J Mater Sci: Mater Med (2014) 25:1129–1136 DOI 10.1007/s10856-013-5129-5
Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering Anne-Marie Haaparanta • Elina Ja¨rvinen • Ibrahim Fatih Cengiz • Ville Ella¨ • Harri T. Kokkonen Ilkka Kiviranta • Minna Kelloma¨ki
•
Received: 11 September 2013 / Accepted: 15 December 2013 / Published online: 28 December 2013 Ó Springer Science+Business Media New York 2013
Abstract In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/ PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a A.-M. Haaparanta (&) I. F. Cengiz V. Ella¨ M. Kelloma¨ki Department of Electronics and Communications Engineering, Tampere University of Technology, Korkeakoulunkatu 3, 33720 Tampere, Finland e-mail: [email protected] A.-M. Haaparanta V. Ella¨ M. Kelloma¨ki BioMediTech, Institute of Biosciences and Medical Technology, Tampere, Finland E. Ja¨rvinen I. Kiviranta Department of Orthopaedics and Traumatology, University of Helsinki and Helsinki University Central Hospital, Haartmaninkatu 8, 00290 Helsinki, Finland H. T. Kokkonen Department of Applied Physics, University of Eastern Finland, Yliopistonranta 1F, 70211 Kuopio, Finland
highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/ PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.
1 Introduction Articular cartilage injuries are common, but the treatment of such injuries remains challenging due to the lack of spontaneous tissue regeneration and the nature of the tissue. Articular cartilage tissue is avascular and there is relatively low cell density and low mitotic activity of chondrocytes [1]. There are various treatment options depending on the injury, but with current repair techniques it is not possible to repair large lesions [1–5]. After treatment, lesions often form fibrous tissue that has poor quality and durability [6–8]. Autologous chondrocyte implantation (ACI) is a promising method for the repair of cartilage defects [9]. The major limitation, however, is the lack of appropriate biomaterial scaffolds that can be used in combination with ACI. The main focus thus far has been on developing threedimensional (3D) scaffolds that have a highly porous structure and an interconnected pore network that supports chondrocyte proliferation and cartilage matrix production [10, 11]. Sufficient mechanical strength is also required to
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withstand the high loads to which the forming repair tissue is subjected. The native extracellular matrix (ECM) of articular cartilage is a complex structure composed of a collagen fiber network filled with proteoglycans. Therefore, a structural protein such as collagen is a potential scaffold material for application in cartilage tissue engineering [12]. Chitosan, a widespread polysaccharide, is a natural polymer derived from chitin that possesses a similar structure to the naturally present glycosaminoglycans found in articular cartilage [13, 14]. However, collagen and chitosan alone cannot be used in load bearing applications because of their low mechanical strength. As a result, composite or hybrid structures containing a synthetic polymer, such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA) with good mechanical properties have been developed [15]. Many of the hybrid scaffolds developed have a two-dimensional synthetic fiber component [16, 17], or a synthetic component that is formed into a 3D structure using a non-fibrous method, for example salt leaching [18, 19]. The biomechanical properties of articular cartilage rely on the ability to restore its shape and regain lost liquids into the structure after loading, which is an essential feature for a scaffold [1, 10]. Therefore, a hybrid scaffold comprising a highly hydrophilic natural component together with a fibrous synthetic component with higher strength could present an optimal scaffold structure for cartilage tissue engineering. The aim of this study was to manufacture novel collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for use in cartilage tissue engineering. The hybrid scaffolds were consequently compared with plain freezedried collagen or chitosan, or collagen–chitosan blend scaffolds. The manufactured scaffolds were studied by determining the microstructure (SEM, microCT), water uptake abilities, mechanical properties, and cell viability and attachment with adult bovine chondrocytes in order to ascertain the suitability of these scaffold types for cartilage tissue engineering applications.
J Mater Sci: Mater Med (2014) 25:1129–1136 Table 1 Manufactured scaffold types and compositions Scaffolds
Hybrid scaffolds
Col
Collagen
ColH
Collagen ? PLA mesh
Chi
Chitosan
ChiH
Chitosan ? PLA mesh
C1C1
Collagen– chitosan 1:1
C1C1H
Collagen–chitosan 1:1 ? PLA mesh
C2C1
Collagen– chitosan 2:1
C2C1H
Collagen–chitosan 2:1 ? PLA mesh
1:10. The pH of the solution was adjusted to 7.20 and the solution was incubated at room temperature (RT) overnight. The initial collagen concentration of the collagen solution was 0.3 wt% and the final concentrations of 0.5 or 1.0 wt% were achieved by centrifuging and then concentrating the solution. Medical grade chitosan (Protasan UP B 90/500, FMC Biopolymer d/b/a NovaMatrix, Sandvika, Norway) with a deacetylation degree of 91 % and a molecular weight of 460,000 g/mol was dissolved in an acetic acid solution at a ratio of 1:1 (w/v), as concentrations of 0.5 or 1.0 wt%. For collagen–chitosan blends, the initial collagen and chitosan solutions were gently mixed together in ratios of 1:1 (v/v) or 2:1 (v/v), respectively. 2.1.2 Fabrication of PLA mesh For hybrids, a PLA 96/4 mesh was manufactured. Medical grade polymer poly(L/D)lactide 96/4 with an intrinsic viscosity of 2.18 dl/g (Purac Biochem, Gorinchem, The Netherlands) was used for fiber manufacture. The polymer was melt-spun into multi-filament fibers (16-ply, average diameter of single fiber *20 lm), using a Gimac microextruder (Gimac, Gastronno, Italy) with a screw diameter 12 mm. The fibers were online oriented using godets. The fibers were cut to staple fibers, at a length of *10 cm, and carded into mesh. The mesh was then cut with a puncher to produce samples with a radius of 8 mm.
