Composite System of PLCL Scaffold and Heparin-Based Hydrogel for

Article pubs.acs.org/Biomac

Composite System of PLCL Scaffold and Heparin-Based Hydrogel for Regeneration of Partial-Thickness Cartilage Defects Mihye Kim,† Bohee Hong,† Jongman Lee,†,⊥ Se Eun Kim,‡ Seong Soo Kang,‡ Young Ha Kim,§ and Giyoong Tae*,† †

School of Materials Science and Engineering and Department of Nanobio Materials and Electronics, Gwangju Institute of Science and Technology, Gwangju, 500-712, Korea ‡ College of Veterinary Medicine, Chonnam National University, Gwang-ju, 500-757, Korea § Department of Chemistry, Chung-Ang University, 221 Heukseok-dong, Dongiak-gu, Seoul 156-755, Korea S Supporting Information *

ABSTRACT: Delivering isolated chondrocytes with matrix is a promising approach to promote the cartilage repair. The present study attempted to combine the advantages of porous scaffold and hydrogel in delivering chondrocytes to partial-thickness cartilage defects. An electrospun, gelatin-incorporated PLCL scaffold mechanically similar to natural cartilage was fabricated, and chondrocytes were seeded using an injectable heparin-based hydrogel for efficient cell seeding. The scaffold/hydrogel composite showed more enhanced expression of chondrogenic genes and production of GAGs than those prepared without hydrogel. In addition, significant cartilage formation showing good integration with surrounding, similar to natural cartilage, was observed by scaffold/hydrogel composite system in partial-thickness defects of rabbit knees while no regeneration was observed in control defects. Although no exogenous chondrogenic factors were added, it was evident that the scaffold/hydrogel composite system was highly effective and better than the scaffold alone system without hydrogel for cartilage regeneration both in vitro and in vivo.

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INTRODUCTION Articular cartilage has a limited regeneration potential due to its avascular nature and insufficient numbers of chondrocyte.1,2 Although several surgical or nonsurgical approaches have been employed to the injured cartilage, the repair of articular cartilage still remains as an important problem and damaged cartilage can lead to further degeneration such as osteoarthritis.3,4 Recently, many reports showed the beneficial effect of biomaterial-based tissue engineering with sufficiently large numbers of chondrocyte.5,6 The desired biomaterial in cartilage tissue engineering is expected to enhance the cell proliferation, maintain the chondrogenic functions, and induce cartilagespecific ECM formation.7 Biodegradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid (PLGA), poly(ε-caprolactone) (PCL), and poly(L-lactide-co-ε-caprolactone) (PLCL) are the major materials applied for cartilage tissue scaffold.8,9 These materials can easily form solid, stable, and porous structures with good mechanical properties and controlled degradation rates via various methods such as particle leaching, phase separation, and electrospinning.8,10 Among these methods, electrospinning has an advantage of producing nanostructured fibers in a simple and economic way.11 Randomly organized electrospun nanofibers can physically mimic the ECM structures of native tissue.12 PLCL has © 2012 American Chemical Society

been applied as a mechano-stimulating tissue engineering scaffold for tendon,13 blood vessel,14 and cartilage,15 due to its highly flexible and elastic properties. In a previous study, we developed the gelatin-incorporated PLCL electrospun nanofiber scaffolds that showed a significantly improved cell proliferation and mechanical strength compared to a bare PLCL scaffold.10 However, solid polymeric scaffolds are associated with many problems including relatively low cellseeding efficiency, nonuniform cell distribution, increased cell dedifferentiation, and poor retention of newly developed ECM.16 Especially, a limited cell penetration across the sheet is the main problem of scaffolds made by electrospinning method.11 Several attempts have been made to increase the pore size of electrospun scaffold, thus to enhance the cell penetration into the scaffold by combining electrospinning with salt-leaching,17 by blending with nano- and microfibers,11 by electrospinning onto ice crystals,18 or by using the melt electrospinning.19 In this paper, we prepared a roll-type scaffold as a new scaffold system for cartilage regeneration that was made by rolling up the gelatin-incorporated PLCL electrospun fiber sheet with a thin, porous PLCL sheet as a spacer to Received: April 5, 2012 Revised: June 26, 2012 Published: July 3, 2012 2287

