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The Impact of Polyethylene Glycol-Modified Chitosan Scaffolds on the Proliferation and Differentiation of Osteoblasts

The Impact of Polyethylene Glycol-Modified Chitosan Scaffolds on the Proliferation and... Hindawi International Journal of Biomaterials Volume 2023, Article ID 4864492, 8 pages https://doi.org/10.1155/2023/4864492 Research Article TheImpactofPolyethyleneGlycol-ModifiedChitosanScaffoldson the Proliferation and Differentiation of Osteoblasts 1 2,3 Wei-Bor Tsai and Ibrahim Nasser Ahmed Department of Chemical Engineering, National Taiwan University, No. 1 Sec. 4 Roosevelt Rd., Taipei 106, Taiwan Nanotechnology Center of Excellence, Addis Ababa Science and Technology University, P.O. Box 16417, Addis Ababa, Ethiopia Department of Industrial Chemistry, College of Applied Sciences, Addis Ababa Science and Technology University, P.O. Box 16417, Addis Ababa, Ethiopia Correspondence should be addressed to Ibrahim Nasser Ahmed; ibrahim.nasser@aastu.edu.et Received 18 August 2022; Revised 20 December 2022; Accepted 24 December 2022; Published 3 January 2023 Academic Editor: Carlo Galli Copyright © 2023 Wei-Bor Tsai and Ibrahim Nasser Ahmed. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te objective of this study was to investigate the infuence of polyethylene glycol (PEG) incorporated chitosan scafolds on osteoblasts proliferation and diferentiation. Te chitosan polymer was initially modifed by the predetermined concentration of the photoreactive azido group for UV-crosslinking and with RGD peptides (N-acetyl-GRGDSPGYG-amide). Te PEG was mixed at diferent ratios (0, 10, and 20 wt%) with modifed chitosan in 96-well tissue culture polystyrene plates to prepare CHI-100, CHI- 90, and CHI-80 scafolds. PEG-containing scafolds exhibited bigger pore size and higher water content compared to unmodifed chitosan scafolds. After 10 days of incubation, the cell number of CHI-90 (1.1 × 106 cells/scafold) surpasses that of CHI-100 (9.2 ×105 cells/scafold) and the cell number of CHI-80 (7.6 ×105 cells/scafold) were signifcantly lower. Te ALP activity of CHI-90 was the highest on the ffth day indicating the favored osteoblasts’ early-stage diferentiation. Moreover, after 14 days of osteogenic culture, calcium deposition in the CHI-90 scafolds (2.7 μmol Ca/scafold) was signifcantly higher than the control (2.2 μmol Ca/scafold) whereas on CHI-80 was 1.9 μmol/scafold. Te results demonstrate that PEG-incorporated chitosan scafolds favored osteoblasts proliferation and diferentiation; however, mixing relatively excess PEG (≥20% wt.) had a negative impact on osteoblasts proliferation and diferentiation. C–H, O–H, and N–H bonds of nearby substance molecules 1. Introduction [6]. Another shortcoming is that chitosan lacks bioactive Chitosan, the deacetylated derivative of chitin, has been signals equivalent to those existing in the extracellular broadly utilized for the fabrication of tissue engineering matrix (ECM) for cell attachment, growth, and diferenti- scafolds due to its nontoxicity, biodegradability, good ation. Incorporation of bioactive signals such as ECM ad- biocompatibility [1], and resemblance to glycosaminogly- hesion proteins and cell-binding peptides into chitosan cans [2]. However, chitosan still possesses some short- substrates can enhance cell adhesion [7–9]. RGD- incorporated and crosslinked chitosan scafolds can be comings for such a purpose, for instance, the mechanical properties of chitosan scafolds may not be suitable to match employed for mesenchymal stem cell proliferation and os- some specifc tissue engineering applications. Previously it teogenic diferentiation [3, 4]. was utilized an azide-based UV-crosslinking mechanism for Tere are many reports aimed at improving chitosan crosslinking chitosan scafolds in order to increase the properties by blending with natural or synthetic molecules. mechanical properties of chitosan scafolds [3–5]. Upon UV Park et al. [10] developed composite chitosan scafolds irradiation, the azido groups are converted into highly re- containing anionic carbohydrates. Te incorporation of active nitrene groups which undergo direct insertion into chondroitin 4-sulfate or alginate in chitosan scafolds 2 International Journal of Biomaterials increased the compressive modulus of the scafolds and 2. Material and Methods enhanced apatite formation. Furthermore, apatite-coated 2.1. Material. Polyethylene glycol M 20,000 and most of the scafolds enhanced the spreading, proliferation, and osteo- n reagents were purchased from Sigma–Aldrich (USA) unless genic diferentiation of bone marrow stromal cells seeded on specifed otherwise. N-(3-dimethylaminopropyl)-N′- the scafolds. A report by Li et al. [11] using a 3- ethylcarbodiimide hydrochloride was purchased from Fluka aminopropyltriethoxysilane treatment for modifcation (USA), and acetic acid was purchased from Baker (USA). and biocompatibility of lyophilized chitosan porous scaf- RGD-peptide (N-acetyl-GRGDSPGYG-amide) was synthe- folds showed the silanization treatment with low 3- sized by Kelowna International Scientifc Inc. (Taipei, Tai- aminopropyltriethoxy silane concentration showed no sig- wan). Te peptide concentration was calculated by nifcant infuence on the morphology of chitosan scafolds, absorbance at 275 nm coming from the tyrosine residue (Y, the porosity, and surface amino densities were increased −1 −1 molar adsorption coefcient 1420 M cm ). after silanization whereas the swelling ratio was reduced, and Osteoblast culture medium contained α-minimum es- the degradation ratio in PBS and antiacid degradation sential medium (α-MEM, HyClone, USA), 10% fetal bovine properties of the silanized chitosan scafolds were signif- serum (JRH Biosciences, Australia), 0.0679% (v/v) 2- cantly improved. Chitosan doped with multiwalled carbon mercaptoethanol, 200 μg/mL gentamicin (GIBCO , Invi- nanotubes has been used to create highly porous conductive trogen, USA), and 25 μg/mL fungizone (GIBCO ), pH 7.4. scafolds [12]. Chitosan can also be modifed by the addition Te osteoblast culture medium supplemented with 1 mM of hydrophobic alkyl chains along the hydrophilic backbone sodium glycerophosphate, 0.1 μM dexamethasone, and of the chitosan polymer and Cooney et al. [13] reported the 50 μg/mL L-ascorbate constituted osteoblast diferentiation scafolds produced from the unmodifed chitosan were more medium. Phosphate bufered saline (PBS) contained stable and rigid and possessed average pore diameters that 137 mM NaCl, 2.7 mM KCl, 10 mM Na HPO , and 1.8 mM were generally smaller than those fabricated from the 2 4 KH PO (pH 7.4). hydrophobically modifed chitosan. Te generally larger 2 4 pores in the butyl-modifed chitosan scafolds might be explained by increased phase separation rates due to the 2.2. Conjugation of Photoreactive Azido Groups or Peptides to introduced hydrophobicity of the chitosan polymer. Te Chitosan. Azido groups were conjugated onto chitosan combination of hydrophobic groups opposed along an (molecular weight 50–190 kDa, 75–85% deacetylation) via otherwise hydrophilic backbone creates internal forces that a reaction forming covalent amide bonds between the amino tend to fold or buckle the polymer chain, creating regions groups of chitosan and the carboxyl groups of an azido- that exhibit micellar behavior [14]. benzoic acid ester. Briefy, 17.6 mg 5-azido-2-nitrobenzoic One major drawback of chitosan in drug delivery is its acid N-hydroxysuccinimide ester was dissolved in 200 μL low solubility. Chitosan is not soluble in aqueous solutions at dimethylsulfoxide and then mixed with chitosan solution neutral or alkaline pH, only soluble in aqueous acid solu- (0.1 g in 4.8 mL of 1% acetic acid), followed by 3 h incubation tions and a few organic solvents [15]. Hence, various chi- at room temperature. Te unreacted azido ester was re- tosan derivatives have been prepared for the purpose of drug moved by dialysis against deionized water through a seam- delivery [15, 16]. Similarly, in this work it was argued that less cellulose tube (MWCO 12,400 Da) in the dark for two chitosan is a relatively hydrophobic material; however, in the days with the changes of deionized water every 12 h. After natural tissues, the extracellular matrix is highly hydrated. freeze-drying, the azido-conjugated chitosan (CHI-g-AZ) Tus, the hydrophobic environment of chitosan scafolds was kept at 4 C until use. might not be suitable for tissue growth. A number of studies RGD peptides were conjugated onto chitosan molecules indicate that both morphology and hydrophilicity infuence via a carbodiimide reaction according to a previously de- the attachment of cells onto the surface of a scafold [17]. veloped procedure [5]. Te graft ratio of RGD to chitosan