2 Materials and methods
2.1.3 Fabrication of different scaffold types
2.1 Scaffold fabrication
The manufactured solutions were then loaded into custommade Teflon sample moulds (diameter 8 mm, height 4 mm). For the plain collagen, chitosan, or collagen–chitosan blend scaffolds, the 1.0 wt% solutions were used. For the hybrids, the 0.5 wt% collagen, chitosan, or collagen–chitosan blend solution was used and the mesh was loaded at the bottom and at the top of the moulds. The samples were then frozen for 24 h at -30 °C prior to freeze-drying for 24 h. All the samples were held under vacuum at RT for a minimum of 48 h before cross-linking of the collagen containing samples or the neutralization of chitosan samples.
Eight scaffold types (Table 1) were manufactured as described below. 2.1.1 Fabrication of plain and blend solutions Type I bovine dermal collagen (PureColÒ, Nutacon B.V., Leimuiden, the Netherlands) fibril formation was carried out as described earlier [20]. Briefly, the collagen–HCl solution was mixed with fibrillogenesis buffer at a ratio of
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The scaffolds containing collagen were cross-linked with 95 % ethanol solution with 14 mM EDC (N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride, SigmaAldrich, Helsinki, Finland) and 6 mM NHS (N-Hydroxysuccinimide, Sigma-Aldrich, Helsinki, Finland) for 4 h at RT. The chitosan scaffolds were neutralized with 99.5 and 70 % ethanol steps for 30 min each. All the samples were washed afterwards with deioniced water, placed into the sample moulds, and re-freeze-dried as described earlier.
immersed in PBS for 24 h at 37 °C. Each sample was compressed at a rate of 0.5 mm/min and a cell load of 1 kN. The corresponding Young’s modulus was determined from the linear elastic region (from 7 to 9 % strain). The stiffness values of each scaffold group were also determined (n = 6).
2.1.4 Scaffold microstructure analysis
Adult bovine primary chondrocytes were isolated from the femoral condyle of the knee of 5–6 month-old male cows (Bos taurus), as described previously [22]. One million chondrocytes were seeded into the 0.5 cm2 scaffold by pipetting. First, 50 ll of cell culture medium containing 500,000 cells were pipetted on top of the scaffold. After 5 min of incubation at RT, the scaffolds were turned around and another 50 ll of cell culture medium containing 500,000 cells were pipetted on the other side of the scaffold. The scaffolds were cultured in the common proliferation medium DMEM/F12 (21331-020 Gibco, Invitrogen, USA) that contains 1 % L-glutamin (25030024, Gibco, Invitrogen, USA), 1 % Penicillin/Streptomycin (15070-063, Gibco, Invitrogen, USA), 1 % Fungizone (15290026, Gibco, Invitrogen, USA), 10 % FBS (CH30160.03, Hyclone, Biofellows, Finland), and 50 lg/ml ascorbic acid (A4034, Sigma, USA) at 37 °C in 5 % CO2 for up to one week.
Scanning electron microscope (SEM) observation was done for the scaffolds at d0 and for the cell-cultured samples at d7. Samples containing cultured chondrocytes were fixed at d7, critical point dried by Bal-Tec CPD 030 (Bal-Tec Union Ltd., Liechtenstein), platinum coated (Quorum Q150TS, Quorum Technologies, UK) and imaged with a scanning electron microscope (SEM, FEI Quanta 250 Field Emission Gun) at a magnification of 5009. At d0, the scaffolds were platinum coated and imaged with SEM at the Electron Microscope Unit, Institute of Biotechnology, Helsinki, Finland. MicroCT analysis of the scaffolds was carried out with a MicroCT scanner (SkyScan 1172, SkyScan, Kontich, Belgium). The tube voltage and voxel size were 40 kV and 30.2 9 30.2 9 30.2 lm3, respectively. The image was averaged 50 times during scanning and no filters were used. 2.1.5 Water uptake
2.1.7 Isolation of bovine primary articular cartilage chondrocytes, seeding, and culture of chondrocytes in the scaffolds
2.1.8 Immunocytochemistry
Water uptake ð%Þ ¼ ½ðWw Wd Þ=Wd 100%;
The chondrocytes were fixed with 4 % paraformaldehyde (PFA) for 10 min at RT and washed with PBS. Blocking was done in 1 % BSA ? 0.1 % TritonX in PBS for 1 h at RT. Cells were incubated in the primary antibody overnight at ?4 °C in blocking buffer. The primary antibodies used were Collagen type II 1:200 (ab34712, Abcam, UK) and Collagen type II 1:200 (ab34712, Abcam, UK). A secondary antibody, alexa-fluor anti-rabbit 488 (A11008, Invitrogen, USA), was diluted in blocking buffer (1:200) and incubated at RT for 1 h. Cells were counterstained in 2 lg/ml Hoechst 33342 (H3570, Invitrogen, USA) for 10 min at RT. Cells were imaged at the Light Microscope Unit, Institute of Biotechnology, Helsinki with a Leica TCS SP5II HCS A confocal microscope using 109 or 209 air objectives. For the imaging of the crosssection, the scaffolds were cut in half with a scalpel and the cross-section was imaged at an approximate depth of 150 lm.
where Ww is the wet weight and Wd is the dry weight of the scaffold.
