Synthesis and Characterisation of Biodegradable Carboxymethyl Cellulose Microcarriers from Oil Palm Empty Fruit Bunch for Therapeutic Applications

Synthesis and Characterisation of Biodegradable Carboxymethyl Cellulose Microcarriers from Oil Palm Empty Fruit Bunch for Therapeutic Applications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and Characterisation of Biodegradable Carboxymethyl Cellulose Microcarriers from Oil Palm Empty Fruit Bunch for Therapeutic Applications Soon Wei To, Rania Hussien Ahmed Al-Ashwal, Nurzila Ab Latif, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4663194/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Nov, 2024 Read the published version in Cellulose → Version 1 posted 8 You are reading this latest preprint version Abstract Microcarrier offers a convenient way to support cell adhesion and proliferation for biomedical applications. However, commercial microcarriers often have high production costs and limited biodegradability. The use of cellulose-rich oil palm empty fruit bunch (OPEFB) for the development of microcarriers could lead to a cheap, sustainable, and biodegradable cell culturing system. In this research, a series of carboxymethyl cellulose (CMC) microcarriers were prepared from OPEFB using FeCl 3 ionic crosslinker at various polymer and crosslinker levels. The microcarriers were characterised by various instrumental techniques, including assessment of gel content, swelling behaviour, mechanical stability, and in vitro degradation test. The resulting OPEFB-derived CMC-microcarriers exhibited an average size ranging from 1105.52 to 1322.25 µm. SEM analysis revealed that the fabricated CMC-microcarriers exhibited ridges and porous surface morphology and the EDX analysis confirmed the successful ionic crosslinking between the OPEFB-derived CMC biopolymer and FeCl 3 solution. In contrast with gel content results that increased from 16.95 to 42.65 %, the swelling behaviours regularly decreased from 385 to 32% with increasing concentrations of polymer and crosslinker. Higher concentrated samples (CMC-3, CMC-6, and CMC-9) demonstrated enhanced mechanical stability and reduced sensitivity to the environment due to the higher degree of crosslinking. Nevertheless, all microcarriers displayed a degree of biodegradability ranging from 40 to 90%. Overall, the findings suggest that OPEFB can serve as a cost-effective, sustainable, and biodegradable source of natural biomaterial for microcarrier development, contributing to advancements in tissue engineering and therapeutic applications. Microcarrier oil palm empty fruit bunch cellulose carboxymethyl cellulose carboxymethylation ionic crosslinking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Microcarrier culture systems, which were first proposed by van Wezel ( 1967 ) in 1967, have gained significant attention in biomedical research for their ability to support cell attachment and growth in three-dimensional (3D) cell culture systems. Unlike conventional methods, microcarriers serve as microscale scaffolds for anchorage-dependent cells enabling increased production capacity (Alkhatib et al., 2017 ), facilitated process scale-up (Bodiou et al., 2020 ), reduced cost requirement (Badenes et al., 2016 ) and improved control in cell culture applications (Chen et al., 2020 ). Early commercial microcarriers were typically synthetic polymer-based such as poly(lactide- co -glycolide) (PLGA), acrylamide, polystyrene, glass, and silica due to their defined chemical composition and adjustable mechanical properties. However, these petroleum-based commercial microcarriers often exhibit drawbacks such as high production costs, limited biodegradability, and restricted nature of the interaction (Alves et al., 2020 ; Tavassoli et al., 2018 ). Additionally, they must be removed from the cell suspension before implantation, leading to the loss of viable cells (Muoio et al., 2021 ). In view of such a predicament, there is growing interest in developing microcarriers from biodegradable natural polymeric materials such as collagen (Steffen et al., 2019 ), gelatine (Nweke and Stegemann, 2022 ), dextran (Rozwadowska et al., 2016 ), cellulose (Kalmer et al., 2019 ), chitosan (Nweke and Stegemann, 2020 ), alginate (Perteghella et al., 2017 )). Among these, cellulose, the most abundant and inexhaustible natural polysaccharide-based biopolymer on Earth, is particularly promising for cellular applications due to its excellent biocompatibility, biodegradability, and good mechanical strength (Courtenay et al., 2018 ; Seddiqi et al., 2021 ). Besides that, cellulose can also be chemically modified into various cellulose derivatives to enhance its usefulness in biomedical applications such as wound dressings and tissue engineering (Hon, 2017 ; Xie et al., 2019 ). Carboxymethyl cellulose (CMC) is among the cellulose derivatives that have gained remarkable attention. It is produced from the cellulose chain through the substitution of its hydroxyl group backbone with the carboxymethyl group (-CH 2 -COOH). CMC is a prominent water-soluble polyelectrolyte cellulose biomaterial which is chemically reactive, non-allergenic, non-toxic, and biodegradable (Huang et al., 2017 ; Kanikireddy et al., 2020 ; Yusup and Mahzan, 2018 ). Consequently, CMC is widely used in cosmetics, pharmaceutical products and biomedical applications (Ciolacu and Suflet, 2018 ; Rahman et al., 2021 ). However, the primary raw materials for CMC preparation are cotton linter and wood pulp which are considered costly agricultural products, increasing the overall production cost of CMC (Abd El-Sayed et al., 2020 ). Hence, the interest in the use of agricultural products and by-products as alternative cellulose resources for CMC preparation is gradually increasing. In view of this, the use of cellulose-rich oil palm empty fruit bunch (OPEFB) presents a useful alternative as a raw material to produce microcarriers. OPEFB is produced in huge quantities in oil palm plantation activities, with 1.07 tons generated per ton of palm oil produced, and the global production of OPEFB in 2018 alone accounted for around 80 million tons (Dolah et al., 2021 ). Malaysia, as the second-largest palm oil producer and the biggest exporter of oil palm products in the world, generates approximately 15 million tons of OPEFB annually (Abdul et al., 2016 ). The amount of OPEFB waste is expected to continue to increase due to the abundant land, cheap labour cost, and high global demand for oil palm products (Ali et al., 2020 ). Since the cellulose content from OPEFB constitutes nearly half its fibre weight, it offers substantial potential as a cellulose source for various applications. Conventionally, OPEFB biomass waste is often burnt or left for the mulching process at the plantation (Faizi et al., 2017 ). Converting OPEFB into cellulose and CMC could harness its potential as a natural biomaterial source for microcarrier development. While many types of lignocellulosic fibres such as canola straws {Zhang, 2023 #71} and sugarcane bagasse {Lam, 2017 #24} have been used for microcarriers and scaffold fabrication, research on the fabrication of cellulose-based microcarriers from OPEFB is limited, which hinders the exploration of this valuable resource and results in forfeiture of substantial economic value (Idris et al., 2021 ). Hence, the study believes the use of OPEFB as the cheaper sustainable cellulose resource for the CMC preparation to produce biodegradable microcarrier would be more acceptable. In this research, OPEFB cellulose is chosen due to the substantial amount of OPEFB biomass waste generated during palm oil production in Malaysia. Therefore, an innovative approach is required to turn OPEFB into a more valuable product that can reduce the negative impact on the environment while increasing the economic use of OPEFB in other non-biodiesel areas in Malaysia. Herein, this study aimed to address this gap by demonstrating the preparation of CMC-microcarriers derived from OPEFB biomass waste through an ionic crosslinking method using an iron (III) chloride solution. The fabricated microcarriers were characterised by physicochemical and morphological properties including particle size, gel content, swelling behaviour, mechanical stability, and in vitro degradation. The findings are expected to advance knowledge in the development of microcarriers from OPEFB, offering a valuable alternative for the application of biodegradable microcarriers in various therapeutic fields. 2 Experimental 2.1 Materials Oil palm empty fruit bunch (OPEFB) was obtained from one palm oil mill facility located in Kulai, Johor, Malaysia. Hydrogen peroxide (H 2 O 2 , 30%), sodium monochloroacetate (SMCA), iron (III) chloride hexahydrate (FeCl 3 .6H 2 O), and phosphate-buffered saline (PBS, pH 7.4) were purchased from Bio Basic Canada Inc. Sodium carboxymethylcellulose (NaCMC; C 8 H 15 O 8 Na) and sodium hydroxide pellet (NaOH) was purchased from Merck. Meanwhile, all chemical solvents such as formic acid (90%), isopropanol (99%), absolute methanol (99%), glacial acetic acid (99%), ethanol (95%), and acetone (99%) were purchased from R & M Chemicals. All chemicals and solvents used in this study are of analytical grade and used without further purification. Distilled water was used in all experiments. 2.2 Preparation of OPEFB-derived CMC OPEFB were pre-treated using the modified eco-friendly procedure (Nazir et al., 2013 ). The OPEFB fibres were cut, dewaxed using a 10% ( w/v ) NaOH solution and autoclaved at 21°C and 1.5 bar for an hour. The dewaxed fibres were then treated with 100 mL hydrogen peroxide (30%) and autoclaved under the same conditions. The delignified fibres were then collected and washed further with 10% formic acid, distilled water, and ethanol to remove excess lignin. The fibres were finally oven-dried at 60°C. The OPEFB-derived CMC was synthesised in two steps (Ab Rasid et al., 2021 ). Briefly, 15 g of OPEFB-cellulose was added in a mixed solution of 50 mL of 30% ( w/v ) NaOH solution and 450 mL isopropanol. After 1.5 hours, 10 mL isopropanol containing pre-dissolved 18 g of SMCA was added to the mixture and the reaction continued for 3 hours at 65°C. The slurry was then filtered and soaked in 100 mL of absolute methanol overnight. OPEFB-derived CMC was neutralised with glacial acetic acid and washed five times with 70% ethanol, followed by a one-time wash with absolute methanol. The OPEFB-derived CMC was oven-dried for 24 h at 60°C and kept in desiccators. 2.3 Fabrication of microcarriers Nine types of CMC-microcarriers were fabricated via ionic crosslinking reactions using FeCl 3 as a crosslinker (Akalin and Pulat, 2018 ). Various concentrations and quantities of OPEFB-derived CMC (4–8% w/v) and FeCl 3 (2–10% w/v) were prepared and termed as CMC-1 to CMC-9. The prepared OPEFB-derived CMC solutions were continuously added dropwise into FeCl 3 , by using a 20-gauge syringe needle. The obtained spherical microcarriers were left to crosslink for 3 hours under mechanical stirring at 200 rpm. The microcarriers were collected, filtered, and rinsed multiple times with distilled water to remove any unreacted FeCl 3 solution before drying at room temperature for 24 hours. Figure 1 shows the schematic illustration of the fabrication of the proposed microcarriers. 2.4 Measurement of microcarrier size For particle size determination, 50 beads of each formulation were spread over the flat surface using a spatula and watched randomly under a light transmission microscope (Model: Nikon Eclipse Ti-S, Japan). The diameter of samples was then analysed from the microscopy images using image analysis software (ImageJ; National Institutes of Health, USA). The particle size of the samples was expressed as the mean value ± standard deviations. 2.5 Scanning electron microscopy (SEM) The images of the sample surface were examined using a scanning microscope (Model: JEOL JSM-IT300LV, Japan). The samples were first freeze-dried, mounted, and sputter-coated with a thin layer of gold. The SEM observations were carried out at an accelerating voltage of 30 kV, with magnification at 50×. Energy-dispersive X-ray (EDX) spectroscopy was performed to collect full-scale elemental quantification data. 2.6 Gel content The gel contents of the samples were determined by immersing the dried samples in deionised water for 72 hours at room temperature. Then, the samples were dried in an oven at 60ºC until a constant weight was obtained. The gel content of the samples was calculated as follows: $$GC\left(\%\right) = \frac{({W}_{i} - {W}_{d})}{{W}_{i}}\times 100\%$$ 1 where GC is the gel content of the sample, W i is the initial weight of the sample, and W d is the weight of the sample after the immersion and drying process. 2.7 Swelling behaviours The swelling behaviours of the samples were conducted under varying time, temperature, and pH. Dried samples were immersed in PBS at room temperature. The samples were periodically removed, dried, and weighed. The test was repeated until a constant weight was obtained. The effect of temperature on swelling was studied by immersing dried samples in PBS at temperatures ranging from 10 to 60°C. The pH effect was studied by immersing dried microcarriers in PBS with pH values ranging from 2.0 to 12.0. All tests were conducted in triplicates to obtain reproducible results. The degree of swelling ( S w ) was determined using the following equation: $${S}_{w}\left(\%\right) = \frac{({W}_{f} - {W}_{0})}{{W}_{0}}\times 100\%$$ 2 where W t and W 0 represent the weight of the swollen and dry samples, respectively. 2.8 Mechanical stability and in vitro degradation test Sample suspension of each formulation ( n = 3) was tested at various centrifugal forces between 100 and 6000x G. After that, the number of the deformed or ruptured microcarriers was identified out of at least 100 microcarriers per set under a light transmission microscope (Aydogdu et al., 2016 ). The deformation rate ( D f ) was then determined by using; D f (%)=(N f /N T ) × 100% $${D}_{f}\left(\%\right) = \frac{\left({N}_{f}\right)}{{N}_{r}}\times 100\%$$ 3 where N f represents the number of deformed samples and N T is the total number of tested samples. 2.9 In vitro degradation test The degradation rate of the samples was determined by putting predetermined weights of sterilised samples into PBS solution followed by incubation at 37°C for 45 days. The swollen samples were removed from the solution after 24 hours, and the weights were reported as the maximum swollen state of the samples. Then, the samples were put into the same bath and weighed at regular intervals. The degradation rate ( D e ) was then calculated using; $${D}_{g}\left(\%\right) = \frac{({W}_{m}-{W}_{f})}{{W}_{m}}\times 100\%$$ 3 where W 0 represents the maximum weight of the samples at the most swollen stage and W f is the wet weight at a certain time point. 2.10 Statistical analysis All the experiments were performed in triplicate and the results were expressed as mean ± standard deviation. A one-way analysis of variance ANOVA followed by Tukey HSD Post Hoc test was then computed to evaluate the significance of the difference between each test using IBM SPSS Statistics version 27 software. All tests were done with a confidence interval of 95% and differences at P > 0.05 were considered statistically significant. 