Terefore, an increase in the hydrophilicity of chitosan (CHI-g-RGD) was estimated as 2.75 mol% with respect to scafolds might improve tissue engineering outcomes. the total moles of the amino groups of chitosan molecules. Polyethylene glycol (PEG) possesses biodegradability, bio- compatibility, less toxicity, and hydrophilicity and has been widely used in biomedical applications, including surface 2.3. Preparation of Chitosan and polyethylene Glycol Mixed modifcation, bioconjugation, drug delivery, and tissue Scafold. A mixture of chitosan (unmodifed, CHI-g-AZ, engineering [18, 19]. Although there are interesting con- and CHI-g-RGD) and PEG with a total concentration of tributions to the preparation, characterization, and aggre- 10 mg/mL in 1% acetic acid was prepared at diferent weight gation behavior of amphiphilic chitosan derivatives having percentages (the composition and abbreviations listed in poly-L-lactic acid side chains [20], there is no report on Table 1). Chitosan-PEG mixed scafolds were prepared by describing the osteogenic proliferation or diferentiation of adding 70 μL/well of the unmodifed chitosan, CHI-g-AZ, cells, from hydrophilically modifed chitosan scafold. CHI-g-RGD, and PEG mixture in 96-well tissue culture Hence, this study aimed to investigate the efect of adding polystyrene (TCPS) plates. Briefy, the mixture was poured polyethylene glycol into the RGD-conjugatedcross-linked into 96-well TCPS plates (70 μL/well for cell culture ex- chitosan scafolds on the proliferation and diferentiation periments), followed by freeze-drying in the dark to form of osteoblasts. scafolds. Subsequently, chitosan substrates were crosslinked International Journal of Biomaterials 3 Table 1: Te weight percentages of the compositions in the exclusion. Te isolated cells were cultured in standard T75 scafolds. fasks to the second passage for the cell experiments. Prior to cell seeding, the chitosan-PEG scafolds were Type of scafolds Chitosan CHI-g-AZ CHI-g-RGD PEG soaked in 70% ethanol for 30 min, followed by rinses with CHI-100 40 50 10 0 sterilized PBS three times. For cell culture on the scafolds, CHI-90 30 50 10 10 20 μL of osteoblast suspension (1.5 ×10 cells/mL) was CHI-80 20 50 10 20 seeded onto scafolds, making the seeding density 3 ×10 cells per scafold. After 1, 5, or 10 days of culture, the by UV irradiation for 30 min (wavelength range cell-inoculated samples were analyzed for cell morphology 280–380 nm). Te UV crosslinking time and the PEG dose cell numbers and alkaline phosphatase (ALP) activities. were selected based on the previous reports [5, 21]. After the cell culture, the morphology of cells in the scafolds after 5 days of incubation was observed by SEM. Te adhered osteoblasts were lysed with 0.1% Triton X-100 2.4. Characterization of Chitosan Scafolds. Te morphology for 30 min. Cell proliferation was determined by the lactate of chitosan-PEG scafolds was observed by scanning electron dehydrogenase (LDH) method according to a reported microscope (SEM) images (JSM-5310, JEOL, Japan). Te protocol [9]. Intracellular alkaline phosphatase activities scafolds were frst dehydrated in graded series of ethanol were assayed by determining the release of p-nitrophenol solutions 30%, 50%, 70%, 90%, 95%, and 100% for 10 min from 4-nitrophenyl phosphate disodium salt at pH 10.2, as each step followed by CO critical point-drying. Samples reported previously [23]. were cut with a scalpel, coated with a gold layer on the Te cell doubling time (T ) was calculated using the section, and then observed with SEM at an acceleration following equation: voltage of 20 kV. Te pore sizes of the scafolds were analyzed using an ∆T T � . (2) NIH Image J. Pores in SEM images were traced manually, 2 log (∆N/No + 1) and the enclosed areas and perimeters of pores were de- termined by the NIH Image J software. Te hydraulic di- No is the number of cells at the beginning of the ob- ameters of the pores were determined by the following servation, and △N is the increase in the number of cells equation: pore diameter (D ) � 4 × area/perimeter [5] more during the period of time of the length △t. Each division than 100 pores were counted for each type of sample. increases the number of cells by adding 1, and △N is also the Te compressive stress-strain properties of the scafolds number of cell divisions during the same period [24]. were determined using a compressive testing machine (FGS- 50V-H, NIDECSIMPO Corporation, Japan) and a digital 2.6. Mineralization Culture of Osteoblast/Scafold Constructs. force gauge (FGP-0.5, NIDECSIMPO Corporation, Japan). Osteoblast/scafold constructs were cultured for 5 days in the Te scafolds were subjected to an unconfned uniaxial osteoblast culture medium, followed by 10 days of miner- compression to 70% strain at a compression velocity of alization culture in the osteoblast diferentiation medium 3 mm/s. Te continuous stress and peak stress were recorded with daily replenishment of L-ascorbate (50 μg/mL). Te and analyzed. total amount of calcium deposition was determined using Dried chitosan-PEG scafolds were soaked in deionized a calcium assay kit (Diagnostic Chemicals Limited, water for 24 h. Te surface water contents on the scafolds USA) [25]. were absorbed by a flter paper. Wet scafolds were weighed (W ), and then placed in a 70 C oven overnight and weighed 2.7. Statistical Analysis. Each experiment has been repeated again (W ). Te equation implemented to calculate water content is shown as follows: at least three times. Te data were presented as mean- ± standard deviation (SD). Te statistical assessment of (Ws − Wd) Water content percentage(%) � (%) × 100%. signifcant variations was performed by Microsoft Excel Wd 2010. Signifcance was assessed by one-way analysis of (1) variance (ANOVA) and two-tailed Student–Newman–Keuls multiple comparisons. Te probability of p ≤ 0.05 was considered as a signifcant diference, where the symbol of ∗ 2.5. Culture of Osteoblasts on Chitosan Scafolds. and ∗∗ marker represent p< 0.05 and p < 0.01, which is of Standard sterile cell culture techniques were used for all cell signifcant diference statistically in 95% and 99% confdence experiments. Te animal procedure was followed by the level, respectively. ethical guidelines of Care and Use of Laboratory Animals (National Taiwan University, National Institutes of Health 3. Results and Discussion Publication No. 85–23, revised 1985) and was approved by the Animal Center Committee of National Taiwan Uni- 3.1. Fabrication and Characterization of Chitosan/PEG versity. Primary osteoblasts were isolated from neonatal rat Scafolds. Chitosan has a structure alike the N- calvariae according to the previously published procedure acetylglucosamine, which exists in hyaluronic acid, is an [22]. Te number and viability of the isolated osteoblasts extracellular macromolecule, and it is vital in wound healing were determined using a hemocytometer with trypan blue [26]. Te morphology of the chitosan scafolds incorporating 4 International Journal of Biomaterials PEG was examined with SEM. All the scafolds exhibited an open pore microstructure with interconnectivity. Te pore structure of the scafolds at diferent PEG concentrations is similar to each other (Figure 1). Te average pore sizes of ** ** CHI-100, CHI-90, and CHI-80 were 33.3 ± 7.4, 42.2 ± 8.2, and 46.9± 8.6 μm, respectively, indicating the pore sizes of the scafolds were signifcantly increased (p < 0.05) with the increasing PEG contents in chitosan scafolds (Figure 1). It was suspected that since PEG is more hydrophilic than chitosan, more water molecules surround PEG and form larger ice crystals during the freezing step than pure chitosan 100/0 90/10 80/20 scafolds. As a result, after lyophilization chitosan/PEG Chitosan (PEG) scafolds contain larger pores compared with pure chitosan Figure 1: SEM micrographs and the corresponding pore size of scafolds. CHI-100 (100/0), CHI-90, and CHI-80 (80/20) scafolds. Te scale Te compressive properties of the chitosan-PEG scaf- bar in the images represents 300 μm. Te values represent folds were next evaluated (Figure 2(a)). Te compressive mean ± standard deviation, n � 4. indicates p< 0.05 vs. PEG-100 stresses of all scafolds increased with increasing strain until and PEG-90 or PEG-100 and PEG-80. a maximum at the end of the compression (70% strain). Te maximum compression stress of CHI-100, CHI-90, and sacrifces the scafold’s stifness. Te Chitosan-PEG scafolds CHI-80 scafolds was 56.1 ± 2.0, 46.9 ± 1.6, and with appropriate hydrophilicity were expected in favor of 41.3 ± 7.0 kPa, respectively. Te incorporation of PEG sig- mass transportation, and then cell proliferation and dif- nifcantly decreased the stifness of chitosan scafolds ferentiation. It was expected that cell