3 Results
2.1.6 Mechanical tests
3.1 Scaffold morphology and porosity
The compression tests were done for both dry and wet scaffolds by using a Lloyd LR30K mechanical tester (Lloyd Instruments Ltd, Hampshire, UK). Before testing, the wet scaffolds were
Figure 1 shows the highly porous structure of the manufactured freeze-dried scaffolds. As the microCT images show, all the scaffolds had interconnected pores and a
The water uptake was measured by immersing the scaffolds (n = 6) into 5 ml phosphate buffered saline (PBS) for 24 h at 37 °C. First, the ability of the scaffold structure as a whole (the material itself with the pore system) to bind water was measured by removing the scaffold from the PBS, shaken gently, and weighed without dripping. Next, the ability of the scaffold material itself (no excess water inside the pore system) to bind water was measured after drying the scaffolds between filter papers to remove the water from the porous structure of the scaffold [21]. The percentage of water uptake was calculated by using the equation:
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relatively homogenous matrix structure (collagen, chitosan, or collagen–chitosan blend). The matrix component in the hybrids also shows highly similar porous structure, as observed in the plain scaffolds (Fig. 1). The SEM images supported the microCT results and the high interlocking between the PLA fibers and the freeze-dried collagen/ chitosan can be seen in the hybrids in Fig. 2. All the samples had high porosity varying from 66.2 to 92.8 % (Table 2) detected from the microCT studies. C1C1 showed only 66.2 % porosity and all the other scaffolds had porosity higher than 85 %. The highest porosity value was in the ColH (92.8 %). The open porosity values of the scaffolds varied from 66.2 to 92.8 %. In all scaffolds, the pores were highly interconnected as the open porosity values of different scaffolds varied maximally only 0.05 % from the total porosity values. 3.2 Water uptake In general, water intake was the highest for collagen component in the scaffolds (Fig. 3). Therefore, the higher
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amount of collagen increased the water uptake of the blends. For chitosan, on the other hand, the water intake was much lower and the scaffolds with chitosan suffered from shrinking when wet. The hybrids followed the same trend as the plain scaffolds in water uptake, but the PLA in the scaffolds lowered the water uptake abilities of the scaffolds. As shown in Fig. 3, the water uptake of the whole scaffold structure (the material itself with the pore system) was much higher than the water uptake of the scaffold material itself (no excess water inside the pore system). 3.3 Mechanical properties The Young’s modulus and stiffness values are listed in Table 3. For all of the scaffolds, elastomeric foam compression curves with an initial linear elastic region, a middle collapse plateau region, and a final densification of the material could be noticed (Fig. 4), as described by Harley et al. [23]. The stress–strain curves (Fig. 4) show
Fig. 1 Cross-section views and 3D reconstructions of microCT images of different scaffold types
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1133 Table 2 Porosity values of different scaffold types collected from microCT data Sample
Total porosity (%)
Open porosity (%)
Total porosity - open porosity (%)
Col
85.92
85.91
0.01
Chi
89.41
89.41
0.00
C1C1 C2C1
66.15 86.64
66.13 86.63
0.02 0.01
ColH
92.76
92.75
0.01
ChiH
88.40
88.38
0.02
C1C1H
85.44
85.39
0.05
C2C1H
88.48
88.47
0.01
Fig. 3 Water uptake properties of the scaffolds after immersion in PBS for 24 h in 37 °C
that the stress values of the dry scaffolds rose steadily after the linear elastic region and the final densification occurred after 75 % of strain. Also, the stress stayed at a relative low level with the wet scaffolds until 60 % of strain for ChiH, C2C1H and C1C1, and 70 % of strain for Col, Chi, C2C1, ColH, and C1C1H. The hybrids showed significantly higher stress values than the Col, Chi and C2C1 after 70 % of strain. C1C1 showed the highest stress values for wet scaffolds after 70 % of strain. The corresponding stiffness of dry scaffolds between the plain and hybrids was over 50 % higher for the hybrids. Also, in wet conditions the stiffness of hybrids was over 70 % higher than for plain scaffolds, except for the C1C1 that had a much higher stiffness than other plain scaffolds. The collagen component in the studied Col and ColH recovered their shapes after the compression test studies almost completely after re-immersing the scaffolds into the PBS for 1 h (data not shown). 3.4 Cell culture studies Fig. 2 SEM micrographs of the scaffolds at d0 (without cells) and scaffolds containing bovine chondrocytes at d7 of culture. Scale bars 100 lm
Seeded cells were evenly distributed on the surfaces of the scaffolds. The cells were viable after one week of culture
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and attached well to the freeze-dried collagen and chitosan networks and the PLA fibers (Fig. 2). The cells expressed collagen type II in the cytoplasm, confirming their chondrogenic phenotype (Fig. 5). There was a high variation in the penetration of the cells into the different scaffolds. As cells were pipetted on top of the dense scaffolds, most cells remained at the top in all
scaffold types. In ColH and C2C1H scaffolds, however, the penetration of the cells was good and cells were detected throughout the scaffold. In Col, Chi, C1C1, and C2C1 scaffolds, the penetration of the cells was weak. In ChiH and C1C1H scaffolds, some penetration was detected.
