PLGA Microsphere Construct Coated with TGF-β 3 Loaded

rMSCs seeded PLGA microsphere constructs were fixed in 4% paraformaldehyde solution, dehydrated, and embedded in paraffin. After deparaffinized, 6 μm...
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PLGA Microsphere Construct Coated with TGF-β 3 Loaded Nanoparticles for Neocartilage Formation Ji Sun Park, Kyeongsoon Park, Dae Gyun Woo, Han Na Yang, Hyung-Min Chung,* and Keun-Hong Park* Pochon CHA University, CHA Stem Cell Institute 606-16, Yeoksam 1-dong, Kangnam-gu, Seoul 135-081, Korea, Biomedical Research Center, Korea Institute of Science and Technology, Korea, and Chabiotech Co., Ltd. 606-16, Yeoksam 1-dong, Korea Received March 7, 2008; Revised Manuscript Received April 27, 2008

Polymeric microsphere system has been widely used in tissue-regeneration matrix and drug delivery systems. To apply these biomaterials as novel cell supporting matrix for stem cell delivery, we have devised a novel method for the fabrication of nanostructured 3D scaffolds that growth factor loaded heparin/poly(L-lysine) nanoparticles were physically attached on the positively charged surface of PLGA microspheres precoated with low molecular weight of poly(ethyleneimmine) (PEI) via a layer-by-layer (LbL) system. Based on a previous study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring heparin/poly(L-lysine) loaded with growth factors. Growth factor loaded heparin/poly(L-lysine) nanoparticles, which were simply produced as polyion complex micelles (PICM) with diameters of 50-150 nm, were fabricated in the first step. Microsphere matrix (size, 20∼80 nm) containing TGF-β 3 showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study in vitro culture of mesenchymal stem cells. Thus, growth factor delivery of PLGA microsphere can be used to engineer synthetic extracellular matrix. This PLGA microsphere matrix containing TGF-β 3 showed promise as coatings for implantable biomedical devices to improve biocompatibility and ensure in vivo performance.

Introduction Microparticulate systems have attracted a great deal of attention over the past few years as carriers for the delivery of cells and proteins in the treatment of defective tissues.1–4 The composition of microparticulates is regarded as being of utmost importance for the successful recruitment of the cells involved in the tissue regeneration process. The design of nanostructures and nanometer-scale fabrication is driven not only by the novel yet unexplored properties associated with nanoscale materials, but also by the continuously increasing demand for further miniaturization of electronic components, optical detectors, and chemical and biochemical sensors and devices. The new type of poly(lactide-co-glycolide) (PLGA) microsphere matrix applied as injectable cell carrier allows one to transplant cells via minimally invasive procedures for cartilage regeneration in vivo.5 It has been known that PLGA and its family as a biocompatible synthetic polymer were broadly applied to tissue engineering due to a long-standing established safety record in humans.6 However, conventional cell culture methodologies using PLGA microspheres are not sufficient for coping with the scale of cell production required for the manufacturing of engineered cartilage tissue products. These efforts have been hampered by the well-known behavior of chondrocytes or mesenchymal stem cells (MSCs) cultured on PLGA microspheres, which undergo a prompt loss of their cartilage-specific phenotype and become fibroblastic.7–9 To make a neocartilage formation using PLGA microsphere matrix, specific drug is needed for chondrogenesis on PLGA microspheres. Many authors studied the specific growth factors and drugs that were contained in the PLGA microspheres and used for neocartilage formation.10–13 * To whom correspondence should be addressed. Phone: 82-2-3468-3392. E-mail: [email protected] (K.-H.P.); [email protected] (H.-M.C.).

