Cartilage regeneration by culturing chondrocytes in scaffolds grafted with TATVHL peptide

Colloids and Surfaces B: Biointerfaces 93 (2012) 235–240

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Cartilage regeneration by culturing chondrocytes in scaffolds grafted with TATVHL peptide Yung-Chih Kuo ∗ , Cheng-Chin Wang Department of Chemical Engineering National Chung Cheng University Chia-Yi, Taiwan 62102, Republic of China

a r t i c l e

i n f o

Article history: Received 15 December 2011 Received in revised form 10 January 2012 Accepted 11 January 2012 Available online 21 January 2012 Keywords: TATVHL peptide Regeneration Cartilage Chondrocyte Scaffold

a b s t r a c t Formation of neocartilage is a critical issue in contemporary regenerative medicine. This study presents the generation of tissue engineering cartilage in TATVHL peptide-grafted scaffolds. Bovine knee chondrocytes were seeded in TATVHL peptide-grafted scaffolds and cultured in a spinner bioreactor. The results revealed that surface TATVHL peptide enhanced the adhesion of bovine knee chondrocytes in scaffolds. However, an increase in the concentration of TATVHL peptide in scaffolds (up to 20 ␮g/mL) did not cause an evident variation in the cell viability. Surface TATVHL peptide was effective in promoting the quantity of cartilaginous components in constructs after dynamic cultivation. Biochemical assay, scanning electron microscope images, and histological staining demonstrated that surface TATVHL peptide accelerated the proliferation of bovine knee chondrocytes in constructs. In addition, the secretion of glycosaminoglycans and production of collagen in TATVHL peptide-grafted constructs were faster than those in TATVHL peptide-free constructs. TATVHL peptide can be a promising bioactive molecule to improve chondrogenesis in porous biomaterials. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Cartilage tissue engineering is one of the focused biomedical fields in the recent decade [1,2]. This development resulted from the surgical demand for repairing degenerated and injured joint, especially for knee diarthrosis. For native cartilage, the tissue is typically composed of a few chondrocytes and plenty of extracellular matrices [3,4]. The cartilaginous extracellular matrix possesses a number of particular biophysical properties, rendering a unique ionic permeability and nutrient transport system [5,6]. In fact, glycosaminoglycans, one of the key components of extracellular matrix in cartilage, are unbranched heteropolysaccharides. The chains of glycosaminoglycans bind covalently to a central protein to form proteoglycan. Since proteoglycans are crucial to arrangement of the extracellular matrix, the quantity of glycosaminoglycans in a cartilage becomes an important index to assess the tissue functionality [7,8]. In addition, interconnected collagen network in articular cartilage can grasp negatively charged proteoglycans and play a fateful role in the force-bearing configuration [9,10]. Therefore, the understanding of chondrocytes, glycosaminoglycans, and collagen are regarded as the most essential issue in neocartilage-related topics [11,12].

Abbreviations: ECM, extracellular matrix; TAT, transactivator of transcription; TATVHL peptide, YGRKKRRQRRRDTLKERCLQVVRSLVK; VHL, von Hippel–Lindau. ∗ Corresponding author. Tel.: +886 5 272 0411x33459; fax: +886 5 272 1206. E-mail address: [email protected] (Y.-C. Kuo). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.01.012

Modification of bioactive molecules on scaffolding substrate is an efficacious strategy for accelerating adhesion and growth of chondrocytes [13,14]. This is because the pore walls of scaffolds are the first contact regions with suspended chondrocytes. In our previous studies, various surface active ingredients, including fibronectin, hydroxyapatite, polyethyleneimine, heparin, lamininderived peptide, albumin, elastin, and poly-l-lysine have been modified on porous biomaterials for improving the formation of neocartilage [6,15–21]. In addition, von Hippel–Lindau (VHL) peptide is a newly developed polypeptide. It has been used in differentiating neuronal progenitor cells, generating cells with dopaminergic phenotype from skin-derived precursors, and repairing the injured spinal cord [22–24]. VHL gene also could guide the differentiation of neuronal progenitor cells toward dopaminergic neurons to reverse the symptoms of Parkinson’s disease in humans [25]. Moreover, transactivator of transcription (TAT) peptide contains 11 amino acids for transduction. When TAT peptide conjugated with VHL peptide, this combined TATVHL peptide could be transplanted and regulated dopamine neuron-like cells in Parkinson’s disease model rats [23]. Besides the high efficacy in neuronal regeneration, TATVHL peptide may have a particular function on chondrogenesis. The aim of this study is to synthesize TATVHL peptide-grafted scaffolds for cartilage regeneration. The capability of TATVHL peptide for chondrocytic adhesion and/or chondrogenesis has not been demonstrated in the literature. Therefore, the potential of TATVHL peptide in cartilage tissue engineering required investigating. We showed the effect of grafted TATVHL peptide on the

