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Transfer of collagen coating from porogen to scaffold: Collagen coating within poly(dl -lactic-co-glycolic acid) scaffold
Composites: Part B 38 (2007) 317–323 www.elsevier.com/locate/compositesb
Transfer of collagen coating from porogen to scaffold: Collagen coating within poly(DL-lactic-co-glycolic acid) scaffold Jiashen Li, Arthur F.T. Mak
*
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China Received 31 March 2006; accepted 2 June 2006 Available online 1 November 2006
Abstract Using paraffin micro-spheres as porogen, this paper addressed a novel method to coat collagen onto the internal pore surface within poly(DL-lactic-co-glycolic acid) (PLGA) scaffold with controlled pore size. The paraffin micro-spheres with desirable size were mixed with collagen solution (0.5–1.0% w/v), molded to form a paraffin micro-sphere scaffold, and dried. The collagen was left on the surface of the paraffin micro-spheres and even among the paraffin micro-spheres. PLGA solution was then cast into the interspace of the paraffin/collagen scaffold and dried. After the paraffin micro-spheres were dissolved and removed, PLGA scaffold with controlled pore size, good interconnectivity and high porosity was obtained. Collagen was transferred from the paraffin micro-spheres to the surfaces of the pore wall. Observation of scanning electron microscopy (SEM) showed that collagen was coated on the paraffin micro-spheres and was on the surfaces of pore wall within PLGA scaffold. Fourier transform infrared spectroscopy (FTIR) also detected the presence of collagen in the PLGA scaffold so formed; and there was no apparent change on the molecular components of collagen during the experimental procedure. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: A. Foams; A. Polymer-matrix composites (PMCs); E. Surface treatments; Collagen
1. Introduction Tissue engineering has focused on the use of synthetic or natural degradable materials as scaffolds for cell transplantation to engine new biological tissues. Biomaterial scaffolds as three-dimensional exogenous extracellular matrices (ECM) to guide tissue regeneration. The biophysical and biomechanical properties of a biomaterial scaffold have much influence on the final outcome of tissue engineering [1–10]. Because their biomechanical properties, such as the compressive modulus and strength, and biodegradabilities, can be adjusted and controlled easily, aliphatic polyesters, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer of poly(DLlactic-co-glycolic acid) (PLGA), have been extensively
*
Corresponding author. E-mail address: [email protected] (A.F.T. Mak).
1359-8368/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.compositesb.2006.06.009
studied [11,12]. The compressive strength and modulus of these polymers can approximate those of soft and hard tissues in human body [13]. Because of a lack of cell-recognizable signals, such materials have little attraction for cell attachment. Collagens are major constituents of bone with rich arginine–glycine– aspartate (RGD) which has been shown to represent the minimal adhesion domain to stimulate cell attachment [14]. Although collagens have been used as a scaffolding material, they in general lack adequate mechanical properties, degradation rate, and batch-to-batch consistency [15]. Using collagen to modify biodegradable polymeric scaffolds seems to be a good way to address the above problems. Collagen fibers were embedded within PLA matrix to strengthen collagen fibers for application in tendon or ligament reconstruction [16]. Recently, a new method was developed to nest collagen micro-sponge in the pores of a polymer sponge [17–21]. This method included two major steps: fabrication of polymeric scaffolds and nesting
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collagen inside polymeric scaffold. After polymeric scaffolds were prepared, they were immersed into an acidic collagen solution under vacuum so that the scaffold pores could be filled with collagen solution. Followed by freezing and lyophilization, the collagen micro-sponges were formed inside the scaffold pores. Such collagen microsponges were crosslinked and blocked. The presence of the collagen micro-sponge increased the wettability of the polymer surface, and facilitated cell attachment in the sponges. However, while the biodegradable polymer was in the collagen solution, the biodegradable polymeric material would likely become degraded to some extent, especially for certain polymers which could degrade relatively fast. The low pH value of the collagen solution may accelerate the polyesters’ degradation rates [22,23]. Although the collagen micro-sponges in the scaffold pore would increase the apparent surface area/volume ratio, the pore size, porosity and interconnectivity