In a previous study, we have recently reported a composite delivery system that is fabricated by dispersing heparinized nanoparticles (NPs) coated on PLGA microspheres for a host of applications, including bioreactor, cell delivery vehicle, and protein or peptide delivery systems.14 In this work, we attempted to devise a simple and highly efficient method for the complete stabilization of threedimensional nanostructures via their embedding into a microspherical matrix. Protein depositions, particularly protein-loaded PLGA microspheres, have been formulated in an attempt to maintain biological activities of protein and structural integrity for long periods. In order to fabricate the suitable matrix, a new type of flexible nanoparticular structure on microsphere matrix was generated via this method. To fabricate the nanoscale structure on a 3D matrix for cell delivery vehicles, heparin/ poly(L-lysine) NPs loaded growth factors were prepared as an initial step. In this step, we considered heparin, a member of the glycosaminoglycan (GAG) family, as a potential candidate for long-term protein stabilization and immobilization of the protein.15–17 These polysaccharides have been shown to interact directly with a number of growth factors, including bone morphogenic proteins (BMPs), basic fibroblast growth factors (bFGF), and transforming growth factor β (TGF-β) via highly negative charged polysaccharide chain.18–21 In this study, we designed more stable delivery of protein and cell differentiation matrix by adsorption of NPs carrying a specific growth factors on polymeric microspheres. By loading on the NPs, the growth factors will be retarded in their bioactivity for stem cell differentiation. Specially, among the sulfate polysaccharide, heparin binds growth factors to form a stable complex that maintains the biological activity and can retard the release pattern.

10.1021/bm800251x CCC: $40.75  2008 American Chemical Society Published on Web 07/17/2008

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Figure 1. Hydrodynamic diameters of the heparinized nanoparticles (NPs) in aqueous solutions measured by dynamic light scattering (A, B), SEM images (C, D), and ζ-potential (E). (*) p < 0.05. (A) Growth factor-free NPs; (B) growth factor-loaded NPs; (C) SEM image of growth factor-free NPs; and (D) SEM image of growth factor-loaded NPs; (E) (a) 0 ng/mL, (b) 10 ng/mL, (c) 50 ng/mL, and (d) 100 ng/mL TGF-β 3-loaded NP size.

Experimental Section Materials. Low molecular weight heparin (LMWH, Fraxiparin), which has an average molecular weight of approximately 4500 Da, was obtained from Sanofi Synthelabo Co. (Gentilly, France). Poly(Llysine) (PLL) (Mw ) 1000∼4000 Da) was purchased from SigmaAldrich (P0879, U.S.A.). A poly(lactide-co-glycolide) (PLGA: Mw ) 33000), a copolymer ratio of lactide to glycolide of 50:50 (R503H, Boehringer Ingelheim, Germany), was used as a wall material for the microspheres. All reagents and organic solvents used were of at least ACS grade. Preparation of Growth Factor-Loaded Heparinized Nanoparticles. The PLL was dissolved in a deionized solution (pH 7.4) to form a 0.5 mg/mL solution. A mixture containing 10, 50, and 100 ng/mL of TGF-β 3 mixed with 0.5 mg/mL of heparin was added to the PEI solution with constant vortexing for 15 s, and the solution was then incubated for 30 min at room temperature, yielding complexes with ( molar charge ratios in a range from 0 to 2.95. In this study, optimal concentration of heparin and PLL (1:1) was chosen because of its low particle size and ζ-potential measurement. The particle size and mean diameter were determined by dynamic light scattering (DLS; Malvern, Worcestershire, U.K.). ζ-Potential Measurement. The ζ-potential (surface charge) of polymers and polyplexes was determined at 25 °C using a Zeta Sizer (Malvern, Worcestershire, U.K.). The samples were prepared in PBS and diluted 1:8 with deionized water to ensure that the measurements had been performed under the conditions of low ionic strength, where the surface charge of the particles can be measured accurately. The final concentration of the polymer was 1 mg/mL. All data refer to 15 measurements from one sample.

