheses of alginate hydrogel and alginate–CaP composite The protocol for synthesizing alginate hydrogel and alginate– CaP composite was very similar to those reported in the literature [11,23,24]. 1.5–2% (w/v) of alginate solution was prepared in ddH2O with a conductivity of 18.3 Ω cm− 1. Plain alginate hydrogel was synthesized by crosslinking alginate solution with a 0.03 M CaCl2 solution. CaCl2 was introduced to the alginate solution drop by drop while stirring intensely to ensure uniform crosslinking. The volume ratio of CaCl2 solution to that of alginate was 1:1. The hydrogel was immersed in CaCl2 overnight followed by immersion in ddH2O overnight. Excess solution was discarded. Alginate–CaP composite was synthesized by first dissolving Na3PO4 into the alginate solution. The crosslinker CaCl2 was then added drop by drop into the mixture while stirring. The Ca/P ratio was also 1.67. The volume ratio of CaCl2 solution to that of (alginate +Na3PO4 solution) was 1:1. The hydrogel was immersed in CaCl2 and then ddH2O in the same manner as the plain hydrogel to remove unreacted salt byproducts. The gel samples were left to dry in an oven at approximately 60 °C prior to characterization. An alternative method of synthesizing the alginate hydrogel was generating alginate in the shape of beads. The alginate solution was added drop by drop into CaCl2 solution, allowing the crosslinking of alginate to occur resulting in spherical beads. The same procedure was used to generate spherical beads of alginate–CaP composites. A solution containing alginate and Na3PO4 was added drop by drop into a CaCl2 solution. The
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observation under SEM. The SEM images were taken on a JEOL 9335 field emission gun SEM. The average pore size (d) was calculated by measuring the pore length (l) and p width ffiffiffiffiffiffiffiffiffiffi(w) ffi from SEM images taken, and using the relation d ¼ l w as previous reported in literature [23,24]. The average pore size for each scaffold was calculated from 3 selected areas of several SEM images, each area containing approximately 5 pores (∼ 15 pores total). 2.8. Swelling behavior study The swelling properties of the hydrogels and the composites were examined by immersing dried samples into distilled water for a period of 48 h. The diameters of the samples before and after the immersion were compared using the equation 1 DS ¼ d2d−d 100%, where DS is the degree of swelling, d1 is 1 the initial diameter, and d2 is the final diameter. The degree of swelling was averaged from 3 samples for each scaffold. Fig. 1. Digital images of agarose and alginate hydrogels. a: Left, plain agarose. Right, agarose–CaP. b: Left, plain alginate. Right, alginate–CaP.
3. Results and discussion
beads were immersed in CaCl2 solution then into ddH2O following similar approaches as the plain hydrogel. The concentrations of the solutions used to synthesize the beads were the same as those used to synthesize the hydrogel.
From the appearances of the hydrogels, plain hydrogels appear transparent or translucent while composites appear white
2.4. Characterization by X-ray Diffraction (XRD) Dried gel samples were collected and XRD patterns were obtained using Philips Analytical, Inc.'s Philips X'Pert PRO X-ray diffraction system using CuKα radiation (λ = 1.5418 Å). The diffractometer was operated at 45 kVand 40 mA over the 2θ range of 20–60°. The step size was 0.03 and the time per step was 50.17 s. 2.5. Calculation of crystallite size The crystallite size of the CaP particles formed within the hydrogel matrix was estimated based on the XRD pattern. The broadening of the peaks was deconvoluted using the Philips Profile Fit software, and the crystallite size was obtained from 0:9k the Debye–Scherrer's formula, t ¼ Bcosh , where t is the crysb tallite size, λ is the wavelength of the X-ray beam, B is one-half the difference between the two extreme angles around the peak at which the intensity is zero, and θb is one-half of the 2θ angle for the peak. The (310) peak (2θ = 40.430°) from three XRD scans was used to calculate the crystallite size. 2.6. Synthesis of porous scaffolds by lyophilization In order to create porosity in the hydrogels and composites, techniques previously described in literature [23,24] were followed. Samples were frozen at a −80 °C freezer and lyophilized for 72 h. 2.7. Characterization by Scanning Electron Microscopy (SEM) Hydrogel and composite samples that were frozen at − 80 °C and lyophilized for 72 h were cut to the appropriate size prior to
Fig. 2. XRD patterns showing CaP peaks in composites. a: As-prepared CaP. b: Agarose–CaP without rinsing off salt. c: Agarose–CaP after rinsing off salt. d: Alginate–CaP.