3 Results and discussion 3.1 Fabrication of OPEFB-CMC Microcarriers This study focuses on the preparation of microcarriers using the reactive functional group of carboxymethylated cellulose derivatives from OPEFB to interact with reactive groups of crosslinkers via an ionic crosslinking process. Among various crosslinkers, trivalent metal ions are identified as an appropriate crosslinker to prepare these types of microcarriers due to their eco-friendly and nontoxic nature (Bulut and Şanlı, 2016 ). Consequently, iron (III) ions were employed as trivalent crosslinkers in the fabrication of CMC microcarriers. The OPEFB-CMC solution, prepared at concentrations of 4%, 6%, and 8%, was incrementally added to aqueous FeCl 3 solutions of varying concentrations (2%, 6%, and 10%) to harden the droplets. As anticipated, the CMC microcarriers formed upon the dropwise addition of aqueous OPEFB-CMC into the FeCl 3 solution, due to ionic crosslinking between the carboxylate ions (COO-) on CMC and Fe 3+ ions from FeCl 3 . Figure 4.4 suggests the general mechanisms for ionic crosslinking between the carboxylic groups of OPEFB-CMC and the iron (III) crosslinker. 3.2 Measurement of Microcarrier Size The average diameter of microcarriers was investigated using a light transmission microscope and image analysis software (Image J) and expressed in Table 1 . Microcarriers exhibited an average size ranging from 1105.52 to 1322.25 µm. Among all the samples, CMC-7 was recorded to have the largest diameter, while CMC-3 had the smallest diameter statistically among all the microcarriers. Interestingly, the study found that the size of the microcarriers increased with the concentration of the biopolymer (OPEFB-derived CMC) increased. This observation aligns with previous research, which suggested that an increased biopolymer concentration leads to higher viscosity and larger microcarriers due to a high degree of crosslinking (Kaur et al., 2018 ). The increase in biopolymer concentration also results in thicker fluid extrusion through the nozzle of the needle which forms larger droplets and ultimately, larger microcarriers (Mehregan Nikoo et al., 2016 ; Takimoto et al., 2022 ). Contrarily, the concentration of FeCl 3 showed an inverse relationship with the particle sizes of the microcarriers. As the FeCl 3 concentration increased. This is consistent with previous studies suggesting that a higher crosslinker concentration results in more compact microcarriers due to increased penetration of crosslinker ions into the droplets, expulsion of water molecules, and tightening of the gel network (Kaur et al., 2018 ). Furthermore, an increase in the polymer concentration and the crosslinking extent also enhances the matrix density of the microcarriers due to a stronger crosslinking reaction, resulting in smaller but more compact microcarriers (Zou et al., 2015 ). Table 1 The diameter size (µm) of microcarriers derived from OPEFB-derived CMC. Microcarriers Concentrations ( w/v , %) Diameter Size (µm) OPEFB derived CMC FeCl 3 CMC-1 4 2 1203.65 ± 229.64 cde CMC-2 4 6 1135.73 ± 169.86 de CMC-3 4 10 1105.52 ± 44.97 e CMC-4 6 2 1302.35 ± 171.31 ab CMC-5 6 6 1262.73 ± 125.15 abcd CMC-6 6 10 1236.98 ± 237.16 bcd CMC-7 8 2 1352.25 ± 68.93 a CMC-8 8 6 1328.29 ± 58.72 abc CMC-9 8 10 1285.22 ± 66.61 abcd All values are mean ± standard deviation ( n > 50). Different superscript letters across a column indicate a significant difference between means ( p < 0.05) 3.3 SEM analysis SEM micrographs of CMC-microcarriers analysed at 50× magnification are shown in (Fig. 3 ). CMC-microcarriers exhibited different morphological shapes and sphericity. However, the shape of the MC became more spherical when the concentration of OPEFB-derived CMC and FeCl 3 increased from 4 to 8% ( w/v ) and 2 to 10% ( w/v ), respectively. These suggested that an increase in both biopolymer and crosslinking agent concentrations facilitates the formation of spherical microcarriers by increasing the homogenous crosslinking network between the polymeric chains. The surface of all CMC-microcarriers appeared to be covered with grooves and ridges which could provide a potential surface for the attachment of the cells. Meanwhile, a more granular structure was observed on the surface at 10000× magnification (Fig. 4 ). The ionic crosslinking occurred intensively on the surface and resulted in the formation of a granulated view on the surface of microcarriers (Akalin and Pulat, 2018 ). All microcarriers possessed a unique three-dimensional spongy structure with irregular sizes and distribution of pores. This porosity serves as an important feature of the MC as it determines the diffusion and absorption of water into the structure which substantially affects the swelling ratio of the microcarriers (Tuan Mohamood et al., 2021 ). In previous studies, it has been observed that the concentration of biopolymer and crosslinker in microcarriers influences the sizes and density of surface pores (Vulpe et al., 2016 ). The observed variations in porosity and pore size among microcarriers can be attributed to the different crosslinking densities resulting from varying amounts of biopolymer and crosslinker (Rahman et al., 2019 ). 3.4 EDX Analysis The elemental analysis was carried out by EDX to confirm the formation of the CMC MC. The EDX spectra of the samples are shown in Fig. 5 . Carbon (C) and oxygen (O) were detected in the EDX analysis of the samples, indicating that they are major components of the fabricated material. The presence of these elements can be attributed to the incorporation of OPEFB-derived CMC, which has the chemical formula (CH 2 COOH) n . All spectra depicted a well-defined peak corresponding to Fe which confirmed the presence of successful ionic crosslinking between FeCl 3 and CMC during the formation of CMC-microcarriers (Bardajee et al., 2014 ). The peak intensity of Fe was increased with an increasing FeCl 3 concentration from 2 to 10% ( w/v ) in 4%, 6%, and 8% ( w/v ) OPEFB-derived CMC solution, respectively. A similar trend was obtained for the atomic fraction values (Table 2 ). This is because more Fe ions are available to be ionically crosslinked to the polymer chains during the MC fabrication process as the FeCl 3 level rises, increasing the proportion of Fe in the structure. A similar conclusion was reported by Akalin and Pulat ( 2018 ). From the results, it could be concluded that the ionic crosslinking between the OPEFB-derived CMC biopolymer and FeCl 3 solution was successful. Table 2 The weight and atomic fractions (%) of Fe elements for the fabricated CMC MC with various formulations Microcarrier Concentration ( w/v . %) Fe OPEFB derived CMC FeCl 3 Weight (%) Atomic (%) CMC-1 4 2 7.03 ± 0.31 1.87 ± 0.08 CMC-2 4 6 14.80 ± 0.82 4.46 ± 0.28 CMC-3 4 10 23.67 ± 0.78 7.61 ± 0.32 CMC-4 6 2 12.97 ± 0.41 3.79 ± 0.14 CMC-5 6 6 16.23 ± 0.49 5.03 ± 0.18 CMC-6 6 10 25.23 ± 1.28 6.71 ± 0.35 CMC-7 8 2 14.60 ± 0.22 4.74 ± 0.15 CMC-8 8 6 18.77 ± 0.05 4.99 ± 0.01 CMC-9 8 10 28.83 ± 7.18 10.32 ± 3.23 All values are mean ± standard deviation ( n = 3) 3.5 Determination of Gel Content Gel content ( GC ) is a measure of crosslinking within microcarrier networks, and it significantly influences their properties. A higher GC indicates greater crosslinking, resulting in more spherical and mechanically robust microcarriers which are advantageous for tissue engineering applications (Rebers et al., 2021 ). Figure 6 depicts the changes in GC of OPEFB-derived CMC-microcarriers at various formulations. The lowest and highest GC values were 16.95% and 42.65% for CMC-1 and CMC-9, respectively. The results showed that GC values increased significantly with the concentration of OPEFB-derived CMC. The increase in GC of the samples was due to enhanced cross-linkages between CMC chains. A similar conclusion was also reached by a prior study reporting that the GC value of CMC hydrogel steadily rose with CMC concentration up to 20% ( w/v ) (Tuan Mohamood et al., 2021 ). However, the study pointed out that an excessively high concentration of CMC can lead to a decrease in GC due to the presence of uncross-linked CMC chains in the sample structure after gelation Despite that, it is not observed at 8% ( w/v ) OPEFB-derived CMC. In terms of FeCl 3 concentration, an increase led to a significant rise in GC . This is attributed to the enhanced electrostatic attraction between anionic charges of polymer chains and multivalent cations, promoting ionic crosslinking of polymers (Sultana et al., 2012 ). A similar pattern of results was obtained in previous literatures (Che Nan et al., 2019 ; Tuan Mohamood et al., 2021 ). The increasing ionic strength of the FeCl 3 crosslinking buffer positively affected CMC cross-linking, as reflected by increased GC . In conclusion, an optimum MC formulation was achieved at 8% OPEFB-derived CMC and 10% FeCl 3 concentration (CMC-9), yielding a GC value of 42.65%. 3.6 Effect of Time, Temperature, and pH on Swelling Behaviour The degree of swelling ( S w ) is a key characteristic of crosslinked microcarriers, influencing their structural changes in response to external stimuli such as temperature, pH, ionic strength, and substance concentration (Dong and Chen, 2020 ). As depicted in Fig. 7 (b), S w values initially increased over time, stabilising at nearly 24 hours. The lowest and highest S w values were observed in CMC-9 (32%) and CMC-1 (385%), respectively. S w values increased with the proportion of OPEFB-derived CMC and iron trivalent ions but were inversely correlating with the gel content of the samples (Fig. 6 ). S w was influenced by the hydrophilicity of the carboxylic groups on the polymer chain which led to swelling when water molecules filled the inert pores in the polymeric chains and created an osmotic pressure within the microcarrier (Zakiah et al., 2015 ) (Sathali and Varun, 2012 ). The swelling of CMC-microcarriers was governed by the degree of crosslinking, with greater cross-linkages reducing the swelling due to limited space available for the diffusion of free water into the polymer network (Che Nan et al., 2019 ; Patil et al., 2016 ; Tuan Mohamood et al., 2021 ). Lower S w was found in samples with higher gel content percentages (CMC-3, CMC-6, and CMC-9), while S w value rose in samples with low gel content (CMC-1, CMC-4, and CMC-7). Thus, S w values were directly linked to the concentration of biopolymer and crosslinker, with samples exhibiting lower swelling behaviour considered more stable due to their reduced likelihood of surface morphology changes upon hydration (de Bournonville et al., 2021 ). Figure 7 (c) illustrates the variations of S w values with temperature at pH 7.0 and 24 h, showing a gradual increase in S w across all samples with different rates of increase. The rise of S w values from 10 ˚C to 60 ˚C can be attributed to the enhanced thermal mobility of polymer chains at higher temperatures. This results in a more relaxed and unrigid polymer network, allowing more water molecules to fill in the accessible void within the structure, thereby increasing S w values (Akalin and Pulat, 2018 ). Conversely, the values of S w decreased as the temperature dropped to 10 ˚C due to the low diffusion rate of water molecules into the hydrophilic polymeric chains. A regular decrease was observed in S w values as the amount of OPEFB-derived CMC and FeCl 3 increased from 4–8%, and 2–10%, respectively. For instance, S w values were determined to be 765% for the CMC-1 MC and 73% for the CMC-9 MC at 60 ˚C. This result can be explained by the enhanced degree of crosslinking (reflected by the gel content percentage) with increased OPEFB-derived CMC and FeCl 3 concentrations, which restricts sample expansion and reduces S w (Swamy and Yun, 2015 ). The ideal CMC MC should maintain stability even near 60 ˚C so that they could be suitable for use in wide temperature ranges (Xiao et al., 2005 ). Thus, samples such as CMC-3, CMC-6, and CMC-9 were identified as the optimal microcarriers in terms of thermal stability due to their comparatively lower increment of S w readings over increasing incubation temperature. Figure 7 (d) shows the swelling ratio for fabricated microcarriers at various at 30 ˚C and 24 h. The S w values rose slightly from pH 2.0 to 6.0 and then followed a sharp increment from pH 7.0 to 12.0. CMC-1 and CMC-9 recorded the highest and lowest increases in S w , respectively. The change in S w across pH levels can be explained by the protonation and deprotonation of carboxylic groups (p Ka = 4.5) of the polymer at different surrounding pH (Thakur and Thakur, 2015 ). Under acidic pH (pH 2.0), most carboxylate anions (COO) of the polymer chains are protonated and converted to COOH form. This eliminates the main anion-anion repulsive forces within the polymer network and causes the degree of swelling to decrease (Ankur and Harish, 2018 ). At pH 7.0, the samples displayed noticeable swelling behaviour due to the neutralisation of protonated carboxylic groups on the polymer chain by counterions present in the environment. This reduces the electrostatic repulsion between the ionised carboxylate, thereby increasing the swelling capacity of the samples (Tavakol et al., 2013 ). As the pH of the environmental solution becomes basic, more carboxylic groups of the polymer are ionised to the COO, maximising the repulsion and swelling (Akalin and Pulat, 2018 ; Ankur and Harish, 2018 ). The S w was found to be inversely proportional to the GC percentage which increases proportionally to the amount of OPEFB-derived CMC and FeCl 3 . Therefore, a regular reduction was observed in S w values as the amount of OPEFB-derived CMC and FeCl 3 increased. In short, CMC-1 and CMC-9 exhibited the highest and lowest pH sensitivity, respectively, while CMC-3, CMC-6, and CMC-9 were identified as the most pH-stable microcarriers. 3.7 Mechanical stability The deformation rate ( D f ) of the samples with various formulations at different centrifugal forces is presented in Fig. 8 (a). The samples with higher concentrations of OPEFB-derived CMC and FeCl 3 exhibited better mechanical stability and less MC failure as reflected by the lower deformation rate recorded by CMC-9, CMC-6, and CMC-4 at the highest centrifuge rate (8000 G). The higher rate of deformation displayed in less-concentrated microcarriers (CMC-1, CMC-4, and CMC-7) could be due to their lower degree of crosslinking which primarily supports and improves the mechanical strength and shape of the developed microcarriers (Jose et al., 2012 ). This was then supported by Takimoto et al. ( 2022 ) who described that the mechanical properties of the microcarriers can be controlled and changed by varying the concentration of biopolymer and crosslinking bath. Their study also concluded that higher-concentration samples were stronger than the lower-concentration samples, which is directly in line with the present findings. In short, the deformation rate was found to be inversely proportional to the concentrations of OPEFB-derived CMC and FeCl 3 . CMC-9 and CMC-1 were discovered to be the most mechanically stable and least mechanically stable microcarriers, respectively, in various centrifugal forces. Therefore, samples with higher mechanical strength or with lower deformation rates such as CMC-3, CMC-6, and CMC-9 are considered the optimum microcarriers used for tissue engineering as limited mechanical properties can affect their applications and cell differentiation (Takimoto et al., 2022 ). 