Hydrolyzed Poly(Butylene Succinate) Scaffolds Coated with Bioactive Agent

Transcription

1 Journal of Metals, Materials and Minerals, Vol.20 No.3 pp.95-99, 2010 Hydrolyzed Poly(Butylene Succinate) Scaffolds Coated with Bioactive Agent Wasana KOSORN, Boonlom THAVORNYUTIKARN, Benjaree PHUMSIRI, Paweena UPPANAN, Preeyapan MEESAP and Wanida JANVIKUL * National Metal and Materials Technology Center, 114 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani Abstract Hydrolyzed poly(butylene succinate) (HPBS) scaffolds coated with a bioactive agent were prepared in this study. Two different bioactive agents, i.e., collagen and hydroxyapatite (HA), were employed at different contents. Chemical crosslinking between amino groups (-NH 2 ) in collagen and carboxylate groups (-COO - ) in HPBS, leading to amide linkages, was evidenced by FT-IR. The contents of collagen or HA coated on the HPBS scaffolds were quantitatively determined by a gravimetric method and elemental analysis. It was found that the higher the content of the bioactive agent initially employed, the greater the content of the bioactive agent apparently coated. Furthermore, the deposition of HA on HPBS was directly influenced by the soaking time of HPBS in HA suspension. Cell proliferation results revealed that, at 35-day culture period, the highest number of chondrocyte cells was observed on the HPBS scaffold coated with 10% w/v HA suspension. In accordance with the proliferation results, RT-PCR analysis showed the highest expression levels of cartilage-specific genes, i.e., aggrecan and type II collagen, on that HA-coated HPBS scaffold. Key words: Hydrolyzed poly(butylene succinate), Hydroxyapatite, Collagen, Scaffolds, Chondrocytes Introduction Biodegradable aliphatic polyesters, e.g., poly (lactic acid) (PLA), poly(glycolic acid) (PGA), poly (ε-caprolactone) (PCL), poly(hydroxybutyrate) (PHB) and poly(butylene succinate) (PBS), have been extensively studied for potential uses in bone tissue engineering applications as cell/tissue culture substrates. (1-2) However, low cell adhesion to biodegradable polymers due to their hydrophobic surfaces is disadvantageous. (3) Recently, several surface treatments of polyesters have been attempted. (4-6) Collagen is a group of naturally occurring proteins and has received most attention as a material for cartilage tissue engineering. Hydroxyapatite (HA) is a ceramic material that is biocompatible due to the chemical similarity with human bones. In this study, the preparation of hydrolyzed poly(butylene succinate) (HPBS) scaffolds coated with a bioactive agent, i.e., collagen or HA, was attempted with a use of different contents of the bioactive agent under various preparative conditions. The resulting scaffolds were characterized by FT-IR, elemental analysis (EA), and energy dispersive X-ray spectrometry (EDS). The proliferation of human articular chondrocytes cultured on the scaffolds was also investigated. Furthermore, the gene expression of aggrecan and type II collagen was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Materials and Experimental Procedures Materials HPBS scaffolds were directly prepared in our laboratory by hydrolysis of PBS scaffolds with 0.3 M NaOH solution at 60 C for 30 minutes. Collagen (Mw 5,000 Da, from T.C. union global public company limited) and HA (from Inui Corporation, Japan) were used as received. All reagent-grade chemicals, e.g., 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS), were also used as received. Preparation of Bioactive Agent-Coated HPBS Scaffolds Collagen-Coated HPBS Scaffolds Collagen was primarily dissolved in phosphate buffer solution at concentrations of 5%, 10% and 20% w/v. Subsequently, EDC and NHS, used as coupling agents, were added into the collagen *Corresponding author wanidaj@mtec.or.th