4 Discussion Table 3 Mechanical test results (Young’s modulus and stiffness values ± SD) of dry and wet scaffolds Samples
Young’s modulus (E, kPa)
Stiffness (N, mm)
Dry
Dry
Wet
Wet
Col
115.1 ± 22.6
5.0 ± 0.5
32.3 ± 2.4
7.8 ± 1.9
Chi
86.0 ± 16.5
4.5 ± 1.4
26.1 ± 2.0
20.2 ± 3.2
C1C1
60.3 ± 14.3
10.0 ± 3.5
35.2 ± 5.9
76.0 ± 6.1
C2C1
130.8 ± 27.6
5.2 ± 1.0
33.0 ± 4.3
17.3 ± 3.8
ColH
22.8 ± 7.8
3.3 ± 1.5
81.0 ± 13.1
65.1 ± 10.9
ChiH
42.5 ± 9.5
9.0 ± 3.6
75.3 ± 5.1
85.7 ± 15.2
C1C1H
48.4 ± 8.5
5.9 ± 2.2
86.8 ± 9.3
95.1 ± 13.6
C2C1H
52.3 ± 6.3
5.3 ± 1.6
83.9 ± 13.6
99.4 ± 10.9
In earlier studies, neither PLA nor collagen has been an optimal scaffold for articular cartilage, as the PLA matrix has shown to be too hard [24], and collagen gels, even though being a fairly good option for cartilage tissue engineering, often suffer from contraction [22]. Therefore, in our present study, we used synthetic PLA to give the scaffolds mechanically a more stable skeleton. The highly hydrophilic components, collagen and chitosan were used to give the scaffolds a better water absorbing ability and to mimic the native ECM components in the cartilage. The studied scaffolds are suturable in order to achieve the mechanical locking of the scaffold in situ. The freezedrying and cross-linking of the collagen component
Fig. 4 Stress–strain curves for a dry and b wet scaffolds showing linear elastic, collapse plateau, and densification regimes
Fig. 5 Evenly distributed and viable chondrocytes show collagen II expression in the scaffolds after one week in culture, indicating that the cells have retained their chondrogenic phenotype in the scaffolds. Green collagen II, blue nuclei of the cells. Scale bar 50 lm (Color figure online)
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prevents the possible undesired contraction of the collagen component. Blends with collagen/chitosan ratio between 2:1 to 1:1 was expected to be ideal as the number of organic components of collagen and the proteoglycans in native articular cartilage are 15–20 wt% of collagen and 10 wt% of proteoglycans [25]. The highly porous structure of a cartilage tissue engineering scaffold is essential for cell migration and the diffusion of oxygen and nutrients, and it also improves the mechanical interlocking of the scaffolds to the surrounding tissue [11]. The structure of the studied scaffolds was highly porous (Fig. 2) and the vast majority of the pores were interconnected, as seen in the microCT images (Fig. 1). The PLA fibers in the hybrids were well interlocked between the matrix polymer. The geometry of each hybrid component remained very stable and did not alter the shape of the meshs or the pore structure of the matrix component. The difference between the hybrid and plain scaffolds varied only 1.0 % for the Chi/ChiH and 1.8 % for the C2C1/C2C1H scaffolds, but the porosity of ColH compared to Col increased by 6.8 % and C1C1H compared to C1C1 by 19.3 %. All scaffolds had a high and similar porosity with the exception of C1C1 that had relatively low porosity values because of the more dense structure. The microCT studies confirmed the highly interconnected pore structure. Collagen showed the best water uptake abilities in the scaffolds. Scaffolds containing chitosan showed the weakest water uptake abilities that lead to the shrinkage of the Chi scaffolds (data not shown). In addition, C1C1 showed considerable shrinking in wet conditions (data not shown), indicating an ineffective neutralization of the chitosan component in C1C1. The PLA was not able to bind the water inside its structure during the wetting period of 24 h and therefore the percentual water uptake of the hybrids was much lower than the values for the plain scaffolds. The compressive modulus of native cartilage varies between the different layers of cartilage [26]. It is known that the stiffness of the scaffold influences the mechanical environment of the cells, which in turn can influence cell differentiation and tissue growth. However, the optimal mechanical properties of a plain scaffold for cartilage tissue engineering are not known, and the engineered tissue may not necessarily be an exact copy of the natural tissue [11]. The cellular microenvironment changes in the scaffold during tissue development in vivo and the mechanical properties of the scaffolds are found to improve compared to in vitro cultivation [22]. In our study, the dry C1C1 showed the lowest Young’s modulus, which could be explained by much lower porosity values (shown in the microCT studies). The lower porosity values were possibly due to a partially-collapsed pore structure during
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processing that lead to a faster pore collapse. The Young’s modulus for the dry hybrids was lower than for the corresponding plain scaffolds. The difference between the wet hybrid scaffolds and the plain scaffolds was more moderate. The lower Young’s modulus of the hybrid scaffolds could be explained by the structure, where the more mechanically stable PLA mesh is at the top and at the bottom of the sandwich structure. Therefore, the initial elastic properties may only be the properties of the softer middle component of the collagen, chitosan, or collagen– chitosan blend. The C1C1 had the highest Young’s modulus for plain wet scaffolds, but this was due to the denser structure of the scaffold, as described earlier. Prominent shrinking of the C1C1 was detected in wet conditions (data not shown), leading to a higher Young’s modulus as well as higher stiffness for C1C1. The hybrid structure improved the stiffness of the scaffolds giving the scaffolds more mechanical strength compared to the plain scaffolds. Even when wet, the hybrids retained their structure after the mechanical loading. Moreover, the ability to reabsorb the removed water after loading is an essential characteristic for articular cartilage tissue engineering scaffolds, as the mechanical loading and unloading is a biological phenomenon in the native tissue. As a consequence, the liquid and nutrition exchange is implemented in the native avascular tissue via the phenomenon [6]. The ColH scaffolds recovered well from the mechanical load after compression, representing the ideal characteristics for a mechanically compatible scaffold. This phenomenon can also be seen in the stress–strain curves of wet scaffolds (Fig. 4) as the ColH scaffolds showed no densification before the compression of 70 %, indicating that no plastic deformation yet exist. Chondrocytes were cultured in the scaffolds to study the viability and attachment of the cells in the scaffolds in vitro. Our results show that cell viability and attachment was good in all scaffolds. The addition of chitosan lowered the penetration of the cells, and the addition of a loose PLA fiber network increased the penetration indicating the positive effect of the used hybrid structure. However, other cell seeding methods, such as injection by needle may be considered as an additional way to improve the even distribution of the cells inside the scaffolds.
5 Conclusion In the present study, we showed the ability of this fabrication method to be used for processing novel hybrid structures with natural and synthetic components. The hybrid collagen scaffolds showed desirable properties for articular cartilage tissue engineering applications, although the composition of the hybrid should be studied more
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detailed to find out an optimal scaffold structure. These scaffolds had high porosity and interconnected pores, improved mechanical strength compared to plain collagen scaffolds, good water uptake, and penetration of chondrocytes indicating this kind of collagen/PLA hybrids to be potential scaffolds for cartilage tissue engineering. Acknowledgments We would like to thank Antti Aula for microCT image editing. The financial support of the Finnish Funding Agency for Technology and Innovation (TEKES) is greatly appreciated (Grant 3110/31/08).