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adhesion, viability, and proliferation of chondrocytes and on the stimulation to synthesize extracellular matrix. 2. Experimental 2.1. Preparation of TATVHL peptide-grafted scaffolds The ternary polyethylene oxide/chitin/chitosan scaffolds were fabricated by the method described previously [8]. Briefly, 3% (w/v) polyethylene oxide (Sigma, St. Louis, MO), chitin (Sigma), and chitosan (Sigma) gels were mixed in a volume ratio 23:26:51 and crosslinked with genipin (Challenge Bioproducts, Taichung, Taiwan) under 365 nm of ultraviolet (UV, Spectronics, Westbury, NY). The wet matrix was frozen in an ultra-low temperature freezer (Sanyo, Osaka, Japan), lyophilized in a freeze-dryer (Eyela, Tokyo, Japan), immersed in liquid nitrogen, and trimmed by a cryostat microtome (Slee, Mainz, Germany). One polyethylene oxide/chitin/chitosan scaffold of 5 × 5 × 4 mm was injected with 90 ␮L of Dulbecco’s phosphate buffered saline (DPBS, Sigma) containing 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (Acros, Morris, NJ) and Nhydroxysuccinimide (Acros), washed with DPBS, dehumidified at 40 ◦ C, injected with 90 ␮L of DPBS containing TATVHL peptide (Kelowna International Scientific, Taipei, Taiwan), centrifuged at centrifuged at 130 × g for 5 min, placed at room temperature for 12 h, washed with DPBS, dried at 40 ◦ C for 12 h, and weighed. The quantity of TATVHL peptide in a scaffold was determined with QuantiPro bicinchoninic acid assay kit (Sigma). Briefly, 150 ␮L of working reagent and 150 ␮L of DPBS containing non-grafted TATVHL peptide in 96-well polystyrene microplates (Nalge Nunc, Rochester, NY) were reacted in an incubator (NuAire, Plymouth, MN) with 95% relative humidity and 5% CO2 at 37 ◦ C for 2 h. The resultant solution was analyzed by an ultraviolet-visible detector (Bio-Tek, Winooski, VT) at 562 nm. Thus, the residual TATVHL peptide in the solution after grafting reaction was estimated. The weight of grafted TATVHL peptide in scaffolds was evaluated by subtracting the weight of total peptide from the weight of residual peptide. 2.2. Isolation of bovine knee chondrocytes Hyaline cartilages were harvested from forearm knees of calves at a local abattoir within 30 min of slaughter. Gathered cartilages were immersed in ice-bathed DPBS containing 1% antibiotic-antimycotic solution (Sigma), transported to our laboratory immediately, cut into tiny cubes about 1 mm3 , digested with 0.18% type II collagenase (Sigma) in the CO2 incubator for 24 h, and centrifuged at 420 × g for 5 min. The pellet was resuspended in 1 mL of Dulbecco’s modified Eagle’s medium (DMEM, Sigma). The concentration and viability of bovine knee chondrocytes were assessed by trypan blue (Sigma) exclusion with a hemocytometer (Neubauer, Marienfeld, Germany) under a phase-contrast biological microscope (Motic, Richmond, BC, Canada). The freshly isolated bovine knee chondrocytes were resuspended in DMEM containing 10% dimethyl sulfoxide (J.T. Baker, Phillipsburg, NJ) and 10% fetal bovine serum (Sigma), refrigerated at 4 ◦ C for 30 min, −4 ◦ C for 2 h, −20 ◦ C for 2 h, and −80 ◦ C for 2 h, and conserved in liquid nitrogen. The cryopreserved bovine knee chondrocytes were unfrozen at 37 ◦ C for 1 min and applied in the following study. 2.3. Adhesion of bovine knee chondrocytes Bovine knee chondrocytes were injected into the scaffolds of 5 × 5 × 2 mm at a density of 5.1 × 106 cells/construct. The constructs were placed in the CO2 incubator for 1 h and 8 h

