Physicochemical Characteristics of Chitosan – Alginate Scaffold Containing Atorvastatin

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Introduction
Osteoporosis is a severe medical condition characterized by low bone mass and deterioration of bone structure, which increase bone fragility and fracture risk [1]. Patients with osteoporosis can lose up to 50% of their bone density at critical skeletal locations [2]. His illness is now treated with RANK ligand antibodies (denosumab), selective estrogen receptor modulators (SERMs), teriparatide, and bisphosphonates [3]. Most of these drugs prevent bone remodeling by reducing osteoclast activity and recruiting new osteoclasts. Although these medications help treat and manage this condition, their long-term use has significant drawbacks. For example, long-term bisphosphonate therapy has been linked to atrial fibrillation, jaw osteonecrosis, and severe bone turnover inhibition [4]. Long-term SERM (raloxifene) use has also been linked to venous thromboembolism and fatal strokes [5]. Therefore, effective therapy is required to improve bone mineral density and generate new bone without significant side effects associated with long-term medication [6,7]. Statins have anti-inflammatory properties, because they lower the levels of interleukin-6 and interleukin-8. Mundy demonstrated for the first time an experimental study in which statins improved bone volume. The trabecular bone volume increased in rats whose ovaries were removed and administered 5-10 mg simvastatin daily for 35 days [8].
Simvastatin, another statin medication, has been shown to increase the amount of bone tissue, the rate at which bone tissue is produced, and the strength of spongy bone tissue [9]. Several processes involved in statin drugs and bone metabolism are of particular interest. These substances stimulate osteoblast activity, which is stimulated by bone morphogenetic protein-2. Statins also inhibit hydroxymethylglutaryl coenzyme-A reductase and lower mevalonate production, thereby interfering with osteoclast function and increasing apoptosis, which prevents bone resorption. Statins have an anti-inflammatory effect, causing bone defects in the affected bone and surrounding tissues. Statin use may also aid bone healing [10].
The effect of atorvastatin (ATV) on the proliferation, migration, and invasiveness of primary smooth muscle cells isolated from human saphenous veins after loading into a hyaluronic acid hydrogel was studied. The results show that the hydrogel is a viable substrate for local drug administration because it allows for the steady release of ATV over hours, resulting in multiple changes in the vascular smooth muscle cells phenotype that are similar to those produced by the drug freely dissolved in the cell environment [11]. Another study used a crosslinked hyaluronic acid hydrogel/poly(lactic-co-glycolic acid) microparticle formulation loaded with ATV to develop a simple drug delivery system to prevent intimal hyperplasia (IH). A carotid artery ligation model was used to validate the in vivo proof of concept. The combination of rapid and continuous ATV release has a synergistic effect on IH development. There was no evidence of harm to the collected tissues [10]. In another study, Özdoğan et al. [12] discovered that combining ATV with chitosan improved anti-inflammatory activity and bone and tissue healing in periodontitis-induced rats. Therefore, using osteoimmunomodulatory biomaterials to create a local immunological environment that promotes bone regeneration and osseointegration may be an appropriate method for redressing this imbalance [13]. Natural sourcebased materials are promising for biomedical applications [14][15][16][17][18][19]. Nanobiomaterials are made using biological resources such as fungi, plants, and algae [20][21][22][23]. Chitin, as a fibrous substance in the cell walls of some fungi, is the source of chitosan. The most important characteristics of chitosan are its nonantigenicity, excellent biodegradability, biocompatibility, ease of processing under mild conditions, interaction with cell matrix components and cell surface receptors, reactive and changeable groups, and chelating capacity. However, its controlled degradation ensured long-term survival of the encapsulated cells. In addition, chitosan can create a dynamic environment for penetration of tissue defects, making it a suitable option for use in delivery systems or matrices for the regeneration of different tissues [24]. In this study, we aim to design a sensitive, fast, and routine method that can be implemented at a low cost in the shortest time, and on the other hand, can be used as a platform that can be developed.
2.2. CS/ALG Hydrogel Fabrication. Chitosan (0.2 g) was dissolved in 10 ml acetic acid (1%) under stirring conditions for 15 min, and the alginate solution was then prepared by dissolving sodium alginate (0.45 g) in deionized water (10 ml, conductivity at 25°C = 0.02 μS/cm) under stirring conditions with magnetic stirrer (1,000 rpm) at room temperature for 15 min. The above solutions were mixed at a specific ratio and stirred overnight with a magnetic stirrer (1,200 rpm) at room temperature to obtain chitosan-alginate (CS/ALG) as the final product.

Preparation of Drug-Loaded Hydrogel.
To load the drug, chitosan (0.1 g) was dissolved in 10 ml of acetic acid (1%), sodium alginate (0.0225 g) in 10 ml of deionized water, TPP (0.005 g) in 2 ml of deionized water, ATV in 10 ml of deionized water, and dimethyl fumarate (3 ml) in 10 ml of deionized water. The solutions were then mixed and stirred using a magnetic stirrer (1,200 rpm) overnight at room temperature. Finally, the CS/ALG/TPP hydrogel containing ATV was obtained.