3.8 In vitro Degradation Analysis In vitro degradation studies are critical to understand the behaviour of fabricated microcarriers in physiological environments. Biodegradable microcarriers offer advantages to the biopharmaceutical industry by allowing precise control over degradation and enabling tailored drug release profiles (Handral et al., 2023 ). The degradation behaviour of the samples at pH 7.0 and 37 ˚C are graphically presented in Fig. 8 (b). A rapid increase in biodegradation rate was recorded for lower concentration samples of (CMC-1, CMC-4, and CMC-7), while higher concentration samples (CMC-3, CMC-6, and CMC-9) exhibited a gradual increase over time. The degradation process is governed by the hydrolytic cleavage of intramolecular and intermolecular hydrogen bonds in polysaccharides leading to a loss of structural stability and gradual degradation over time (Yan et al., 2019 ). Therefore, it is influenced by the number of intermolecular bonds in the polymer. The findings align with the previous study, indicating that higher crosslinking density had a decelerated degradation process (Akalin and Pulat, 2018 ). A faster degradation rate was obtained in all low-concentration samples due to the lower crosslinking density within the structure. This is because disintegration of network structure occurs more quickly in relatively loose networks (Lee et al., 2016 ). The degradation of all samples implies that CMC derived from OPEFB retains the biodegradability properties of cellulose and is suitable to be extensively used in tissue engineering (Kalmer et al., 2019 ). Despite having biodegradability, suitable microcarriers should maintain high structural stability for long-term applications (Akalin and Pulat, 2018 ). Among the tested samples, CMC-3, CMC-6, and CMC-9 exhibited better structural stability in physiological environments, making them suitable for long-term applications. These samples can also be modified to have an accelerated degradation rate according to the application needs by adding an external esterase enzyme in the medium (Lee et al., 2016 ). 4 Conclusions In conclusion, this study successfully demonstrated the synthesis of biodegradable CMC-microcarriers from OPEFB using FeCl 3 as the crosslinking agent. The fabricated CMC-microcarriers, with sizes ranging from 1105.52 to 1322.25 µm, exhibited a porous structure and significant mechanical stability, making them suitable for various biomedical applications. The microcarriers showed promising biodegradability and swelling behaviours, which can be tailored by adjusting the concentrations of OPEFB-derived CMC and FeCl 3 . Notably, samples with higher crosslinking densities displayed enhanced structural integrity and controlled degradation rates, which is essential for long-term therapeutic applications. These findings highlight the feasibility of using OPEFB, an agricultural waste product as a cost-effective, sustainable, and eco-friendly resource for developing advanced biodegradable biomaterials, paving the way for greener and more efficient solutions in the biomedical industry. Future research should focus on optimising the functional properties of these microcarriers to further expand their application in cell culture systems and tissue engineering. Declarations Acknowledgements The authors gratefully acknowledge the support of time and facilities from Universiti Teknologi Malaysia for this study. Funding The work was supported by Universiti Teknologi Malaysia under the Fundamental Research Grant Scheme (Grant No: 20H78). Author information Authors and Affiliations Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, Johor Darul Ta‘Zim, 81310, Skudai, Malaysia Soon Wei To, Nurzila Ab Latif, Mohd Helmi Sani School of Biomedical Engineering and Health Sciences, Faculty of Engineering, Universiti Teknologi Malaysia, Johor Darul Ta‘Zim, 81310, Skudai, Malaysia Rania Hussien Ahmed Al-Ashwal Contributions Soon Wei To: conceptualization, methodology, formal analysis and investigation, and writing – original draft preparation. Rania Hussien Ahmed Al-Ashwal: writing – review and editing. Nurzila Ab Latif: writing – review and editing. Mohd Helmi Sani: funding acquisition, project administration, resources, writing – review and editing and supervision. All authors have given approval to the final version of the manuscript. Corresponding author Correspondence to Mohd Helmi Sani Ethics declarations Ethical approval The research meets the ethical approval, including adherence to the legal requirements of the study country. Consent to participate The article has been written by the stated authors who are all aware of its content and approve its submission. Human and animal rights We declare that there are no animal studies or human participants involved in the study. Conflict of interest No conflict of interest to declare. References Ab Rasid, N. S., Zainol, M. M., & Amin, N. A. S. (2021). Synthesis and characterization of carboxymethyl cellulose derived from empty fruit bunch. Sains Malaysiana, 50 (9), 2523-2535. doi:10.17576/jsm-2021-5009-03 Abd El-Sayed, E. S., El-Sakhawy, M., & El-Sakhawy, M. A.-M. (2020). Non-wood fibers as raw material for pulp and paper industry. Nordic Pulp & Paper Research Journal, 35 (2), 215-230. doi:10.1515/npprj-2019-0064 Abdul, P. M., Jahim, J. M., Harun, S., Markom, M., Lutpi, N. A., Hassan, O. , et al. (2016). Effects of changes in chemical and structural characteristic of ammonia fibre expansion (AFEX) pretreated oil palm empty fruit bunch fibre on enzymatic saccharification and fermentability for biohydrogen. Bioresource Technology, 211 , 200-208. doi:10.1016/j.biortech.2016.02.135 Akalin, G. O., & Pulat, M. (2018). Preparation and characterization of nanoporous sodium carboxymethyl cellulose hydrogel beads. Journal of Nanomaterials, 2018 . doi:10.1155/2018/9676949 Ali, M. M., Muhadi, N. A., Hashim, N., Abdullah, A. F., & Mahadi, M. R. (2020). Pulp and paper production from oil palm empty fruit bunches: A current direction in Malaysia. Journal of Agricultural and Food Engineering, 2 , 1-9. Alkhatib, R., Hilal-Alnaqbi, A., Naciri, M., Al-Majmaie, R., Saseedharan, P., Karam, S. M., & Al-Rubeai, M. (2017). 3D culture of mouse gastric stem cells using porous microcarriers. Frontiers in Bioscience, 9 (1), 172-179. doi:10.2741/s481 Alves, T. F. R., Morsink, M., Batain, F., Chaud, M. V., Almeida, T., Fernandes, D. A. , et al. (2020). Applications of natural, semi-synthetic, and synthetic polymers in cosmetic formulations. Cosmetics, 7 (4), 75. doi:10.3390/cosmetics7040075 Ankur, G., & Harish, K. (2018). Synthesis and swelling behavior of superabsorbent hydrogels acquired from CMC for efficient drug-delivery. Research Journal of Chemistry and Environment, 22 (5), 5. Aydogdu, H., Keskin, D., Baran, E. T., & Tezcaner, A. (2016). Pullulan microcarriers for bone tissue regeneration. Mater Sci Eng C Mater Biol Appl, 63 , 439-449. doi:10.1016/j.msec.2016.03.002 Badenes, S., Fernandes-Platzgummer, A., Rodrigues, C., Diogo, M., da Silva, C., & Cabral, J. (2016). Microcarrier culture systems for stem cell manufacturing. Stem Cell Manufacturing , 77. Bardajee, G. R., Hooshyar, Z., Asli, M. J., Shahidi, F. E., & Dianatnejad, N. (2014). Synthesis of a novel supermagnetic iron oxide nanocomposite hydrogel based on graft copolymerization of poly((2-dimethylamino)ethyl methacrylate) onto salep for controlled release of drug. Materials Science and Engineering C-Materials for Biological Applications, 36 , 277-286. doi:10.1016/j.msec.2013.11.022 Bodiou, V., Moutsatsou, P., & Post, M. (2020). Microcarriers for Upscaling Cultured Meat Production. Frontiers in Nutrition, 7 , 10. Bulut, E., & Şanlı, O. (2016). Novel ionically crosslinked acrylamide-grafted poly(vinyl alcohol)/sodium alginate/sodium carboxymethyl cellulose pH-sensitive microspheres for delivery of Alzheimer's drug donepezil hydrochloride: Preparation and optimization of release conditions. Artificial Cells, Nanomedicine, and Biotechnology, 44 (2), 431-442. doi:10.3109/21691401.2014.962741 Che Nan, N. F., Zainuddin, N., & Ahmad, M. (2019). Preparation and swelling study of CMC hydrogel as potential superabsorbent. Pertanika Journal of Science & Technology, 27 (1). Chen, X. Y., Chen, J. Y., Tong, X. M., Mei, J. G., Chen, Y. F., & Mou, X. Z. (2020). Recent advances in the use of microcarriers for cell cultures and their ex vivo and in vivo applications. Biotechnology Letters, 42 (1), 1-10. doi:10.1007/s10529-019-02738-7 Ciolacu, D. E., & Suflet, D. M. (2018). 11 - cellulose-based hydrogels for medical/pharmaceutical applications. In V. Popa & I. Volf (Eds.), Biomass as Renewable Raw Material to Obtain Bioproducts of High-Tech Value (pp. 401-439): Elsevier. Courtenay, J. C., Deneke, C., Lanzoni, E. M., Costa, C. A., Bae, Y., Scott, J. L., & Sharma, R. I. (2018). Modulating cell response on cellulose surfaces; tunable attachment and scaffold mechanics. Cellulose, 25 (2), 925-940. doi:10.1007/s10570-017-1612-3 de Bournonville, S., Geris, L., & Kerckhofs, G. (2021). Micro computed tomography with and without contrast enhancement for the characterization of microcarriers in dry and wet state. Sci Rep, 11 (1), 2819. doi:10.1038/s41598-021-81998-8 Dolah, R., Karnik, R., & Hamdan, H. (2021). A comprehensive review on biofuels from oil palm empty bunch (EFB): Current status, potential, barriers and way forward. Sustainability, 13 (18), 10210. doi:10.3390/su131810210 Dong, M., & Chen, Y. (2020). Chapter 7 - The stimuli-responsive properties of hydrogels based on natural polymers. In Y. Chen (Ed.), Hydrogels Based on Natural Polymers (pp. 173-222): Elsevier. Faizi, M., Shahriman, A., Majid, M. A., Shamsul, B., Ng, Y., Basah, S. , et al. (2017). An overview of the oil palm empty fruit bunch (OPEFB) potential as reinforcing fibre in polymer composite for energy absorption applications. Paper presented at the MATEC web of conferences. Handral, H. K., Wyrobnik, T. A., & Lam, A. T. (2023). Emerging Trends in Biodegradable Microcarriers for Therapeutic Applications. Polymers (Basel), 15 (6), 1487. doi:10.3390/polym15061487 Hon, D. N.-S. (2017). Cellulose and its derivatives: structures, reactions, and medical uses. In Polysaccharides In Medicinal Applications (pp. 87-105): Routledge. Huang, C. M. Y., Chia, P. X., Lim, C. S. S., Nai, J. Q., Ding, D. Y., Seow, P. B. , et al. (2017). Synthesis and characterisation of carboxymethyl cellulose from various agricultural wastes. Cellulose Chemistry and Technology, 51 (7-8), 665-672. Idris, R., Chong, W. W. F., Ali, A., Idris, S., Hasan, M. F., Ani, F. N., & Chong, C. T. (2021). Phenol-rich bio-oil derivation via microwave-induced fast pyrolysis of oil palm empty fruit bunch with activated carbon. Environmental Technology & Innovation, 21 , 101291. doi:10.1016/j.eti.2020.101291 Jose, S., Fangueiro, J. F., Smitha, J., Cinu, T. A., Chacko, A. J., Premaletha, K., & Souto, E. B. (2012). Cross-linked chitosan microspheres for oral delivery of insulin: Taguchi design and in vivo testing. Colloids and Surfaces B: Biointerfaces, 92 , 175-179. doi:10.1016/j.colsurfb.2011.11.040 Kalmer, R. R., Mohammadi, M., Karimi, A., Najafpour, G., & Haghighatnia, Y. (2019). Fabrication and evaluation of carboxymethylated diethylaminoethyl cellulose microcarriers as support for cellular applications. Carbohydrate Polymers, 226 , 115284. doi:10.1016/j.carbpol.2019.115284 Kanikireddy, V., Varaprasad, K., Jayaramudu, T., Karthikeyan, C., & Sadiku, R. (2020). Carboxymethyl cellulose-based materials for infection control and wound healing: A review. International Journal of Biological Macromolecules, 164 , 963-975. doi:10.1016/j.ijbiomac.2020.07.160 Kaur, N., Singh, B., & Sharma, S. (2018). Hydrogels for potential food application: Effect of sodium alginate and calcium chloride on physical and morphological properties. The Pharma Innovation Journal, 7 (7), 142-148. Lee, S., Park, Y. H., & Ki, C. S. (2016). Fabrication of PEG-carboxymethylcellulose hydrogel by thiol-norbornene photo-click chemistry. International Journal of Biological Macromolecules, 83 , 1-8. doi:10.1016/j.ijbiomac.2015.11.050 Mehregan Nikoo, A., Kadkhodaee, R., Ghorani, B., Razzaq, H., & Tucker, N. (2016). Controlling the morphology and material characteristics of electrospray generated calcium alginate microhydrogels. Journal of Microencapsulation, 33 (7), 605-612. doi:10.1080/02652048.2016.1228707 Muoio, F., Panella, S., Lindner, M., Jossen, V., Harder, Y., Moccetti, T. , et al. (2021). Development of a biodegradable microcarrier for the cultivation of human adipose stem cells (hASCs) with a defined xeno- and serum-free medium. Applied Sciences, 11 (3), 925. doi:10.3390/app11030925 Nazir, M. S., Wahjoedi, B. A., Yussof, A. W., & Abdullah, M. A. (2013). Eco-friendly extraction and characterization of cellulose from oil palm empty fruit bunches. Bioresources, 8 (2), 2161-2172. doi:10.15376/BIORES.8.2.2161-2172 Nweke, C. E., & Stegemann, J. P. (2020). Modular microcarrier technologies for cell-based bone regeneration. Journal of Biomedical Materials Research Part B: Applied Biomaterials 8 (18), 3972-3984. doi:10.1039/d0tb00116c Nweke, C. E., & Stegemann, J. P. (2022). Fabrication and characterization of osteogenic function of progenitor cell-laden gelatin microcarriers. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 110 (6), 1265-1278. doi:10.1002/jbm.b.34998 Patil, J. S., Kole, S. G., Gurav, P. B., & Vilegave, K. V. (2016). Natural polymer based mucoadhesive hydrogel beads of nizatidine: Preparation, characterization and evaluation. Indian Journal of Pharmaceutical Education and Research, 50 (1), 159-169. doi:10.5530/ijper.50.1.20 Perteghella, S., Martella, E., de Girolamo, L., Perucca Orfei, C., Pierini, M., Fumagalli, V. , et al. (2017). Fabrication of innovative silk/alginate microcarriers for mesenchymal stem cell delivery and tissue regeneration. International Journal of Molecular Sciences, 18 (9), 1829. doi:10.3390/ijms18091829 Rahman, M., Hasan, M., Nitai, A. S., Nam, S., Karmakar, A. K., Ahsan, M. , et al. (2021). Recent developments of carboxymethyl cellulose. Polymers, 13 (8), 1345. Rahman, M. S., Islam, M. M., Islam, M. S., Zaman, A., Ahmed, T., Biswas, S. , et al. (2019). Morphological characterization of hydrogels. Cellulose-Based Superabsorbent Hydrogels , 819-863. doi:10.1007/978-3-319-77830-3_28 Rebers, L., Reichsöllner, R., Regett, S., Tovar, G. E. M., Borchers, K., Baudis, S., & Southan, A. (2021). Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels. Scientific Reports, 11 (1), 3256. doi:10.1038/s41598-021-82393-z Rozwadowska, N., Malcher, A., Baumann, E., Kolanowski, T. J., Rucinski, M., Mietkiewski, T. , et al. (2016). In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3(R). Folia Histochemica et Cytobiologica, 54 (2), 81-90. doi:10.5603/FHC.a2016.0010 Sathali, A., & Varun, J. (2012). Formulation, development and in vitro evaluation of candesartan cilexetil mucoadhesive microbeads. International Journal of Current Pharmaceutical Research, 4 (3), 109-118. Seddiqi, H., Oliaei, E., Honarkar, H., Jin, J. F., Geonzon, L. C., Bacabac, R. G., & Klein-Nulend, J. (2021). Cellulose and its derivatives: towards biomedical applications. Cellulose, 28 (4), 1893-1931. doi:10.1007/s10570-020-03674-w Steffen, F., Bertolo, A., Affentranger, R., Ferguson, S. J., & Stoyanov, J. (2019). Treatment of naturally degenerated canine lumbosacral intervertebral discs with autologous mesenchymal stromal cells and collagen microcarriers: A prospective clinical study. Cell Transplant, 28 (2), 201-211. doi:10.1177/0963689718815459 Sultana, S., Islam, M., Dafader, N., Haque, M. E. u., Nagasawa, N., & Tamada, M. (2012). Effect of mono- and divalent salts on the properties of carboxymethyl cellulose hydrogel under irradiation technique. International Journal of Chemical Sciences, 10 . Swamy, B. Y., & Yun, Y. S. (2015). In vitro release of metformin from iron (III) cross-linked alginate-carboxymethyl cellulose hydrogel beads. International Journal of Biological Macromolecules, 77 , 114-119. doi:10.1016/j.ijbiomac.2015.03.019 Takimoto, K., Arahira, T., & Todo, M. (2022). Development and characterization of three-dimensional layered structures with gel beads for bone tissue engineering. Results in Materials, 16 , 100317. doi:10.1016/j.rinma.2022.100317 Tavakol, M., Vasheghani-Farahani, E., & Hashemi-Najafabadi, S. (2013). The effect of polymer and CaCl(2) concentrations on the sulfasalazine release from alginate-N,O-carboxymethyl chitosan beads. Prog Biomater, 2 (1), 10. doi:10.1186/2194-0517-2-10 Tavassoli, H., Alhosseini, S. N., Tay, A., Chan, P. P. Y., Weng Oh, S. K., & Warkiani, M. E. (2018). Large-scale production of stem cells utilizing microcarriers: A biomaterials engineering perspective from academic research to commercialized products. Biomaterials, 181 , 333-346. doi:10.1016/j.biomaterials.2018.07.016 Thakur, V. K., & Thakur, M. K. (2015). Handbook of polymers for pharmaceutical technologies, structure and chemistry (Vol. 1): John Wiley & Sons. Tuan Mohamood, N. F. A.-Z., Abdul Halim, A. H., & Zainuddin, N. (2021). Carboxymethyl cellulose hydrogel from biomass waste of oil palm empty fruit bunch using calcium chloride as crosslinking agent. Polymers, 13 (23), 4056. doi:10.3390/polym13234056. van Wezel, A. L. (1967). Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature, 216 (5110), 64-65. doi:10.1038/216064a0 Vulpe, R., Popa, M., Picton, L., Balan, V., Dulong, V., Butnaru, M., & Verestiuc, L. (2016). Crosslinked hydrogels based on biological macromolecules with potential use in skin tissue engineering. Int J Biol Macromol, 84 , 174-181. doi:10.1016/j.ijbiomac.2015.12.019 Xiao, X. C., Chu, L. Y., Chen, W. M., & Zhu, J. H. (2005). Monodispersed thermoresponsive hydrogel microspheres with a volume phase transition driven by hydrogen bonding. Polymer, 46 (9), 3199-3209. doi:10.1016/j.polymer.2005.01.075 Xie, H., Pan, Y., Xiao, H., & Liu, H. (2019). Preparation and characterization of amphoteric cellulose–montmorillonite composite beads with a controllable porous structure. Journal of Applied Polymer Science, 136 (37), 47941. Yan, J., Wang, Y., Zhang, X., Zhao, X., Ma, J., Pu, X. , et al. (2019). Snakegourd root/Astragalus polysaccharide hydrogel preparation and application in 3D printing. International Journal of Biological Macromolecules, 121 , 309-316. doi:10.1016/j.ijbiomac.2018.10.008 Yusup, E. M., & Mahzan, S. B. (2018). The physical characteristics of composite bone scaffold fabricated from OPEFB-CMC, chitosan and HA. International Journal of Mechanical Engineering and Robotics Research, 7 (5), 466-470. doi:10.18178/ijmerr.7.5.466-470 Zakiah, J., Mansor, A., Kamaruddin, H., & Norhazlin, Z. (2015). Sago starch based hydrogel prepared using electron beam irradiation technique for controlled release application. Malaysian Journal of Analytical Sciences, 19 (3), 503-512. Zou, Q., Li, J., & Li, Y. (2015). Preparation and characterization of vanillin-crosslinked chitosan therapeutic bioactive microcarriers. International Journal of Biological Macromolecules (79), 736-747. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Nov, 2024 Read the published version in Cellulose → Version 1 posted Editorial decision: Revision requested 11 Aug, 2024 Reviews received at journal 31 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 21 Jul, 2024 Reviewers invited by journal 21 Jul, 2024 Editor assigned by journal 05 Jul, 2024 Submission checks completed at journal 05 Jul, 2024 First submitted to journal 30 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4663194","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333440205,"identity":"5cac3c10-9baf-44a5-b3f2-71fc1db03a31","order_by":0,"name":"Soon Wei To","email":"","orcid":"","institution":"Universiti Teknologi Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Soon","middleName":"Wei","lastName":"To","suffix":""},{"id":333440207,"identity":"86014493-bc27-46c6-99e3-b7e535f3f1db","order_by":1,"name":"Rania Hussien Ahmed Al-Ashwal","email":"","orcid":"","institution":"Universiti Teknologi Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Rania","middleName":"Hussien Ahmed","lastName":"Al-Ashwal","suffix":""},{"id":333440208,"identity":"0420a790-203f-48e0-848d-9e2a9da80906","order_by":2,"name":"Nurzila Ab Latif","email":"","orcid":"","institution":"Universiti Teknologi Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nurzila","middleName":"Ab","lastName":"Latif","suffix":""},{"id":333440209,"identity":"b7534df1-7b8d-4016-bb24-d0030adcea3d","order_by":3,"name":"Mohd Helmi Sani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACAwbmBoYEIIONvYFoLYxQLTwHGInTBNYCBhIJRGoxZ29s3fDgj10en+Qb80c3GLYlEtRn2XOw7UZiW3Ixm3SOYXMOw23CWgyA6m8kNjAnthGv5f7DthsJf+oT2yTPEG0LI1AL2+HENgkeYrWcATms7XhiG09a4ewcg9vGhLUcP3zs5o8/1Ynz2w9v+JxTcVuWoBZ0ExgcSdXCwGBPso5RMApGwSgY9gAAxrlGRrgYZN4AAAAASUVORK5CYII=","orcid":"","institution":"Universiti Teknologi Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Mohd","middleName":"Helmi","lastName":"Sani","suffix":""}],"badges":[],"createdAt":"2024-06-30 14:36:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4663194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4663194/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10570-024-06269-x","type":"published","date":"2024-11-13T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61410627,"identity":"7093fbc3-18d4-4501-873d-0463306a74cc","added_by":"auto","created_at":"2024-07-30 11:53:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177140,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the fabrication of the OPEFB-derived CMC-microcarriers\u003cstrong\u003e \u003c/strong\u003evia carboxymethylation and ionic crosslinking process\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/5ade7c351ad549c00d14bf3f.png"},{"id":61409955,"identity":"2707a39f-9d24-4ed6-8c0c-05c093c62bef","added_by":"auto","created_at":"2024-07-30 11:45:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158453,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of ionic crosslinking formation through CMC and FeCl\u003csub\u003e3\u003c/sub\u003e. Three ionic bonds are formed between one Fe\u003csup\u003e3+\u003c/sup\u003e ion and three carboxylate groups on two CMC molecules. The colour changed from off-white to orange when (a) CMC was crosslinked with to Fe\u003csup\u003e2+\u003c/sup\u003eions to form (b) CMC-microcarriers\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/b57757479c9b369694fb24a4.png"},{"id":61410625,"identity":"004d9950-e651-4005-ab35-129796af008f","added_by":"auto","created_at":"2024-07-30 11:53:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228164,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of (a) CMC-1, (b) CMC-2, (c) CMC-3, (d) CMC-4, (e) CMC-5, (f) CMC-6, (g) CMC-7, (h) CMC-8, and (i) CMC-9 at magnifications of 50×\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/13aac996e6890ba49fb3c3b3.png"},{"id":61411205,"identity":"824a4508-8036-407b-9fe8-7277ba36cb67","added_by":"auto","created_at":"2024-07-30 12:01:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268424,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of (a) CMC-1, (b) CMC-2, (c) CMC-3, (d) CMC-4, (e) CMC-5, (f) CMC-6, (g) CMC-7, (h) CMC-8, and (i) CMC-9 at magnifications of 10000×\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/69a5a7f13345e71febf25055.png"},{"id":61409962,"identity":"067faec9-3bac-4fe8-8ba8-4badac2e773d","added_by":"auto","created_at":"2024-07-30 11:45:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55109,"visible":true,"origin":"","legend":"\u003cp\u003eThe EDX spectra of (a) CMC-1, (b) CMC-2, (c) CMC-3, (d) CMC-4, (e) CMC-5, (f) CMC-6, (g) CMC-7, (h) CMC-8, and (i) CMC-9 with different formulations\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/23b594695b8c5882270a7223.png"},{"id":61409963,"identity":"c6999f22-1ace-4041-bf68-a72e5637f880","added_by":"auto","created_at":"2024-07-30 11:45:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":25257,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in gel content (%) of microcarriers formulated with various OPEFB-derived CMC concentrations (4 %, 6 %, and 8 %) and FeCl\u003csub\u003e3\u003c/sub\u003e, concentrations (2 %, 6 % and 10 %), in triplicates. Error bars represent the standard deviation of the mean and different superscript letters above the error bars indicate a significant difference between means (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/c92d1bfd2f8e5a6b74a216c6.png"},{"id":61409960,"identity":"d2b3e27f-8358-4a5e-aa8b-41ae918e53c2","added_by":"auto","created_at":"2024-07-30 11:45:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":117189,"visible":true,"origin":"","legend":"\u003cp\u003e(a) OPEFB-derived CMC-microcarriers after swelling, the effect of (a) time, (b) temperature, and (c) pH on the \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e \u003c/em\u003e(%) of OPEFB-derived CMC microcarriers, in triplicates. Markers and bars represent mean ± standard deviation\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/f4b53de06b16e0303a76b068.png"},{"id":61409965,"identity":"6e01e0d3-89c3-4eb9-af3a-956f6d4b0966","added_by":"auto","created_at":"2024-07-30 11:45:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":24363,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The variation of \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e (%)\u003cem\u003e \u003c/em\u003ewith centrifuge rate and (b) the variations of degradation rate (%)\u003cem\u003e \u003c/em\u003ewith time (day) at differently formulated microcarriers\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Markers and bars represent mean ± standard deviation\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/ac432720a4d01e81bd084107.png"},{"id":69274745,"identity":"86515ae7-71cc-4c7a-b3db-066a157ed51d","added_by":"auto","created_at":"2024-11-18 16:21:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1933293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4663194/v1/fd1f2640-dd4e-423b-a3e8-e5adde134156.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and Characterisation of Biodegradable Carboxymethyl Cellulose Microcarriers from Oil Palm Empty Fruit Bunch for Therapeutic Applications","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMicrocarrier culture systems, which were first proposed by van Wezel (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1967\u003c/span\u003e) in 1967, have gained significant attention in biomedical research for their ability to support cell attachment and growth in three-dimensional (3D) cell culture systems. Unlike conventional methods, microcarriers serve as microscale scaffolds for anchorage-dependent cells enabling increased production capacity (Alkhatib et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), facilitated process scale-up (Bodiou et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), reduced cost requirement (Badenes et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and improved control in cell culture applications (Chen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Early commercial microcarriers were typically synthetic polymer-based such as poly(lactide-\u003cem\u003eco\u003c/em\u003e-glycolide) (PLGA), acrylamide, polystyrene, glass, and silica due to their defined chemical composition and adjustable mechanical properties. However, these petroleum-based commercial microcarriers often exhibit drawbacks such as high production costs, limited biodegradability, and restricted nature of the interaction (Alves et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tavassoli et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, they must be removed from the cell suspension before implantation, leading to the loss of viable cells (Muoio et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn view of such a predicament, there is growing interest in developing microcarriers from biodegradable natural polymeric materials such as collagen (Steffen et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), gelatine (Nweke and Stegemann, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), dextran (Rozwadowska et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), cellulose (Kalmer et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), chitosan (Nweke and Stegemann, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), alginate (Perteghella et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)). Among these, cellulose, the most abundant and inexhaustible natural polysaccharide-based biopolymer on Earth, is particularly promising for cellular applications due to its excellent biocompatibility, biodegradability, and good mechanical strength (Courtenay et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Seddiqi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides that, cellulose can also be chemically modified into various cellulose derivatives to enhance its usefulness in biomedical applications such as wound dressings and tissue engineering (Hon, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Carboxymethyl cellulose (CMC) is among the cellulose derivatives that have gained remarkable attention. It is produced from the cellulose chain through the substitution of its hydroxyl group backbone with the carboxymethyl group (-CH\u003csub\u003e2\u003c/sub\u003e-COOH). CMC is a prominent water-soluble polyelectrolyte cellulose biomaterial which is chemically reactive, non-allergenic, non-toxic, and biodegradable (Huang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kanikireddy et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yusup and Mahzan, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, CMC is widely used in cosmetics, pharmaceutical products and biomedical applications (Ciolacu and Suflet, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rahman et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the primary raw materials for CMC preparation are cotton linter and wood pulp which are considered costly agricultural products, increasing the overall production cost of CMC (Abd El-Sayed et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hence, the interest in the use of agricultural products and by-products as alternative cellulose resources for CMC preparation is gradually increasing.