and immersed in culture medium with a volume of 0.2 mL per construct for releasing bovine knee chondrocytes. The quantity of free cells was evaluated by the hemocytometer and the phase-contrast biological microscope. The adhesion efficiency of bovine knee chondrocytes in TATVHL peptide-grafted scaffolds was defined as [(5.1 × 106 − number of released bovine knee chondrocytes)/(5.1 × 106 )] × 100%. 2.4. Viability of bovine knee chondrocytes After adhesion for 4 h, one construct was incubated in 0.4 mL of culture medium, placed in the CO2 incubator for 8 h, washed with DPBS, centrifuged at 420 × g, reacted with 100 ␮L of MTT solution with a concentration of 5 mg/mL in darkness, placed at room temperature for 4 h, and mixed with 900 ␮L of MTT solubilization solution in darkness for 10 min. The liquid sample was placed in 96-well polystyrene microplates (Nalge Nunc, Rochester, NY) and determined by an ultraviolet-visible detector (Bio-Tek, Winooski, VT) for absorbance at 570 nm. The viability of bovine knee chondrocytes in TATVHL peptide-grafted scaffolds was calculated by A12 /A4 × 100%, where A4 and A12 are the light absorbance after adhesion for 4 h and that after incubation for 8 h, respectively. 2.5. Culture of bovine knee chondrocytes Bovine knee chondrocytes in TATVHL peptide-grafted scaffolds were cultured by the method described previously [4]. Briefly, bovine knee chondrocytes were seeded in a scaffold at a density of 4.8 × 106 cells/construct, cultured with 250 mL of DMEM supplemented with 50 ␮g/mL penicillin G (Sigma), 50 ␮g/mL streptomycin sulfate (Sigma), 10 mM 4-2-hydroxyethyl1-piperazineethanesulfonic acid (J.T. Baker), 50 ␮g/mL l-(+)ascorbic acid (J.T. Baker), 10% fetal bovine serum, 0.1 mM MEM non-essential amino acid (Sigma), and 0.4 mM l-proline (Sigma), stirred in a spinner system (Integra Biosciences, Wallisellen, Switzerland) at 60 rpm, and placed in the CO2 incubator. A half the medium was replaced with fresh medium every 3 days. 2.6. Surface morphology of cultured construct The microstructure of cultured constructs was visualized by an SEM (Jeol, Tokyo, Japan). The porous biomaterials were sliced, washed with DPBS, treated with 2.5% glutaraldehyde (Fluka, Buchs, Switzerland) for 4 h, dehydrated with ethanol (Tedia, Fairfield, OH) stepwise from 70% to 99.8% (v/v), vacuum-dried for 10 min, and sputter-coated with gold at 5 mA associated with an accelerating voltage at 1 kV for 10 min. 2.7. Biochemical analysis One construct was digested with 95.24 ␮g papain (Sigma), 55 mM trisodium citrate (Hanawa, Osaka, Japan), 150 mM sodium chloride (J.T. Baker), 5 mM cysteine (Fluka), and 5 mM ethylenediaminetetraacetic acid (Riedel-de Haën) in 2 mL of tris (hydroxymethyl) aminomethane (Riedel-de Haën, Seelze, Germany) at 60 ◦ C for 24 h. The digested solution for quantifying bovine knee chondrocytes was reacted with Hoechst No. 33258 (Sigma), analyzed by a fluorescent spectrophotometer (F-4500, Hitachi, Tokyo, Japan) at 365 nm (excitation) and 458 nm (emission), and calibrated with suspended bovine knee chondrocytes. The weight of bovine knee chondrocytes was estimated by the fact that the average weight of a chondrocyte is 1 ng [26]. The digested solution for quantifying glycosaminoglycans was reacted with

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13

90

80

70

5

4 9

3

5 2

1

Percentage of ECM (% dry construct weight)

Percentage of chondrocytes (% dry construct weight)

Adhesion efficiency in scaffolds (%)

100

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1 0

5

10

15

20

Concentration of TATVHL peptide ( μ g/mL)

60 0

5

10

15

20

Concentration of TATVHL peptide (μg/mL) Fig. 1. Effect of TATVHL peptide on the adhesion of bovine knee chondrocytes in scaffolds. (): Adhesion for 1 h; (): adhesion for 8 h. n = 3.