dx.doi.org/10.1021/bm3005353 | Biomacromolecules 2012, 13, 2287−2298

Biomacromolecules

Article

previously.10 More detailed information about the copolymerization and characterization of PLCL was previously reported.10 Then, gelatinincorporated PLCL scaffold was prepared by combining an electrospun fiber sheet and a porous sheet. Electrospun gelatin-incorporated PLCL fiber sheet was fabricated according to the procedures previously reported.10 Briefly, PLCL/gelatin solution was prepared by dissolving them in HFIP at 3 wt %. PLCL90/gelatin10 solution was delivered by a syringe pump (KDS 100, KD Scientific, Holliston, MA, U.S.A.) at a constant flow rate (2 mL/h). The blunt-ended 22 gauge needle was clamped to the positive electrode of a high voltage power supply (Chungpa EMT, Korea) generating 18 kV of electric field, and the negative electrode was connected to Al foil collector with an air gap distance of 15 cm. The PLCL/gelatin fiber sheets were treated with 50 mM EDC in ethanol for 24 h at room temperature. Then, the sheets were washed with distilled water three times. All the crosslinked fiber sheets were freeze-dried overnight and stored in a desiccator until subsequent uses. Porous PLCL sheets with high porosity were prepared by an extrusion-salt leaching method as previously described.13 A PLCL solution in chloroform at a concentration of 10 wt % was mixed with NaCl particles (425−600 μm, 90 wt % of salt content) and extruded into a sheet shape using a homemade piston extrusion tool. The residual chloroform was evaporated at room temperature for 2 days and further removed under vacuum for 1 week. A possible skin layer on the outer surface of scaffolds was removed by sandpaper treatment. After that, the electrospun gelatin-incorporated PLCL sheet was rolled up with the porous PLCL sheet, which was used as a spacer to provide space between the electrospun sheets so that cell-carrying hydrogel can be injected into the scaffold. After cutting the scaffold into a desired size (5 mm in diameter and 3 mm in height), the rolltype scaffolds were sterilized with 70% ethanol and UV irradiation for 2−3 h. The scaffolds were prewetted with PBS before cell culture. Sterilization of scaffolds was mainly done by immersing in 70% ethanol. UV sterilization was done by drying the scaffold under UV lamp in a laminar hood for cell culture. So, UV irradiation used was not strong enough to change the surface properties of the scaffold. We checked the contact angle of scaffolds before and after UV irradiation, and it did not show any change in water contact angle, supporting no apparent change in surface properties by UV treatment (Supporting Information, Figure 1). Characterization of Gelatin-Incorporated PLCL Scaffold. The morphology of gelatin-incorporated PLCL scaffold was examined by using a scanning electron microscope (SEM; Hitachi S-4700, Japan). The samples were coated with Pt using a sputter coater (Hitachi E1030, Japan), and the microscope was operated at a voltage of 5 kV. The mean fiber diameter was calculated by selecting 20 single fibers randomly observed on the SEM images. The porosity was determined by using the following equation (n = 5):

overcome the cell penetration issue. Furthermore, to increase the cell-seeding efficiency with uniform distribution and to provide the good chondrogenic environment, a cell-carrying heparin-based hydrogel was injected into the roll-type scaffold. Previously, we demonstrated that an injectable heparin-based hydrogel, formed by Michael-type reaction between thiolated heparin and diacrylated poly(ethyleneglycol) (PEG-DA),20 was an excellent matrix for cultivation of articular chondrocytes.21,22 A good cell proliferation and proper GAGs production of chondrocytes while maintaining chondrogenic phenotype were observed in the heparin-based hydrogels.21 Also, the heparinbased hydrogel was very effective to induce the redifferentiation of dedifferentiated chondrocytes under a normal cell culture condition and to form a new cartilage tissue for partialthickness cartilage defects in rabbits.22 However, a relatively poor mechanical strength of the hydrogel cannot support the heavy load, thus, might limit its application in practice. Therefore, we employed a composite system made of gelatinincorporated PLCL scaffold and heparin-based hydrogel to combine their advantages: (1) a high cell seeding efficiency and uniform cell distribution, (2) suitable mechanical strength for nature load, and (3) biomimetic environment to cells. The present study investigated the synergetic effect of this complex system as a 3-D matrix for cell proliferation and chondrogenic functions under a normal cell culture condition (no growth factors or chondrogenic components). Then the cartilage tissue regeneration was investigated by implantation of cell-carrying scaffold on partial-thickness cartilage defects in rabbits.