2 96 KOSORN, W. et al. solution with a weight ratio of EDC:NHS:collagen of 0.15:0.09:1. HPBS scaffolds were then dipped into the collagen solutions heated at 80 C for 2 hours. Afterwards, the samples were thoroughly washed with water to remove unreacted collagen before being lyophilized. The product code was assigned based on the concentration of collagen initially used, e.g., HPBS/10Col prepared from 10% w/v collagen solution. HA-Coated HPBS Scaffolds HA was initially suspended in water at concentrations of 5%, 10% and 20% w/v. HPBS scaffolds were then immersed into the HA suspensions for given times, i.e., 30, 60 and 120 minutes. Afterwards, the HA-coated HPBS scaffolds were thoroughly extracted with water to remove unreacted HA before being freeze-dried. The product code was assigned based on the HA concentration initially used and soaking time, e.g., HPBS/10HA/60 prepared from 10% w/v HA suspension with a soaking time of 60 minutes. Characterization The chemical compositions of the HPBS scaffolds before and after being coated were analyzed by FT-IR spectroscopy (Perkin Elmer System 2000 FTIR) and EA (Leco TruSpec CHN/CHNS). To elucidate the coating efficiency, the contents of either collagen or HA coated on the HPBS samples, so-called weight gain (WG), were directly determined by a gravimetric method, using the following equation: %WG = (Wa-Wb)/Wa x 100 Where Wa and Wb are the weights of a scaffold after and before being coated, respectively. 200 μl of the medium from each scaffold were read at absorbance of 530 and 590 nm. RT-PCR Analysis Cartilage-specific gene, i.e., aggrecan and type II collagen, expression was determined by RT-PCR analysis. In brief, after 35-day culture period, total RNA was extracted from chondrocytes cultured on the scaffolds using TRIZOL reagent (GibcoBRL). 1 µg of total RNA was reversetranscribed into cdna using the RevertAid First Strand cdna synthesis Kit (Fermentas). RT-PCR analysis was conducted for both genes of interest and glyceraldehydes-3-phosphate dehydrogenase gene (GAPDH). The GAPDH mrna levels were used as internal controls. The PCR products were identified by electrophoresis on 2% agarose gels. Results and Discussions Collagen-Coated HPBS Scaffolds Figure 1 shows the overlaid FT-IR spectra of the collagen-coated HPBS scaffolds and the starting HPBS scaffold. The spectrum of the HPBS scaffold (Figure 1(a)) clearly revealed the characteristic peaks at 1720 and 3 cm -1, corresponding to carbonyl (>C=O) and hydroxyl (-OH) stretchings, respectively. The spectra of the collagen-coated HPBS scaffolds (Figure 1(b-d)), on the other hand, revealed additional absorption peaks at 1650 cm -1 (amide I band (-CONH-)) and 1597 cm -1 (-NH 2 deformation), indicating the formation of amide linkages (-CONH-) via a reaction between amine (-NH 2 ) groups in collagen and carboxylate (-COO - ) groups in HPBS. HA coated on the HPBS samples was also qualitatively examined by EDS (Oxford model INCA 300). Evaluation of Biological Properties Cell Proliferation Assay Cell proliferation on the scaffolds was assessed by Alamar Blue assay, which is based on the detection of metabolic activity. Typically, after given incubation periods, the cells cultured on each scaffold were further incubated in a medium containing resazurin dye for 4 hours. Aliquots of Figure 1. FT-IR spectra of (a) HPBS, (b) HPBS/5Col, (c) HPBS/10Col and (d) HPBS/20Col scaffolds.

3 Hydrolyzed Poly(Butylene Succinate) Scaffolds Coated with Bioactive Agent 97 As revealed in Table 1, it was noted that the higher the collagen concentration initially employed, the greater the content of collagen apparently coated. The content of collagen found on the PBS/10Col sample was lower than that of the HPBS/10Col sample, suggesting that collagen was physically trapped in the porous PBS scaffold; there was no chemical linkage between collagen and PBS. Table 1. The % weight gain (WG) of scaffolds coated with concentration-varied collagen solutions. Sample code Collagen concentration (%w/v) %WG 1 PBS/10Col ± HPBS/5Col ± HPBS/10Col ± HPBS/20Col ±0.69 The results on the determination of collagen content coated on the scaffolds by elemental analysis are reported in Table 2. Nitrogen is one of the essential elements found in collagen. The quantitative results obtained from EA were in a good accordance with those determined by a gravimetric method (%WG). Table 2. The content of collagen coated on scaffolds, determined by elemental analysis. %Weight of each Sample code element C H N 1 PBS HPBS Collagen PBS/10Col HPBS/5Col HPBS/10Col HPBS/20Col HA-Coated HPBS Scaffolds %Collagen by weight After extraction in water, the content of HA binding on the HPBS scaffolds, prepared under different concentrations of HA solution and soaking times, was determined by a gravimetric method, in terms of %WG. It was noted that the higher the HA content initially used and the longer the soaking time employed, the greater the amount of HA apparently coated, as shown in Table 3. Table 3. Effects of HA concentration and soaking time on the amount of HA coated on scaffolds. Sample code Coating condition [HA] (% w/v) Soaking time (min) %WG 1 PBS/10HA/ ± HPBS/5HA/ ± HPBS/5HA/ ± HPBS/5HA/ ± HPBS/10HA/ ± HPBS/10HA/ ± HPBS/10HA/ ± HPBS/20HA/ ± HPBS/20HA/ ± HPBS/20HA/ ±0.22 The content of HA coated on the HPBS scaffolds was also determined by EDS. It was found that, as shown in Table 4, %Ca increased with increasing concentration of HA and soaking time used. This suggested that HPBS did interact with HA, resulting in the formation of ionic bonding, supported by a report in the literature revealing that carboxylic (-COOH) groups could bind with Ca 2+ ions of HA, forming electrostatic forces. (5) The calcium found in the PBS/10HA/60 sample ( 1) mainly arose from the physical deposition of HA on the scaffold. The calcium content was much lower than that detected on HPBS/10HA/60 ( 3); both samples were prepared under the same coating condition. Table 4. The % calcium found on HA-coated scaffolds, determined by EDS. Sample code Ca (wt%) 1 PBS/10HA/ ± HPBS/10HA/ ± HPBS/10HA/ ± HPBS/10HA/ ± HPBS/5HA/ ± HPBS/20HA/ ±3.12 Figure 2 shows the proliferation of chondrocytes cultured on the scaffolds at different incubation periods. It was found that, after 7-day incubation period, the number of chondrocytes cultured on the collagen-coated HPBS scaffold was not much different from that observed on the pure HPBS scaffold. In contrast, a considerably greater proliferation of chondrocytes was found on the HA-coated HPBS scaffolds, compared with that of the pure HPBS scaffold. When the incubation time prolonged, the number of cells in