Fig. 2. Enhancement in chondrogenesis of bovine knee chondrocytes in scaffolds grafted with TATVHL peptide. (): Bovine knee chondrocytes; (): glycosaminoglycans (one of ECMs); (): collagen (one of ECMs). n = 3.

3. Results and discussion 1,9-dimethyl-methylene blue (Sigma), analyzed by an ultravioletvisible detector (Bio-Tek, Winooski, VT) at 525 nm, and calibrated with chondroitin sulfate (Sigma). The digested solution for quantifying collagen was acidolyzed with hydrochloric acid of 6 N (Hanawa), oxidized with n-chloro-p-toluene-sulfonamide (Sigma), reacted with 4-dimethylamino-benzaldehyde (Sigma), analyzed by an ultraviolet-visible detector (Bio-Tek) at 550 nm, and calibrated with l-4-hydroxyproline (Fluka). The weight of collagen was calculated by the relation that the average ratio of hydroxyproline to collagen is 0.1 [27].

3.1. Adhesion in TATVHL peptide-grafted scaffolds Fig. 1 shows the effect of the concentration of TATVHL peptide on the adhesion efficiency of bovine knee chondrocytes in scaffolds.

2.8. Staining of cartilaginous components The constructs were fixed with 10% (v/v) neutral buffered formalin (Sigma) for 1 h, dehydrated with increasing ethanol concentration from 70% to 99.8% (v/v), immersed in o-xylene (Fluka) for 3 h, embedded in paraffin, and sliced by a sliding microtome (Leica, Nussloch, Germany) into samples with thickness of 4 ␮m. Paraffin was removed by treating with o-xylene. For H&E staining, the samples were immersed in hematoxylin (Sigma) for 2 min, rinsed with 0.5% hydrochloric acid for 1 s, immersed in eosin (Sigma) for 2 min. For staining of glycosaminoglycans, the samples were immersed in safranin-O (Sigma) overnight. For staining against type II collagen, the samples were immersed in 30% (w/w) hydrogen peroxide (Sigma) for 10 min, treated with serum-blocking solution (Zymed, South San Francisco, CA) for 10 min, reacted with primary antibody of collagen II Ab-2 (Lab. Vision, Fremont, CA) for 1 h, incubated with the secondary antibody (Zymed) for 10 min and peroxidase-conjugated tertiary antibody (Zymed) for 10 min, and stained with 3,3 -diaminobenzidine-plus substrate kit (Zymed) for 5 min in complete darkness. The staining images were observed under an inverted phase-contrast fluoromicroscope (TE2000-U, Nikon, Tokyo, Japan) at 1000×. 2.9. Statistics The data shown are mean ± standard deviation. Statistical significance of the datum groups was assessed by a one-way analysis of variance (ANOVA) followed by Tukey’s HSD test with a significant level of p < 0.05.

Fig. 3. Surface morphology of the cultivated constructs. (a) Construct without TATVHL peptide; (b) construct with TATVHL peptide of 10 ␮g/mL. n = 3.

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Fig. 4. Staining images of cartilaginous components after cultivation over 4 weeks. (a) H&E stain, construct without TATVHL peptide; (b) H&E stain, construct with TATVHL peptide of 10 ␮g/mL; (c) safranin-O stain, construct without TATVHL peptide; (d) safranin-O stain, construct with TATVHL peptide of 10 ␮g/mL; (e) type II collagen stain, construct without TATVHL peptide; (f) type II collagen stain, construct with TATVHL peptide of 10 ␮g/mL. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)

As indicated in this figure, the adhesion efficiency enhanced when the concentration of TATVHL peptide increased. In addition, the adhesion efficiency increased almost in a linear relation with the concentration of TATVHL peptide from 0 to 20 ␮g/mL. This suggested that surface TATVHL peptide played a proper and effective role in recruiting bovine knee chondrocytes to the pore surface of scaffolds. This was because the colonizing cells were likely to migrate and yield space for further chondrocytic occupation and form compact cell structure on the scaffolding surface grafted with TATVHL peptide. As revealed in Fig. 1, the adhesion efficiency for 8 h was higher than that for 1 h. This suggested that a longer period could benefit the adhesion of bovine knee chondrocytes in TATVHL peptide-grafted surface. In fact, an adhesion for 1 h did not reach the attaching saturation for cells onto the surface. However, an adhesion for 8 h could be sufficient for bovine knee chondrocytes to recognize the surface motifs, adopt the ligands, and accommodate themselves in scaffolds. Especially, when the

porous biomaterials grafted with 20 ␮g/mL of TATVHL peptide, a high seeding (higher than 95% adhesion efficiency) of bovine knee chondrocytes was obtained after incubation for 8 h. This result demonstrated that surface TATVHL peptide could induce a certain attractive pathways toward chondrocytic occupation [28,29].

