Document Type : Original Research Article

Authors

1 Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran

3 School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran 3 Applied Biotechnology Research Center, Baqiyatallah University of Medical Science, Tehran, Iran.

4 Dental School, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

In this study, the purpose of this study was to fabricate a porous nanocomposite chitosan-gelatin along with different weight percentages of bioglass using the freeze-drying method. In this study, the Platelet-rich plasma (PRP) technique was used for the first time to strengthen bone grafting. Tensile strength, elastic modulus, and percentage of porosity were evaluated. Also, the samples were examined in simulated body fluid (SBF) and phosphate buffer saline (PBS) to study bone growth and bone dissolution rate.  Also, the porosity percentage was about 54% with 39% bone-like apatite formation for the third sample. The early absorption and late absorption rate of bone substitutes and the main volume of bone substitutes can be used for orthopedic approaches. The specimen were abalysized using X-ray diffraction (XRD) and scanning electron microscope (SEM) technique. Porous membranes were examined under SEM tools to estimate pore size and morphological behavior. The pore diameter can be controlled by adjusting the ethanol percentage in the range of 10-30 micrometers. The formation of needle bioglass crystals on the membrane surface after 7 days of immersion in biological solution was also evaluated using SEM images. In general, the third sample can be used to repair the bone tissue of the knee with the PRP technique.

Keywords

INTRODUCTION
Tissue engineering is an emerging technology that has been developed to regenerate tissues and organs with increasing interest in the field of biomaterials [1-2]. Bioactive glass (BG) ceramics were made by Hanch et al in the 1960s, as a biomaterial for repairing bone defects. BG is widely used in dentistry and orthopedic applications. Different types of synthetic polymers have been used to make absorbable membranes for bone tissue repairment. The most important of these polymers are known as Polylactice Acid (PLA), poly(glycolic acid) (PGA) and copolymers of these two compounds. PGA has weaker biomechanical properties than PLA, while PGA is more hydrophilic and PLA has hydrophobic properties [3-8]. The elasticity of PGA is much higher than PLA and the adsorption and adsorption rate is slower. Therefore, these two combinations are usually used as a network-like and interwoven microstructure. To improve the mechanical and chemical properties of the copolymer, the two compounds sometimes use a third polymer, such as trimethylene carbonate [6-8]. The main part of the process of decomposition and adsorption of these membranes is a non-enzymatic process that causes the production of PLA and PGA.  
Although, researchers believe that the degradation and adsorption stages of these membranes are associated with the production of acid in the newly formed tissue. There are several studies that have shown the positive performance of these membranes in orthopedic surgeries [9-12]. The adsorption of these membranes can also be adjusted by changing the amount of cross-linking polymer. The complete absorption time of some types of these membranes can last up to about more than a year. It should be noted that collagen membranes are contraindicated in people who are allergic to collagen or animal products such as cattle or pigs. In fact, the importance and value of allografts or xenografts or alloplasts is due to their ability to attach these molecules to their surface [13-15]. In implant osteointegration, the bone is not formed directly on the surface of the titanium alloy, but these cell adhesion molecules are first placed on the surface of the titanium alloy and form the appropriate biological matrix [16-19]. In fact, bone binds through collagen fibrils to materials resembling a natural cement composed of protein and osteopontin located on the surface of the implant fixture. In the field of implant therapies and bone regenerations, bone marrow is considered the gold standard because it has an osteoconductive matrix, together with growth signaling cells and molecules [20-24]. 
In many bone diseases such as Multiple Myeloma, Fibrous Dysplasin, Paget’s disease, or diseases such as uncontrolled diabetes that affects bone repair, bone grafts or osteointegration of dental and orthopedic implants. Natural polymers including collagen, alginate, agarose, fibrin, and chitosan is widely used in the field of bone and neuro tissue science [25-34]. These materials are known as biocompatible and show similar environment to natural tissues [26-38], blood vessels [23] and nerve. However, the main problems of natural polymers are poor mechanical properties, lack of cellular interaction and uncontrolled degradation [39-45]. To enhance the mechanical properties, some polymer composites have been developed for bone tissue engineering. Gelatin is added to the membrane system to increase its physical and mechanical properties [45-52]. Chitosan-gelatin membrane bioactivity must be improved for specific tissues like many polymers. To improve membrane bioactivity, it is often combined with other bioactive substances [53-57]. Chitosan-gelatin membranes can be molded into various shapes and by freeze drying technique, it can create a porous microstructure [58-59]. In this paper, we describe the preparation of bioactive glass cement and chitosan-gelatin membranes and their properties related to the engineering applications of bone tissue.

MATERIALS AND METHODS
The materials used in this article is bioactive glass (BG) cement derived from sol-gel and chitosan-gelatin membrane. BG cement was synthesized by combining SiO2 (64%), CaO (26%), P2O5 (8%) and AgNO3 (2%) by sol-gel method in which that the solution was prepared using specific amount of Tetraethyl orthosilicate to 0.1 mol nitric and then mixed to continue for 4 hours at room temperature for hydrolysis. Triethyl phosphate and calcium nitrate tetrahydrate were combined to the solution and stirred for 1 hour. The solution was then kept at 37°C for 10 days to gelatinize and for removing the water, the gel was heated at 70°C for 3 days. The dried gel was then heated to 700°C at 3°C/min and reminded for 1 day at 700°C for stabilization. Finally, BG powder was prepared in a high energy ball mill for 30 minutes. To prepare the chitosan-gelatin membrane, 2 wt% of chitosan-gelatin (mass ratio 70 to 30) was added to 20 ml of distilled water and stirred on magnetic stirrer. Two samples were obtained by adding ethanol 1% and 2% to the solution. The samples were then frozen and the samples were insert into the freeze-drying machine at 0.01 mbar and -45°C. The samples were tested using tensile strength by Hounsfield machine with 0.2 mm/min speed rate. The stress-strain diagram presents the tensile strength and elastic modulus of the four samples. The samples then soaked in the simulated body fluid and phosphate buffer saline for bioactivity analysis and biodegrdatuin rate after 21 days in water bath.