\u003c/p\u003e \u003cp\u003eIn view of this, the use of cellulose-rich oil palm empty fruit bunch (OPEFB) presents a useful alternative as a raw material to produce microcarriers. OPEFB is produced in huge quantities in oil palm plantation activities, with 1.07 tons generated per ton of palm oil produced, and the global production of OPEFB in 2018 alone accounted for around 80\u0026nbsp;million tons (Dolah et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Malaysia, as the second-largest palm oil producer and the biggest exporter of oil palm products in the world, generates approximately 15\u0026nbsp;million tons of OPEFB annually (Abdul et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The amount of OPEFB waste is expected to continue to increase due to the abundant land, cheap labour cost, and high global demand for oil palm products (Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Since the cellulose content from OPEFB constitutes nearly half its fibre weight, it offers substantial potential as a cellulose source for various applications. Conventionally, OPEFB biomass waste is often burnt or left for the mulching process at the plantation (Faizi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Converting OPEFB into cellulose and CMC could harness its potential as a natural biomaterial source for microcarrier development. While many types of lignocellulosic fibres such as canola straws {Zhang, 2023 #71} and sugarcane bagasse {Lam, 2017 #24} have been used for microcarriers and scaffold fabrication, research on the fabrication of cellulose-based microcarriers from OPEFB is limited, which hinders the exploration of this valuable resource and results in forfeiture of substantial economic value (Idris et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, the study believes the use of OPEFB as the cheaper sustainable cellulose resource for the CMC preparation to produce biodegradable microcarrier would be more acceptable.\u003c/p\u003e \u003cp\u003eIn this research, OPEFB cellulose is chosen due to the substantial amount of OPEFB biomass waste generated during palm oil production in Malaysia. Therefore, an innovative approach is required to turn OPEFB into a more valuable product that can reduce the negative impact on the environment while increasing the economic use of OPEFB in other non-biodiesel areas in Malaysia. Herein, this study aimed to address this gap by demonstrating the preparation of CMC-microcarriers derived from OPEFB biomass waste through an ionic crosslinking method using an iron (III) chloride solution. The fabricated microcarriers were characterised by physicochemical and morphological properties including particle size, gel content, swelling behaviour, mechanical stability, and \u003cem\u003ein vitro\u003c/em\u003e degradation. The findings are expected to advance knowledge in the development of microcarriers from OPEFB, offering a valuable alternative for the application of biodegradable microcarriers in various therapeutic fields.\u003c/p\u003e"},{"header":"2 Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eOil palm empty fruit bunch (OPEFB) was obtained from one palm oil mill facility located in Kulai, Johor, Malaysia. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 30%), sodium monochloroacetate (SMCA), iron (III) chloride hexahydrate (FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO), and phosphate-buffered saline (PBS, pH 7.4) were purchased from Bio Basic Canada Inc. Sodium carboxymethylcellulose (NaCMC; C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eNa) and sodium hydroxide pellet (NaOH) was purchased from Merck. Meanwhile, all chemical solvents such as formic acid (90%), isopropanol (99%), absolute methanol (99%), glacial acetic acid (99%), ethanol (95%), and acetone (99%) were purchased from R \u0026amp; M Chemicals. All chemicals and solvents used in this study are of analytical grade and used without further purification. Distilled water was used in all experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of OPEFB-derived CMC\u003c/h2\u003e \u003cp\u003eOPEFB were pre-treated using the modified eco-friendly procedure (Nazir et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The OPEFB fibres were cut, dewaxed using a 10% (\u003cem\u003ew/v\u003c/em\u003e) NaOH solution and autoclaved at 21\u0026deg;C and 1.5 bar for an hour. The dewaxed fibres were then treated with 100 mL hydrogen peroxide (30%) and autoclaved under the same conditions. The delignified fibres were then collected and washed further with 10% formic acid, distilled water, and ethanol to remove excess lignin. The fibres were finally oven-dried at 60\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe OPEFB-derived CMC was synthesised in two steps (Ab Rasid et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Briefly, 15 g of OPEFB-cellulose was added in a mixed solution of 50 mL of 30% (\u003cem\u003ew/v\u003c/em\u003e) NaOH solution and 450 mL isopropanol. After 1.5 hours, 10 mL isopropanol containing pre-dissolved 18 g of SMCA was added to the mixture and the reaction continued for 3 hours at 65\u0026deg;C. The slurry was then filtered and soaked in 100 mL of absolute methanol overnight. OPEFB-derived CMC was neutralised with glacial acetic acid and washed five times with 70% ethanol, followed by a one-time wash with absolute methanol. The OPEFB-derived CMC was oven-dried for 24 h at 60\u0026deg;C and kept in desiccators.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fabrication of microcarriers\u003c/h2\u003e \u003cp\u003eNine types of CMC-microcarriers were fabricated via ionic crosslinking reactions using FeCl\u003csub\u003e3\u003c/sub\u003e as a crosslinker (Akalin and Pulat, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Various concentrations and quantities of OPEFB-derived CMC (4\u0026ndash;8% w/v) and FeCl\u003csub\u003e3\u003c/sub\u003e (2\u0026ndash;10% w/v) were prepared and termed as CMC-1 to CMC-9. The prepared OPEFB-derived CMC solutions were continuously added dropwise into FeCl\u003csub\u003e3\u003c/sub\u003e, by using a 20-gauge syringe needle. The obtained spherical microcarriers were left to crosslink for 3 hours under mechanical stirring at 200 rpm. The microcarriers were collected, filtered, and rinsed multiple times with distilled water to remove any unreacted FeCl\u003csub\u003e3\u003c/sub\u003e solution before drying at room temperature for 24 hours. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the schematic illustration of the fabrication of the proposed microcarriers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Measurement of microcarrier size\u003c/h2\u003e \u003cp\u003eFor particle size determination, 50 beads of each formulation were spread over the flat surface using a spatula and watched randomly under a light transmission microscope (Model: Nikon Eclipse Ti-S, Japan). The diameter of samples was then analysed from the microscopy images using image analysis software (ImageJ; National Institutes of Health, USA). The particle size of the samples was expressed as the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe images of the sample surface were examined using a scanning microscope (Model: JEOL JSM-IT300LV, Japan). The samples were first freeze-dried, mounted, and sputter-coated with a thin layer of gold. The SEM observations were carried out at an accelerating voltage of 30 kV, with magnification at 50\u0026times;. Energy-dispersive X-ray (EDX) spectroscopy was performed to collect full-scale elemental quantification data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Gel content\u003c/h2\u003e \u003cp\u003eThe gel contents of the samples were determined by immersing the dried samples in deionised water for 72 hours at room temperature. Then, the samples were dried in an oven at 60\u0026ordm;C until a constant weight was obtained. The gel content of the samples was calculated as follows:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$GC\\left(\\%\\right) = \\frac{({W}_{i} - {W}_{d})}{{W}_{i}}\\times 100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eGC\u003c/em\u003e is the gel content of the sample, \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the initial weight of the sample, and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e is the weight of the sample after the immersion and drying process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Swelling behaviours\u003c/h2\u003e \u003cp\u003eThe swelling behaviours of the samples were conducted under varying time, temperature, and pH. Dried samples were immersed in PBS at room temperature. The samples were periodically removed, dried, and weighed. The test was repeated until a constant weight was obtained. The effect of temperature on swelling was studied by immersing dried samples in PBS at temperatures ranging from 10 to 60\u0026deg;C. The pH effect was studied by immersing dried microcarriers in PBS with pH values ranging from 2.0 to 12.0. All tests were conducted in triplicates to obtain reproducible results. The degree of swelling (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e) was determined using the following equation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${S}_{w}\\left(\\%\\right) = \\frac{({W}_{f} - {W}_{0})}{{W}_{0}}\\times 100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e represent the weight of the swollen and dry samples, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Mechanical stability and \u003cem\u003ein vitro\u003c/em\u003e degradation test\u003c/h2\u003e \u003cp\u003eSample suspension of each formulation (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) was tested at various centrifugal forces between 100 and 6000x G. After that, the number of the deformed or ruptured microcarriers was identified out of at least 100 microcarriers per set under a light transmission microscope (Aydogdu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The deformation rate (\u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e) was then determined by using; \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(%)=(N\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/N\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e) \u0026times; 100%\u003c/em\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$${D}_{f}\\left(\\%\\right) = \\frac{\\left({N}_{f}\\right)}{{N}_{r}}\\times 100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e represents the number of deformed samples and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e is the total number of tested samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 \u003cem\u003eIn vitro\u003c/em\u003e degradation test\u003c/h2\u003e \u003cp\u003eThe degradation rate of the samples was determined by putting predetermined weights of sterilised samples into PBS solution followed by incubation at 37\u0026deg;C for 45 days. The swollen samples were removed from the solution after 24 hours, and the weights were reported as the maximum swollen state of the samples. Then, the samples were put into the same bath and weighed at regular intervals. The degradation rate (\u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e) was then calculated using;\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$${D}_{g}\\left(\\%\\right) = \\frac{({W}_{m}-{W}_{f})}{{W}_{m}}\\times 100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e represents the maximum weight of the samples at the most swollen stage and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e is the wet weight at a certain time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were performed in triplicate and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A one-way analysis of variance ANOVA followed by Tukey HSD Post Hoc test was then computed to evaluate the significance of the difference between each test using IBM SPSS Statistics version 27 software. All tests were done with a confidence interval of 95% and differences at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Fabrication of OPEFB-CMC Microcarriers\u003c/h2\u003e\n \u003cp\u003eThis study focuses on the preparation of microcarriers using the reactive functional group of carboxymethylated cellulose derivatives from OPEFB to interact with reactive groups of crosslinkers via an ionic crosslinking process. Among various crosslinkers, trivalent metal ions are identified as an appropriate crosslinker to prepare these types of microcarriers due to their eco-friendly and nontoxic nature (Bulut and Şanlı, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, iron (III) ions were employed as trivalent crosslinkers in the fabrication of CMC microcarriers. The OPEFB-CMC solution, prepared at concentrations of 4%, 6%, and 8%, was incrementally added to aqueous FeCl\u003csub\u003e3\u003c/sub\u003e solutions of varying concentrations (2%, 6%, and 10%) to harden the droplets. As anticipated, the CMC microcarriers formed upon the dropwise addition of aqueous OPEFB-CMC into the FeCl\u003csub\u003e3\u003c/sub\u003e solution, due to ionic crosslinking between the carboxylate ions (COO-) on CMC and Fe\u003csup\u003e3+\u003c/sup\u003e ions from FeCl\u003csub\u003e3\u003c/sub\u003e. Figure 4.4 suggests the general mechanisms for ionic crosslinking between the carboxylic groups of OPEFB-CMC and the iron (III) crosslinker.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Measurement of Microcarrier Size\u003c/h2\u003e\n \u003cp\u003eThe average diameter of microcarriers was investigated using a light transmission microscope and image analysis software (Image J) and expressed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Microcarriers exhibited an average size ranging from 1105.52 to 1322.25 \u0026micro;m. Among all the samples, CMC-7 was recorded to have the largest diameter, while CMC-3 had the smallest diameter statistically among all the microcarriers. Interestingly, the study found that the size of the microcarriers increased with the concentration of the biopolymer (OPEFB-derived CMC) increased. This observation aligns with previous research, which suggested that an increased biopolymer concentration leads to higher viscosity and larger microcarriers due to a high degree of crosslinking (Kaur et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The increase in biopolymer concentration also results in thicker fluid extrusion through the nozzle of the needle which forms larger droplets and ultimately, larger microcarriers (Mehregan Nikoo et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Takimoto et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Contrarily, the concentration of FeCl\u003csub\u003e3\u003c/sub\u003e showed an inverse relationship with the particle sizes of the microcarriers. As the FeCl\u003csub\u003e3\u003c/sub\u003e concentration increased. This is consistent with previous studies suggesting that a higher crosslinker concentration results in more compact microcarriers due to increased penetration of crosslinker ions into the droplets, expulsion of water molecules, and tightening of the gel network (Kaur et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, an increase in the polymer concentration and the crosslinking extent also enhances the matrix density of the microcarriers due to a stronger crosslinking reaction, resulting in smaller but more compact microcarriers (Zou et al.,\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe diameter size (\u0026micro;m) of microcarriers derived from OPEFB-derived CMC.