proliferation would be (p< 0.05). Tis situation is most visible in the CHI-80. It is much improved by increasing the hydrophilicity of the not surprising because PEG is less stifness material com- three-dimensional scafolds, which even outweighed the pared to chitosan [27]. A similar argument by Cheng et al. disadvantages of the weaker mechanical property. Next, it [28] explains the blend of PNIPAM with PEG hydrogels was examined the efect of PEG incorporation in the culture exhibits a lower mechanical strength than pure PNIPAM. of osteoblasts. Tanuma et al. [29] reported that the PEG-cross-linked chitosan hydrogel flm swelling ratio increases with the decrease of molecular weight of PEG with the same content 3.2. Osteoblast Culture on the Chitosan Scafolds. Te non- sample, and the degradation rate of chitosan component was toxicity of the Chitosan scafold has been afrmed [34]. In found to be infuenced by the content and molecular weight this study, the chitosan-PEG scafold showed good cell of PEG. An increase in the total PEG content resulted in adhesion on all the used scafold formulations (Figure 3). Te cells on the pure chitosan scafold (Figure 3(a)) are few a considerable increase in the degradation rate. Te water contents in CHI-100, CHI-90, and CHI-80 and separately adhered on the surface, while the cells on scafolds were next determined. Te water uptake of the chitosan-PEG (Figures 3(b) and 3(c)) are more aggregated chitosan scafolds was signifcantly (p < 0.05) increased with which indicated the favored environment for cells pro- increasing PEG contents from 4476 to 6025% (Figure 2(b)) liferation. Cell proliferation is the process of multiplying the dry weight basis. Besides the hydrophilicity of the added number of cells, and in this process, mitochondria gained PEG, chitosan-PEG scafolds have higher pore size and more a central role in the regulation of cell proliferation [35]. It water storage space as a result the ratio of water absorption was found that the addition of PEG decreased one-day cell had a signifcant diference (p < 0.05) with unmodifed adhesion to the chitosan-based scafolds (Figure 4(a)). It is chitosan. Te previous study on incorporating PEG into not surprising because PEG is a well-known nonadhesive Alginate/Elastin composite matrix indicates water content material [36, 37]. After fve days of incubation (Figure 4(a)), it was observed that the trend of cell number was still the increased with an increase in PEG content [30]. Similarly, Wan et al. [31] reported that the introduction of PEG same on the frst day; however, the cell number in all segments enhanced the surface hydrophilicity of the poly-l- scafolds signifcantly (p < 0.05) improved and the doubling lactide-polyethylene glycol copolymers. Likewise, several time of cells were 41.5, 22.6, and 23.3 h on CHI-100, CHI-90, modifcations (chemical, mechanical, and structural) of and CHI-80 scafolds, respectively. After 10 days of in- hyaluronic acid hydrogels have been conducted in the cubation, the cell numbers of CHI-90 (1.1 × 10 cells/scaf- fabrication of artifcial extracellular matrix [32]. Since fold) surpass that of CHI-100 (9.2 ×10 cells/scafold), while hyaluronic acid has negative charges, it can absorb large the cell number of CHI-80 (7.6 ×10 cells/scafold) was amounts of water and swell up to 1000 times in volume [33], signifcantly lower than the cell numbers of CHI-100 and However, chitosan is claimed for inadequate moisture CHI-90 (p < 0.001). During this period, the doubling time of availability, thus this study is the frst on improving the cells of CHI-100, CHI-90, and CHI-80 scafolds was 66.4, hydrophilicity of chitosan scafold via hydrophilic polymer 21.5, and 926 h, respectively, indicating that the rate of cell along with the increase of pore size and water content. proliferation of CHI-90 remained fast. However, the cell Overall, the incorporation of PEG increases the pore proliferation rate of CHI-100 and CHI-80 decreased, es- sizes and the water-uptake ability of chitosan scafolds but pecially CHI-80. Pore size (μm) International Journal of Biomaterials 5 7000.0 6000.0 50 * * 5000.0 4000.0 3000.0 2000.0 10 1000.0 0.0 100/0 90/10 80/20 100/0 90/10 80/20 Chitosan (PEG) Chitosan (PEG) (a) (b) Figure 2: (a) Compressive modulus of CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p < 0.05 vs. PEG-100 and PEG-90; PEG-100, and PEG-80. (b) Te dry-based water content of CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p< 0.05 vs. PEG-100 and PEG-90; PEG-100, and PEG-80. (a) (b) (c) Figure 3: Te morphology of the cells on (a) CHI-100, (b) CHI-90, and (c) CHI-80 scafolds. Te scale bar in the images represents 60 μm. PEG in chitosan scafolds provides well hydration en- osteoblast diferentiation is mineralization, at which vironment. As a result, it may enhance the difusion of a mineral matrix containing mainly calcium phosphate is nutrients, bio-factors, and wastes. Hence, it might be the secreted and deposited by mature osteoblasts. main reason CHI-90 scafolds could maintain low doubling In this study, after osteogenic culture for one day, the time. On the other hand, during incubation, it was observed cellular ALP activity of CHI-80 was the highest, followed by that the CHI-80 scafold was too soft that it might afect the CHI-90 and CHI-100 (Figure 4(b)). However, after fve days cell proliferation of osteoblasts. It was reported before by of incubation, the ALP activity of CHI-100 and CHI-90 Tanuma et al. [29] that the degradation rate of the chitosan increased signifcantly (p < 0.001) compared to their frst day, respectively, and exceeded the values of CHI-80. After component was found to be infuenced by the content and molecular weight of PEG. An increase in total PEG content ten days of incubation, the ALP activity was decreased in all resulted in a considerable increase in the degradation rate. the samples. Te ALP activity of CHI-90 was the highest on Te osteogenic diferentiation of osteoblasts on the the ffth day, indicating the favored osteoblasts’ early-stage chitosan-PEG scafolds was investigated by early and late diferentiation. osteogenic markers. Alkaline phosphatase (ALP), an es- After the osteoblasts were cultured in the osteogenic sential enzyme for ossifcation, is an early bone marker medium for 2 weeks, the total amounts of calcium in CHI-90 protein, and one of the most frequently used markers to and CHI-100 were quantifed as 2.7 and 2.2 μmol/scafold; demonstrate osteoblast diferentiation [38]. Te fnal stage of whereas, the amount in CHI-80 was 1.9 μmol/scafold Compressive stress (KPa) Water content (%) 6 International Journal of Biomaterials ** ** ** ** ff 100/0 90/10 80/20 100/0 90/10 80/20 Chitosan (PEG) Chitosan (PEG) Day 1 Day 1 Day 5 Day 5 Day 10 Day 10 (a) (b) Figure 4: (a) Cell number of osteoblast after 1, 5, and 10 days of seeding on CHI-100, CHI-90, and CHI-80 scafolds. Te values represent ∗ ∗∗ mean ± standard deviation, n � 4. indicates p< 0.05; indicated p< 0.001 vs. Day 1 and Day 5. (b) ALP activity of osteoblast after 1, 5, and 10 days of seeding on CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p< 0.05; ∗∗ indicated p< 0.001 vs. Day 1 and Day 5. on the PEG-chitosan scafold showed better cell pro- liferation and diferentiation than that of the chitosan scafold. However, adding more PEG (≥20% wt.) into the scafolds has no beneft on the proliferation and diferen- tiation of osteoblast. Taken together, these results indicate that adding hydrophilic molecules such as polyethylene glycol at an optimum amount (10% wt) into chitosan changed the characteristic of the scafolds and improved the proliferation and diferentiation of osteoblast. Te bio- compatibility, safety, and biodegradability of the chitosan make it an excellent scafold candidate, and in the near 100/0 90/10 80/20 future will witness its crucial role in biomaterials and tissue Chitosan (PEG) engineering. Figure 5: Calcium deposition of osteoblast following 15 days in osteogenic media in the CHI-100, CHI-90, and CHI-80 scafolds. Data Availability Te values represent mean ± standard deviation, n � 4. indicates ∗∗ p< 0.05; indicated p< 0.001 vs. PEG-100 and PEG-90; PEG-100 All data used to support the fndings of this study are in- and PEG-80. cluded within the article. Conflicts of Interest (Figure 5). Te results indicate that calcium deposition between the CHI-100 and CHI-90 had a signifcant difer- Te authors declare that there are no conficts of interest. ence (p < 0.05), suggesting that osteoblast diferentiation is enhanced with the optimal amount of PEG (10%). However, Acknowledgments excess PEG (20%) signifcantly decreased osteoblasts min- eralization. For future work, it is suggested to investigate Te authors acknowledge National Taiwan University for optimizing