3.2. Cell viability in TATVHL peptide-grafted scaffolds Table 1 lists the viability of bovine knee chondrocytes in scaffolds. The variation in cell viability was not obvious when the concentration of TATVHL peptide increased. This was mainly because the contact time for bovine knee chondrocytes and scaffolds was not long enough. The incubation for 12 h could avoid cell duplication and was applied in the cytotoxicity experiment. Therefore, the variation in the concentration of TATVHL peptide in biocompatible substrate, including polyethylene oxide, chitin, and

Y.-C. Kuo, C.-C. Wang / Colloids and Surfaces B: Biointerfaces 93 (2012) 235–240 Table 1 Viability of bovine knee chondrocytes in scaffolds grafted with TATVHL peptide. n = 3. Concentration of TATVHL peptide (␮g/mL)

Viability of bovine knee chondrocytes (%)

0 5 10 15 20

92.74 94.37 93.15 94.72 95.68

± ± ± ± ±

4.57 2.89 4.16 4.85 2.93

chitosan, could hardly alter the adaption of bovine knee chondrocytes in scaffolds within this time frame. 3.3. Assessment of chondrogenesis in TATVHL peptide-grafted constructs Fig. 2 shows the cartilaginous components in constructs after cultivation over 4 weeks. As indicated in this figure, an increase in the concentration of TATVHL peptide enhanced the quantity of bovine knee chondrocytes, glycosaminoglycans, and collagen in constructs. These results suggested that hybridization of the substrate matrix with functional TATVHL peptide could astonishingly improved the biological performance of bovine knee chondrocytes in constructs. As revealed in Fig. 2, the slope of the curve in cell number increased when the concentration of TATVHL peptide increased. This suggested that TATVHL peptide could associate with chondrocyte-secreted growth factors to synergistically stimulate the growth of bovine knee chondrocytes in constructs. As displayed in Fig. 2, an increase in the concentration of TATVHL peptide accelerated the accumulation of extracellular glycosaminoglycans in a fashion close to linear growth. The secretion of glycosaminoglycans could be prosperous in constructs. However, glycosaminoglycans might be released from the boundaries of constructs into the culture medium [30]. Therefore, the capability of biomaterials to maintain glycosaminoglycans in constructs was not as high as that to maintain chondrocytes when surface TATVHL peptide increased at high concentrations such as 10–20 ␮g/mL. As exhibited in Fig. 2, the enhancement in the quantity of collagen with increasing the concentration of TATVHL peptide was similar to that in chondrocytes. This was because the quantity of produced collagen was fewer than that of secreted glycosaminoglycans. In addition, collagen could be probably synthesized to form a dense network around bovine knee chondrocytes [31]. Therefore, the release of collagen into the culture medium during cultivation might be minor. 3.4. Morphological images of cultured constructs Fig. 3 shows the typical SEM micrographs of constructs after cultivation. As indicated in Fig. 3(a), bovine knee chondrocytes in forms of tiny clusters (cell aggregates) were scattered on the pore surface. These chondrocytes attached on the substrate displayed a characteristic oval shape. In addition, the cell distribution on the surface was quite uniform. As revealed in Fig. 3(b), a compact colony of bovine knee chondrocytes appeared in the middle bottom of the image. These cells could contact with each other and extend densely in the pore. The number of bovine knee chondrocytes in Fig. 3(b) is larger than that in Fig. 3(a), indicating that the graft of TATVHL peptide favored chondrogenesis. Thus, the SEM morphology shown in Fig. 3 was consistent with the quantitative results shown in Fig. 2. 3.5. Staining images of generated neocartilage in TATVHL peptide-grafted constructs Fig. 4 shows the staining photomicrographs of regenerated bovine knee chondrocytes and extracellular matrices. As indicated