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Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering Anne-Marie Haaparanta • Elina Ja¨rvinen • Ibrahim Fatih Cengiz • Ville Ella¨ • Harri T. Kokkonen Ilkka Kiviranta • Minna Kelloma¨ki
•
Received: 11 September 2013 / Accepted: 15 December 2013 / Published online: 28 December 2013 Ó Springer Science+Business Media New York 2013
Abstract In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/ PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a A.-M. Haaparanta (&) I. F. Cengiz V. Ella¨ M. Kelloma¨ki Department of Electronics and Communications Engineering, Tampere University of Technology, Korkeakoulunkatu 3, 33720 Tampere, Finland e-mail: [email protected] A.-M. Haaparanta V. Ella¨ M. Kelloma¨ki BioMediTech, Institute of Biosciences and Medical Technology, Tampere, Finland E. Ja¨rvinen I. Kiviranta Department of Orthopaedics and Traumatology, University of Helsinki and Helsinki University Central Hospital, Haartmaninkatu 8, 00290 Helsinki, Finland H. T. Kokkonen Department of Applied Physics, University of Eastern Finland, Yliopistonranta 1F, 70211 Kuopio, Finland
highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/ PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.
1 Introduction Articular cartilage injuries are common, but the treatment of such injuries remains challenging due to the lack of spontaneous tissue regeneration and the nature of the tissue. Articular cartilage tissue is avascular and there is relatively low cell density and low mitotic activity of chondrocytes [1]. There are various treatment options depending on the injury, but with current repair techniques it is not possible to repair large lesions [1–5]. After treatment, lesions often form fibrous tissue that has poor quality and durability [6–8]. Autologous chondrocyte implantation (ACI) is a promising method for the repair of cartilage defects [9]. The major limitation, however, is the lack of appropriate biomaterial scaffolds that can be used in combination with ACI. The main focus thus far has been on developing threedimensional (3D) scaffolds that have a highly porous structure and an interconnected pore network that supports chondrocyte proliferation and cartilage matrix production [10, 11]. Sufficient mechanical strength is also required to
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withstand the high loads to which the forming repair tissue is subjected. The native extracellular matrix (ECM) of articular cartilage is a complex structure composed of a collagen fiber network filled with proteoglycans. Therefore, a structural protein such as collagen is a potential scaffold material for application in cartilage tissue engineering [12]. Chitosan, a widespread polysaccharide, is a natural polymer derived from chitin that possesses a similar structure to the naturally present glycosaminoglycans found in articular cartilage [13, 14]. However, collagen and chitosan alone cannot be used in load bearing applications because of their low mechanical strength. As a result, composite or hybrid structures containing a synthetic polymer, such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA) with good mechanical properties have been developed [15]. Many of the hybrid scaffolds developed have a two-dimensional synthetic fiber component [16, 17], or a synthetic component that is formed into a 3D structure using a non-fibrous method, for example salt leaching [18, 19]. The biomechanical properties of articular cartilage rely on the ability to restore its shape and regain lost liquids into the structure after loading, which is an essential feature for a scaffold [1, 10]. Therefore, a hybrid scaffold comprising a highly hydrophilic natural component together with a fibrous synthetic component with higher strength could present an optimal scaffold structure for cartilage tissue engineering. The aim of this study was to manufacture novel collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for use in cartilage tissue engineering. The hybrid scaffolds were consequently compared with plain freezedried collagen or chitosan, or collagen–chitosan blend scaffolds. The manufactured scaffolds were studied by determining the microstructure (SEM, microCT), water uptake abilities, mechanical properties, and cell viability and attachment with adult bovine chondrocytes in order to ascertain the suitability of these scaffold types for cartilage tissue engineering applications.
J Mater Sci: Mater Med (2014) 25:1129–1136 Table 1 Manufactured scaffold types and compositions Scaffolds
Hybrid scaffolds
Col
Collagen
ColH
Collagen ? PLA mesh
Chi
Chitosan
ChiH
Chitosan ? PLA mesh
C1C1
Collagen– chitosan 1:1
C1C1H
Collagen–chitosan 1:1 ? PLA mesh
C2C1
Collagen– chitosan 2:1
C2C1H
Collagen–chitosan 2:1 ? PLA mesh
1:10. The pH of the solution was adjusted to 7.20 and the solution was incubated at room temperature (RT) overnight. The initial collagen concentration of the collagen solution was 0.3 wt% and the final concentrations of 0.5 or 1.0 wt% were achieved by centrifuging and then concentrating the solution. Medical grade chitosan (Protasan UP B 90/500, FMC Biopolymer d/b/a NovaMatrix, Sandvika, Norway) with a deacetylation degree of 91 % and a molecular weight of 460,000 g/mol was dissolved in an acetic acid solution at a ratio of 1:1 (w/v), as concentrations of 0.5 or 1.0 wt%. For collagen–chitosan blends, the initial collagen and chitosan solutions were gently mixed together in ratios of 1:1 (v/v) or 2:1 (v/v), respectively. 2.1.2 Fabrication of PLA mesh For hybrids, a PLA 96/4 mesh was manufactured. Medical grade polymer poly(L/D)lactide 96/4 with an intrinsic viscosity of 2.18 dl/g (Purac Biochem, Gorinchem, The Netherlands) was used for fiber manufacture. The polymer was melt-spun into multi-filament fibers (16-ply, average diameter of single fiber *20 lm), using a Gimac microextruder (Gimac, Gastronno, Italy) with a screw diameter 12 mm. The fibers were online oriented using godets. The fibers were cut to staple fibers, at a length of *10 cm, and carded into mesh. The mesh was then cut with a puncher to produce samples with a radius of 8 mm.
