Bone tissue engineering is now used as an alternative for the treatment of bone-related diseases. In this study, chitosan-gelatin scaffold with olibanum microspheres containing dexamethasone has been produced by the freeze-drying method. SEM and FTIR were used to characterize the scaffolds synthesized. The rate of drug release from these scaffolds was measured by a UV spectrophotometer. The synthesized scaffold was measured in terms of the swelling and degradation rates. In vitro studies were conducted to evaluate bioactivity and the environmental compatibility of the scaffold with an MTT test. A good controlled release was achieved. The bioactivity analysis confirmed the formation of apatite. The results of the MTT test showed that the synthesized scaffold was biocompatible and had an appropriate interaction with the cell. The results showed that the produced scaffold had the properties necessary to regenerate and repair bones.

Keywords: Olibanum, microsphere, scaffold, freeze-drying, dexamethasone

Various factors such as diseases, aging, accident, etc., can cause bone damage affecting bone function. Recovery and improvement of bone function are important issues in the medical field. Therefore, autograft and allograft sources are used, but there are problems in these methods such as transmission of diseases, rejection by the immune system, and resource shortages. Another solution is the use of bio-compatible degradable scaffolds that can handle bone problems.^1,2 Consequently, tissue engineering is used to treat lost tissues and tissues that are in trouble. Scaffolds are three-dimensional and porous structures that can mimic extra cellular matrix (ECM) due to this structure. ECM is required for cell functions such as adhesion, growth, migration, differentiation, and the formation of new tissues. Scaffolding design is one of the most important research areas.³
Chitosan,^4-9 alginate,² collagen, gelatin^5-9 and poly-lactic glycolic acid,^8-11 and poly-caprolactone,⁴ as natural or synthetic biodegradable polymers, are used for tissue engineering applications, due to their excellent biological properties, low prices, and high availability.² Chitosan is a natural biopolymer that is structurally similar to glycosaminoglycan (GAG). GAG has an important structure for ECM regeneration. Chitosan has good properties such as high biocompatibility, hydrophilicity, antibacterial properties, biodegradability, non-toxicity, and nonallergenicity. It is used in wound healing applications, scaffolding, drug release, and gene therapy to treat cancers.^3,6,12,13 Gelatin is a natural macromolecule that is also widely used in the biomedical and biotechnology fields due to its low antigenicity and its physical and chemical stability. In addition, it has an arginine-glycine-asparagine (RGD) sequence, which is essential for cell adhesion, differentiation, and cell proliferation. ^5,6,14,15 Today, biodegradable polymers are useful options in drug delivery systems. In advanced drug delivery technologies, there are new plant-based approaches. The use of plants is very practical and useful because it has the advantages of being affordable, economical, biodegradable, non-toxic, and chemically neutral. Recent views are that these materials are a substitute for synthetic materials, because, in addition to the above properties, such as synthetic polymers, they do not pollute the environment and have the potential for chemical optimization. Their very important feature is their biocompatibility.¹⁶ One of these natural polymers is olibanum. Olibanum is composed of three main parts: essential oil (5%-9%), resins (65%– 85%) and gum (21%-22%).^17-21 Several studies have been done on olibanum in the field of drug release. These studies are related to the synthesis of drug-carrying microspheres and microcapsules,^22,23 the coating of olibanum on the drug loaded microspheres,^24-26 and the synthesis of drug tablets.^25,27-29
Here are some ways to synthesize the tissue engineering scaffolds: Freeze-drying,^5,7,8,15,30 electro-spinning,^31-33 freezecasting, ^2,34 phase-separation³⁵ and gas-foaming.¹¹ Freeze-drying is a useful method for the synthesis of polymer scaffolds.
There are three stages in the freeze-drying procedure: Freezing, initial drying, and secondary drying. The freezing process occurs through the placing of the liquid sample in a cold environment (freezer or liquid nitrogen), and then the sample is placed in a freeze dryer. In this step, the solvent is frozen, sublimated, and, the solvent is removed from the vacuum created. The freezing step in this protocol is very important because it plays an important role in the production of a porous structure. During the freezing, first, solvent crystals are formed, and then they grow and complete freezing occurs, and the solvent is removed. In this method, the freezing temperature, solution concentration, and solvent type have an effect on the porosity structure. The faster the freezing process, the smaller is the porosity. By applying the freeze-drying method, a structure with a porosity of more than 90% and a thickness of 20 to 200 μm can be achieved.³⁶ In the production of this composite system, different cross-link methods have been used, such as physical or chemical cross-linking.⁵ One of the most important crosslinker is (3-glycidoxypropyl) methyldiethoxysilane (GPTMS).
GPTMS is a silane-coupling agent having epoxy and methoxysilane groups. The epoxy rings on the GPTMS molecules react with the amino groups on the gelatin and chitosan chains.¹⁵
J. S. Patil et al. demonstrated that the synthesized olibanum microspheres coated with Rifampicin displayed a slow release from the drug and kept the drug for a long time.²⁵ Satyajit Panda et al. showed that in microcapsules containing zidovudine and coated with olibanum resin, drug release was performed at a slow and controlled rate.²³ I. R. Serra et al. showed that using a freeze drying technique, porous and three-dimensional scaffolds with open porosity and a high percentage of porosity could be obtained that are suitable candidates for the reconstruction of bone tissue.⁵ P. Gentile et al. examined the chitosan-gelatin scaffold with the microspheres carrying the drug Simvastatin and proved that this scaffold with the drug carrier microspheres was a suitable system for drug delivery and was used in bone tissue engineering. This scaffold was synthesized by a freeze-drying technique.⁶ C. Tonda-Turo et al. compared the effect of Genipin and GPTMS cross-linkers on gelatin scaffolds, and it was observed that cross-linking scaffolds with GPTMS had greater mechanical properties because GPTMS was able to bind to amino groups on the gelatin chains.¹⁵ Martins et al. showed that dexamethasone caused the differentiation of bone marrow mesenchymal stem cells due to the osteogenic effect of this drug.³⁷ Son et al. showed that dexamethasone had osteoinductivity property on the HA/DEX-loaded PLA scaffolds. In samples containing dexamethasone, an increase in alkaline phosphate concentration, an increase in proteins, and calcification of bone tissue was observed.³⁸
According to the review of these articles, it is observed that all attempts are focused to increase the efficacy of the drug, the comfort of the patient, the reduction of the rate of drug readministration and, ultimately, the reduction of the side effects of inappropriate dosages. These points highlight the importance of the research ahead. In addition, many efforts have been made to build drug scaffolds in bone tissue engineering, and research to achieve a proper scaffold with controlled drug release rates continues. One of the challenges ahead is the mechanism for entering the drug into the structure of the scaffold. The amounts of drug loading and the complete destruction of the drug are the effective parameters in this field. Accordingly, if it is possible to control the release rate of the drug by controlling the scaffold parameters, then the proper functioning of the scaffold can be expected. In addition, the achievement of a field of knowledge that demonstrates the impact of different parameters in the dynamics of drug release is one of the important gap and necessities in this field of research that is being addressed in this study.
In this research, a chitosan-gelatin scaffold with dexamethasone-containing olibanum microspheres was synthesized by the freeze-drying method and the synthesized scaffold was evaluated. Bioactivity of the scaffold was studied for bone tissue engineering applications.