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in Fig. 4(a) and (b), the purple dots were the multiplied chondrocytes. The number of bovine knee chondrocytes in Fig. 4(b) was larger than that in Fig. 4(a), suggesting that surface TATVHL peptide could promote the mitosis of chondrocytes. As revealed in Fig. 4(c) and (d), the crimson patches were the secreted glycosaminoglycans and the red stripes were the pore walls of scaffolds. The intensity of crimson area in Fig. 4(d) was stronger than that in Fig. 4(c), suggesting that surface TATVHL peptide favored the synthesis of glycosaminoglycans. As exhibited in Fig. 4(e) and (f), the brown speckles were the produced type II collagen. The quantity of brown stains in Fig. 4(f) was larger than that in Fig. 4(e), suggesting that surface TATVHL peptide could upregulate the generation of type II collagen. The staining images in Fig. 4 were consistent with the biochemical assay in Fig. 2. In addition, cartilaginous extracellular matrix is sustained by continuous secretion of chondrocytes. However, degeneration and mechanical injury yield, respectively, a gradual and sudden disappearance of tissue. These damages may further lead to the proteolytic decomposition of extracellular matrix and alter the behavior of articular stress [32]. 4. Conclusions TATVHL peptide-grafted scaffolds were applied to produce neocartilage. Surface TATVHL peptide could favor the adhesion of bovine knee chondrocytes in scaffolds without an apparent variation in cell viability. The formation of cartilaginous components, including chondrocytes, glycosaminoglycans, and collagen, in TATVHL peptide-grafted constructs was faster than that in TATVHL peptide-free constructs. TATVHL peptide-grafted scaffolds can be competent biomaterials for regenerating cartilage in future in vivo trials. Acknowledgment This work was supported by the National Science Council of the Republic of China. References [1] J.S. Temenoff, A.G. Mikos, Review: tissue engineering for regeneration of articular cartilage, Biomaterials 21 (2000) 431–440. [2] Y.C. Kuo, Y.T. Tsai, Inverted colloidal crystal scaffolds for uniform cartilage regeneration, Biomacromolecules 11 (2010) 731–739. [3] P.K. Levangie, C.C. Norkin, Joint Structure and Function: A Comprehensive Analysis, 5th ed., F.A. Davis Co., Philadelphia, 2011. [4] Y.C. Kuo, C.Y. Chung, Application of bovine pituitary extract and polyglycolide/poly(lactide-co-glycolide) scaffold to the cultivation of bovine knee chondrocytes, Biotechnol. Prog. 21 (2005) 1708–1715. [5] B. Obradovic, R.L. Carrier, G. Vunjak-Novakovic, L.E. Freed, Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage, Biotechnol. Bioeng. 63 (1999) 197–205. [6] Y.C. Kuo, I.N. Ku, Effects of gel concentration, human fibronectin, and cation supplement on the tissue-engineered cartilage, Biotechnol. Prog. 23 (2007) 238–245. [7] B.O. Enobakhare, D.L. Bader, D.A. Lee, Quantification of sulfated glycosaminoglycans in chondrocyte/alginate culture by use of 1,9-dimethylmethylene blue, Anal. Biochem. 243 (1996) 189–191. [8] Y.C. Kuo, I.N. Ku, Cartilage regeneration by novel polyethylene oxide/chitin/chitosan scaffolds, Biomacromolecules 9 (2008) 2662–2669. [9] L.E. Freed, G. Vunjak-Novakovic, Tissue engineering of cartilage, in: J.D. Brozino (Ed.), The Biomedical Engineering Handbook, Vol. II, 2nd ed., CRC Press, Boca Raton, 2000, pp. 124–127. [10] Y.C. Kuo, Y.R. Hsu, Tissue-engineered polyethylene oxide/chitosan scaffolds as potential substitutes for articular cartilage, J. Biomed. Mater. Res. A 91 (2009) 277–287. [11] J.K.F. Suh, H.W.T. Matthew, Application of chitosan based polysaccharide biomaterials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589–2598. [12] Y.C. Kuo, C.C. Wang, Effect of bovine pituitary extract on the formation of neocartilage in chitosan/gelatin scaffolds, J. Taiwan Inst. Chem. Eng. 41 (2010) 150–156. [13] Y.L. Cui, A.D. Qi, W.G. Liu, X.H. Wang, H. Wang, D.M. Ma, K.D. Yao, Biomimetic surface modification of poly(l-latic acid) with chitosan and its effects on articular chondrocytes in vitro, Biomaterials 24 (2003) 3859–3868.

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