hydrophilic polymer doping onto a chitosan funding this work. scafold. References 4. Conclusion [1] P. J. VandeVord, H. W. T. Matthew, S. P. DeSilva, L. Mayton, B. Wu, and P. H. Wooley, “Evaluation of the biocompatibility Te impact of PEG-incorporated chitosan scafolds on os- of a chitosan scafold in mice,” Journal of Biomedical Materials teoblasts diferentiation and proliferation has been dem- Research, vol. 59, no. 3, pp. 585–590, 2002. onstrated in this study. Te characteristic analysis of PEG- [2] T. Majima, T. Funakosi, N. Iwasaki et al., “Alginate and containing scafolds exhibited bigger pore size, weaker chitosan polyion complex hybrid fbers for scafolds in liga- mechanical properties, and higher water content compared ment and tendon tissue engineering,” Journal of Orthopaedic to unmodifed chitosan substrates. Te cultured osteoblasts Science, vol. 10, no. 3, pp. 302–307, 2005. Calcium deposition (μmol/scafold) 10 Cells/scaold ALP (μmol/cell/h) International Journal of Biomaterials 7 [3] W. B. Tsai, Y. R. Chen, W. T. Li, J. Y. Lai, and H. L. Liu, “RGD- [19] J. H. Lee, H. B. Lee, and J. D. Andrade, “Blood compatibility of conjugated UV-crosslinked chitosan scafolds inoculated with polyethylene oxide surfaces,” Progress in Polymer Science, vol. 20, no. 6, pp. 1043–1079, 1995. mesenchymal stem cells for bone tissue engineering,” Car- [20] G. Li, Y. Zhuang, Q. Mu, M. Wang, and Y. E. Fang, “Prep- bohydrate Polymers, vol. 89, no. 2, pp. 379–387, 2012. aration, characterization and aggregation behavior of am- [4] W. B. Tsai, Y. R. Chen, and H. L. Liu, “RGD-conjugated phiphilic chitosan derivative having poly (l-lactic acid) side crosslinked chitosan scafolds for culture and osteogenic chains,” Carbohydrate Polymers, vol. 72, no. 1, pp. 60–66, diferentiation of mesenchymal stem cells,” Journal of the Taiwan Institute of Chemical Engineers, vol. 44, no. 1, pp. 1–7, [21] Y. C. Kuo, H. F. Ku, and R. Rajesh, “Chitosan/c-poly(glutamic acid) scafolds with surface-modifed albumin, elastin and [5] W. B. Tsai, Y. R. Chen, H. L. Liu, and J. Y. Lai, “Fabrication of poly- l -lysine for cartilage tissue engineering,” Materials UV-crosslinked chitosan scafolds with conjugation of RGD Science and Engineering: C, vol. 78, no. 1, pp. 265–277, 2017. peptides for bone tissue engineering,” Carbohydrate Polymers, [22] P. Y. Wang, W. B. Tsai, and N. H. Voelcker, “Screening of rat vol. 85, no. 1, pp. 129–137, 2011. mesenchymal stem cell behaviour on polydimethylsiloxane [6] M. Das and C. F. Fox, “Chemical cross-linking in biology,” stifness gradients,” Acta Biomaterialia, vol. 8, no. 2, Annual Review of Biophysics & Bioengineering, vol. 8, no. 1, pp. 519–530, 2012. pp. 165–193, 1979. [23] W. B. Tsai, R. P. Y. Chen, K. L. Wei et al., “Polyelectrolyte [7] T. W. Chung, Y. F. Lu, H. Y. Wang et al., “Growth of human multilayer flms functionalized with peptides for promoting endothelial cells on diferent concentrations of gly-arg-gly-asp osteoblast functions,” Acta Biomaterialia, vol. 5, no. 9, grafted chitosan surface,” Artifcial Organs, vol. 27, no. 2, pp. 3467–3477, 2009. pp. 155–161, 2003. [24] A. Korzynskai and M. Zychowicz, “A method of estimation of [8] W. Wang, M. Fan, L. Zhang, S. H. Liu, L. Sun, and C. Y. Wang, the cell doubling time on basis of the cell culture monitoring “Compatibility of hyaluronic acid hydrogel and skeletal data,” Biocybernetics and Biomedical Engineering, vol. 28, muscle myoblasts,” Biomedical Materials, vol. 4, no. 2, Article no. 4, pp. 75–82, 2008. ID 025011, 2009. [25] W. B. Tsai, R. P. Y. Chen, K. L. Wei, S. F. Tan, and J. Y. Lai, [9] P. Y. Wang, H. H. Chow, J. Y. Lai, H. L. Liu, and W. B. Tsai, “Modulation of RGD-functionalized polyelectrolyte multi- “Dynamic compression modulates chondrocyte proliferation layer membranes for promoting osteoblast function,” Journal and matrix biosynthesis in chitosan/gelatin scafolds,” Journal of Biomaterials Science, Polymer Edition, vol. 21, no. 3, of Biomedical Materials Research Part B: Applied Biomaterials, pp. 377–394, 2010. vol. 91B, no. 1, pp. 143–152, 2009. [26] B. Vigani, S. Rossi, G. Sandri, M. C. Bonferoni, [10] H. Park, B. Choi, J. Nguyen et al., “Anionic carbohydrate- C. M. Caramella, and F. Ferrari, “Hyaluronic acid and containing chitosan scafolds for bone regeneration,” Car- chitosan-based nanosystems: a new dressing generation for bohydrate Polymers, vol. 97, no. 2, pp. 587–596, 2013. wound care,” Expert Opinion on Drug Delivery, vol. 16, no. 7, [11] G. Li, L. Zhang, C. Wang et al., “Efect of silanization on pp. 715–740, 2019. chitosan porous scafolds for peripheral nerve regeneration,” [27] S. Dumitriu, Polymeric Biomaterials, Marcel Dekker, Man- Carbohydrate Polymers, vol. 101, pp. 718–726, 2014. hattan, N. Y, 2002. [12] C. Lau, M. J. Cooney, and P. Atanassov, “Conductive mac- [28] D. Cheng, Y. Wu, Y. Guan, and Y. Zhang, “Tuning properties roporous composite Chitosan−Carbon nanotube scafolds,” of injectable hydrogel scafold by PEG blending,” Polymer, Langmuir, vol. 24, no. 13, pp. 7004–7010, 2008. vol. 53, no. 22, pp. 5124–5131, 2012. [13] M. J. Cooney, J. Petermann, C. Lau, and S. D. Minteer, [29] H. Tanuma, T. Saito, K. Nishikawa, T. Dong, K. Yazawa, and “Characterization and evaluation of hydrophobically modi- Y. Inoue, “Preparation and characterization of PEG-cross- fed chitosan scafolds: towards design of enzyme immobilized linked chitosan hydrogel flms with controllable swelling and fow-through electrodes,” Carbohydrate Polymers, vol. 75, enzymatic degradation behavior,” Carbohydrate Polymers, no. 3, pp. 428–435, 2009. vol. 80, no. 1, pp. 260–265, 2010. [14] G. B. Jiang, D. Quan, K. Liao, and H. Wang, “Novel polymer [30] T. Chandy, G. H. R. Rao, R. F. Wilson, and G. S. Das, “Te micelles prepared from chitosan grafted hydrophobic pal- development of porous alginate/elastin/PEG composite ma- mitoyl groups for drug delivery,” Molecular Pharmaceutics, trix for cardiovascular engineering,” Journal of Biomaterials vol. 3, no. 2, pp. 152–160, 2006. Applications, vol. 17, no. 4, pp. 287–301, 2003. [15] C. K. S. Pillai, W. Paul, and C. P. Sharma, “Chitin and chitosan [31] Y. Wan, W. Chen, J. Yang, J. Bei, and S. Wang, “Biodegradable polymers: chemistry, solubility and fber formation,” Progress poly(l-lactide)-poly(ethylene glycol) multiblock copolymer: in Polymer Science, vol. 34, no. 7, pp. 641–678, 2009. synthesis and evaluation of cell afnity,” Biomaterials, vol. 24, [16] M. Sugimoto, M. Morimoto, H. Sashiwa, H. Saimoto, and no. 13, pp. 2195–2203, 2003. Y. Shigemasa, “Preparation and characterization of water- [32] E. Ahmadian, S. M. Dizaj, A. Eftekhari et al., “Te potential soluble chitin and chitosan derivatives,” Carbohydrate Poly- applications of hyaluronic acid hydrogels in biomedicine,” mers, vol. 36, no. 1, pp. 49–59, 1998. Drug Research, vol. 70, no. 01, pp. 6–11, 2020. [17] N. Idota, T. Tsukahara, K. Sato, T. Okano, and T. Kitamori, [33] X. Xu, A. K. Jha, D. A. Harrington, M. C. Farach-Carson, “Te use of electron beam lithographic graft-polymerization X. Jia, and X. Jia, “Hyaluronic acid-based hydrogels: from on thermoresponsive polymers for regulating the di- a natural polysaccharide to complex networks,” Soft Matter, rectionality of cell attachment and detachment,” Biomaterials, vol. 8, no. 12, pp. 3280–3294, 2012. vol. 30, no. 11, pp. 2095–2101, 2009. [34] M. Rodr´ıguez-Vazquez, ´ B. Vega-Ruiz, R. Ramos-Zuñiga, ´ [18] N. A. Alcantar, E. S. Aydil, and J. N. Israelachvili, “Poly- D. A. Saldaña-Koppel, and L. F. Quiñones-Olvera, “Chitosan ethylene glycol–coated biocompatible surfaces,” Journal of and its potential use as a scafold for tissue engineering in Biomedical Materials Research, vol. 51, no. 3, pp. 343–351, regenerative medicine,” BioMed Research International, 2000. vol. 2015, pp. 1–15, Article ID 821279, 2015. 8 International Journal of Biomaterials [35] V. G. Antico Arciuch, M. E. Elguero, J. J. Poderoso, and M. C. Carreras, “Mitochondrial regulation of cell cycle and proliferation,” Antioxidants and Redox Signaling, vol. 16, no. 10, pp. 1150–1180, 2012. [36] J. G. Archambault and J. L. Brash, “Protein resistant poly- urethane surfaces by chemical grafting of PEO: amino- terminated PEO as grafting reagent,” Colloids and Surfaces B: Biointerfaces, vol. 39, no. 1-2, pp. 9–16, 2004. [37] W. H. Kuo, M. J. Wang, C. W. Chang et al., “Improvement of hemocompatibility on materials by photoimmobilization of poly(ethylene glycol),” Journal of Materials Chemistry, vol. 22, no. 19, pp. 9991–9999, 2012. [38] E. E. Golub and K. Boesze-Battaglia, Te Role of Alkaline Phosphatase in Mineralization, pp. 444–448, Lippincott Williams & Wilkins, Philadelphia U. S, 2008. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Biomaterials Hindawi Publishing Corporation