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MATERIALS AND METHODS

Materials. L-Lactide (LA; Purac Biochem) was used as received without purification. ε-Caprolactone (CL; Aldrich) was purified by drying over calcium hydride (CaH2) for 24 h and then distilled under reduced pressure (ca. 0.3 mmHg) at 55 °C. Stannous octoate (Sigma) was purified by vacuum distillation at 175 °C (ca. 0.2 mmHg). Toluene was completely dried by distillation over calcium hydride (CaH2). Gelatin (bovine skin, type B powder) was purchased from Sigma (St. Louis, MO, U.S.A.). 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP; Wako, Japan) was used as a solvent for dissolving PLCL and gelatin. Heparin (sodium salt, from porcine intestinal mucosa, Mw 12 kDa) was purchased from Cellsus Ins. (Cincinnati, OH, U.S.A.). Poly(ethylene glycol) diacrylate (PEG-DA, Mw 6 kDa, degree of substitution 98%) was purchased from Sunbio Inc. (Anyang, Korea). Sodium chloride, potassium phosphate monobasic, sodium phosphate dibasic, potassium chloride, glycine, papain, sodium phosphate, sodium-EDTA, cysteine-HCl, chondroitin sulfate, agarose, fast green, and Safranin-O were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). PBS (phosphate buffered saline, 0.01 mol/L PBS solution with 0.138 mol/L NaCl and 0.0027 mol/L KCl, pH = 7.4) was prepared with potassium phosphate monobasic and sodium phosphate dibasic. As a cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM) with 4500 mg/L of D-glucose and glutamine, fetal bovine serum (certified), penicillin G, and streptomycin were used (all from Gibco, NY, U.S.A.). TrypsinEDTA (ethylenediaminetetraacetic acid; 0.25%) was also purchased from Gibco. Trireagent was obtained from Molecular Research Center Inc. (Cincinnati, OH, U.S.A.). Antirabbit TGF-it antibody was obtained from Novus International Inc. (St. Charles, MO, U.S.A.). Anti-TGF-β1 detection antibody, Avidin-HRP conjugate, and ABTS liquid substrate were purchased from PeproTech (Rocky Hill, NJ, U.S.A.). Rabbit antigoat IgG-H&L (TR) was obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA, U.S.A.). Preparation of Gelatin-Incorporated PLCL Scaffold. To prepare the PLCL scaffold, PLCL (typical composition was 60:40 mol %) was synthesized by reacting L-lactide (0.555 mol) and εcaprolactone (0.37 mol) in a 250 mL glass ampule, as reported

porosity(%) = [(Vt − Vrp)/Vt] × 100(%) where Vt is the total volume and Vrp is the volume after removing porosity of the scaffold. The compressive moduli of scaffold samples (5 mm in diameter, 3 mm in height) were measured by using a uniaxial testing machine (Instron 5566, company name, city, state, nation) with a 1 kN load cell under a cross-head speed of 1 mm/min (n = 5). The compressive moduli of scaffold samples were obtained from the initial linear slope in the elastic region of stress−strain curves. Preparation of Injectable Heparin-Based Hydrogel. Heparinbased hydrogels were prepared by a Michael-type addition reaction between thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA).20 Thiol derivative of heparin (Hep-SH) with 40% conversion of COOH group to thiol group and 6 kDa PEG-DA (1:1 molar ratio of thiol group and acrylate group) were dissolved in cell culture medium to make 10% (w/v) solution and filtered through a 0.2 μM sterile filter to sterilize the precursor solution. Gelation occurred within 10 min at 37 °C in pH 7.4.20 Chondrocyte Culture in PLCL Scaffold/Hydrogel Composite System. Rabbit articular chondrocytes were isolated from the cartilage 2288