Release Study in Phosphate-Buffered Saline.
Three samples (average weight = 19.66 g) of CS/ALG/TPP loaded with ART were dispersed in 25 ml of phosphate-buffered saline (PBS) (pH = 7.4). The hydrogels were then placed in a shaker incubator (80 rpm) at 37°C. At specific times, PBS (3 ml) containing ART in the buffer solution was removed, measured using a UV-visible at 241.5 nm, and resupplemented with a fresh 3 ml of PBS. A set of standard solutions was used to calibrate the amount of ART present at each time point. The actual drug concentration was determined using Equation (1) [26]: where n, n th concentration measurement; Cn, corrected drug concentration; Cn:meas, the acquired drug concentration at the n th measurement; Δv, the withdrawn volume at each measurement; and Vt, the total volume of solution.

Physicochemical Properties of Hydrogels
3.1. Fourier Transform Infrared Spectroscopy. Fourier transform infrared spectroscopy (FTIR) (Equinox 55 LS 101, Bruker, Germany) was used to assess the chemical characteristics of the prepared CS/ALG/TPP. We used a wavelength range of 400-4,000 cm −1 , as in our previous work [27]. The Infrared (IR) spectra of all KBr-pressed samples were recorded in CO 2 nitrogen gas at room temperature [27].

Scanning Electron Microscopy of CS/ALG/TPP Hydrogel
Loaded with ART. Scanning electron microscope (SEM) (LEO 1430 VP, Germany) was used to evaluate the surface morphology and structure of prepared material according to our previous works [28] at 15 kV acceleration voltage. Materials were sectioned into thin slices and vacuum-coated with Au-Pd thin layer by a Polaron SC7620 sputter coater as used previously [27].

Moisture Content and Swelling Ratio.
To evaluate the swelling performance, freeze-dried hydrogel samples were immersed in PBS (pH 7.4) at 37°C and evaluated immediately after the liquid was removed from the hydrogel surface using a wet sponge. This process was repeated until total hydration was achieved. The swelling ratio (SR, g/g) was calculated using Equation (2) [25]: where W d , hydrogel weight before floating in buffer (dry hydrogel) and W s , weight of hydrogel after swelling in buffer (wet hydrogel).  Advances in Polymer Technology crosslinkers is TPP, which is widely used in the synthesis of scaffolds, membranes, nanoparticles, and various composites for medical purposes [29].

Results and Discussion
In addition, because phosphate groups are necessary for bone mineralization, TPP is widely used as a crosslinker in the development of biomimetic polymer systems for bone regeneration.
The degree of crosslinking can affect the homogeneity, porosity, hydrophilicity, and mechanical properties of scaffolds [30]. Consequently, a precise duration of the crosslinking stage is crucial for obtaining systems with exceptional performance. This negative phosphate crosslinker is frequently used to form positive ammonium chitosan crosslinks. Because instant gelation results in the formation of precipitates, simple mixing of chitosan and TPP solutions in tissue engineering applications is not an appropriate method for regulating the crosslinking reaction. Consequently, numerous methods for preparing CS-TPP systems with a controlled degree of crosslinking have been developed, such as those based on the ionic strength of the CS solvent in the presence of NaCl or the slow diffusion of TPP in the CS solution. This was used to create uniform membranes. In this study, CS/ALG scaffolds were crosslinked by immersion in TPP solution. To investigate the effects of various experimental conditions on scaffold performance, scaffolds were prepared with various concentrations of CS (1% and 2% w/w) and alginate, as well as two concentrations of TPP (1% and 2% w/w), and various reaction times (2, 4, and 8 hr). FTIR spectral range: the formation of chitosan crosslinks was confirmed by the crosslink scaffold composed of CS/ELG/TPP. Figure 2(a)-2(c) depicts the FTIR spectra of chitosan, alginate, pentasodium TPP, and their mixtures [31].
Except for TPP, all spectra exhibited a strong and broad antisymmetric band at approximately 3,430 cm −1 , which was caused by the overlap of O-H and N-H stretching vibrations of the functional groups involved in hydrogen bonding. Chitosan possesses characteristic bands at 1,650 cm −1 (associated with the stretching vibrations of carbonyl amide 1), 1,598 cm −1 (associated with the bending vibrations of the first type of nonacetylated aminoglycoside), and 1,560 cm −1 (related to the bending vibrations of the N-H group of amide type 2).
Chitosan's saccharide structure is characterized by absorption bands at 2,253 cm −1 (asymmetric stretching vibrations of C-O-C bonds), 1,083 cm −1 , and 1,031 cm −1 (skeletal vibrations including C-O bonds). When chitosan was crosslinked with TPP, a new absorption band was observed at 1,635 cm −1 , owing to the antisymmetric vibration of N-H in the NH 2 groups. After the ion crosslinking of chitosan with TPP, additional changes in the IR spectrum were observed. The absorption at 1,215 cm −1 was due to the stretching vibrations of the C = O bond of the crossphosphate linker groups [25].