The Impact of Polyethylene Glycol-Modified Chitosan Scaffolds on the Proliferation and Differentiation of Osteoblasts

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Hindawi International Journal of Biomaterials Volume 2023, Article ID 4864492, 8 pages https://doi.org/10.1155/2023/4864492 Research Article TheImpactofPolyethyleneGlycol-ModifiedChitosanScaffoldson the Proliferation and Differentiation of Osteoblasts 1 2,3 Wei-Bor Tsai and Ibrahim Nasser Ahmed Department of Chemical Engineering, National Taiwan University, No. 1 Sec. 4 Roosevelt Rd., Taipei 106, Taiwan Nanotechnology Center of Excellence, Addis Ababa Science and Technology University, P.O. Box 16417, Addis Ababa, Ethiopia Department of Industrial Chemistry, College of Applied Sciences, Addis Ababa Science and Technology University, P.O. Box 16417, Addis Ababa, Ethiopia Correspondence should be addressed to Ibrahim Nasser Ahmed; ibrahim.nasser@aastu.edu.et Received 18 August 2022; Revised 20 December 2022; Accepted 24 December 2022; Published 3 January 2023 Academic Editor: Carlo Galli Copyright © 2023 Wei-Bor Tsai and Ibrahim Nasser Ahmed. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te objective of this study was to investigate the infuence of polyethylene glycol (PEG) incorporated chitosan scafolds on osteoblasts proliferation and diferentiation. Te chitosan polymer was initially modifed by the predetermined concentration of the photoreactive azido group for UV-crosslinking and with RGD peptides (N-acetyl-GRGDSPGYG-amide). Te PEG was mixed at diferent ratios (0, 10, and 20 wt%) with modifed chitosan in 96-well tissue culture polystyrene plates to prepare CHI-100, CHI- 90, and CHI-80 scafolds. PEG-containing scafolds exhibited bigger pore size and higher water content compared to unmodifed chitosan scafolds. After 10 days of incubation, the cell number of CHI-90 (1.1 × 106 cells/scafold) surpasses that of CHI-100 (9.2 ×105 cells/scafold) and the cell number of CHI-80 (7.6 ×105 cells/scafold) were signifcantly lower. Te ALP activity of CHI-90 was the highest on the ffth day indicating the favored osteoblasts’ early-stage diferentiation. Moreover, after 14 days of osteogenic culture, calcium deposition in the CHI-90 scafolds (2.7 μmol Ca/scafold) was signifcantly higher than the control (2.2 μmol Ca/scafold) whereas on CHI-80 was 1.9 μmol/scafold. Te results demonstrate that PEG-incorporated chitosan scafolds favored osteoblasts proliferation and diferentiation; however, mixing relatively excess PEG (≥20% wt.) had a negative impact on osteoblasts proliferation and diferentiation. C–H, O–H, and N–H bonds of nearby substance molecules 1. Introduction [6]. Another shortcoming is that chitosan lacks bioactive Chitosan, the deacetylated derivative of chitin, has been signals equivalent to those existing in the extracellular broadly utilized for the fabrication of tissue engineering matrix (ECM) for cell attachment, growth, and diferenti- scafolds due to its nontoxicity, biodegradability, good ation. Incorporation of bioactive signals such as ECM ad- biocompatibility [1], and resemblance to glycosaminogly- hesion proteins and cell-binding peptides into chitosan cans [2]. However, chitosan still possesses some short- substrates can enhance cell adhesion [7–9]. RGD- incorporated and crosslinked chitosan scafolds can be comings for such a purpose, for instance, the mechanical properties of chitosan scafolds may not be suitable to match employed for mesenchymal stem cell proliferation and os- some specifc tissue engineering applications. Previously it teogenic diferentiation [3, 4]. was utilized an azide-based UV-crosslinking mechanism for Tere are many reports aimed at improving chitosan crosslinking chitosan scafolds in order to increase the properties by blending with natural or synthetic molecules. mechanical properties of chitosan scafolds [3–5]. Upon UV Park et al. [10] developed composite chitosan scafolds irradiation, the azido groups are converted into highly re- containing anionic carbohydrates. Te incorporation of active nitrene groups which undergo direct insertion into chondroitin 4-sulfate or alginate in chitosan scafolds 2 International Journal of Biomaterials increased the compressive modulus of the scafolds and 2. Material and Methods enhanced apatite formation. Furthermore, apatite-coated 2.1. Material. Polyethylene glycol M 20,000 and most of the scafolds enhanced the spreading, proliferation, and osteo- n reagents were purchased from Sigma–Aldrich (USA) unless genic diferentiation of bone marrow stromal cells seeded on specifed otherwise. N-(3-dimethylaminopropyl)-N′- the scafolds. A report by Li et al. [11] using a 3- ethylcarbodiimide hydrochloride was purchased from Fluka aminopropyltriethoxysilane treatment for modifcation (USA), and acetic acid was purchased from Baker (USA). and biocompatibility of lyophilized chitosan porous scaf- RGD-peptide (N-acetyl-GRGDSPGYG-amide) was synthe- folds showed the silanization treatment with low 3- sized by Kelowna International Scientifc Inc. (Taipei, Tai- aminopropyltriethoxy silane concentration showed no sig- wan). Te peptide concentration was calculated by nifcant infuence on the morphology of chitosan scafolds, absorbance at 275 nm coming from the tyrosine residue (Y, the porosity, and surface amino densities were increased −1 −1 molar adsorption coefcient 1420 M cm ). after silanization whereas the swelling ratio was reduced, and Osteoblast culture medium contained α-minimum es- the degradation ratio in PBS and antiacid degradation sential medium (α-MEM, HyClone, USA), 10% fetal bovine properties of the silanized chitosan scafolds were signif- serum (JRH Biosciences, Australia), 0.0679% (v/v) 2- cantly improved. Chitosan doped with multiwalled carbon mercaptoethanol, 200 μg/mL gentamicin (GIBCO , Invi- nanotubes has been used to create highly porous conductive trogen, USA), and 25 μg/mL fungizone (GIBCO ), pH 7.4. scafolds [12]. Chitosan can also be modifed by the addition Te osteoblast culture medium supplemented with 1 mM of hydrophobic alkyl chains along the hydrophilic backbone sodium glycerophosphate, 0.1 μM dexamethasone, and of the chitosan polymer and Cooney et al. [13] reported the 50 μg/mL L-ascorbate constituted osteoblast diferentiation scafolds produced from the unmodifed chitosan were more medium. Phosphate bufered saline (PBS) contained stable and rigid and possessed average pore diameters that 137 mM NaCl, 2.7 mM KCl, 10 mM Na HPO , and 1.8 mM were generally smaller than those fabricated from the 2 4 KH PO (pH 7.4). hydrophobically modifed chitosan. Te generally larger 2 4 pores in the butyl-modifed chitosan scafolds might be explained by increased phase separation rates due to the 2.2. Conjugation of Photoreactive Azido Groups or Peptides to introduced hydrophobicity of the chitosan polymer. Te Chitosan. Azido groups were conjugated onto chitosan combination of hydrophobic groups opposed along an (molecular weight 50–190 kDa, 75–85% deacetylation) via otherwise hydrophilic backbone creates internal forces that a reaction forming covalent amide bonds between the amino tend to fold or buckle the polymer chain, creating regions groups of chitosan and the carboxyl groups of an azido- that exhibit micellar behavior [14]. benzoic acid ester. Briefy, 17.6 mg 5-azido-2-nitrobenzoic One major drawback of chitosan in drug delivery is its acid N-hydroxysuccinimide ester was dissolved in 200 μL low solubility. Chitosan is not soluble in aqueous solutions at dimethylsulfoxide and then mixed with chitosan solution neutral or alkaline pH, only soluble in aqueous acid solu- (0.1 g in 4.8 mL of 1% acetic acid), followed by 3 h incubation tions and a few organic solvents [15]. Hence, various chi- at room temperature. Te unreacted azido ester was re- tosan derivatives have been prepared for the purpose of drug moved by dialysis against deionized water through a seam- delivery [15, 16]. Similarly, in this work it was argued that less cellulose tube (MWCO 12,400 Da) in the dark for two chitosan is a relatively hydrophobic material; however, in the days with the changes of deionized water every 12 h. After natural tissues, the extracellular matrix is highly hydrated. freeze-drying, the azido-conjugated chitosan (CHI-g-AZ) Tus, the hydrophobic environment of chitosan scafolds was kept at 4 C until use. might not be suitable for tissue growth. A number of studies RGD peptides were conjugated onto chitosan molecules indicate that both morphology and hydrophilicity infuence via a carbodiimide reaction according to a previously de- the attachment of cells onto the surface of a scafold [17]. veloped procedure [5]. Te graft ratio of RGD to chitosan Terefore, an increase in the hydrophilicity of chitosan (CHI-g-RGD) was estimated as 2.75 mol% with respect to scafolds