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMicrocarriers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eConcentrations (\u003cem\u003ew/v\u003c/em\u003e, %)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDiameter Size (\u0026micro;m)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOPEFB derived CMC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFeCl\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1203.65\u0026thinsp;\u0026plusmn;\u0026thinsp;229.64\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1135.73\u0026thinsp;\u0026plusmn;\u0026thinsp;169.86\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1105.52\u0026thinsp;\u0026plusmn;\u0026thinsp;44.97\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1302.35\u0026thinsp;\u0026plusmn;\u0026thinsp;171.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1262.73\u0026thinsp;\u0026plusmn;\u0026thinsp;125.15\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1236.98\u0026thinsp;\u0026plusmn;\u0026thinsp;237.16\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1352.25\u0026thinsp;\u0026plusmn;\u0026thinsp;68.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1328.29\u0026thinsp;\u0026plusmn;\u0026thinsp;58.72\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1285.22\u0026thinsp;\u0026plusmn;\u0026thinsp;66.61\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;50). Different superscript letters across a column indicate a significant difference between means (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 SEM analysis\u003c/h2\u003e\n \u003cp\u003eSEM micrographs of CMC-microcarriers analysed at 50\u0026times; magnification are shown in (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). CMC-microcarriers exhibited different morphological shapes and sphericity. However, the shape of the MC became more spherical when the concentration of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e increased from 4 to 8% (\u003cem\u003ew/v\u003c/em\u003e) and 2 to 10% (\u003cem\u003ew/v\u003c/em\u003e), respectively. These suggested that an increase in both biopolymer and crosslinking agent concentrations facilitates the formation of spherical microcarriers by increasing the homogenous crosslinking network between the polymeric chains. The surface of all CMC-microcarriers appeared to be covered with grooves and ridges which could provide a potential surface for the attachment of the cells.\u003c/p\u003e\n \u003cp\u003eMeanwhile, a more granular structure was observed on the surface at 10000\u0026times; magnification (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The ionic crosslinking occurred intensively on the surface and resulted in the formation of a granulated view on the surface of microcarriers (Akalin and Pulat, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). All microcarriers possessed a unique three-dimensional spongy structure with irregular sizes and distribution of pores. This porosity serves as an important feature of the MC as it determines the diffusion and absorption of water into the structure which substantially affects the swelling ratio of the microcarriers (Tuan Mohamood et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). In previous studies, it has been observed that the concentration of biopolymer and crosslinker in microcarriers influences the sizes and density of surface pores (Vulpe et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The observed variations in porosity and pore size among microcarriers can be attributed to the different crosslinking densities resulting from varying amounts of biopolymer and crosslinker (Rahman et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 EDX Analysis\u003c/h2\u003e\n \u003cp\u003eThe elemental analysis was carried out by EDX to confirm the formation of the CMC MC. The EDX spectra of the samples are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Carbon (C) and oxygen (O) were detected in the EDX analysis of the samples, indicating that they are major components of the fabricated material. The presence of these elements can be attributed to the incorporation of OPEFB-derived CMC, which has the chemical formula (CH\u003csub\u003e2\u003c/sub\u003eCOOH)\u003csub\u003en\u003c/sub\u003e. All spectra depicted a well-defined peak corresponding to Fe which confirmed the presence of successful ionic crosslinking between FeCl\u003csub\u003e3\u003c/sub\u003e and CMC during the formation of CMC-microcarriers (Bardajee et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The peak intensity of Fe was increased with an increasing FeCl\u003csub\u003e3\u003c/sub\u003e concentration from 2 to 10% (\u003cem\u003ew/v\u003c/em\u003e) in 4%, 6%, and 8% (\u003cem\u003ew/v\u003c/em\u003e) OPEFB-derived CMC solution, respectively. A similar trend was obtained for the atomic fraction values (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This is because more Fe ions are available to be ionically crosslinked to the polymer chains during the MC fabrication process as the FeCl\u003csub\u003e3\u003c/sub\u003e level rises, increasing the proportion of Fe in the structure. A similar conclusion was reported by Akalin and Pulat (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). From the results, it could be concluded that the ionic crosslinking between the OPEFB-derived CMC biopolymer and FeCl\u003csub\u003e3\u003c/sub\u003e solution was successful.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe weight and atomic fractions (%) of Fe elements for the fabricated CMC MC with various formulations\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMicrocarrier\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eConcentration (\u003cem\u003ew/v\u003c/em\u003e. %)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFe\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOPEFB derived CMC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFeCl\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeight (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAtomic (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMC-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.32\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eAll values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Determination of Gel Content\u003c/h2\u003e\n \u003cp\u003eGel content (\u003cem\u003eGC\u003c/em\u003e) is a measure of crosslinking within microcarrier networks, and it significantly influences their properties. A higher GC indicates greater crosslinking, resulting in more spherical and mechanically robust microcarriers which are advantageous for tissue engineering applications (Rebers et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e depicts the changes in \u003cem\u003eGC\u003c/em\u003e of OPEFB-derived CMC-microcarriers at various formulations. The lowest and highest \u003cem\u003eGC\u003c/em\u003e values were 16.95% and 42.65% for CMC-1 and CMC-9, respectively. The results showed that \u003cem\u003eGC\u003c/em\u003e values increased significantly with the concentration of OPEFB-derived CMC. The increase in \u003cem\u003eGC\u003c/em\u003e of the samples was due to enhanced cross-linkages between CMC chains. A similar conclusion was also reached by a prior study reporting that the \u003cem\u003eGC\u003c/em\u003e value of CMC hydrogel steadily rose with CMC concentration up to 20% (\u003cem\u003ew/v\u003c/em\u003e) (Tuan Mohamood et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the study pointed out that an excessively high concentration of CMC can lead to a decrease in \u003cem\u003eGC\u003c/em\u003e due to the presence of uncross-linked CMC chains in the sample structure after gelation Despite that, it is not observed at 8% (\u003cem\u003ew/v\u003c/em\u003e) OPEFB-derived CMC. In terms of FeCl\u003csub\u003e3\u003c/sub\u003e concentration, an increase led to a significant rise in \u003cem\u003eGC\u003c/em\u003e. This is attributed to the enhanced electrostatic attraction between anionic charges of polymer chains and multivalent cations, promoting ionic crosslinking of polymers (Sultana et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). A similar pattern of results was obtained in previous literatures (Che Nan et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tuan Mohamood et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The increasing ionic strength of the FeCl\u003csub\u003e3\u003c/sub\u003e crosslinking buffer positively affected CMC cross-linking, as reflected by increased \u003cem\u003eGC\u003c/em\u003e. In conclusion, an optimum MC formulation was achieved at 8% OPEFB-derived CMC and 10% FeCl\u003csub\u003e3\u003c/sub\u003e concentration (CMC-9), yielding a GC value of 42.65%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Effect of Time, Temperature, and pH on Swelling Behaviour\u003c/h2\u003e\n \u003cp\u003eThe degree of swelling (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e) is a key characteristic of crosslinked microcarriers, influencing their structural changes in response to external stimuli such as temperature, pH, ionic strength, and substance concentration (Dong and Chen, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e(b), \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values initially increased over time, stabilising at nearly 24 hours. The lowest and highest \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values were observed in CMC-9 (32%) and CMC-1 (385%), respectively. \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values increased with the proportion of OPEFB-derived CMC and iron trivalent ions but were inversely correlating with the gel content of the samples (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e was influenced by the hydrophilicity of the carboxylic groups on the polymer chain which led to swelling when water molecules filled the inert pores in the polymeric chains and created an osmotic pressure within the microcarrier (Zakiah et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) (Sathali and Varun, \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). The swelling of CMC-microcarriers was governed by the degree of crosslinking, with greater cross-linkages reducing the swelling due to limited space available for the diffusion of free water into the polymer network (Che Nan et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Patil et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tuan Mohamood et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lower \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e was found in samples with higher gel content percentages (CMC-3, CMC-6, and CMC-9), while \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e value rose in samples with low gel content (CMC-1, CMC-4, and CMC-7). Thus, \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values were directly linked to the concentration of biopolymer and crosslinker, with samples exhibiting lower swelling behaviour considered more stable due to their reduced likelihood of surface morphology changes upon hydration (de Bournonville et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e(c) illustrates the variations of \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values with temperature at pH 7.0 and 24 h, showing a gradual increase in \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e across all samples with different rates of increase. The rise of \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values from 10 ˚C to 60 ˚C can be attributed to the enhanced thermal mobility of polymer chains at higher temperatures. This results in a more relaxed and unrigid polymer network, allowing more water molecules to fill in the accessible void within the structure, thereby increasing \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values (Akalin and Pulat, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Conversely, the values of \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e decreased as the temperature dropped to 10 ˚C due to the low diffusion rate of water molecules into the hydrophilic polymeric chains. A regular decrease was observed in \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values as the amount of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e increased from 4\u0026ndash;8%, and 2\u0026ndash;10%, respectively. For instance, \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values were determined to be 765% for the CMC-1 MC and 73% for the CMC-9 MC at 60 ˚C. This result can be explained by the enhanced degree of crosslinking (reflected by the gel content percentage) with increased OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e concentrations, which restricts sample expansion and reduces \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e (Swamy and Yun, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The ideal CMC MC should maintain stability even near 60 ˚C so that they could be suitable for use in wide temperature ranges (Xiao et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). Thus, samples such as CMC-3, CMC-6, and CMC-9 were identified as the optimal microcarriers in terms of thermal stability due to their comparatively lower increment of \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e readings over increasing incubation temperature.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e(d) shows the swelling ratio for fabricated microcarriers at various at 30 ˚C and 24 h. The \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values rose slightly from pH 2.0 to 6.0 and then followed a sharp increment from pH 7.0 to 12.0. CMC-1 and CMC-9 recorded the highest and lowest increases in \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e, respectively. The change in \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e across pH levels can be explained by the protonation and deprotonation of carboxylic groups (p\u003cem\u003eKa\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.5) of the polymer at different surrounding pH (Thakur and Thakur, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Under acidic pH (pH 2.0), most carboxylate anions (COO) of the polymer chains are protonated and converted to COOH form. This eliminates the main anion-anion repulsive forces within the polymer network and causes the degree of swelling to decrease (Ankur and Harish, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). At pH 7.0, the samples displayed noticeable swelling behaviour due to the neutralisation of protonated carboxylic groups on the polymer chain by counterions present in the environment. This reduces the electrostatic repulsion between the ionised carboxylate, thereby increasing the swelling capacity of the samples (Tavakol et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). As the pH of the environmental solution becomes basic, more carboxylic groups of the polymer are ionised to the COO, maximising the repulsion and swelling (Akalin and Pulat, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ankur and Harish, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e was found to be inversely proportional to the \u003cem\u003eGC\u003c/em\u003e percentage which increases proportionally to the amount of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e. Therefore, a regular reduction was observed in \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e values as the amount of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e increased. In short, CMC-1 and CMC-9 exhibited the highest and lowest pH sensitivity, respectively, while CMC-3, CMC-6, and CMC-9 were identified as the most pH-stable microcarriers.