2 Materials and methods
2.1.3 Fabrication of different scaffold types
2.1 Scaffold fabrication
The manufactured solutions were then loaded into custommade Teflon sample moulds (diameter 8 mm, height 4 mm). For the plain collagen, chitosan, or collagen–chitosan blend scaffolds, the 1.0 wt% solutions were used. For the hybrids, the 0.5 wt% collagen, chitosan, or collagen–chitosan blend solution was used and the mesh was loaded at the bottom and at the top of the moulds. The samples were then frozen for 24 h at -30 °C prior to freeze-drying for 24 h. All the samples were held under vacuum at RT for a minimum of 48 h before cross-linking of the collagen containing samples or the neutralization of chitosan samples.
Eight scaffold types (Table 1) were manufactured as described below. 2.1.1 Fabrication of plain and blend solutions Type I bovine dermal collagen (PureColÒ, Nutacon B.V., Leimuiden, the Netherlands) fibril formation was carried out as described earlier [20]. Briefly, the collagen–HCl solution was mixed with fibrillogenesis buffer at a ratio of
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The scaffolds containing collagen were cross-linked with 95 % ethanol solution with 14 mM EDC (N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride, SigmaAldrich, Helsinki, Finland) and 6 mM NHS (N-Hydroxysuccinimide, Sigma-Aldrich, Helsinki, Finland) for 4 h at RT. The chitosan scaffolds were neutralized with 99.5 and 70 % ethanol steps for 30 min each. All the samples were washed afterwards with deioniced water, placed into the sample moulds, and re-freeze-dried as described earlier.
immersed in PBS for 24 h at 37 °C. Each sample was compressed at a rate of 0.5 mm/min and a cell load of 1 kN. The corresponding Young’s modulus was determined from the linear elastic region (from 7 to 9 % strain). The stiffness values of each scaffold group were also determined (n = 6).
2.1.4 Scaffold microstructure analysis
Adult bovine primary chondrocytes were isolated from the femoral condyle of the knee of 5–6 month-old male cows (Bos taurus), as described previously [22]. One million chondrocytes were seeded into the 0.5 cm2 scaffold by pipetting. First, 50 ll of cell culture medium containing 500,000 cells were pipetted on top of the scaffold. After 5 min of incubation at RT, the scaffolds were turned around and another 50 ll of cell culture medium containing 500,000 cells were pipetted on the other side of the scaffold. The scaffolds were cultured in the common proliferation medium DMEM/F12 (21331-020 Gibco, Invitrogen, USA) that contains 1 % L-glutamin (25030024, Gibco, Invitrogen, USA), 1 % Penicillin/Streptomycin (15070-063, Gibco, Invitrogen, USA), 1 % Fungizone (15290026, Gibco, Invitrogen, USA), 10 % FBS (CH30160.03, Hyclone, Biofellows, Finland), and 50 lg/ml ascorbic acid (A4034, Sigma, USA) at 37 °C in 5 % CO2 for up to one week.
Scanning electron microscope (SEM) observation was done for the scaffolds at d0 and for the cell-cultured samples at d7. Samples containing cultured chondrocytes were fixed at d7, critical point dried by Bal-Tec CPD 030 (Bal-Tec Union Ltd., Liechtenstein), platinum coated (Quorum Q150TS, Quorum Technologies, UK) and imaged with a scanning electron microscope (SEM, FEI Quanta 250 Field Emission Gun) at a magnification of 5009. At d0, the scaffolds were platinum coated and imaged with SEM at the Electron Microscope Unit, Institute of Biotechnology, Helsinki, Finland. MicroCT analysis of the scaffolds was carried out with a MicroCT scanner (SkyScan 1172, SkyScan, Kontich, Belgium). The tube voltage and voxel size were 40 kV and 30.2 9 30.2 9 30.2 lm3, respectively. The image was averaged 50 times during scanning and no filters were used. 2.1.5 Water uptake
2.1.7 Isolation of bovine primary articular cartilage chondrocytes, seeding, and culture of chondrocytes in the scaffolds
2.1.8 Immunocytochemistry
Water uptake ð%Þ ¼ ½ðWw Wd Þ=Wd 100%;
The chondrocytes were fixed with 4 % paraformaldehyde (PFA) for 10 min at RT and washed with PBS. Blocking was done in 1 % BSA ? 0.1 % TritonX in PBS for 1 h at RT. Cells were incubated in the primary antibody overnight at ?4 °C in blocking buffer. The primary antibodies used were Collagen type II 1:200 (ab34712, Abcam, UK) and Collagen type II 1:200 (ab34712, Abcam, UK). A secondary antibody, alexa-fluor anti-rabbit 488 (A11008, Invitrogen, USA), was diluted in blocking buffer (1:200) and incubated at RT for 1 h. Cells were counterstained in 2 lg/ml Hoechst 33342 (H3570, Invitrogen, USA) for 10 min at RT. Cells were imaged at the Light Microscope Unit, Institute of Biotechnology, Helsinki with a Leica TCS SP5II HCS A confocal microscope using 109 or 209 air objectives. For the imaging of the crosssection, the scaffolds were cut in half with a scalpel and the cross-section was imaged at an approximate depth of 150 lm.
where Ww is the wet weight and Wd is the dry weight of the scaffold.