Preparation of PLGA Microspheres. The PLGA microspheres were prepared using a solvent evaporation in an oil-in-water emulsion. In brief, PLGA (4 g) was dissolved in 30 mL of dichloromethane. Using a glass syringe and needle (needle gauge; 20G), the polymer solution was dropped into 300 mL of aqueous solution containing 2 w/v % of poly(vinyl alcohol) (PVA) while mixing, using a magnetic stirrer at 600 rpm. The suspension was then gently stirred for 2 to 3 h at 35 °C with a magnetic stirrer at 600 rpm to evaporate the dichloromethane, and the microspheres were collected via 2 min of centrifugation at 1500 rpm. The collected microspheres were washed four times in distilled water and were then lyophilized. The size of the microspheres, as measured by SEM, ranged between 20∼80 µm. Immobilization of Heparin/Poly(L-lysine) NPs Loaded with Growth Factors onto PLGA Microspheres. First, the PLGA microspheres were coated with positively charged poly(ethyleneimmine) (PEI: Mw, 1800). The coating was conducted at the native pH (7.4) of the polymer solution, with no additional salt. Under the same conditions, the PEI side chain amines (pKa ≈ 10) would be extensively protonated. The microspheres (1 g) were then soaked for 12 h in the PEI solution (1 mg/mL, 0.1 w/v %) with gentle stirring, collected through 2 min of centrifugation at 1500 rpm, rinsed three times in distilled water, and then dipped into the heparin/poly(L-lysine) NPs loaded with growth factors solution (1 mg/mL) for 24 h with gentle stirring. Heparin/ poly(Llysine) NPs loaded with growth factor-coated PLGA microspheres were then collected via 2 min of centrifugation at 1000 rpm and rinsed three times in distilled water. Finally, the heparin/poly(L-lysine) NPs loaded with growth factor-coated PLGA microspheres were washed four times in distilled water and lyophilized.

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Figure 2. The SEM images of PLGA microspheres fabricated with heparin/poly(L-lysine) nanoparticles. (A and B) Control PLGA microspheres and (C and D) NP immobilized PLGA microspheres.

Growth Factor Released from PLGA Microspheres. TGF-β 3 (100, 70, and 50 ng/mL) coated microspheres (100 mg) in dialysis membrane bag were evaluated, and release characteristics, relocated to conical polypropylene tubes in the l mL of R-MEM, were placed on an orbital shaker. Blank PLGA microspheres were used as controls. The medium was removed, frozen, and replenished at 1, 3, 5, 7, 10, 14, 21, and 28 days. ELISA (enzyme-linked immunosorbent assay) for TGF-β 3 was performed to determine the concentration of immunoreactive protein. Circular Dichroism Spectroscopy (CD) Analysis. CD spectra were measured at 4 °C on a Jasco J-750 spectropolarimeter (Jasco Inc., Easton, MD). Samples were equilibrated at the desired temperature for 30 min prior to data collection; equilibration was indicated by the absence of further changes in the CD signal at longer equilibration times. All CD spectra were taken in a 1 mm path length quartz cuvette, at wavelengths from 190 to 260 nm. Data points were recorded at every nanometer with a 4.0 s response time. The concentrations of TGF-β 3 loaded in nanoparticles were determined via amino acid analysis for calculation of mean residue ellipticities. Cell Seeding and Growth on Nanoparticles Loaded TGFCoated Polymeric Microspheres In Vitro. Rabbit bone marrowderived mesenchymal stem cells (rMSCs) were isolated from white New Zealand rabbits. rMSC were grown in monolayer culture in highglucose Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) containing L-glutamine and 1.5 g/L sodium bicarbonate and supplementedwith10%fetalbovineserum(FBS)andantibiotic-antimyotic solutions (streptomycin at 100 mg/mL, penicillin at 100 IU/mL) from Gibco (Grand Island, NY). For chondrogenic cultures, rMSC (passage 5) and microspheres were incubated in a 15 mL polypropylene round tube, with gentle shaking, in the serum-free condition in vitro. After 2 h of incubation, the unattached cells were removed, and the 15 mL polypropylene round tube was incubated for cell growth. Chondrogenic cultures consisted of cells and microspheres incubated in culture medium without TGF-β 3. Culture medium consisting of serum-free

Figure 3. Gross views of neocartilage formed by cultivation of rabbit MSCs adhered onto a microsphere matrix coated with TGF-β 3. (A and D) Control; (B and E) NP; (C and F) NP-TGF-β 3.