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Mechanical stability\u003c/h2\u003e\n \u003cp\u003eThe deformation rate (\u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e) of the samples with various formulations at different centrifugal forces is presented in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e(a). The samples with higher concentrations of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e exhibited better mechanical stability and less MC failure as reflected by the lower deformation rate recorded by CMC-9, CMC-6, and CMC-4 at the highest centrifuge rate (8000 G). The higher rate of deformation displayed in less-concentrated microcarriers (CMC-1, CMC-4, and CMC-7) could be due to their lower degree of crosslinking which primarily supports and improves the mechanical strength and shape of the developed microcarriers (Jose et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). This was then supported by Takimoto et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) who described that the mechanical properties of the microcarriers can be controlled and changed by varying the concentration of biopolymer and crosslinking bath. Their study also concluded that higher-concentration samples were stronger than the lower-concentration samples, which is directly in line with the present findings. In short, the deformation rate was found to be inversely proportional to the concentrations of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e. CMC-9 and CMC-1 were discovered to be the most mechanically stable and least mechanically stable microcarriers, respectively, in various centrifugal forces. Therefore, samples with higher mechanical strength or with lower deformation rates such as CMC-3, CMC-6, and CMC-9 are considered the optimum microcarriers used for tissue engineering as limited mechanical properties can affect their applications and cell differentiation (Takimoto et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 \u003cem\u003eIn vitro\u003c/em\u003e Degradation Analysis\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e degradation studies are critical to understand the behaviour of fabricated microcarriers in physiological environments. Biodegradable microcarriers offer advantages to the biopharmaceutical industry by allowing precise control over degradation and enabling tailored drug release profiles (Handral et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The degradation behaviour of the samples at pH 7.0 and 37 ˚C are graphically presented in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e(b). A rapid increase in biodegradation rate was recorded for lower concentration samples of (CMC-1, CMC-4, and CMC-7), while higher concentration samples (CMC-3, CMC-6, and CMC-9) exhibited a gradual increase over time. The degradation process is governed by the hydrolytic cleavage of intramolecular and intermolecular hydrogen bonds in polysaccharides leading to a loss of structural stability and gradual degradation over time (Yan et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, it is influenced by the number of intermolecular bonds in the polymer. The findings align with the previous study, indicating that higher crosslinking density had a decelerated degradation process (Akalin and Pulat, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). A faster degradation rate was obtained in all low-concentration samples due to the lower crosslinking density within the structure. This is because disintegration of network structure occurs more quickly in relatively loose networks (Lee et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The degradation of all samples implies that CMC derived from OPEFB retains the biodegradability properties of cellulose and is suitable to be extensively used in tissue engineering (Kalmer et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite having biodegradability, suitable microcarriers should maintain high structural stability for long-term applications (Akalin and Pulat, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among the tested samples, CMC-3, CMC-6, and CMC-9 exhibited better structural stability in physiological environments, making them suitable for long-term applications. These samples can also be modified to have an accelerated degradation rate according to the application needs by adding an external esterase enzyme in the medium (Lee et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn conclusion, this study successfully demonstrated the synthesis of biodegradable CMC-microcarriers from OPEFB using FeCl\u003csub\u003e3\u003c/sub\u003e as the crosslinking agent. The fabricated CMC-microcarriers, with sizes ranging from 1105.52 to 1322.25 \u0026micro;m, exhibited a porous structure and significant mechanical stability, making them suitable for various biomedical applications. The microcarriers showed promising biodegradability and swelling behaviours, which can be tailored by adjusting the concentrations of OPEFB-derived CMC and FeCl\u003csub\u003e3\u003c/sub\u003e. Notably, samples with higher crosslinking densities displayed enhanced structural integrity and controlled degradation rates, which is essential for long-term therapeutic applications. These findings highlight the feasibility of using OPEFB, an agricultural waste product as a cost-effective, sustainable, and eco-friendly resource for developing advanced biodegradable biomaterials, paving the way for greener and more efficient solutions in the biomedical industry. Future research should focus on optimising the functional properties of these microcarriers to further expand their application in cell culture systems and tissue engineering.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors\u0026nbsp;gratefully acknowledge the support of time and facilities from\u0026nbsp;Universiti Teknologi Malaysia for\u0026nbsp;this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by Universiti Teknologi Malaysia under the Fundamental Research Grant Scheme (Grant No: 20H78).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, Johor Darul Ta‘Zim, 81310, Skudai, Malaysia\u003c/p\u003e\n\u003cp\u003eSoon Wei To,\u0026nbsp;Nurzila Ab Latif, Mohd Helmi Sani\u003c/p\u003e\n\u003cp\u003eSchool of Biomedical Engineering and Health Sciences, Faculty of Engineering, Universiti Teknologi Malaysia, Johor Darul Ta‘Zim, 81310, Skudai, Malaysia\u003c/p\u003e\n\u003cp\u003eRania Hussien Ahmed Al-Ashwal\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoon Wei To: conceptualization, methodology, formal analysis and investigation, and writing – original draft preparation. Rania Hussien Ahmed Al-Ashwal: writing – review and editing. Nurzila Ab Latif: writing – review and editing. Mohd Helmi Sani: funding acquisition, project administration, resources, writing – review and editing and supervision. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Mohd Helmi Sani\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research meets the ethical approval, including adherence to the legal requirements of the study country.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe article has been written by the stated authors who are all aware of its content and approve its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and animal rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that there are no animal studies or human participants involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAb Rasid, N. S., Zainol, M. M., \u0026amp; Amin, N. A. S. (2021). Synthesis and characterization of carboxymethyl cellulose derived from empty fruit bunch. \u003cem\u003eSains Malaysiana, 50\u003c/em\u003e(9), 2523-2535. doi:10.17576/jsm-2021-5009-03\u003c/li\u003e\n \u003cli\u003eAbd El-Sayed, E. S., El-Sakhawy, M., \u0026amp; El-Sakhawy, M. A.-M. (2020). Non-wood fibers as raw material for pulp and paper industry. \u003cem\u003eNordic Pulp \u0026amp; Paper Research Journal, 35\u003c/em\u003e(2), 215-230. doi:10.1515/npprj-2019-0064\u003c/li\u003e\n \u003cli\u003eAbdul, P. M., Jahim, J. M., Harun, S., Markom, M., Lutpi, N. A., Hassan, O.\u003cem\u003e, et al.\u003c/em\u003e (2016). Effects of changes in chemical and structural characteristic of ammonia fibre expansion (AFEX) pretreated oil palm empty fruit bunch fibre on enzymatic saccharification and fermentability for biohydrogen. \u003cem\u003eBioresource Technology, 211\u003c/em\u003e, 200-208. doi:10.1016/j.biortech.2016.02.135\u003c/li\u003e\n \u003cli\u003eAkalin, G. O., \u0026amp; Pulat, M. (2018). Preparation and characterization of nanoporous sodium carboxymethyl cellulose hydrogel beads. \u003cem\u003eJournal of Nanomaterials, 2018\u003c/em\u003e. doi:10.1155/2018/9676949\u003c/li\u003e\n \u003cli\u003eAli, M. M., Muhadi, N. A., Hashim, N., Abdullah, A. F., \u0026amp; Mahadi, M. R. (2020). Pulp and paper production from oil palm empty fruit bunches: A current direction in Malaysia. \u003cem\u003eJournal of Agricultural and Food Engineering, 2\u003c/em\u003e, 1-9.\u003c/li\u003e\n \u003cli\u003eAlkhatib, R., Hilal-Alnaqbi, A., Naciri, M., Al-Majmaie, R., Saseedharan, P., Karam, S. M., \u0026amp; Al-Rubeai, M. (2017). 3D culture of mouse gastric stem cells using porous microcarriers. \u003cem\u003eFrontiers in Bioscience, 9\u003c/em\u003e(1), 172-179. doi:10.2741/s481\u003c/li\u003e\n \u003cli\u003eAlves, T. F. R., Morsink, M., Batain, F., Chaud, M. V., Almeida, T., Fernandes, D. A.\u003cem\u003e, et al.\u003c/em\u003e (2020). Applications of natural, semi-synthetic, and synthetic polymers in cosmetic formulations. \u003cem\u003eCosmetics, 7\u003c/em\u003e(4), 75. doi:10.3390/cosmetics7040075\u003c/li\u003e\n \u003cli\u003eAnkur, G., \u0026amp; Harish, K. (2018). Synthesis and swelling behavior of superabsorbent hydrogels acquired from CMC for efficient drug-delivery. \u003cem\u003eResearch Journal of Chemistry and Environment, 22\u003c/em\u003e(5), 5.\u003c/li\u003e\n \u003cli\u003eAydogdu, H., Keskin, D., Baran, E. T., \u0026amp; Tezcaner, A. (2016). Pullulan microcarriers for bone tissue regeneration. \u003cem\u003eMater Sci Eng C Mater Biol Appl, 63\u003c/em\u003e, 439-449. doi:10.1016/j.msec.2016.03.002\u003c/li\u003e\n \u003cli\u003eBadenes, S., Fernandes-Platzgummer, A., Rodrigues, C., Diogo, M., da Silva, C., \u0026amp; Cabral, J. (2016). Microcarrier culture systems for stem cell manufacturing. \u003cem\u003eStem Cell Manufacturing\u003c/em\u003e, 77.\u003c/li\u003e\n \u003cli\u003eBardajee, G. R., Hooshyar, Z., Asli, M. J., Shahidi, F. E., \u0026amp; Dianatnejad, N. (2014). Synthesis of a novel supermagnetic iron oxide nanocomposite hydrogel based on graft copolymerization of poly((2-dimethylamino)ethyl methacrylate) onto salep for controlled release of drug. \u003cem\u003eMaterials Science and Engineering C-Materials for Biological Applications, 36\u003c/em\u003e, 277-286. doi:10.1016/j.msec.2013.11.022\u003c/li\u003e\n \u003cli\u003eBodiou, V., Moutsatsou, P., \u0026amp; Post, M. (2020). Microcarriers for Upscaling Cultured Meat Production. \u003cem\u003eFrontiers in Nutrition, 7\u003c/em\u003e, 10.\u003c/li\u003e\n \u003cli\u003eBulut, E., \u0026amp; Şanlı, O. (2016). Novel ionically crosslinked acrylamide-grafted poly(vinyl alcohol)/sodium alginate/sodium carboxymethyl cellulose pH-sensitive microspheres for delivery of Alzheimer\u0026apos;s drug donepezil hydrochloride: Preparation and optimization of release conditions. \u003cem\u003eArtificial Cells, Nanomedicine, and Biotechnology, 44\u003c/em\u003e(2), 431-442. doi:10.3109/21691401.2014.962741\u003c/li\u003e\n \u003cli\u003eChe Nan, N. F., Zainuddin, N., \u0026amp; Ahmad, M. (2019). Preparation and swelling study of CMC hydrogel as potential superabsorbent. \u003cem\u003ePertanika Journal of Science \u0026amp; Technology, 27\u003c/em\u003e(1).\u003c/li\u003e\n \u003cli\u003eChen, X. Y., Chen, J. Y., Tong, X. M., Mei, J. G., Chen, Y. F., \u0026amp; Mou, X. Z. (2020). Recent advances in the use of microcarriers for cell cultures and their ex vivo and in vivo applications. \u003cem\u003eBiotechnology Letters, 42\u003c/em\u003e(1), 1-10. doi:10.1007/s10529-019-02738-7\u003c/li\u003e\n \u003cli\u003eCiolacu, D. E., \u0026amp; Suflet, D. M. (2018). 11 - cellulose-based hydrogels for medical/pharmaceutical applications. In V. Popa \u0026amp; I. Volf (Eds.), \u003cem\u003eBiomass as Renewable Raw Material to Obtain Bioproducts of High-Tech Value\u003c/em\u003e (pp. 401-439): Elsevier.\u003c/li\u003e\n \u003cli\u003eCourtenay, J. C., Deneke, C., Lanzoni, E. M., Costa, C. A., Bae, Y., Scott, J. L., \u0026amp; Sharma, R. I. (2018). Modulating cell response on cellulose surfaces; tunable attachment and scaffold mechanics. \u003cem\u003eCellulose, 25\u003c/em\u003e(2), 925-940. doi:10.1007/s10570-017-1612-3\u003c/li\u003e\n \u003cli\u003ede Bournonville, S., Geris, L., \u0026amp; Kerckhofs, G. (2021). Micro computed tomography with and without contrast enhancement for the characterization of microcarriers in dry and wet state. \u003cem\u003eSci Rep, 11\u003c/em\u003e(1), 2819. doi:10.1038/s41598-021-81998-8\u003c/li\u003e\n \u003cli\u003eDolah, R., Karnik, R., \u0026amp; Hamdan, H. (2021). A comprehensive review on biofuels from oil palm empty bunch (EFB): Current status, potential, barriers and way forward. \u003cem\u003eSustainability, 13\u003c/em\u003e(18), 10210. doi:10.3390/su131810210\u003c/li\u003e\n \u003cli\u003eDong, M., \u0026amp; Chen, Y. (2020). Chapter 7 - The stimuli-responsive properties of hydrogels based on natural polymers. In Y. Chen (Ed.), \u003cem\u003eHydrogels Based on Natural Polymers\u003c/em\u003e (pp. 173-222): Elsevier.\u003c/li\u003e\n \u003cli\u003eFaizi, M., Shahriman, A., Majid, M. A., Shamsul, B., Ng, Y., Basah, S.\u003cem\u003e, et al.\u003c/em\u003e (2017). \u003cem\u003eAn overview of the oil palm empty fruit bunch (OPEFB) potential as reinforcing fibre in polymer composite for energy absorption applications.\u003c/em\u003e Paper presented at the MATEC web of conferences.\u003c/li\u003e\n \u003cli\u003eHandral, H. K., Wyrobnik, T. A., \u0026amp; Lam, A. T. (2023). Emerging Trends in Biodegradable Microcarriers for Therapeutic Applications. \u003cem\u003ePolymers (Basel), 15\u003c/em\u003e(6), 1487. doi:10.3390/polym15061487\u003c/li\u003e\n \u003cli\u003eHon, D. N.-S. (2017). Cellulose and its derivatives: structures, reactions, and medical uses. In \u003cem\u003ePolysaccharides In Medicinal Applications\u003c/em\u003e (pp. 87-105): Routledge.