3 Results
2.1.6 Mechanical tests
3.1 Scaffold morphology and porosity
The compression tests were done for both dry and wet scaffolds by using a Lloyd LR30K mechanical tester (Lloyd Instruments Ltd, Hampshire, UK). Before testing, the wet scaffolds were
Figure 1 shows the highly porous structure of the manufactured freeze-dried scaffolds. As the microCT images show, all the scaffolds had interconnected pores and a
The water uptake was measured by immersing the scaffolds (n = 6) into 5 ml phosphate buffered saline (PBS) for 24 h at 37 °C. First, the ability of the scaffold structure as a whole (the material itself with the pore system) to bind water was measured by removing the scaffold from the PBS, shaken gently, and weighed without dripping. Next, the ability of the scaffold material itself (no excess water inside the pore system) to bind water was measured after drying the scaffolds between filter papers to remove the water from the porous structure of the scaffold [21]. The percentage of water uptake was calculated by using the equation:
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relatively homogenous matrix structure (collagen, chitosan, or collagen–chitosan blend). The matrix component in the hybrids also shows highly similar porous structure, as observed in the plain scaffolds (Fig. 1). The SEM images supported the microCT results and the high interlocking between the PLA fibers and the freeze-dried collagen/ chitosan can be seen in the hybrids in Fig. 2. All the samples had high porosity varying from 66.2 to 92.8 % (Table 2) detected from the microCT studies. C1C1 showed only 66.2 % porosity and all the other scaffolds had porosity higher than 85 %. The highest porosity value was in the ColH (92.8 %). The open porosity values of the scaffolds varied from 66.2 to 92.8 %. In all scaffolds, the pores were highly interconnected as the open porosity values of different scaffolds varied maximally only 0.05 % from the total porosity values. 3.2 Water uptake In general, water intake was the highest for collagen component in the scaffolds (Fig. 3). Therefore, the higher
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amount of collagen increased the water uptake of the blends. For chitosan, on the other hand, the water intake was much lower and the scaffolds with chitosan suffered from shrinking when wet. The hybrids followed the same trend as the plain scaffolds in water uptake, but the PLA in the scaffolds lowered the water uptake abilities of the scaffolds. As shown in Fig. 3, the water uptake of the whole scaffold structure (the material itself with the pore system) was much higher than the water uptake of the scaffold material itself (no excess water inside the pore system). 3.3 Mechanical properties The Young’s modulus and stiffness values are listed in Table 3. For all of the scaffolds, elastomeric foam compression curves with an initial linear elastic region, a middle collapse plateau region, and a final densification of the material could be noticed (Fig. 4), as described by Harley et al. [23]. The stress–strain curves (Fig. 4) show
Fig. 1 Cross-section views and 3D reconstructions of microCT images of different scaffold types
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1133 Table 2 Porosity values of different scaffold types collected from microCT data Sample
Total porosity (%)
Open porosity (%)
Total porosity - open porosity (%)
Col
85.92
85.91
0.01
Chi
89.41
89.41
0.00
C1C1 C2C1
66.15 86.64
66.13 86.63
0.02 0.01
ColH
92.76
92.75
0.01
ChiH
88.40
88.38
0.02
C1C1H
85.44
85.39
0.05
C2C1H
88.48
88.47
0.01
Fig. 3 Water uptake properties of the scaffolds after immersion in PBS for 24 h in 37 °C
that the stress values of the dry scaffolds rose steadily after the linear elastic region and the final densification occurred after 75 % of strain. Also, the stress stayed at a relative low level with the wet scaffolds until 60 % of strain for ChiH, C2C1H and C1C1, and 70 % of strain for Col, Chi, C2C1, ColH, and C1C1H. The hybrids showed significantly higher stress values than the Col, Chi and C2C1 after 70 % of strain. C1C1 showed the highest stress values for wet scaffolds after 70 % of strain. The corresponding stiffness of dry scaffolds between the plain and hybrids was over 50 % higher for the hybrids. Also, in wet conditions the stiffness of hybrids was over 70 % higher than for plain scaffolds, except for the C1C1 that had a much higher stiffness than other plain scaffolds. The collagen component in the studied Col and ColH recovered their shapes after the compression test studies almost completely after re-immersing the scaffolds into the PBS for 1 h (data not shown). 3.4 Cell culture studies Fig. 2 SEM micrographs of the scaffolds at d0 (without cells) and scaffolds containing bovine chondrocytes at d7 of culture. Scale bars 100 lm
Seeded cells were evenly distributed on the surfaces of the scaffolds. The cells were viable after one week of culture
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and attached well to the freeze-dried collagen and chitosan networks and the PLA fibers (Fig. 2). The cells expressed collagen type II in the cytoplasm, confirming their chondrogenic phenotype (Fig. 5). There was a high variation in the penetration of the cells into the different scaffolds. As cells were pipetted on top of the dense scaffolds, most cells remained at the top in all
scaffold types. In ColH and C2C1H scaffolds, however, the penetration of the cells was good and cells were detected throughout the scaffold. In Col, Chi, C1C1, and C2C1 scaffolds, the penetration of the cells was weak. In ChiH and C1C1H scaffolds, some penetration was detected.