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of two-week-old New Zealand white rabbits (Fine Corp., Gwangju, Korea) as described previously.23 Briefly, 40% thiolated Hep-SH and 6 kDa PEG-DA were dissolved in cell culture medium to make 10% (w/ v) solution and filtered through a 0.2 μM sterile filter. Chondrocytes (passage 1, 1 × 107 cells/mL) were mixed with the precursors of the hydrogel. The mixture was then injected into the roll-type scaffold before gelation in situ. Two different groups (Group I, scaffold alone without hydrogel, “PLCL scaffold”; and Group II, hydrogel-scaffold composite, “PLCL scaffold + Hydrogel”) were used. Then, the scaffolds were placed on the bottom of a 96-well plate (Corning, VWR, Fontenay-sous-Bois, France) and incubated for 2 h at 37 °C for gel formation and stabilization. Then, 200 μL of cell culture medium was added on the wells containing scaffolds and cultured in a standard cell culture condition (at 37 °C with 5% CO2 supply). The medium was replaced with the fresh one after every 2−3 days. Cell Proliferation in PLCL Scaffold/Hydrogel Composite System. The proliferation of chondrocytes inside the composite system was assessed by measuring cell metabolic activity with Cell Proliferation Reagent WST-1 assay (Roche Ltd., Basel, Switzerland). The WST-1 is a tetrazolium salt that is converted into the soluble formazan salt by succinate-tetrazolium reductase of the respiratory chain of active mitochondria of proliferating viable cells.24 Briefly, 15 μL of WST-1 reagent was added to each of the 96-well plate containing samples with 150 μL of culture medium at predetermined culture time interval. The cells were incubated at 37 °C in humidified atmosphere of 5% CO2 for 4 h in the dark. Absorbance at 450 nm was measured by using a scanning multiwell spectrophotometer (FL600, Bio-Tek, Vermont, U.S.A.). Analyses of Real-Time PCR. Total RNAs in the sample was isolated using Trireagent. Purified RNA was quantified with a spectrophotometer and also checked by the electrophoresis in 1% agarose gel. A total of 1 μg/μL of RNA was reverse-transcribed with ImProm-IITM reverse transcriptase (Promega, WI, U.S.A.) according to the manufacturer’s protocol25 by using iQ5 real-time PCR detection system (Bio-Rad, CA, U.S.A.) for 5 min annealing at 25 °C and for 60 min extending the first strand at 42 °C, as described previously.26 The final cDNAs were then subjected to real-time PCR to determine the expression of genes for type I collagen, type II collagen, and aggrecan. PCR reaction was carried out with specific primers and iQ SYBR Green Supermix (Bio-Rad) containing dNTPs, iTaq DNA polymerase, MgCl2, and SYBR Green I fluorescein for an initial denaturation at 95 °C for 5 min, followed by 40 cycles of PCR. Each cycle was proceeded at 95 °C for 20 s, 62 °C for 20 s, and 72 °C for 40 s, in accordance with the manufacturer’s recommendation. Relative quantification was calculated by using 2 − (ΔΔCt) method and all gene expressions were normalized to the expression of glyceraldehyde-3-phosphatase dehydrogenase (GAPDH), a house-keeping gene. The sequence of primers for GAPDH, type I collagen, type II collagen, and aggrecan were as follows: GAPDH: 5-TCACCATCTTCCAGGAGCGA-3, 5CACAATGCCGAAGTGGTCGT-3; type I collagen: 5GGCTTTCCTGGAGAGAAAGG-3, 5-ATAGAACCAGCAGGGCCAGG-3; type II collagen: 5-AACACTGCCAACGTCCAGAT-3, 5CTGCAGCACGGTATAGGTGA-3; PCR products were electrophoresed in 1% agarose gels and visualized after ethidium bromide staining. Quantification of Sulphated-Glycosaminoglycans (GAGs). The amounts of sulphated-glcosaminoglycans (GAGs) in the samples were estimated by using dimethylmethylene blue (DMMB) metachromatic assay (Sigma).27 In brief, the color reagent was prepared by dissolving 16 mg of DMMB in a solution (1000 mL, pH 3.0) containing 3.04 g of glycine, 2.37 g of NaCl, and 95 mL of 0.1 mol/L HCl. The chondrocyte-cultured PLCL scaffold or PLCL scaffold/hydrogel were immersed in a digestion solution with 500 μg/ mL papain in 0.1 M sodium phosphate (pH 6.2) with 5 mM Na2EDTA and 5 mM cysteine-HCl. After overnight incubation at 60 °C for digestion, they were centrifuged at 13000 g for 15 min. The supernatant was collected and stored at −20 °C until the GAG assay was performed. Total amounts of GAGs were determined by measuring an absorbance at 525 nm and comparing with a standard curve of shark chondroitin sulfate in the range of 0−50 μg/mL.