\u003c/li\u003e\n \u003cli\u003eHuang, C. M. Y., Chia, P. X., Lim, C. S. S., Nai, J. Q., Ding, D. Y., Seow, P. B.\u003cem\u003e, et al.\u003c/em\u003e (2017). Synthesis and characterisation of carboxymethyl cellulose from various agricultural wastes. \u003cem\u003eCellulose Chemistry and Technology, 51\u003c/em\u003e(7-8), 665-672.\u003c/li\u003e\n \u003cli\u003eIdris, R., Chong, W. W. F., Ali, A., Idris, S., Hasan, M. F., Ani, F. N., \u0026amp; Chong, C. T. (2021). Phenol-rich bio-oil derivation via microwave-induced fast pyrolysis of oil palm empty fruit bunch with activated carbon. \u003cem\u003eEnvironmental Technology \u0026amp; Innovation, 21\u003c/em\u003e, 101291. doi:10.1016/j.eti.2020.101291\u003c/li\u003e\n \u003cli\u003eJose, S., Fangueiro, J. F., Smitha, J., Cinu, T. A., Chacko, A. J., Premaletha, K., \u0026amp; Souto, E. B. (2012). Cross-linked chitosan microspheres for oral delivery of insulin: Taguchi design and in vivo testing.\u003cem\u003e\u0026nbsp;Colloids and Surfaces B: Biointerfaces, 92\u003c/em\u003e, 175-179. doi:10.1016/j.colsurfb.2011.11.040\u003c/li\u003e\n \u003cli\u003eKalmer, R. R., Mohammadi, M., Karimi, A., Najafpour, G., \u0026amp; Haghighatnia, Y. (2019). Fabrication and evaluation of carboxymethylated diethylaminoethyl cellulose microcarriers as support for cellular applications. \u003cem\u003eCarbohydrate Polymers, 226\u003c/em\u003e, 115284. doi:10.1016/j.carbpol.2019.115284\u003c/li\u003e\n \u003cli\u003eKanikireddy, V., Varaprasad, K., Jayaramudu, T., Karthikeyan, C., \u0026amp; Sadiku, R. (2020). Carboxymethyl cellulose-based materials for infection control and wound healing: A review. \u003cem\u003eInternational Journal of Biological Macromolecules, 164\u003c/em\u003e, 963-975. doi:10.1016/j.ijbiomac.2020.07.160\u003c/li\u003e\n \u003cli\u003eKaur, N., Singh, B., \u0026amp; Sharma, S. (2018). Hydrogels for potential food application: Effect of sodium alginate and calcium chloride on physical and morphological properties. \u003cem\u003eThe Pharma Innovation Journal, 7\u003c/em\u003e(7), 142-148.\u003c/li\u003e\n \u003cli\u003eLee, S., Park, Y. H., \u0026amp; Ki, C. S. (2016). Fabrication of PEG-carboxymethylcellulose hydrogel by thiol-norbornene photo-click chemistry. \u003cem\u003eInternational Journal of Biological Macromolecules, 83\u003c/em\u003e, 1-8. doi:10.1016/j.ijbiomac.2015.11.050\u003c/li\u003e\n \u003cli\u003eMehregan Nikoo, A., Kadkhodaee, R., Ghorani, B., Razzaq, H., \u0026amp; Tucker, N. (2016). Controlling the morphology and material characteristics of electrospray generated calcium alginate microhydrogels. \u003cem\u003eJournal of Microencapsulation, 33\u003c/em\u003e(7), 605-612. doi:10.1080/02652048.2016.1228707\u003c/li\u003e\n \u003cli\u003eMuoio, F., Panella, S., Lindner, M., Jossen, V., Harder, Y., Moccetti, T.\u003cem\u003e, et al.\u003c/em\u003e (2021). Development of a biodegradable microcarrier for the cultivation of human adipose stem cells (hASCs) with a defined xeno- and serum-free medium. \u003cem\u003eApplied Sciences, 11\u003c/em\u003e(3), 925. doi:10.3390/app11030925\u003c/li\u003e\n \u003cli\u003eNazir, M. S., Wahjoedi, B. A., Yussof, A. W., \u0026amp; Abdullah, M. A. (2013). Eco-friendly extraction and characterization of cellulose from oil palm empty fruit bunches. \u003cem\u003eBioresources, 8\u003c/em\u003e(2), 2161-2172. doi:10.15376/BIORES.8.2.2161-2172\u003c/li\u003e\n \u003cli\u003eNweke, C. E., \u0026amp; Stegemann, J. P. (2020). Modular microcarrier technologies for cell-based bone regeneration. \u003cem\u003eJournal of Biomedical Materials Research Part B: Applied Biomaterials 8\u003c/em\u003e(18), 3972-3984. doi:10.1039/d0tb00116c\u003c/li\u003e\n \u003cli\u003eNweke, C. E., \u0026amp; Stegemann, J. P. (2022). Fabrication and characterization of osteogenic function of progenitor cell-laden gelatin microcarriers. \u003cem\u003eJournal of Biomedical Materials Research Part B: Applied Biomaterials, 110\u003c/em\u003e(6), 1265-1278. doi:10.1002/jbm.b.34998\u003c/li\u003e\n \u003cli\u003ePatil, J. S., Kole, S. G., Gurav, P. B., \u0026amp; Vilegave, K. V. (2016). Natural polymer based mucoadhesive hydrogel beads of nizatidine: Preparation, characterization and evaluation. \u003cem\u003eIndian Journal of Pharmaceutical Education and Research, 50\u003c/em\u003e(1), 159-169. doi:10.5530/ijper.50.1.20\u003c/li\u003e\n \u003cli\u003ePerteghella, S., Martella, E., de Girolamo, L., Perucca Orfei, C., Pierini, M., Fumagalli, V.\u003cem\u003e, et al.\u003c/em\u003e (2017). Fabrication of innovative silk/alginate microcarriers for mesenchymal stem cell delivery and tissue regeneration. \u003cem\u003eInternational Journal of Molecular Sciences, 18\u003c/em\u003e(9), 1829. doi:10.3390/ijms18091829\u003c/li\u003e\n \u003cli\u003eRahman, M., Hasan, M., Nitai, A. S., Nam, S., Karmakar, A. K., Ahsan, M.\u003cem\u003e, et al.\u003c/em\u003e (2021). Recent developments of carboxymethyl cellulose. \u003cem\u003ePolymers, 13\u003c/em\u003e(8), 1345.\u003c/li\u003e\n \u003cli\u003eRahman, M. S., Islam, M. M., Islam, M. S., Zaman, A., Ahmed, T., Biswas, S.\u003cem\u003e, et al.\u003c/em\u003e (2019). Morphological characterization of hydrogels. \u003cem\u003eCellulose-Based Superabsorbent Hydrogels\u003c/em\u003e, 819-863. doi:10.1007/978-3-319-77830-3_28\u003c/li\u003e\n \u003cli\u003eRebers, L., Reichs\u0026ouml;llner, R., Regett, S., Tovar, G. E. M., Borchers, K., Baudis, S., \u0026amp; Southan, A. (2021). Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels. \u003cem\u003eScientific Reports, 11\u003c/em\u003e(1), 3256. doi:10.1038/s41598-021-82393-z\u003c/li\u003e\n \u003cli\u003eRozwadowska, N., Malcher, A., Baumann, E., Kolanowski, T. J., Rucinski, M., Mietkiewski, T.\u003cem\u003e, et al.\u003c/em\u003e (2016). In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3(R). \u003cem\u003eFolia Histochemica et Cytobiologica, 54\u003c/em\u003e(2), 81-90. doi:10.5603/FHC.a2016.0010\u003c/li\u003e\n \u003cli\u003eSathali, A., \u0026amp; Varun, J. (2012). Formulation, development and in vitro evaluation of candesartan cilexetil mucoadhesive microbeads. \u003cem\u003eInternational Journal of Current Pharmaceutical Research, 4\u003c/em\u003e(3), 109-118.\u003c/li\u003e\n \u003cli\u003eSeddiqi, H., Oliaei, E., Honarkar, H., Jin, J. F., Geonzon, L. C., Bacabac, R. G., \u0026amp; Klein-Nulend, J. (2021). Cellulose and its derivatives: towards biomedical applications. \u003cem\u003eCellulose, 28\u003c/em\u003e(4), 1893-1931. doi:10.1007/s10570-020-03674-w\u003c/li\u003e\n \u003cli\u003eSteffen, F., Bertolo, A., Affentranger, R., Ferguson, S. J., \u0026amp; Stoyanov, J. (2019). Treatment of naturally degenerated canine lumbosacral intervertebral discs with autologous mesenchymal stromal cells and collagen microcarriers: A prospective clinical study. \u003cem\u003eCell Transplant, 28\u003c/em\u003e(2), 201-211. doi:10.1177/0963689718815459\u003c/li\u003e\n \u003cli\u003eSultana, S., Islam, M., Dafader, N., Haque, M. E. u., Nagasawa, N., \u0026amp; Tamada, M. (2012). Effect of mono- and divalent salts on the properties of carboxymethyl cellulose hydrogel under irradiation technique. \u003cem\u003eInternational Journal of Chemical Sciences, 10\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eSwamy, B. Y., \u0026amp; Yun, Y. S. (2015). In vitro release of metformin from iron (III) cross-linked alginate-carboxymethyl cellulose hydrogel beads. \u003cem\u003eInternational Journal of Biological Macromolecules, 77\u003c/em\u003e, 114-119. doi:10.1016/j.ijbiomac.2015.03.019\u003c/li\u003e\n \u003cli\u003eTakimoto, K., Arahira, T., \u0026amp; Todo, M. (2022). Development and characterization of three-dimensional layered structures with gel beads for bone tissue engineering. \u003cem\u003eResults in Materials, 16\u003c/em\u003e, 100317. doi:10.1016/j.rinma.2022.100317\u003c/li\u003e\n \u003cli\u003eTavakol, M., Vasheghani-Farahani, E., \u0026amp; Hashemi-Najafabadi, S. (2013). The effect of polymer and CaCl(2) concentrations on the sulfasalazine release from alginate-N,O-carboxymethyl chitosan beads. \u003cem\u003eProg Biomater, 2\u003c/em\u003e(1), 10. doi:10.1186/2194-0517-2-10\u003c/li\u003e\n \u003cli\u003eTavassoli, H., Alhosseini, S. N., Tay, A., Chan, P. P. Y., Weng Oh, S. K., \u0026amp; Warkiani, M. E. (2018). Large-scale production of stem cells utilizing microcarriers: A biomaterials engineering perspective from academic research to commercialized products. \u003cem\u003eBiomaterials, 181\u003c/em\u003e, 333-346. doi:10.1016/j.biomaterials.2018.07.016\u003c/li\u003e\n \u003cli\u003eThakur, V. K., \u0026amp; Thakur, M. K. (2015). \u003cem\u003eHandbook of polymers for pharmaceutical technologies, structure and chemistry\u003c/em\u003e (Vol. 1): John Wiley \u0026amp; Sons.\u003c/li\u003e\n \u003cli\u003eTuan Mohamood, N. F. A.-Z., Abdul Halim, A. H., \u0026amp; Zainuddin, N. (2021). Carboxymethyl cellulose hydrogel from biomass waste of oil palm empty fruit bunch using calcium chloride as crosslinking agent. \u003cem\u003ePolymers, 13\u003c/em\u003e(23), 4056. doi:10.3390/polym13234056.\u003c/li\u003e\n \u003cli\u003evan Wezel, A. L. (1967). Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. \u003cem\u003eNature, 216\u003c/em\u003e(5110), 64-65. doi:10.1038/216064a0\u003c/li\u003e\n \u003cli\u003eVulpe, R., Popa, M., Picton, L., Balan, V., Dulong, V., Butnaru, M., \u0026amp; Verestiuc, L. (2016). Crosslinked hydrogels based on biological macromolecules with potential use in skin tissue engineering. \u003cem\u003eInt J Biol Macromol, 84\u003c/em\u003e, 174-181. doi:10.1016/j.ijbiomac.2015.12.019\u003c/li\u003e\n \u003cli\u003eXiao, X. C., Chu, L. Y., Chen, W. M., \u0026amp; Zhu, J. H. (2005). Monodispersed thermoresponsive hydrogel microspheres with a volume phase transition driven by hydrogen bonding. \u003cem\u003ePolymer, 46\u003c/em\u003e(9), 3199-3209. doi:10.1016/j.polymer.2005.01.075\u003c/li\u003e\n \u003cli\u003eXie, H., Pan, Y., Xiao, H., \u0026amp; Liu, H. (2019). Preparation and characterization of amphoteric cellulose\u0026ndash;montmorillonite composite beads with a controllable porous structure. \u003cem\u003eJournal of Applied Polymer Science, 136\u003c/em\u003e(37), 47941.\u003c/li\u003e\n \u003cli\u003eYan, J., Wang, Y., Zhang, X., Zhao, X., Ma, J., Pu, X.\u003cem\u003e, et al.\u003c/em\u003e (2019). Snakegourd root/Astragalus polysaccharide hydrogel preparation and application in 3D printing. \u003cem\u003eInternational Journal of Biological Macromolecules, 121\u003c/em\u003e, 309-316. doi:10.1016/j.ijbiomac.2018.10.008\u003c/li\u003e\n \u003cli\u003eYusup, E. M., \u0026amp; Mahzan, S. B. (2018). The physical characteristics of composite bone scaffold fabricated from OPEFB-CMC, chitosan and HA. \u003cem\u003eInternational Journal of Mechanical Engineering and Robotics Research, 7\u003c/em\u003e(5), 466-470. doi:10.18178/ijmerr.7.5.466-470\u003c/li\u003e\n \u003cli\u003eZakiah, J., Mansor, A., Kamaruddin, H., \u0026amp; Norhazlin, Z. (2015). Sago starch based hydrogel prepared using electron beam irradiation technique for controlled release application. \u003cem\u003eMalaysian Journal of Analytical Sciences, 19\u003c/em\u003e(3), 503-512.\u003c/li\u003e\n \u003cli\u003eZou, Q., Li, J., \u0026amp; Li, Y. (2015). Preparation and characterization of vanillin-crosslinked chitosan therapeutic bioactive microcarriers. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e(79), 736-747.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microcarrier, oil palm empty fruit bunch, cellulose, carboxymethyl cellulose, carboxymethylation, ionic crosslinking","lastPublishedDoi":"10.21203/rs.3.rs-4663194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4663194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrocarrier offers a convenient way to support cell adhesion and proliferation for biomedical applications. However, commercial microcarriers often have high production costs and limited biodegradability. The use of cellulose-rich oil palm empty fruit bunch (OPEFB) for the development of microcarriers could lead to a cheap, sustainable, and biodegradable cell culturing system. In this research, a series of carboxymethyl cellulose (CMC) microcarriers were prepared from OPEFB using FeCl\u003csub\u003e3\u003c/sub\u003e ionic crosslinker at various polymer and crosslinker levels. The microcarriers were characterised by various instrumental techniques, including assessment of gel content, swelling behaviour, mechanical stability, and \u003cem\u003ein vitro\u003c/em\u003e degradation test. The resulting OPEFB-derived CMC-microcarriers exhibited an average size ranging from 1105.52 to 1322.25 µm. SEM analysis revealed that the fabricated CMC-microcarriers exhibited ridges and porous surface morphology and the EDX analysis confirmed the successful ionic crosslinking between the OPEFB-derived CMC biopolymer and FeCl\u003csub\u003e3\u003c/sub\u003e solution. In contrast with gel content results that increased from 16.95 to 42.65 %, the swelling behaviours regularly decreased from 385 to 32% with increasing concentrations of polymer and crosslinker. Higher concentrated samples (CMC-3, CMC-6, and CMC-9) demonstrated enhanced mechanical stability and reduced sensitivity to the environment due to the higher degree of crosslinking. Nevertheless, all microcarriers displayed a degree of biodegradability ranging from 40 to 90%. Overall, the findings suggest that OPEFB can serve as a cost-effective, sustainable, and biodegradable source of natural biomaterial for microcarrier development, contributing to advancements in tissue engineering and therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Synthesis and Characterisation of Biodegradable Carboxymethyl Cellulose Microcarriers from Oil Palm Empty Fruit Bunch for Therapeutic Applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 11:45:14","doi":"10.21203/rs.3.rs-4663194/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-11T20:39:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-31T10:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164022105600803354392153328139868841732","date":"2024-07-22T09:05:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142982821492367264830466928676830673519","date":"2024-07-22T00:01:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-21T17:50:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T04:03:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-05T04:03:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellulose","date":"2024-06-30T14:32:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a4d3e1ee-538b-488f-8a22-db003ea106f6","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T15:59:08+00:00","versionOfRecord":{"articleIdentity":"rs-4663194","link":"https://doi.org/10.1007/s10570-024-06269-x","journal":{"identity":"cellulose","isVorOnly":false,"title":"Cellulose"},"publishedOn":"2024-11-13 15:56:55","publishedOnDateReadable":"November 13th, 2024"},"versionCreatedAt":"2024-07-30 11:45:14","video":"","vorDoi":"10.1007/s10570-024-06269-x","vorDoiUrl":"https://doi.org/10.1007/s10570-024-06269-x","workflowStages":[]},"version":"v1","identity":"rs-4663194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4663194","identity":"rs-4663194","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}