4 Discussion Table 3 Mechanical test results (Young’s modulus and stiffness values ± SD) of dry and wet scaffolds Samples
Young’s modulus (E, kPa)
Stiffness (N, mm)
Dry
Dry
Wet
Wet
Col
115.1 ± 22.6
5.0 ± 0.5
32.3 ± 2.4
7.8 ± 1.9
Chi
86.0 ± 16.5
4.5 ± 1.4
26.1 ± 2.0
20.2 ± 3.2
C1C1
60.3 ± 14.3
10.0 ± 3.5
35.2 ± 5.9
76.0 ± 6.1
C2C1
130.8 ± 27.6
5.2 ± 1.0
33.0 ± 4.3
17.3 ± 3.8
ColH
22.8 ± 7.8
3.3 ± 1.5
81.0 ± 13.1
65.1 ± 10.9
ChiH
42.5 ± 9.5
9.0 ± 3.6
75.3 ± 5.1
85.7 ± 15.2
C1C1H
48.4 ± 8.5
5.9 ± 2.2
86.8 ± 9.3
95.1 ± 13.6
C2C1H
52.3 ± 6.3
5.3 ± 1.6
83.9 ± 13.6
99.4 ± 10.9
In earlier studies, neither PLA nor collagen has been an optimal scaffold for articular cartilage, as the PLA matrix has shown to be too hard [24], and collagen gels, even though being a fairly good option for cartilage tissue engineering, often suffer from contraction [22]. Therefore, in our present study, we used synthetic PLA to give the scaffolds mechanically a more stable skeleton. The highly hydrophilic components, collagen and chitosan were used to give the scaffolds a better water absorbing ability and to mimic the native ECM components in the cartilage. The studied scaffolds are suturable in order to achieve the mechanical locking of the scaffold in situ. The freezedrying and cross-linking of the collagen component
Fig. 4 Stress–strain curves for a dry and b wet scaffolds showing linear elastic, collapse plateau, and densification regimes
Fig. 5 Evenly distributed and viable chondrocytes show collagen II expression in the scaffolds after one week in culture, indicating that the cells have retained their chondrogenic phenotype in the scaffolds. Green collagen II, blue nuclei of the cells. Scale bar 50 lm (Color figure online)
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prevents the possible undesired contraction of the collagen component. Blends with collagen/chitosan ratio between 2:1 to 1:1 was expected to be ideal as the number of organic components of collagen and the proteoglycans in native articular cartilage are 15–20 wt% of collagen and 10 wt% of proteoglycans [25]. The highly porous structure of a cartilage tissue engineering scaffold is essential for cell migration and the diffusion of oxygen and nutrients, and it also improves the mechanical interlocking of the scaffolds to the surrounding tissue [11]. The structure of the studied scaffolds was highly porous (Fig. 2) and the vast majority of the pores were interconnected, as seen in the microCT images (Fig. 1). The PLA fibers in the hybrids were well interlocked between the matrix polymer. The geometry of each hybrid component remained very stable and did not alter the shape of the meshs or the pore structure of the matrix component. The difference between the hybrid and plain scaffolds varied only 1.0 % for the Chi/ChiH and 1.8 % for the C2C1/C2C1H scaffolds, but the porosity of ColH compared to Col increased by 6.8 % and C1C1H compared to C1C1 by 19.3 %. All scaffolds had a high and similar porosity with the exception of C1C1 that had relatively low porosity values because of the more dense structure. The microCT studies confirmed the highly interconnected pore structure. Collagen showed the best water uptake abilities in the scaffolds. Scaffolds containing chitosan showed the weakest water uptake abilities that lead to the shrinkage of the Chi scaffolds (data not shown). In addition, C1C1 showed considerable shrinking in wet conditions (data not shown), indicating an ineffective neutralization of the chitosan component in C1C1. The PLA was not able to bind the water inside its structure during the wetting period of 24 h and therefore the percentual water uptake of the hybrids was much lower than the values for the plain scaffolds. The compressive modulus of native cartilage varies between the different layers of cartilage [26]. It is known that the stiffness of the scaffold influences the mechanical environment of the cells, which in turn can influence cell differentiation and tissue growth. However, the optimal mechanical properties of a plain scaffold for cartilage tissue engineering are not known, and the engineered tissue may not necessarily be an exact copy of the natural tissue [11]. The cellular microenvironment changes in the scaffold during tissue development in vivo and the mechanical properties of the scaffolds are found to improve compared to in vitro cultivation [22]. In our study, the dry C1C1 showed the lowest Young’s modulus, which could be explained by much lower porosity values (shown in the microCT studies). The lower porosity values were possibly due to a partially-collapsed pore structure during
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processing that lead to a faster pore collapse. The Young’s modulus for the dry hybrids was lower than for the corresponding plain scaffolds. The difference between the wet hybrid scaffolds and the plain scaffolds was more moderate. The lower Young’s modulus of the hybrid scaffolds could be explained by the structure, where the more mechanically stable PLA mesh is at the top and at the bottom of the sandwich structure. Therefore, the initial elastic properties may only be the properties of the softer middle component of the collagen, chitosan, or collagen– chitosan blend. The C1C1 had the highest Young’s modulus for plain wet scaffolds, but this was due to the denser structure of the scaffold, as described earlier. Prominent shrinking of the C1C1 was detected in wet conditions (data not shown), leading to a higher Young’s modulus as well as higher stiffness for C1C1. The hybrid structure improved the stiffness of the scaffolds giving the scaffolds more mechanical strength compared to the plain scaffolds. Even when wet, the hybrids retained their structure after the mechanical loading. Moreover, the ability to reabsorb the removed water after loading is an essential characteristic for articular cartilage tissue engineering scaffolds, as the mechanical loading and unloading is a biological phenomenon in the native tissue. As a consequence, the liquid and nutrition exchange is implemented in the native avascular tissue via the phenomenon [6]. The ColH scaffolds recovered well from the mechanical load after compression, representing the ideal characteristics for a mechanically compatible scaffold. This phenomenon can also be seen in the stress–strain curves of wet scaffolds (Fig. 4) as the ColH scaffolds showed no densification before the compression of 70 %, indicating that no plastic deformation yet exist. Chondrocytes were cultured in the scaffolds to study the viability and attachment of the cells in the scaffolds in vitro. Our results show that cell viability and attachment was good in all scaffolds. The addition of chitosan lowered the penetration of the cells, and the addition of a loose PLA fiber network increased the penetration indicating the positive effect of the used hybrid structure. However, other cell seeding methods, such as injection by needle may be considered as an additional way to improve the even distribution of the cells inside the scaffolds.
5 Conclusion In the present study, we showed the ability of this fabrication method to be used for processing novel hybrid structures with natural and synthetic components. The hybrid collagen scaffolds showed desirable properties for articular cartilage tissue engineering applications, although the composition of the hybrid should be studied more
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detailed to find out an optimal scaffold structure. These scaffolds had high porosity and interconnected pores, improved mechanical strength compared to plain collagen scaffolds, good water uptake, and penetration of chondrocytes indicating this kind of collagen/PLA hybrids to be potential scaffolds for cartilage tissue engineering. Acknowledgments We would like to thank Antti Aula for microCT image editing. The financial support of the Finnish Funding Agency for Technology and Innovation (TEKES) is greatly appreciated (Grant 3110/31/08).
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