of the scaffold decreased, making it more difficult for the cells to move into the scaffolds. Similarly, diffusion of nutrients and waste products into and out of the scaffold would also be limited. Furthermore, the mechanical property of collagen micro-sponges inside the polymeric scaffold was likely to be as weak as a pure collagen scaffold; potential collapses could be harmful to cell attachment and proliferation. This paper developed a new method to introduce collagen into the polymeric scaffold to address some of the above issues. Using paraffin micro-spheres as porogen to fabricate scaffold, it is easy to control the pore size by the size of paraffin spheres and the pore interconnectivity by changing the treating temperature and duration [24]. In this paper, paraffin micro-spheres were also used to introduce collagen coating onto the pore wall throughout the whole biodegradable polymer scaffold. 2. Materials and methods
poured into 1000 mL PVA/water solution (0.5% w/v, 60– 70 °C) with stirring. The molten paraffin was dispersed to form a suspension of paraffin micro-drops. The suspension was immediately poured into ice water to solidify the paraffin micro-drops as micro-spheres. The micro spheres were washed by deionized water for three times and separated using standard sieves (100, 150, 180, 250, 280, 400, and 600 lm) into different sizes. 2.3. Preparation of paraffin micro-spheres/collagen scaffold Collagen solution with different concentrations (from 0.1 to 1.5% in 0.3% malonic acid) was mixed with paraffin micro-spheres of desirable size. Air bulbs trapped in the suspension and among the paraffin micro-spheres were eliminated under low air pressure. A plastic syringe without a needle end was used as a mould of the paraffin scaffold. To prepare paraffin scaffold of about 3 mm high, the piston was pulled to leave a gap of about 5 mm between the piston and the syringe end. The gap was filled with paraffin microspheres/collagen suspension. Caution was taken to make sure that no air bubbles were trapped in the mould during this step. The syringe end was placed vertically on a piece of rubber plate; and the piston was pushed down gently. The extra collagen solution was extruded from the gap between the syringe end and the rubber plate. When no more collagen solution was observed coming out, the pressure on the piston was released. Then the whole paraffin scaffold with collagen was pushed out by the piston and dried at 37 °C in air for 12 h. Subsequently, the sample was immersed into 20 mL crosslinking agent for 4 h with shaking. Finally, the collagen coated paraffin scaffolds were washed with deionized water for three times and dried again.
2.1. Materials
2.4. Preparation of PLGA/collagen scaffold
Poly(DL-lactic-co-glycolic acid) (50/50) (PLGA50/50) was purchased from PURAC (Netherlands). The average molecular weight for PLGA50/50 is 50,000 g/mol. Poly(vinyl alcohol) (PVA) (88% hydrolyzed, average molecular weight 25,000 g/mol), paraffin (melting point 53–57 °C), pyridine (boiling point 115 °C), malonic acid, and cyclohexane (melting point 7 °C) were purchased from Acros (Belgium). Standard sieves were purchased from Lantian Ltd Co., China. Bovine type I collagen was obtained from Tsinghua University, China. Deionized water was obtained with nanopure diamond ultrapure water systems from Barnstead, USA. The collagen crosslinking agent was a solution of formaldehyde (37%) and acetone (1/5, w/w).
PLGA was dissolved in pyridine for the preparation of a solution with a desired concentration. PLGA solution was cast into the paraffin micro-sphere scaffold drop by drop until the inter space among paraffin micro-spheres was fully filled with PLGA solution. The samples were maintained under low vacuum for 2 days to evaporate the pyridine away. After they are dried, the PLGA was left among the paraffin micro-spheres with collagen. If necessary, additional casting and vacuum-drying steps could be repeated to produce scaffold with higher polymeric content. The dried paraffin micro-spheres/collagen/PLGA sample was removed from the vacuum box, and immersed in 20 mL of cyclohexane to dissolve the paraffin at room temperature for 6 h with shaking. Cyclohexane was changed every 1.5 h. Then the sample was placed into a freezer and frozen at 20 °C for about 6 h. Subsequently, they were freeze-dried at 10 °C for 2 days and dried at room temperature under vacuum to remove any remaining
2.2. Preparation of paraffin micro-spheres The method to prepare paraffin micro-spheres has been reported before [25,26]. Briefly, 20 g melted paraffin was
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solvent. The PLGA scaffold with collagen coating was kept in desiccators until characterization.
coating onto the polymeric scaffold. The untreated and treated collagen films were studied using FTIR.