might improve tissue engineering outcomes. the total moles of the amino groups of chitosan molecules. Polyethylene glycol (PEG) possesses biodegradability, bio- compatibility, less toxicity, and hydrophilicity and has been widely used in biomedical applications, including surface 2.3. Preparation of Chitosan and polyethylene Glycol Mixed modifcation, bioconjugation, drug delivery, and tissue Scafold. A mixture of chitosan (unmodifed, CHI-g-AZ, engineering [18, 19]. Although there are interesting con- and CHI-g-RGD) and PEG with a total concentration of tributions to the preparation, characterization, and aggre- 10 mg/mL in 1% acetic acid was prepared at diferent weight gation behavior of amphiphilic chitosan derivatives having percentages (the composition and abbreviations listed in poly-L-lactic acid side chains [20], there is no report on Table 1). Chitosan-PEG mixed scafolds were prepared by describing the osteogenic proliferation or diferentiation of adding 70 μL/well of the unmodifed chitosan, CHI-g-AZ, cells, from hydrophilically modifed chitosan scafold. CHI-g-RGD, and PEG mixture in 96-well tissue culture Hence, this study aimed to investigate the efect of adding polystyrene (TCPS) plates. Briefy, the mixture was poured polyethylene glycol into the RGD-conjugatedcross-linked into 96-well TCPS plates (70 μL/well for cell culture ex- chitosan scafolds on the proliferation and diferentiation periments), followed by freeze-drying in the dark to form of osteoblasts. scafolds. Subsequently, chitosan substrates were crosslinked International Journal of Biomaterials 3 Table 1: Te weight percentages of the compositions in the exclusion. Te isolated cells were cultured in standard T75 scafolds. fasks to the second passage for the cell experiments. Prior to cell seeding, the chitosan-PEG scafolds were Type of scafolds Chitosan CHI-g-AZ CHI-g-RGD PEG soaked in 70% ethanol for 30 min, followed by rinses with CHI-100 40 50 10 0 sterilized PBS three times. For cell culture on the scafolds, CHI-90 30 50 10 10 20 μL of osteoblast suspension (1.5 ×10 cells/mL) was CHI-80 20 50 10 20 seeded onto scafolds, making the seeding density 3 ×10 cells per scafold. After 1, 5, or 10 days of culture, the by UV irradiation for 30 min (wavelength range cell-inoculated samples were analyzed for cell morphology 280–380 nm). Te UV crosslinking time and the PEG dose cell numbers and alkaline phosphatase (ALP) activities. were selected based on the previous reports [5, 21]. After the cell culture, the morphology of cells in the scafolds after 5 days of incubation was observed by SEM. Te adhered osteoblasts were lysed with 0.1% Triton X-100 2.4. Characterization of Chitosan Scafolds. Te morphology for 30 min. Cell proliferation was determined by the lactate of chitosan-PEG scafolds was observed by scanning electron dehydrogenase (LDH) method according to a reported microscope (SEM) images (JSM-5310, JEOL, Japan). Te protocol [9]. Intracellular alkaline phosphatase activities scafolds were frst dehydrated in graded series of ethanol were assayed by determining the release of p-nitrophenol solutions 30%, 50%, 70%, 90%, 95%, and 100% for 10 min from 4-nitrophenyl phosphate disodium salt at pH 10.2, as each step followed by CO critical point-drying. Samples reported previously [23]. were cut with a scalpel, coated with a gold layer on the Te cell doubling time (T ) was calculated using the section, and then observed with SEM at an acceleration following equation: voltage of 20 kV. Te pore sizes of the scafolds were analyzed using an ∆T T � . (2) NIH Image J. Pores in SEM images were traced manually, 2 log (∆N/No + 1) and the enclosed areas and perimeters of pores were de- termined by the NIH Image J software. Te hydraulic di- No is the number of cells at the beginning of the ob- ameters of the pores were determined by the following servation, and △N is the increase in the number of cells equation: pore diameter (D ) � 4 × area/perimeter [5] more during the period of time of the length △t. Each division than 100 pores were counted for each type of sample. increases the number of cells by adding 1, and △N is also the Te compressive stress-strain properties of the scafolds number of cell divisions during the same period [24]. were determined using a compressive testing machine (FGS- 50V-H, NIDECSIMPO Corporation, Japan) and a digital 2.6. Mineralization Culture of Osteoblast/Scafold Constructs. force gauge (FGP-0.5, NIDECSIMPO Corporation, Japan). Osteoblast/scafold constructs were cultured for 5 days in the Te scafolds were subjected to an unconfned uniaxial osteoblast culture medium, followed by 10 days of miner- compression to 70% strain at a compression velocity of alization culture in the osteoblast diferentiation medium 3 mm/s. Te continuous stress and peak stress were recorded with daily replenishment of L-ascorbate (50 μg/mL). Te and analyzed. total amount of calcium deposition was determined using Dried chitosan-PEG scafolds were soaked in deionized a calcium assay kit (Diagnostic Chemicals Limited, water for 24 h. Te surface water contents on the scafolds USA) [25]. were absorbed by a flter paper. Wet scafolds were weighed (W ), and then placed in a 70 C oven overnight and weighed 2.7. Statistical Analysis. Each experiment has been repeated again (W ). Te equation implemented to calculate water content is shown as follows: at least three times. Te data were presented as mean- ± standard deviation (SD). Te statistical assessment of (Ws − Wd) Water content percentage(%) � (%) × 100%. signifcant variations was performed by Microsoft Excel Wd 2010. Signifcance was assessed by one-way analysis of (1) variance (ANOVA) and two-tailed Student–Newman–Keuls multiple comparisons. Te probability of p ≤ 0.05 was considered as a signifcant diference, where the symbol of ∗ 2.5. Culture of Osteoblasts on Chitosan Scafolds. and ∗∗ marker represent p< 0.05 and p < 0.01, which is of Standard sterile cell culture techniques were used for all cell signifcant diference statistically in 95% and 99% confdence experiments. Te animal procedure was followed by the level, respectively. ethical guidelines of Care and Use of Laboratory Animals (National Taiwan University, National Institutes of Health 3. Results and Discussion Publication No. 85–23, revised 1985) and was approved by the Animal Center Committee of National Taiwan Uni- 3.1. Fabrication and Characterization of Chitosan/PEG versity. Primary osteoblasts were isolated from neonatal rat Scafolds. Chitosan has a structure alike the N- calvariae according to the previously published procedure acetylglucosamine, which exists in hyaluronic acid, is an [22]. Te number and viability of the isolated osteoblasts extracellular macromolecule, and it is vital in wound healing were determined using a hemocytometer with trypan blue [26]. Te morphology of the chitosan scafolds incorporating 4 International Journal of Biomaterials PEG was examined with SEM. All the scafolds exhibited an open pore microstructure with interconnectivity. Te pore structure of the scafolds at diferent PEG concentrations is similar to each other (Figure 1). Te average pore sizes of ** ** CHI-100, CHI-90, and CHI-80 were 33.3 ± 7.4, 42.2 ± 8.2, and 46.9± 8.6 μm, respectively, indicating the pore sizes of the scafolds were signifcantly increased (p < 0.05) with the increasing PEG contents in chitosan scafolds (Figure 1). It was suspected that since PEG is more hydrophilic than chitosan, more water molecules surround PEG and form larger ice crystals during the freezing step than pure chitosan 100/0 90/10 80/20 scafolds. As a result, after lyophilization chitosan/PEG Chitosan (PEG) scafolds contain larger pores compared with pure chitosan Figure 1: SEM micrographs and the corresponding pore size of scafolds. CHI-100 (100/0), CHI-90, and CHI-80 (80/20) scafolds. Te scale Te compressive properties of the chitosan-PEG scaf- bar in the images represents 300 μm. Te values represent folds were next evaluated (Figure 2(a)). Te compressive mean ± standard deviation, n � 4. indicates p< 0.05 vs. PEG-100 stresses of all scafolds increased with increasing strain until and PEG-90 or PEG-100 and PEG-80. a maximum at the end of the compression (70% strain). Te maximum compression stress of CHI-100, CHI-90, and sacrifces the scafold’s stifness. Te Chitosan-PEG scafolds CHI-80 scafolds was 56.1 ± 2.0, 46.9 ± 1.6, and with appropriate hydrophilicity were expected in favor of 41.3 ± 7.0 kPa, respectively. Te incorporation of PEG sig- mass transportation, and then cell proliferation and dif- nifcantly decreased the stifness of chitosan scafolds ferentiation. It was expected that cell proliferation would be (p< 0.05). Tis situation is most visible in the CHI-80. It is much improved by increasing the hydrophilicity