medium contains antibiotic-antimyotic solutions (streptomycin at 100 mg/mL, penicillin at 100 IU/mL). Biochemical Assays for MSC Proliferation and GAG Production. At each time point, the samples and negative controls from each time point were extracted, rinsed in 2.5 mL of PBS, homogenized with a pellet grinder (Fisher Scientific), and digested in 500 mL of a proteinase K solution (1 mg/mL proteinase K, 10 mg/mL pepstatin A, and 185 mg/mL iodoacetamide) in PBE buffer (6.055 mg/mL Tris(hydroxymethyl aminomethane), 0.372 mg/mL EDTA, pH 7.6, adjusted by HCl) at 60 °C for 16 h. After collection and digestion of all samples and controls, the specimens underwent three repetitions of a freeze/ thaw/sonication cycle (30 min at -80 °C, 30 min at room temperature, 30 min of sonication) for the complete extraction of DNA from the cell cytoplasm. The DNA and GAG assays were run in triplicate for each experimental and control group at each time point. The number of cells was determined by measuring the double-stranded DNA content using a PicoGreen assay (Molecular Probes, Eugene, OR) according

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Figure 4. Cumulative nanoparticle-loaded TGF-β 3 release profiles from PLGA microsphere matrix and the concentration of TGF-β 3 released from PLGA microsphere matrix by ELISA (A). Bioactivity test of TGF-β 3 released from PLGA microsphere (B), GAG/DNA (C), and collagen/ DNA (D) contents from rMSCs embedded in PLGA microsphere construct. rMSCs were cultured in 3-D microspheres for 1 week (9) and 4 weeks (0) in basal medium. Error bars represent standard error of the mean (*p < 0.05). Error bars each show the mean ( SD (n ) 3). (A) 100 ng/mL (2), 70 ng/mL (9), and 50 ng/mL (•); (B) Bioactivity test (1, native 100 ng of TGF-β 3; 2, 1 day; 3, 7 day; 4, 10 day; 5, 14 day; 6, 21 day); (C) GAG/DNA assay; and (D) collagen/DNA assay. (a) Control, (b) NP-coated PLGA microsphere, and (c) NP-TGF-β 3 coated PLGA microspheres.

to the instructions of the manufacturer. The fluorescence of negative, cell-free controls was subtracted from the fluorescence values of the experimental groups to account for the fluorescence of the material alone. Similarly, the GAG content was determined using the dimethylmethylene blue dye (DMMB) assay. Briefly, 50 mL of papain digested sample was incubated with 2 mL of DMMB dye, and the reaction was observed on a spectrophotometer at 520 nm, with shark chondroitin sulfate C (Sigma) used as a standard. Total amount of GAG was normalized versus the total amount of DNA. Collagen Contents. The retrieved samples from the nude mice were digested with papain digestion solution (125 µg/mL papain, 5 mM L-cystein, 100 mM Na2HPO4, 5 mM EDTA, pH 6.8) at 60 °C for 16 h. Total collagen of each specimen was measured. A dye solution (pH 3.5) was prepared with Sirius red dissolved in picric acid saturated solution (1.3%, Sigma) to a final concentration of 1 mg/mL. The digested samples were dried at 37 °C in a 96-well plate for 24 h and then reacted with the dye solution for 1 h on a shaker. Washed five times with 0.01 N HCl, the dye sample complex in each well was resolved in 0.1 N NaOH. The absorbance was read at 550 nm wavelength using ELISA Reader (BIO-TEK Instruments Inc., Winooski, VT). The total collagen in each sample was extrapolated using a standard plot of bovine collagen (Sigma) in the range of 0-10 µg/mL. RT-PCR and Real-Time QPCR. The isolated RNA samples were converted to cDNA using reverse transcriptase (SuperScript III, Invitrogen, Carlsbad, CA) and oligo (dT) primers and amplified by PCR using platinum Taq Polymerase (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommendations. PCR were performed by 35 cycles of 94 °C for 30 s, 58 °C for 45 s, and 72 °C for 45 s. Expression of the following genes was examined: collagen type II (Col II), (forward) 5′- GCACCCATGGACATTGGAGGG-3′ and (reverse) 5′- GACACGGAGTAGCACCATCG-3′ (366bp); collagen type XI (Col XI), (forward) 5′- GGAAAGGACGAAGTTGGTCTGC-3′ and (reverse) 5′-TTCTCCACGCTGATTGCTACCC-3′ (590bp); aggrecan, (forward) 5′- CCTTGGAGGTCGTGGTGAAAGG-3′ and (reverse) 5′-