2.5. Characterizations
3. Results and discussion
Collagen coatings on paraffin micro-spheres and on the pore wall surface of PLGA scaffold were investigated under a LEICA scanning electron microscope (Model Stereoscan 440) after gold coating. FTIR was used to detect the molecular components of scaffold and coating. The transmission spectra of the PLGA scaffolds with and without collagen coating were measured with a Perkin-Elmer FTIR Spectrometer. In order to examine if there were any changes in the collagen molecule before and after the material processing, a piece of crosslinked collagen film was immersed into pyridine, cyclohexane for the same period as collagen
3.1. SEM morphology of collagen coating on paraffin micro-spheres and PLGA scaffold SEM photomicrograph of the paraffin micro-spheres without collagen (Fig. 1a) showed that the contact between two paraffin micro-spheres was more focal. For the paraffin spheres with collagen coating, collagen could be clearly observed on the surface of the micro-spheres (Fig. 1b), and between neighboring micro-spheres (Fig. 1c). It could be noted that some collagen fibers were left spanning between two adjacent paraffin micro-spheres (Fig. 1d).
Fig. 1. SEM micrographs of paraffin micro-spheres without and with collagen coating. (a) Paraffin micro-spheres without collagen coating. (b) and (c) Collagen coating on the paraffin micro-spheres. Coating could be easily observed at and around the contacts between the neighboring micro-spheres. (d) Collagen fibers between the paraffin micro-spheres.
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These fibers would be embedded into the PLGA after the PLGA casting and would help anchoring the collagen coating into the PLGA matrix when the paraffin micro-spheres were removed. The amount of collagen coating on the paraffin microspheres could be adjusted by varying the concentration of the collagen solution. Apparently, the higher the concentration of the collagen solution was, the more the collagen coating was left on the surface of paraffin spheres. For effective collagen coating, it was found that the concentration of collagen solution should be from 0.5 to 1.0% (w/v). When the collagen concentration was 0.3% (w/v), there was not enough collagen to cover the whole surface of paraffin micro-spheres (Fig. 2a). When the concentration of collagen solution was 1.2% (w/v), some interspace among the paraffin micro-spheres were blocked by the collagen and could not be filled with polymer solution in the subsequent casting procedure (Fig. 2b).
Fig. 2. SEM micrographs paraffin micro-spheres with different amounts of collagen coating. The concentrations of collagen solution was 0.3% (a) and 1.2% (b), respectively.
3.2. SEM morphology of PLGA/collagen scaffold The PLGA scaffolds prepared by paraffin micro-spheres leaching method were highly porous and well interconnected. A typical example was shown in Fig. 3. After the paraffin micro-spheres were dissolved and removed by solvent, the PLGA originally among and on the paraffin micro-spheres became the pore wall of the PLGA scaffold. Some spherical pores could be observed inside the scaffold with dimensions (about 280 lm) corresponding to the size of paraffin micro-spheres (250–280 lm). The contact areas between two adjacent paraffin micro-spheres became small holes connecting the adjacent bigger pores. They contributed to the interconnectivity of the PLGA scaffold. The diameter of these holes was about 100 lm, which served as the channels for cells, nutrients and waste to transport through the scaffold. The pore size of the scaffold could be adjusted by choosing paraffin micro-spheres with different diameters (from 50 to 500 lm). After the paraffin micro-spheres were removed, collagen coating was transferred to the surface of the pore wall of the PLGA scaffold (Figs. 4 and 5). Some collagen network could be observed clearly on the surface of PLGA scaffold with collagen coating (0.5% collagen solution). The film of collagen coating was too thin to stand against the surface tension; they contracted and crinkled to form a collagen network with some round holes on the pore wall (Fig. 4). This kind of collagen structure increased its contrast and could be seen more readily under SEM. Due to the fact that some collagen fibers were embedded inside the PLGA matrix, most of the collagen network appeared to be attached quite securely on the pore wall surface after the whole preparation process. Fig. 5 were SEM micrographs of PLGA scaffold with collagen coating by 1.0% collagen solution. More collagen was coated on paraffin micro-spheres and was transferred
Fig. 3. SEM micrograph of PLGA scaffold. After paraffin micro-spheres were removed, spherical pores were left inside the PLGA scaffold. The pore size (280 lm) corresponded with the size range of paraffin microspheres (250–280 lm).
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Fig. 4. SEM micrographs of PLGA scaffold with collagen coating (0.5% collagen solution). Some areas of collagen coating were clearly observed as a collagen network.