Swelling
Study of CS/ALG/TPP Hydrogel. The SR was measured to evaluate the swelling performance of the prepared CS/ALG/TPP hydrogel containing ART and quantify its water uptake ability. The SR values of the prepared hydrogels are shown in Figure 3. Swelling is caused by liquid absorption by the polymer, and can be calculated by measuring the hydrogel mass. The mass SR data revealed that the Chit/Alg/TPP hydrogels exhibited 700%-800% swelling in the first few hours and 1,000% swelling after 30 hr, before decreasing to approximately 800% [32]. The swelling behavior and structural stability of hydrogels are critical for their practical use in tissue engineering. Most natural polymers, including chitosan, rapidly swell in biological fluids. Previous research indicates that early swelling is favorable and that the resulting increase in pore size promotes cell adhesion and development in a three-dimensional structure [33]. On the other hand, persistent swelling may result in loss of mechanical integrity and maceration of the surrounding tissue [32].
4.3. SEM Study of CS/ALG/TPP Hydrogel. SEM was used to investigate the morphological and surface properties of CS/ALG/TPP-containing ART hydrogels. Figure 4 shows the SEM images of the hydrogels. Images captured by SEM of the CS/ALG/TPP freeze-dried hydrogel containing ART revealed the morphology of the hydrogel. Examination of the hydrogel architecture revealed that the scaffold had a highly porous structure with interconnected pores generated by phase separation during lyophilization. The hydrogel structure is completely permeable. The holes are approximately 30 μm in size, which facilitates bone cell adhesion and development and is advantageous for cell attachment and migration [34].

Porosity
Determination of CS/ALG/TPP Hydrogel. SEM was used to investigate the morphological and surface properties of CS/ALG/TPP-containing ART hydrogels. Figure 4 shows the SEM images of the hydrogels. Images captured by SEM of the CS/ALG/TPP freeze-dried hydrogel containing ART revealed the morphology of the hydrogel. Examination of the hydrogel architecture revealed that the scaffold had a highly porous structure with interconnected pores generated by phase separation during lyophilization. The hydrogel structure is completely permeable. The holes are approximately 30 μm in size, which facilitates bone cell adhesion and development and is advantageous for cell attachment and migration [35]: where V 1 = 9, V 2 = 9.05, V 3 = 8.4, and P = 92%. After crosslinking, the hydrogel was 92% porous (Figure 3), and after 24 hr in water, it swelled by a shock of 98.6%.
Numerous studies have reported the amount of water absorption and swelling of alginate and chitosan hydrogels crosslinked with TPP. After approximately 24 hr, the maximum swelling percentage of the crosslinked CS/ALG hydrogel was 98.6%, which is greater than the values reported in the literature [36,37]. The results showed that using a TPP crosslinker is beneficial, and because the structure of the hydrogel is highly porous (92%), the expansion percentage can be significantly increased.     there was no significant increase in the percentage of drug released ( Figure 5(b)). The gradual release of ATV from the alginate-chitosan microspheres was studied. Because TPP crosslinking does not occur in this system, the swelling percentage and amount of drug loading are lower, and drug release from these microspheres is slow and complete after 25 hr [38]. This suggests that the drug was retained in the three-dimensional network formed by TPP, and that the drug molecules entrapped within the porosity of the hydrogel were slowly released. During the first hour, 20% of the drug attached to the hydrogel is released via surface absorption. The percentage of crosslinkers significantly influenced the encapsulation gain and release profiles. Increasing the proportion of crosslinkers is expected to increase the proportion of drug loading while decreasing and limiting the proportion of drug release; thus, optimizing the proportion of crosslinkers is critical.

Conclusion
CS/ALG hydrogels were prepared and loaded with ATV at various ratios and percentages of TPP crosslinker. The results showed that the drug was loaded with an approximately 100% gain during crosslinking. The drug release profile from the hydrogel was investigated, and the results showed that the percentage of drug release was relatively low owing to drug retention in the three-dimensional TPP network. The results of this study demonstrated that the crosslinked CS/ALG hydrogel could be a suitable scaffold for loading all types of drugs, macromolecules, and cells because of its swelling percentage and high porosity. The drug loading and percentage of release are expected to be controlled by adjusting the ratio of the two polymers and crosslinker. Future studies should consider scaffold cell compatibility tests, such as hemolysis and cytotoxicity. Furthermore, stability studies, biodegradability determination, and modification of the drug release profile via a change in the crosslinker ratio will help to improve this study. Finally, the scaffolds mentioned above can be studied further in cell and animal studies for bone tissue engineering and stem cell differentiation in the presence of ATV.

Data Availability
The data used to support the findings of this study have been included in this article.