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due to the presence of CaP. Fig. 1a shows the digital images of plain agarose and agarose–CaP composite. The hydrogels originally covered the entire area of the Petri dishes. However, during drying, the hydrogels started shrinking uniformly toward the center of the Petri dishes as water evaporated. Fig. 1b shows the digital images of beads of plain alginate and alginate–CaP composite.
peaks can be seen and appear amorphous (not shown). Fig. 2d shows the XRD patterns for alginate–CaP composite. Similar to the agarose–CaP composite, CaP peaks are also seen in the XRD patterns, while the pattern for alginate also appears amorphous (not shown), demonstrating CaP's presence in the composites for both hydrogels. 3.2. Calculation of crystallite size
3.1. Characterization by X-ray diffraction XRD was used to confirm the successful in situ synthesis of CaP and the incorporation of CaP into the hydrogels. Fig. 2a shows the XRD patterns for the as-prepared CaP. The broad peaks are characteristic of a lack of crystallinity in the asprepared CaP. The major peaks in the XRD pattern appear to match well with HA. The broad nature of the peaks, however, does not preclude the presence of other CaP phases. Hence, we refer to phase present in the XRD pattern as CaP. Fig. 2b shows the XRD pattern for agarose–CaP composite. Without removing the NaCl byproduct formed during the in situ synthesis of CaP, NaCl was trapped within the hydrogel and resulted in the formation of NaCl crystals upon drying. This was evident in Fig. 2b, where major NaCl peaks centered at 2θ = 31.70° and 45.45° [(200) and (220) planes, respectively] overlap the much lower CaP peaks. The agarose–CaP composite samples were then immersed in ddH2O for 72 h, replacing the water multiple times, and then dried again for XRD analysis. Fig. 2c shows the XRD patterns for agarose–CaP composite after rinsing off the NaCl residue. NaCl peaks are no longer present, and broad, overlapping CaP peaks centered around 2θ = 32–34° [(112), (300), (202) planes] are seen. Agarose is not crystalline, thus no
The crystallite size was determined from the agarose–CaP XRD pattern shown in Fig. 2c. The (310) peak (2θ =40.430°) from the XRD pattern was used since it contains the least overlap of the broadened peaks. The calculation of the crystallite size from 3 sample runs yielded an approximate crystallite size of 7.20 (±0.34) nm. The size calculation demonstrated and confirmed the nano-structured nature of the CaP within the hydrogel matrix. 3.3. Characterization by Scanning Electron Microscopy SEM images were taken to determine the structure of the pores and to ascertain the approximate size of the pores created after the hydrogels were lyophilized for 72 h. Fig. 3a shows the SEM images of agarose–CaP composite taken at two different magnifications (250× and 350×). An interconnected, porous structure could be seen from the SEM images. The average pore size for each scaffold was calculated from 3 selected areas of several SEM images, each area containing approximately 5 pores (∼15 pores total). The average pore size for the agarose–CaP composite was calculated to be 41.4 (±7.3) μm. Fig. 3b shows the SEM images of alginate–CaP composite taken at two different magnifications (95× and 150×). A similar porous structure to that
Fig. 3. SEM images of agarose and alginate–CaP composites. a: Agarose–CaP composite. b: Alginate–CaP composite.
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of agarose–CaP could also be seen in the SEM. The size of the pores appears to be larger than that of the agarose–CaP composite, and the average pore size calculation yields 103 (±20) μm. With the porous structure containing pores large enough for cells to infiltrate, and the potential to manipulate the pore size by adjusting the freezing temperature, freezing medium, duration, etc, these composites could become suitable candidates as scaffolds in numerous tissue engineering applications. 3.4. Swelling behavior study The swelling properties of agarose hydrogel, alginate hydrogel, and the composites were examined by comparing the change in diameter before and after immersion in water. The degree of swelling, calculated from 3 samples for each scaffold, shows average diameter increases of 4.78 (±0.51) %, 10.64 (±0.42) %, 9.38 (±1.1) %, and 11.98 (±0.65) % for plain agarose, CaP– agarose, plain alginate, and CaP–alginate, respectively. It should be noted that the composites underwent a larger reduction in diameter than the hydrogels during drying. Upon rehydration, the degree of swelling for the composites was much greater compared to that of the plain hydrogels. These results demonstrate that both agarose and alginate have limited swelling behavior. It was also observed that upon incorporation of CaP, the hydrogels exhibit a larger shrinkage and correspondingly, a larger swelling upon rehydration. 4. Conclusion In this report, methods to fabricate agarose–CaP and alginate– CaP in situ have been described. CaP incorporation into the hydrogel matrix was verified by XRD patterns showing peaks characteristic of CaP. By lyophilizing the composites, porous structures were observed by SEM, creating an environment with sufficient pore size for cells to infiltrate. The average pore sizes for agarose– CaP and alginate–CaP were 41.4 (±7.3) μm and 103 (±20) μm, respectively. Both of the composites also exhibited swelling properties, showing average diameter increases of 10.64 (±0.42) % and 11.98 (±0.65) % for CaP–agarose and CaP–alginate, respectively. Agarose–CaP and alginate–CaP composites are thus a promising approach to enhance the integrity of plain hydrogels. CaP's other characteristics such as osteoconductivity and ability to degrade at physiological pH can also render the composite to be one of the more superior scaffolds for use in tissue engineering.