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Fig. 5. SEM micrographs of PLGA scaffold with collagen coating (1.0% collagen solution). The pore wall surface of the PLGA scaffold was rather rough with small collagen knobs.
to the PLGA scaffold than 0.5% collagen solution. The pore wall of PLGA scaffold with collagen coating was rather rough. It appeared that the knobs on the surface were collagen (Fig. 5b). These collagen knobs significantly increase the roughness and the surface area of the scaffold. It could be useful for cell attachment and proliferation later during the subsequent cell seeding and culture processes. 3.3. FTIR spectra During the fabrication of collagen/PLGA scaffold, the collagen coating was in contact with some other chemicals. If the chemical structure of collagen molecule was somehow affected during these steps, it might be possible for collagen to lose its cell affinity. FTIR spectra of collagen films are shown in Fig. 6 before and after they were exposed to pyridine and cyclohexane. Amide bands at the wavenumber of 3082, 1680–1630, 1570–1510 and 1242 cm 1 could be observed (Fig. 6a). Generally, amide I bands
Fig. 6. FTIR spectra of collagen before (a) and after (b) a similar coating transfer process. No change in the spectra was observed before and after the process.
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References
Fig. 7. FTIR spectra of PLGA scaffold with collagen coating (a) and without collagen coating (b). Two peaks (indicated by arrows) showed that collagen was coated on the PLGA scaffold.
(1659 cm 1) originated from C@O stretching vibrations coupled to N–H bending vibration. The amide II bands (1553 cm 1) arise from the N–H bending vibrations coupled to C–N stretching vibrations [27–29]. After the collagen was treated by chemicals, no difference could be observed for the values of frequencies of these bands (Fig. 6b). It could be concluded that FTIR did not detect any significant change in the collagen molecular components before and after the coating process. Amide peaks were also revealed in the FTIR spectrum of PLGA scaffold with collagen coating at 1553 cm 1 for amide II bands and from 1680–1630 cm 1 (Fig. 7a). No such peaks could be observed in FTIR spectrum of PLGA scaffold without collagen coating (Fig. 7b). These analyses showed that the collagen was coated on PLGA scaffold. 4. Conclusions Using paraffin micro-spheres as porogens, biodegradable PLGA scaffolds were prepared with controlled pore size, good interconnectivity and high porosity. Collagen was transferred from paraffin micro-spheres to the pore wall surface within PLGA scaffold. Collagen fibers interpenetrated into PLGA matrix and could help to enhance the anchorage of the coating onto its substrate. The effective concentration of the collagen solution was from 0.5 to 1.0% (w/v). Collagen coating on the PLGA scaffold increased the roughness and surface area which could be benefit for cell attachment and proliferation. FTIR detected the presence of collagen in PLGA scaffold after the removal of the porogens, and did not show any changes in the molecular components of collagen after going through the entire transfer process. Using this coating method, the biodegradable polymer material did not contact with any aqueous solutions; unnecessary degradation was avoided.
[1] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. [2] Nerem RM, Sambanis A. Tissue engineering: From biology to biological substitutes. Tissue Eng 1995;1:3–13. [3] Rotter N, Aigner J, Naumann A, Planck H, Hammer C, Burmester G, et al. Cartilage reconstruction in head and neck surgery: comparison of resorbable polymer scaffolds for tissue engineering of human septal cartilage. J Biomed Mater Res 1998;42:347–56. [4] Freed LE, Grand DA, Lingbin Z, Emmanual J, Marquis JC, Langer R. Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J Biomed Mater Res 1994;28:891–9. [5] Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AC, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 1993;27:11–23. [6] Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G. In vitro engineering of human skin-like tissue. J Biomed Mater Res 1998;40:187–94. [7] Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res 1997;36:17–28. [8] Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs for ACL reconstruction. J Biomed Mater Res 1995;29:1363–71. [9] Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol 1998;16:224–30. [10] Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg Am 2004;86:1541–58. [11] Thomson RC, Yaszemski MJ, Powers JM, Mikos AG. Fabrication of biodegradable polymer scaffolds to engineer trabecular bone. J Biomater Sci, Polym Edn 1995;7:23–38. [12] Mikos AG, Sarakinos G, Leite SM, Vacanti JP, Langer R. Laminated three-dimensional biodegradable forms for use in tissue engineering. Biomaterials 1993;14:323–30. [13] Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. Polymer concepts in tissue engineering. J Biomed Mater Res 1998;43:422–7. [14] Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30–3. [15] Ma PX, Zhang RY. Synthetic nano-scale fibrous extracellular matrix. J Biomed Mater Res 1999;46:60–72. [16] Dunn MG, Bellincampi LD, Tria AJ, Zawadsky JP. Preliminary development of a collagen-PLA composite for ACL reconstruction. J Appl Polym Sci 1997;63:1423–8. [17] Chen G, Ushida T, Tateishi T. A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res 2000;51:273–9. [18] Chen G, Ushida T, Tateishi T. Hybrid biomaterials for tissue engineering: a preparative method for PLA or PLGA-collagen hybrid sponges. Adv Mater 2000;12:455–7. [19] Ochi K, Chen G, Ushida T, Gojo S, Segawa K, Tai H, et al. Use of isolated mature osteoblasts in abundance acts as desired-shaped bone regeneration in combination with a modified poly-DL-lactic-coglycolic acid (PLGA)-collagen sponge. J Cell Physiol 2002;194:45–53. [20] Chen G, Ushida T, Tateishi T. A hybrid network of synthetic polymer mesh and collagen sponge. Chem Commun 2000:1505–6. [21] Ma ZW, Gao CY, Gong YH, Shen JC. Paraffin micro-spheres as porogen to fabricate poly(L-lactic acid) scaffolds with improved cytocompatibility for cartilage tissue engineering. J Biomed Mater Res 2003;67:610–7. [22] Vert M, Mauduit J, Li S. Biodegradation of PLA/GA polymers: increasing complexity. Biomaterials 1994;15(15):1209–13. [23] Zhang R, Ma PX. Poly(a-hydroxyl acids)/hydroxyapatite porous composites for bone–tissue engineering. I. Preparation and morphology. J Biomed Mater Res 1999;44:446–55.