of the not surprising because PEG is less stifness material com- three-dimensional scafolds, which even outweighed the pared to chitosan [27]. A similar argument by Cheng et al. disadvantages of the weaker mechanical property. Next, it [28] explains the blend of PNIPAM with PEG hydrogels was examined the efect of PEG incorporation in the culture exhibits a lower mechanical strength than pure PNIPAM. of osteoblasts. Tanuma et al. [29] reported that the PEG-cross-linked chitosan hydrogel flm swelling ratio increases with the decrease of molecular weight of PEG with the same content 3.2. Osteoblast Culture on the Chitosan Scafolds. Te non- sample, and the degradation rate of chitosan component was toxicity of the Chitosan scafold has been afrmed [34]. In found to be infuenced by the content and molecular weight this study, the chitosan-PEG scafold showed good cell of PEG. An increase in the total PEG content resulted in adhesion on all the used scafold formulations (Figure 3). Te cells on the pure chitosan scafold (Figure 3(a)) are few a considerable increase in the degradation rate. Te water contents in CHI-100, CHI-90, and CHI-80 and separately adhered on the surface, while the cells on scafolds were next determined. Te water uptake of the chitosan-PEG (Figures 3(b) and 3(c)) are more aggregated chitosan scafolds was signifcantly (p < 0.05) increased with which indicated the favored environment for cells pro- increasing PEG contents from 4476 to 6025% (Figure 2(b)) liferation. Cell proliferation is the process of multiplying the dry weight basis. Besides the hydrophilicity of the added number of cells, and in this process, mitochondria gained PEG, chitosan-PEG scafolds have higher pore size and more a central role in the regulation of cell proliferation [35]. It water storage space as a result the ratio of water absorption was found that the addition of PEG decreased one-day cell had a signifcant diference (p < 0.05) with unmodifed adhesion to the chitosan-based scafolds (Figure 4(a)). It is chitosan. Te previous study on incorporating PEG into not surprising because PEG is a well-known nonadhesive Alginate/Elastin composite matrix indicates water content material [36, 37]. After fve days of incubation (Figure 4(a)), it was observed that the trend of cell number was still the increased with an increase in PEG content [30]. Similarly, Wan et al. [31] reported that the introduction of PEG same on the frst day; however, the cell number in all segments enhanced the surface hydrophilicity of the poly-l- scafolds signifcantly (p < 0.05) improved and the doubling lactide-polyethylene glycol copolymers. Likewise, several time of cells were 41.5, 22.6, and 23.3 h on CHI-100, CHI-90, modifcations (chemical, mechanical, and structural) of and CHI-80 scafolds, respectively. After 10 days of in- hyaluronic acid hydrogels have been conducted in the cubation, the cell numbers of CHI-90 (1.1 × 10 cells/scaf- fabrication of artifcial extracellular matrix [32]. Since fold) surpass that of CHI-100 (9.2 ×10 cells/scafold), while hyaluronic acid has negative charges, it can absorb large the cell number of CHI-80 (7.6 ×10 cells/scafold) was amounts of water and swell up to 1000 times in volume [33], signifcantly lower than the cell numbers of CHI-100 and However, chitosan is claimed for inadequate moisture CHI-90 (p < 0.001). During this period, the doubling time of availability, thus this study is the frst on improving the cells of CHI-100, CHI-90, and CHI-80 scafolds was 66.4, hydrophilicity of chitosan scafold via hydrophilic polymer 21.5, and 926 h, respectively, indicating that the rate of cell along with the increase of pore size and water content. proliferation of CHI-90 remained fast. However, the cell Overall, the incorporation of PEG increases the pore proliferation rate of CHI-100 and CHI-80 decreased, es- sizes and the water-uptake ability of chitosan scafolds but pecially CHI-80. Pore size (μm) International Journal of Biomaterials 5 7000.0 6000.0 50 * * 5000.0 4000.0 3000.0 2000.0 10 1000.0 0.0 100/0 90/10 80/20 100/0 90/10 80/20 Chitosan (PEG) Chitosan (PEG) (a) (b) Figure 2: (a) Compressive modulus of CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p < 0.05 vs. PEG-100 and PEG-90; PEG-100, and PEG-80. (b) Te dry-based water content of CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p< 0.05 vs. PEG-100 and PEG-90; PEG-100, and PEG-80. (a) (b) (c) Figure 3: Te morphology of the cells on (a) CHI-100, (b) CHI-90, and (c) CHI-80 scafolds. Te scale bar in the images represents 60 μm. PEG in chitosan scafolds provides well hydration en- osteoblast diferentiation is mineralization, at which vironment. As a result, it may enhance the difusion of a mineral matrix containing mainly calcium phosphate is nutrients, bio-factors, and wastes. Hence, it might be the secreted and deposited by mature osteoblasts. main reason CHI-90 scafolds could maintain low doubling In this study, after osteogenic culture for one day, the time. On the other hand, during incubation, it was observed cellular ALP activity of CHI-80 was the highest, followed by that the CHI-80 scafold was too soft that it might afect the CHI-90 and CHI-100 (Figure 4(b)). However, after fve days cell proliferation of osteoblasts. It was reported before by of incubation, the ALP activity of CHI-100 and CHI-90 Tanuma et al. [29] that the degradation rate of the chitosan increased signifcantly (p < 0.001) compared to their frst day, respectively, and exceeded the values of CHI-80. After component was found to be infuenced by the content and molecular weight of PEG. An increase in total PEG content ten days of incubation, the ALP activity was decreased in all resulted in a considerable increase in the degradation rate. the samples. Te ALP activity of CHI-90 was the highest on Te osteogenic diferentiation of osteoblasts on the the ffth day, indicating the favored osteoblasts’ early-stage chitosan-PEG scafolds was investigated by early and late diferentiation. osteogenic markers. Alkaline phosphatase (ALP), an es- After the osteoblasts were cultured in the osteogenic sential enzyme for ossifcation, is an early bone marker medium for 2 weeks, the total amounts of calcium in CHI-90 protein, and one of the most frequently used markers to and CHI-100 were quantifed as 2.7 and 2.2 μmol/scafold; demonstrate osteoblast diferentiation [38]. Te fnal stage of whereas, the amount in CHI-80 was 1.9 μmol/scafold Compressive stress (KPa) Water content (%) 6 International Journal of Biomaterials ** ** ** ** ff 100/0 90/10 80/20 100/0 90/10 80/20 Chitosan (PEG) Chitosan (PEG) Day 1 Day 1 Day 5 Day 5 Day 10 Day 10 (a) (b) Figure 4: (a) Cell number of osteoblast after 1, 5, and 10 days of seeding on CHI-100, CHI-90, and CHI-80 scafolds. Te values represent ∗ ∗∗ mean ± standard deviation, n � 4. indicates p< 0.05; indicated p< 0.001 vs. Day 1 and Day 5. (b) ALP activity of osteoblast after 1, 5, and 10 days of seeding on CHI-100, CHI-90, and CHI-80 scafolds. Te values represent mean ± standard deviation, n � 4. indicates p< 0.05; ∗∗ indicated p< 0.001 vs. Day 1 and Day 5. on the PEG-chitosan scafold showed better cell pro- liferation and diferentiation than that of the chitosan scafold. However, adding more PEG (≥20% wt.) into the scafolds has no beneft on the proliferation and diferen- tiation of osteoblast. Taken together, these results indicate that adding hydrophilic molecules such as polyethylene glycol at an optimum amount (10% wt) into chitosan changed the characteristic of the scafolds and improved the proliferation and diferentiation of osteoblast. Te bio- compatibility, safety, and biodegradability of the chitosan make it an excellent scafold candidate, and in the near 100/0 90/10 80/20 future will witness its crucial role in biomaterials and tissue Chitosan (PEG) engineering. Figure 5: Calcium deposition of osteoblast following 15 days in osteogenic media in the CHI-100, CHI-90, and CHI-80 scafolds. Data Availability Te values represent mean ± standard deviation, n � 4. indicates ∗∗ p< 0.05; indicated p< 0.001 vs. PEG-100 and PEG-90; PEG-100 All data used to support the fndings of this study are in- and PEG-80. cluded within the article. Conflicts of Interest (Figure 5). Te results indicate that calcium deposition between the CHI-100 and CHI-90 had a signifcant difer- Te authors declare that there are no conficts of interest. ence (p < 0.05), suggesting that osteoblast diferentiation is enhanced with the optimal amount of PEG (10%). However, Acknowledgments excess PEG (20%) signifcantly decreased osteoblasts min- eralization. For future work, it is suggested to investigate Te authors acknowledge National Taiwan University for optimizing hydrophilic polymer doping onto a chitosan funding this work. scafold. References 