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Acknowledgement This work was supported by the Defense University Research Initiative on Nanotechnology (DURINT) program administered by the Office of Naval Research under Grant N00014-0110715. This work was also supported by the Carnegie Mellon University, the Pittsburgh Tissue Engineering Initiative (PTEI), NSF-NIRT (CTS0210238), NIH-NIBIB (1R01EB002706-01), NIHNIDC (1R03DE015905-01) and the National Science Foundation (grants CTS-0000563 and DMR-0073586). References [1] R. Valani, Queen's Health Sci. J. 1 (1998) 1. [2] K. Lundstorm, Trends Biotech. 21 (2003) 117. [3] D.W. Hutmacher, J.C.H. Goh, S.H. Teoh, Ann. Acad. Med. Singap. 30 (2001) 183. [4] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337. [5] S. Yang, K.F. Leong, Z. Du, C.K. Chua, Tissue Eng. 7 (2001) 679. [6] B.D. Ratner, S.J. Bryant, Annu. Rev. Biomed. Eng. 6 (2004) 41. [7] X. Liu, P.X. Ma, Ann. Biomed. Eng. 32 (2004) 477. [8] A.P. Balgude, X. Yu, A. Szymanski, R.V. Bellamkonda, Biomaterials 22 (2001) 1077. [9] J.C. Hu, K.A. Athanasiou, Biomaterials 26 (2005) 2001. [10] A.C. Aufderheide, K.A. Athanasiou, Tissue Eng. 11 (2005) 1095. [11] H.R. Lin, Y.J. Yeh, J. Biomed. Mater. Res. B 71 (2004) 52. [12] R. Glicklis, L. Shapiro, R. Agbaria, J.C. Merchuk, S. Cohen, Biotechnol. Bioeng. 67 (2000) 344. [13] S. Gerecht-Nir, S. Cohen, A. Ziskind, J. Itskovitz-Eldor, Biotechnol. Bioeng. 88 (2004) 313. [14] T. Sone, E. Nagamori, T. Ikeuchi, A. Mizukami, Y. Takakura, S. Kajiyama, E. Fukusaki, S. Harashima, A. Kobayashi, K. Fukui, J. Biosci. Bioeng. 94 (2002) 87. [15] T. Higashi, E. Nagamori, T. Sone, S. Matsunaga, K. Fukui, J. Biosci. Bioeng. 97 (2004) 191. [16] M. Sivakumar, K.P. Rao, J. Biomed. Mater. Res. A 65 (2003) 222. [17] A. Perets, Y. Baruch, F. Weisbuch, G. Shaoshany, G. Neufeld, S. Cohen, J. Biomed. Mater. Res. A 65 (2003) 489. [18] K. Burg, S. Porter, J. Kellam, Biomaterials 21 (2000) 2347. [19] R.Z. LeGeros, Clin. Orthop. Relat. Res. 395 (2002) 81. [20] K.G. Marra, J.W. Szem, P.N. Kumta, P.A. DiMilla, L.E. Weiss, J. Biomed. Mater. Res. 47 (1999) 324. [21] A. Nussinovitch, N. Jaffe, M. Gillilov, Food Hydrocoll. 18 (2004) 825. [22] D. Choi, P.N. Kumta, J. Amer. Cer. Soc. 89 (2006) 444. [23] L. Shapiro, S. Cohen, Biomaterials 18 (1997) 583. [24] S. Zmora, R. Glicklis, S. Cohen, Biomaterials 23 (2002) 4087.