J. Li, A.F.T. Mak / Composites: Part B 38 (2007) 317–323 [24] Ma PX, Choi JW. Biodegradable polymer scaffolds with well-defined interconnected spherical pore network. Tissue Eng 2001;7:23–33. [25] Shum AWT, Li JS, Mak AFT. Fabrication and structural characterization of porous biodegradable poly(DL-lactic-co-glycolic acid) scaffolds with controlled range of pore sizes. Polym Degrad Stabil 2005;87(3):487–93. [26] Li JS, Beaussart A, Mak AFT. Transfer of apatite coating from porogens to scaffolds: Uniform apatite coating within porous poly(DL-lactic-co-glycolic acid) scaffold in vitro. J Biomed Mater Res (Accepted).
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[27] Lazarev YA, Grishkovsky BA, Khromova TB. Amide I bond of IR spectrum and structure of collagen and related polypeptides. Biopolymers 1985;24:1449–78. [28] Camacho NP, West P, Torzilli PA, Mendelsohn R. FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage. Biopolymers 2001;62:1–8. [29] Chen Y, Mak AFT, Wang M, Li JS. Composite coating of bonelike apatite particles and collagen fibers on poly L-lactic acid formed through an accelerated biomimetic coprecipitation process. J Biomed Mater Res 2006.
Transfer of collagen coating from porogen to scaffold: Collagen coating within poly(DL-lactic-co-glycolic acid) scaffold Jiashen Li, Arthur F.T. Mak
*
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China Received 31 March 2006; accepted 2 June 2006 Available online 1 November 2006
Abstract Using paraffin micro-spheres as porogen, this paper addressed a novel method to coat collagen onto the internal pore surface within poly(DL-lactic-co-glycolic acid) (PLGA) scaffold with controlled pore size. The paraffin micro-spheres with desirable size were mixed with collagen solution (0.5–1.0% w/v), molded to form a paraffin micro-sphere scaffold, and dried. The collagen was left on the surface of the paraffin micro-spheres and even among the paraffin micro-spheres. PLGA solution was then cast into the interspace of the paraffin/collagen scaffold and dried. After the paraffin micro-spheres were dissolved and removed, PLGA scaffold with controlled pore size, good interconnectivity and high porosity was obtained. Collagen was transferred from the paraffin micro-spheres to the surfaces of the pore wall. Observation of scanning electron microscopy (SEM) showed that collagen was coated on the paraffin micro-spheres and was on the surfaces of pore wall within PLGA scaffold. Fourier transform infrared spectroscopy (FTIR) also detected the presence of collagen in the PLGA scaffold so formed; and there was no apparent change on the molecular components of collagen during the experimental procedure. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: A. Foams; A. Polymer-matrix composites (PMCs); E. Surface treatments; Collagen
1. Introduction Tissue engineering has focused on the use of synthetic or natural degradable materials as scaffolds for cell transplantation to engine new biological tissues. Biomaterial scaffolds as three-dimensional exogenous extracellular matrices (ECM) to guide tissue regeneration. The biophysical and biomechanical properties of a biomaterial scaffold have much influence on the final outcome of tissue engineering [1–10]. Because their biomechanical properties, such as the compressive modulus and strength, and biodegradabilities, can be adjusted and controlled easily, aliphatic polyesters, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer of poly(DLlactic-co-glycolic acid) (PLGA), have been extensively
*
Corresponding author. E-mail address: [email protected] (A.F.T. Mak).