4 98 KOSORN, W. et al. all scaffolds continuously increased. At 35-day culture period, the greatest number of chondrocyte cells was found on the HPBS/10HA/60 scaffold. Fluorescense Intensity HPBS/5HA/60 HPBS/10HA/60 HPBS/20HA/60 HPBS/10Col HPBS/20Col Figure 2. Proliferation of chondrocytes cultured on various scaffolds after 7, 14, 21 and 35 days of incubation period. As reported, chondrocytes grown in a monolayer culture dedifferentiated and gradually lost their ability to express cartilage-specific genes, e.g., aggrecan and type II collagen. (7) Hence, chondrocyte phenotype must be essentially investigated after cell culture. The gene expression of chondrogenic markers was examined by RT-PCR analysis in this study. The mrna profiles of aggrecan and type II collagen from chondrocytes cultured on the scaffolds for 35 days are illustrated in Figure 3. It was found that the dedifferentiated chondrocytes could express the aggrecan mrna in every tested scaffold. The highest aggrecan expression level was observed in the HPBS coated with 10% w/v HA suspension, HPBS/10HA/60. However, the expression of type II collagen gene could be detected only when the chondrocytes were cultured on the HPBS/10HA/60 scaffold. Figure 3. RT-PCR profiles of mrna for GAPDH, aggrecan and type II collagen from chondrocytes cultured on scaffolds for 35 days. Lane 1: HPBS/5HA/60, Lane 2: HPBS/10HA/60, Lane 3: HPBS/20HA/60, Lane 4: HPBS/10Col, Lane 5: HPBS/20Col, Lane 6: HPBS, M=marker. HPBS D7 D14 D21 D35 GAPDH Product size 496 bp Aggrecan Product size 501 bp Type II collagen Product size 621 bp The HPBS/10HA/60 scaffold was found to be the only sample that could induce the dedifferentiated chondrocytes to redifferentiate into their cartilaginous characteristics. The SEM micrographs (not shown here) also revealed chondrocytes with round morphology when being cultured on this scaffold. On the other scaffolds, fibroblast-like cells were predominantly observed. When the HPBS scaffold coated with a higher content of HA, i.e., in HPBS/20HA/60, was used, the dedifferentiated chondrocytes were not more redifferentiated. Too many HA particles coated clogged the pores of the scaffold, preventing the cells to migrate throughout the whole scaffold. Therefore, the cells just adhered and well spread on the surface of the scaffold in the fibroblast-like structure. Conclusions The bioactive agents, i.e., collagen or HA, were successfully coated on the surfaces of the HPBS scaffolds via the crosslinking reaction between amino groups in collagen and carboxylate groups in HPBS or the ionic interaction between calcium ions of HA and carboxylate groups in HPBS, respectively. The contents of the bioactive agents coated on the scaffolds were directly influenced by the initial concentration of the bioactive agents used and the soaking time (in HA case). After being cultured for 35 days, the chondrocytes cultured on the HPBS coated with 10% HA suspension appeared to proliferate most efficiently. The greatest number of cells and the highest expression levels of aggrecan and type II collagen were observed on this scaffold. Acknowledgments This research was financially supported by National Metal and Materials Technology Center (project code: MT-B-52-BMD I). References 1. Lee, J.B., Lee, S.H., Yu, S.M., Park, J.C., Choi, J.B. & Kim, J.K. (2008). PLGA scaffold incorporated with hydroxyapatite for cartilage regeneration. Surf. Coat. Tech. 202(22-23) : Zhang, P., Hong, Z., Yu, T., Chen, X. & Jing, X. (2009). In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-coglycolide) and hydroxyapatite surface-grafted with poly(l-lactide). Biomaterials 30(1) :

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