4. Conclusion [1] P. J. VandeVord, H. W. T. Matthew, S. P. DeSilva, L. Mayton, B. Wu, and P. H. Wooley, “Evaluation of the biocompatibility Te impact of PEG-incorporated chitosan scafolds on os- of a chitosan scafold in mice,” Journal of Biomedical Materials teoblasts diferentiation and proliferation has been dem- Research, vol. 59, no. 3, pp. 585–590, 2002. onstrated in this study. Te characteristic analysis of PEG- [2] T. Majima, T. Funakosi, N. Iwasaki et al., “Alginate and containing scafolds exhibited bigger pore size, weaker chitosan polyion complex hybrid fbers for scafolds in liga- mechanical properties, and higher water content compared ment and tendon tissue engineering,” Journal of Orthopaedic to unmodifed chitosan substrates. Te cultured osteoblasts Science, vol. 10, no. 3, pp. 302–307, 2005. Calcium deposition (μmol/scafold) 10 Cells/scaold ALP (μmol/cell/h) International Journal of Biomaterials 7 [3] W. B. Tsai, Y. R. Chen, W. T. Li, J. Y. Lai, and H. L. Liu, “RGD- [19] J. H. Lee, H. B. Lee, and J. D. Andrade, “Blood compatibility of conjugated UV-crosslinked chitosan scafolds inoculated with polyethylene oxide surfaces,” Progress in Polymer Science, vol. 20, no. 6, pp. 1043–1079, 1995. mesenchymal stem cells for bone tissue engineering,” Car- [20] G. Li, Y. Zhuang, Q. Mu, M. Wang, and Y. E. Fang, “Prep- bohydrate Polymers, vol. 89, no. 2, pp. 379–387, 2012. aration, characterization and aggregation behavior of am- [4] W. B. Tsai, Y. R. Chen, and H. L. Liu, “RGD-conjugated phiphilic chitosan derivative having poly (l-lactic acid) side crosslinked chitosan scafolds for culture and osteogenic chains,” Carbohydrate Polymers, vol. 72, no. 1, pp. 60–66, diferentiation of mesenchymal stem cells,” Journal of the Taiwan Institute of Chemical Engineers, vol. 44, no. 1, pp. 1–7, [21] Y. C. Kuo, H. F. Ku, and R. Rajesh, “Chitosan/c-poly(glutamic acid) scafolds with surface-modifed albumin, elastin and [5] W. B. Tsai, Y. R. Chen, H. L. Liu, and J. Y. Lai, “Fabrication of poly- l -lysine for cartilage tissue engineering,” Materials UV-crosslinked chitosan scafolds with conjugation of RGD Science and Engineering: C, vol. 78, no. 1, pp. 265–277, 2017. peptides for bone tissue engineering,” Carbohydrate Polymers, [22] P. Y. Wang, W. B. Tsai, and N. H. Voelcker, “Screening of rat vol. 85, no. 1, pp. 129–137, 2011. mesenchymal stem cell behaviour on polydimethylsiloxane [6] M. Das and C. F. Fox, “Chemical cross-linking in biology,” stifness gradients,” Acta Biomaterialia, vol. 8, no. 2, Annual Review of Biophysics & Bioengineering, vol. 8, no. 1, pp. 519–530, 2012. pp. 165–193, 1979. [23] W. B. Tsai, R. P. Y. Chen, K. L. Wei et al., “Polyelectrolyte [7] T. W. Chung, Y. F. Lu, H. Y. Wang et al., “Growth of human multilayer flms functionalized with peptides for promoting endothelial cells on diferent concentrations of gly-arg-gly-asp osteoblast functions,” Acta Biomaterialia, vol. 5, no. 9, grafted chitosan surface,” Artifcial Organs, vol. 27, no. 2, pp. 3467–3477, 2009. pp. 155–161, 2003. [24] A. Korzynskai and M. Zychowicz, “A method of estimation of [8] W. Wang, M. Fan, L. Zhang, S. H. Liu, L. Sun, and C. Y. Wang, the cell doubling time on basis of the cell culture monitoring “Compatibility of hyaluronic acid hydrogel and skeletal data,” Biocybernetics and Biomedical Engineering, vol. 28, muscle myoblasts,” Biomedical Materials, vol. 4, no. 2, Article no. 4, pp. 75–82, 2008. ID 025011, 2009. [25] W. B. Tsai, R. P. Y. Chen, K. L. Wei, S. F. Tan, and J. Y. Lai, [9] P. Y. Wang, H. H. Chow, J. Y. Lai, H. L. Liu, and W. B. Tsai, “Modulation of RGD-functionalized polyelectrolyte multi- “Dynamic compression modulates chondrocyte proliferation layer membranes for promoting osteoblast function,” Journal and matrix biosynthesis in chitosan/gelatin scafolds,” Journal of Biomaterials Science, Polymer Edition, vol. 21, no. 3, of Biomedical Materials Research Part B: Applied Biomaterials, pp. 377–394, 2010. vol. 91B, no. 1, pp. 143–152, 2009. [26] B. Vigani, S. Rossi, G. Sandri, M. C. Bonferoni, [10] H. Park, B. Choi, J. Nguyen et al., “Anionic carbohydrate- C. M. Caramella, and F. Ferrari, “Hyaluronic acid and containing chitosan scafolds for bone regeneration,” Car- chitosan-based nanosystems: a new dressing generation for bohydrate Polymers, vol. 97, no. 2, pp. 587–596, 2013. wound care,” Expert Opinion on Drug Delivery, vol. 16, no. 7, [11] G. Li, L. Zhang, C. Wang et al., “Efect of silanization on pp. 715–740, 2019. chitosan porous scafolds for peripheral nerve regeneration,” [27] S. Dumitriu, Polymeric Biomaterials, Marcel Dekker, Man- Carbohydrate Polymers, vol. 101, pp. 718–726, 2014. hattan, N. Y, 2002. [12] C. Lau, M. J. Cooney, and P. Atanassov, “Conductive mac- [28] D. Cheng, Y. Wu, Y. Guan, and Y. Zhang, “Tuning properties roporous composite Chitosan−Carbon nanotube scafolds,” of injectable hydrogel scafold by PEG blending,” Polymer, Langmuir, vol. 24, no. 13, pp. 7004–7010, 2008. vol. 53, no. 22, pp. 5124–5131, 2012. [13] M. J. Cooney, J. Petermann, C. Lau, and S. D. Minteer, [29] H. Tanuma, T. Saito, K. Nishikawa, T. Dong, K. Yazawa, and “Characterization and evaluation of hydrophobically modi- Y. Inoue, “Preparation and characterization of PEG-cross- fed chitosan scafolds: towards design of enzyme immobilized linked chitosan hydrogel flms with controllable swelling and fow-through electrodes,” Carbohydrate Polymers, vol. 75, enzymatic degradation behavior,” Carbohydrate Polymers, no. 3, pp. 428–435, 2009. vol. 80, no. 1, pp. 260–265, 2010. [14] G. B. Jiang, D. Quan, K. Liao, and H. Wang, “Novel polymer [30] T. Chandy, G. H. R. Rao, R. F. Wilson, and G. S. Das, “Te micelles prepared from chitosan grafted hydrophobic pal- development of porous alginate/elastin/PEG composite ma- mitoyl groups for drug delivery,” Molecular Pharmaceutics, trix for cardiovascular engineering,” Journal of Biomaterials vol. 3, no. 2, pp. 152–160, 2006. Applications, vol. 17, no. 4, pp. 287–301, 2003. [15] C. K. S. Pillai, W. Paul, and C. P. Sharma, “Chitin and chitosan [31] Y. Wan, W. Chen, J. Yang, J. Bei, and S. Wang, “Biodegradable polymers: chemistry, solubility and fber formation,” Progress poly(l-lactide)-poly(ethylene glycol) multiblock copolymer: in Polymer Science, vol. 34, no. 7, pp. 641–678, 2009. synthesis and evaluation of cell afnity,” Biomaterials, vol. 24, [16] M. Sugimoto, M. Morimoto, H. Sashiwa, H. Saimoto, and no. 13, pp. 2195–2203, 2003. Y. Shigemasa, “Preparation and characterization of water- [32] E. Ahmadian, S. M. Dizaj, A. Eftekhari et al., “Te potential soluble chitin and chitosan derivatives,” Carbohydrate Poly- applications of hyaluronic acid hydrogels in biomedicine,” mers, vol. 36, no. 1, pp. 49–59, 1998. Drug Research, vol. 70, no. 01, pp. 6–11, 2020. [17] N. Idota, T. Tsukahara, K. Sato, T. Okano, and T. Kitamori, [33] X. Xu, A. K. Jha, D. A. Harrington, M. C. Farach-Carson, “Te use of electron beam lithographic graft-polymerization X. Jia, and X. Jia, “Hyaluronic acid-based hydrogels: from on thermoresponsive polymers for regulating the di- a natural polysaccharide to complex networks,” Soft Matter, rectionality of cell attachment and detachment,” Biomaterials, vol. 8, no. 12, pp. 3280–3294, 2012. vol. 30, no. 11, pp. 2095–2101, 2009. [34] M. Rodr´ıguez-Vazquez, ´ B. Vega-Ruiz, R. Ramos-Zuñiga, ´ [18] N. A. Alcantar, E. S. Aydil, and J. N. Israelachvili, “Poly- D. A. Saldaña-Koppel, and L. F. Quiñones-Olvera, “Chitosan ethylene glycol–coated biocompatible surfaces,” Journal of and its potential use as a scafold for tissue engineering in Biomedical Materials Research, vol. 51, no. 3, pp. 343–351, regenerative medicine,” BioMed Research International, 2000. vol. 2015, pp. 1–15, Article ID 821279, 2015. 8 International Journal of Biomaterials [35] V. G. Antico Arciuch, M. E. Elguero, J. J. Poderoso, and M. C. Carreras, “Mitochondrial regulation of cell cycle and proliferation,” Antioxidants and Redox Signaling, vol. 16, no. 10, pp. 1150–1180, 2012. [36] J. G. Archambault and J. L. Brash, “Protein resistant poly- urethane surfaces by chemical grafting of PEO: amino- terminated PEO as grafting reagent,” Colloids and Surfaces B: Biointerfaces, vol. 39, no. 1-2, pp. 9–16, 2004. [37] W. H. Kuo, M. J. Wang, C. W. Chang et al., “Improvement of hemocompatibility on materials by photoimmobilization of poly(ethylene glycol),” Journal of Materials Chemistry, vol. 22, no. 19, pp. 9991–9999, 2012. [38] E. E. Golub and K. Boesze-Battaglia, Te Role of Alkaline Phosphatase in Mineralization, pp. 444–448, Lippincott Williams & Wilkins, Philadelphia U. S, 2008.

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International Journal of Biomaterials – Hindawi Publishing Corporation

Published: Jan 3, 2023

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