1359-8368/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.compositesb.2006.06.009
studied [11,12]. The compressive strength and modulus of these polymers can approximate those of soft and hard tissues in human body [13]. Because of a lack of cell-recognizable signals, such materials have little attraction for cell attachment. Collagens are major constituents of bone with rich arginine–glycine– aspartate (RGD) which has been shown to represent the minimal adhesion domain to stimulate cell attachment [14]. Although collagens have been used as a scaffolding material, they in general lack adequate mechanical properties, degradation rate, and batch-to-batch consistency [15]. Using collagen to modify biodegradable polymeric scaffolds seems to be a good way to address the above problems. Collagen fibers were embedded within PLA matrix to strengthen collagen fibers for application in tendon or ligament reconstruction [16]. Recently, a new method was developed to nest collagen micro-sponge in the pores of a polymer sponge [17–21]. This method included two major steps: fabrication of polymeric scaffolds and nesting
318
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collagen inside polymeric scaffold. After polymeric scaffolds were prepared, they were immersed into an acidic collagen solution under vacuum so that the scaffold pores could be filled with collagen solution. Followed by freezing and lyophilization, the collagen micro-sponges were formed inside the scaffold pores. Such collagen microsponges were crosslinked and blocked. The presence of the collagen micro-sponge increased the wettability of the polymer surface, and facilitated cell attachment in the sponges. However, while the biodegradable polymer was in the collagen solution, the biodegradable polymeric material would likely become degraded to some extent, especially for certain polymers which could degrade relatively fast. The low pH value of the collagen solution may accelerate the polyesters’ degradation rates [22,23]. Although the collagen micro-sponges in the scaffold pore would increase the apparent surface area/volume ratio, the pore size, porosity and interconnectivity of the scaffold decreased, making it more difficult for the cells to move into the scaffolds. Similarly, diffusion of nutrients and waste products into and out of the scaffold would also be limited. Furthermore, the mechanical property of collagen micro-sponges inside the polymeric scaffold was likely to be as weak as a pure collagen scaffold; potential collapses could be harmful to cell attachment and proliferation. This paper developed a new method to introduce collagen into the polymeric scaffold to address some of the above issues. Using paraffin micro-spheres as porogen to fabricate scaffold, it is easy to control the pore size by the size of paraffin spheres and the pore interconnectivity by changing the treating temperature and duration [24]. In this paper, paraffin micro-spheres were also used to introduce collagen coating onto the pore wall throughout the whole biodegradable polymer scaffold. 2. Materials and methods
poured into 1000 mL PVA/water solution (0.5% w/v, 60– 70 °C) with stirring. The molten paraffin was dispersed to form a suspension of paraffin micro-drops. The suspension was immediately poured into ice water to solidify the paraffin micro-drops as micro-spheres. The micro spheres were washed by deionized water for three times and separated using standard sieves (100, 150, 180, 250, 280, 400, and 600 lm) into different sizes. 2.3. Preparation of paraffin micro-spheres/collagen scaffold Collagen solution with different concentrations (from 0.1 to 1.5% in 0.3% malonic acid) was mixed with paraffin micro-spheres of desirable size. Air bulbs trapped in the suspension and among the paraffin micro-spheres were eliminated under low air pressure. A plastic syringe without a needle end was used as a mould of the paraffin scaffold. To prepare paraffin scaffold of about 3 mm high, the piston was pulled to leave a gap of about 5 mm between the piston and the syringe end. The gap was filled with paraffin microspheres/collagen suspension. Caution was taken to make sure that no air bubbles were trapped in the mould during this step. The syringe end was placed vertically on a piece of rubber plate; and the piston was pushed down gently. The extra collagen solution was extruded from the gap between the syringe end and the rubber plate. When no more collagen solution was observed coming out, the pressure on the piston was released. Then the whole paraffin scaffold with collagen was pushed out by the piston and dried at 37 °C in air for 12 h. Subsequently, the sample was immersed into 20 mL crosslinking agent for 4 h with shaking. Finally, the collagen coated paraffin scaffolds were washed with deionized water for three times and dried again.
2.1. Materials
2.4. Preparation of PLGA/collagen scaffold
Poly(DL-lactic-co-glycolic acid) (50/50) (PLGA50/50) was purchased from PURAC (Netherlands). The average molecular weight for PLGA50/50 is 50,000 g/mol. Poly(vinyl alcohol) (PVA) (88% hydrolyzed, average molecular weight 25,000 g/mol), paraffin (melting point 53–57 °C), pyridine (boiling point 115 °C), malonic acid, and cyclohexane (melting point 7 °C) were purchased from Acros (Belgium). Standard sieves were purchased from Lantian Ltd Co., China. Bovine type I collagen was obtained from Tsinghua University, China. Deionized water was obtained with nanopure diamond ultrapure water systems from Barnstead, USA. The collagen crosslinking agent was a solution of formaldehyde (37%) and acetone (1/5, w/w).