AGGTGAACTTCTCTGGCGACGT-3′ (364bp); cartilage oligomeric protein (Comp), (forward) 5′-CAGGACGACTTTGATGCAGA-3′ and (reverse) 5′-AAGCTGGAGCTGTCCTGGTA-3′(314bp); and the housekeeping gene glyceradehyde-3-phosphate dehydrogenase (GAPDH), (forward) 5′- TCACAATCTTCCAGAGCGA-3′ and (reverse) 5′CACAATGCCGAAGTGGTCGT-3′ (293bp). The concentration of GAPDH was used to control input RNA (determined once for each cDNA sample) and to normalize all other genes tested from the same cDNA sample. The copy ratio of each analyzed cDNA was determined as the mean of three experiments. PCR products were separated by electrophoresis at 100 V on a 2% agarose gel in TAE buffer. In addition, collagen type II (Col II), aggrecan, and cartilage oligomeric protein (Comp) gene expression were measured by real-time QPCR in an ExiCycler (Bioneer, Daejeon, Korea, http://www.bioneer.com). A total of 1 µL of each RT reaction was amplified in a 20 µL PCR assay volume containing 2.0 mM MgCl2, 0.5 µM each primer, and 1× Greenstar PCR Master Mix (Bioneer). Samples were incubated in the ExiCycler for an initial denaturation at 94 °C for 10 min followed by 45 PCR cycles. Each cycle proceeded at 94 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. Relative quantification was calculated using the 2-(CT) method, according to the manufacturer’s recommendation. To confirm amplification of specific transcripts, melting curve profiles (cooling the sample to 40 °C and heating slowly to 95 °C with continuous measurement of fluorescence) were produced at the end of each PCR. Histology and Immunohistochemistry. rMSCs seeded PLGA microsphere constructs were fixed in 4% paraformaldehyde solution, dehydrated, and embedded in paraffin. After deparaffinized, 6 µm sections were processed and stained with eosin and hematoxylin. Deparaffined sections were incubated with 0.5% Triton X-100/ phosphate-buffered saline (PBS) solution for 30 min and washed with PBS three times. Embedded sections were stained with Safranin-O and Alcian blue staining for histological evaluation. Nonspecific binding sites were blocked with normal horse serum diluted 1:10 in 0.3% bovine

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Figure 5. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time QPCR analysis of gene expression from rMSCs cultured onto PLGA microspheres. Expression of specific genes was analyzed by RT-PCR at rMSCs differentiation after 4 weeks on the surfaces of the PLGA microspheres modified by heparin bound TGF-β 3 (A); (B) quantification of aggrecan; (C) quantification of COMP; and (D) quantification of COL II. Error bars each show the mean ( SD (n ) 3).

serum albumin for 30-60 min and then incubated for 2 h at 4 °C in mouse antiserum against collagen type II (Chemicon) in 1:200 in a humid environment. After rinsing in Tris-buffered saline (TBS), sections were incubated in FITC-conjugated rabbit antimouse immunoglobulin G (Amersham Pharmacia Biotech, Piscataway, NJ) secondary antibody was applied, and after three PBS washes, samples were incubated with 1:200 propidium iodide (PI, P4170; Sigma) stain for 5 min. To determine the quantification of immuno-stained COL II released from differentiated MSCs embedded in porous scaffolds were calculated in each section of randomly selected four areas of entire PLGA microsphere constructs by confocal laser microscope (Carl Zeiss, Axiovert 200 M). The digital images, two-dimensional digital images were 262144 pixels (512 × 512 pixels), were analyzed by using the LSM 510 Meta software (Jena, Germany) to calculate the ratios of FITC-labeled area/FITC-unlabeled area fluorescence profiles. Statistical Analyses. The statistical significance of the differences between the experimental groups was assessed using a two-tailed Student’s t-test. A p value