Absorbance values of negative control groups (PLCL scaffold without cells and PLCL scaffold/hydrogel without cells at 525 nm) were subtracted for each group to consider only the accumulated GAG amounts by cell culture. Histology and Immunohistochemistry. The characteristics of chondrocytes cultured in the PLCL scaffold or PLCL scaffold/ hydrogel were analyzed colorimetrically after Safranin-O/fast green staining. Briefly, samples were embedded in OCT compound (TissueTeks; Sakura Finetek, Tyoto, Japan) and frozen. The cell-cultured scaffold or scaffold composition was cut into 10 μm-thick sections at −20 °C, and mounted on glass slides. The Safranin-O/fast green staining was done by the modified protocol as previously described.28 Evidence of chondrogenesis was also assayed based on the immunohistochemical detection of type II collagen and aggrecan, the primary matrix proteins of articular cartilage, and type I collagen as a negative control. Cryo-sectioned samples (10 μm) after culturing were placed in a 10% formalin solution for 30 min, and were fixed on the slide. After washing with PBS, the samples were blocked with 3% H2O2 for 10 min. The sections were then placed in 1% BSA for 30 min at 37 °C. The primary antibodies (Chemicon, CA, U.S.A.), such as type II collagen, aggrecan, and type I collagen, were applied and allowed to react overnight at 4 °C. The sections were then incubated with the secondary biotinylated antimouse/antigoat antibodies and peroxidase labeled streptavidin (LSAB2 System, Dako Corp, CA, U.S.A.). After color development using Histonstain-SP with AEC (Dako Corp., city, nation), the coverslips were applied and the slides were optically monitored (TE2000-U, Nikon Co., Tokyo, Japan). Measurement of Endogenous TGF-β1 in PLCL Scaffold/ Hydrogel Composite System. Characterization of endogenous TGF-β1 accumulated inside the cell cultured PLCL scaffold or PLCL scaffold/hydrogel construct was carried out with TGF-β1 immunofluorescence staining. For TGF-β1 immunofluorescence staining, cryosectioned samples (10 μm) after fixation were incubated with antirabbit TGF-β1 antibody (diluted to a concentration of 5 μg/ mL) and rabbit antigoat IgG-H&L (Texas Red; diluted in 1:200 in blocking solution). Samples were also counterstained with DAPI to determine the location of nuclei. Samples stained without primary antibody was used as a negative control. Stained cells were visualized and imaged using a fluorescence microscope (TE2000-U, Nikon Co., Tokyo, Japan). In Vivo Implantation on Partial-Thickness Cartilage Defect in Rabbit Knees. All animal procedures were followed by the animal care and use committee of Chonnam National University-approved protocols, and the animals were cared for in accordance with the Guidelines for Animal Experiments of Chonnam National University. A total of 16 New Zealand white rabbits (16−18 weeks old, weighing 3.0−3.4 kg; Samtako Bio Korea, Osan, Korea) were selected for the animal study. Animals were mature and had a tidemark junction. The knee joint was exposed and a partial cartilage defect (5 mm of diameter,