PLGA was dissolved in pyridine for the preparation of a solution with a desired concentration. PLGA solution was cast into the paraffin micro-sphere scaffold drop by drop until the inter space among paraffin micro-spheres was fully filled with PLGA solution. The samples were maintained under low vacuum for 2 days to evaporate the pyridine away. After they are dried, the PLGA was left among the paraffin micro-spheres with collagen. If necessary, additional casting and vacuum-drying steps could be repeated to produce scaffold with higher polymeric content. The dried paraffin micro-spheres/collagen/PLGA sample was removed from the vacuum box, and immersed in 20 mL of cyclohexane to dissolve the paraffin at room temperature for 6 h with shaking. Cyclohexane was changed every 1.5 h. Then the sample was placed into a freezer and frozen at 20 °C for about 6 h. Subsequently, they were freeze-dried at 10 °C for 2 days and dried at room temperature under vacuum to remove any remaining
2.2. Preparation of paraffin micro-spheres The method to prepare paraffin micro-spheres has been reported before [25,26]. Briefly, 20 g melted paraffin was
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solvent. The PLGA scaffold with collagen coating was kept in desiccators until characterization.
coating onto the polymeric scaffold. The untreated and treated collagen films were studied using FTIR.
2.5. Characterizations
3. Results and discussion
Collagen coatings on paraffin micro-spheres and on the pore wall surface of PLGA scaffold were investigated under a LEICA scanning electron microscope (Model Stereoscan 440) after gold coating. FTIR was used to detect the molecular components of scaffold and coating. The transmission spectra of the PLGA scaffolds with and without collagen coating were measured with a Perkin-Elmer FTIR Spectrometer. In order to examine if there were any changes in the collagen molecule before and after the material processing, a piece of crosslinked collagen film was immersed into pyridine, cyclohexane for the same period as collagen
3.1. SEM morphology of collagen coating on paraffin micro-spheres and PLGA scaffold SEM photomicrograph of the paraffin micro-spheres without collagen (Fig. 1a) showed that the contact between two paraffin micro-spheres was more focal. For the paraffin spheres with collagen coating, collagen could be clearly observed on the surface of the micro-spheres (Fig. 1b), and between neighboring micro-spheres (Fig. 1c). It could be noted that some collagen fibers were left spanning between two adjacent paraffin micro-spheres (Fig. 1d).
Fig. 1. SEM micrographs of paraffin micro-spheres without and with collagen coating. (a) Paraffin micro-spheres without collagen coating. (b) and (c) Collagen coating on the paraffin micro-spheres. Coating could be easily observed at and around the contacts between the neighboring micro-spheres. (d) Collagen fibers between the paraffin micro-spheres.
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These fibers would be embedded into the PLGA after the PLGA casting and would help anchoring the collagen coating into the PLGA matrix when the paraffin micro-spheres were removed. The amount of collagen coating on the paraffin microspheres could be adjusted by varying the concentration of the collagen solution. Apparently, the higher the concentration of the collagen solution was, the more the collagen coating was left on the surface of paraffin spheres. For effective collagen coating, it was found that the concentration of collagen solution should be from 0.5 to 1.0% (w/v). When the collagen concentration was 0.3% (w/v), there was not enough collagen to cover the whole surface of paraffin micro-spheres (Fig. 2a). When the concentration of collagen solution was 1.2% (w/v), some interspace among the paraffin micro-spheres were blocked by the collagen and could not be filled with polymer solution in the subsequent casting procedure (Fig. 2b).
Fig. 2. SEM micrographs paraffin micro-spheres with different amounts of collagen coating. The concentrations of collagen solution was 0.3% (a) and 1.2% (b), respectively.
3.2. SEM morphology of PLGA/collagen scaffold The PLGA scaffolds prepared by paraffin micro-spheres leaching method were highly porous and well interconnected. A typical example was shown in Fig. 3. After the paraffin micro-spheres were dissolved and removed by solvent, the PLGA originally among and on the paraffin micro-spheres became the pore wall of the PLGA scaffold. Some spherical pores could be observed inside the scaffold with dimensions (about 280 lm) corresponding to the size of paraffin micro-spheres (250–280 lm). The contact areas between two adjacent paraffin micro-spheres became small holes connecting the adjacent bigger pores. They contributed to the interconnectivity of the PLGA scaffold. The diameter of these holes was about 100 lm, which served as the channels for cells, nutrients and waste to transport through the scaffold. The pore size of the scaffold could be adjusted by choosing paraffin micro-spheres with different diameters (from 50 to 500 lm). After the paraffin micro-spheres were removed, collagen coating was transferred to the surface of the pore wall of the PLGA scaffold (Figs. 4 and 5). Some collagen network could be observed clearly on the surface of PLGA scaffold with collagen coating (0.5% collagen solution). The film of collagen coating was too thin to stand against the surface tension; they contracted and crinkled to form a collagen network with some round holes on the pore wall (Fig. 4). This kind of collagen structure increased its contrast and could be seen more readily under SEM. Due to the fact that some collagen fibers were embedded inside the PLGA matrix, most of the collagen network appeared to be attached quite securely on the pore wall surface after the whole preparation process. Fig. 5 were SEM micrographs of PLGA scaffold with collagen coating by 1.0% collagen solution. More collagen was coated on paraffin micro-spheres and was transferred
Fig. 3. SEM micrograph of PLGA scaffold. After paraffin micro-spheres were removed, spherical pores were left inside the PLGA scaffold. The pore size (280 lm) corresponded with the size range of paraffin microspheres (250–280 lm).
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Fig. 4. SEM micrographs of PLGA scaffold with collagen coating (0.5% collagen solution). Some areas of collagen coating were clearly observed as a collagen network.
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Fig. 5. SEM micrographs of PLGA scaffold with collagen coating (1.0% collagen solution). The pore wall surface of the PLGA scaffold was rather rough with small collagen knobs.
to the PLGA scaffold than 0.5% collagen solution. The pore wall of PLGA scaffold with collagen coating was rather rough. It appeared that the knobs on the surface were collagen (Fig. 5b). These collagen knobs significantly increase the roughness and the surface area of the scaffold. It could be useful for cell attachment and proliferation later during the subsequent cell seeding and culture processes. 3.3. FTIR spectra During the fabrication of collagen/PLGA scaffold, the collagen coating was in contact with some other chemicals. If the chemical structure of collagen molecule was somehow affected during these steps, it might be possible for collagen to lose its cell affinity. FTIR spectra of collagen films are shown in Fig. 6 before and after they were exposed to pyridine and cyclohexane. Amide bands at the wavenumber of 3082, 1680–1630, 1570–1510 and 1242 cm 1 could be observed (Fig. 6a). Generally, amide I bands
Fig. 6. FTIR spectra of collagen before (a) and after (b) a similar coating transfer process. No change in the spectra was observed before and after the process.
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References
Fig. 7. FTIR spectra of PLGA scaffold with collagen coating (a) and without collagen coating (b). Two peaks (indicated by arrows) showed that collagen was coated on the PLGA scaffold.
(1659 cm 1) originated from C@O stretching vibrations coupled to N–H bending vibration. The amide II bands (1553 cm 1) arise from the N–H bending vibrations coupled to C–N stretching vibrations [27–29]. After the collagen was treated by chemicals, no difference could be observed for the values of frequencies of these bands (Fig. 6b). It could be concluded that FTIR did not detect any significant change in the collagen molecular components before and after the coating process. Amide peaks were also revealed in the FTIR spectrum of PLGA scaffold with collagen coating at 1553 cm 1 for amide II bands and from 1680–1630 cm 1 (Fig. 7a). No such peaks could be observed in FTIR spectrum of PLGA scaffold without collagen coating (Fig. 7b). These analyses showed that the collagen was coated on PLGA scaffold. 4. Conclusions Using paraffin micro-spheres as porogens, biodegradable PLGA scaffolds were prepared with controlled pore size, good interconnectivity and high porosity. Collagen was transferred from paraffin micro-spheres to the pore wall surface within PLGA scaffold. Collagen fibers interpenetrated into PLGA matrix and could help to enhance the anchorage of the coating onto its substrate. The effective concentration of the collagen solution was from 0.5 to 1.0% (w/v). Collagen coating on the PLGA scaffold increased the roughness and surface area which could be benefit for cell attachment and proliferation. FTIR detected the presence of collagen in PLGA scaffold after the removal of the porogens, and did not show any changes in the molecular components of collagen after going through the entire transfer process. Using this coating method, the biodegradable polymer material did not contact with any aqueous solutions; unnecessary degradation was avoided.
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