X-ray diffraction analysis
The samples microstructure containing various amount of BG was analyzed using X-ray diffraction (XRD) method by Philips PW 3710. The XRD voltage of 30 kV and Cu-Kα radiation was applied.  

Scanning electron microscopy analysis
The samples containimg various amount of BG in CHI-GN membrane were coated with a thin layer of gold (EMITECH K450X, England) and after that the morphological investigation of the prepared specimen membranes were observed using a scanning electron microscope (SEM; STEREOSCAN S 360).
RESULT AND DISCUSSION
The characterization of nanocomposite sample was performed with XRD tools. The XRD pattern shows in Fig. 1. It shows the XRD pattern of bioactive glass which present the straight base line and sharp diffraction peaks of the sample as a well crystallized nanoparticle.  The result of XRD for bioglass nanoparticles is shown in Fig. 1, which are shown at angles of 20, 25 and 32°.
The use of BG nanoparticles to enhance the mechanical properties of bone scaffolds is proposed for knee joint repair with PRP technique which can improve cell growth. The XRD pattern shows the crystal structure is similar to that of human bone. Fig. 2 shows the first and second samples with a lower bioglass weight percentage to evaluate the lower tensile strength compared to the third and fourth samples. In general, it can be said that the elastic modulus for the fourth sample is about 1.1 MPa which increased 0.3 MPa compared to the pure sample. Fig. 3 indicates the porosity of the samples and the bone growth rate using the results of SEM images for four samples. 
Fig. 4 shows that with the increase of bioglass from 0 wt% increase the porosity percentage from 48.5% to 57% which has a significant effect on the apatite formation of the porous scaffold. Therefore, the apatite formation increases from 33% to 40% by addition of PRP. The obtained results show that the increase in porosity has the same effect on an increase in apatite formation. In general, it can be said that with increasing porosity, bone growth, calcium and phosphorus deposition increase which are the most important apatite formation and created inside porous cavities. Fig. 4 demonstrate the elastic modulus of the porous samples so that the elastic modulus has increased from 0.8 MPa to 1.3 MPa due to the addition of BG nanoparticles which have a spherical microstructure with high mechanical strength. 
The SEM images was used to examine chitosan-gelatin membranes containing 0, 1 and 2% ethanol and to observe their morphology, especially needle HA nanocrystals in samples taken in ringer solution and to estimate the shape and diameter of porosity. The bone’s response to a simple fracture differs from that in which a gap is created at a distance between two pieces of bone. Therefore, the bone response is different according to the type of injury [55-58]. A significant proportion of advanced implant therapies are clearly associated with maxillofacial bone surgery. Therefore, correct understanding and sufficient knowledge of bone and effective factors in bone response to treatment are the background of advanced implant therapies. Bone is made by osteoblasts or osteoplasts and bone-forming cells are derived from osteogenic progenitor cells during a complex evolutionary pathway, and osteogenic progenitor cells either directly by cell differentiation in mesenchymal stem cells (MSCs) [57-64].   
With increasing ethanol percentage, SEM results showed an increase in porous size. Meanwhile, as can be seen, the porosity forms become spherical with increasing percentage of ethanol. The SEM images showed the formation of nano-sized needle-like hydroxyapatite crystals on the membrane surface after 7 days of immersion. 
Fig. 5 demonstarte a SEM image for 4 samples immersed in a SBF after 21 days which shows by decreasing the weight percentage of BG, the microstructure and chemical stability decrease and the dissolution rate increases, which causes a corrolation between bone formation and bone dissolution. Since it is possible to use collagen membranes in two or three layers during the surgical phase, the surgeon can extend the presence and function of the membrane by increasing the number of layers of collagen membrane, depending on the required treatment. At present, the maximum time for complete absorption of collagen membranes is about 6 to 8 months (26 to 28 weeks). It should be noted that these images do not indicate how long the gelatin membrane has membrane function. It seems that as the gelatin membrane maintains its membrane integrity for one to two months.

CONCLUSION
The use of platelet-rich plasma due to growth factor in the lesion with bioactive glass causes faster healing, repair, very high biocompatibility as anti-inflammatory. The bioactive glass and chitosan/gelatin membrane can be suitable choice for in vivo tests for bone lesions and regenerates the jaw. The mean pore size of chitosan-gelatin membrane with 2% ethanol was 30 ± 6.3 μm. The membrane with the optimal percentage of ethanol is very suitable for tissue engineering with sufficient pore size for cell penetration. One of the disadvantages of collagen as a membrane is that the materials completely degraded before the process of bone formation and the process of its decomposition and absorption is formed along with partial inflammation in the tissue.

ACKLOWDGEMENT
The authors of the article are grateful for the valuable guidance of the professors at the Islamic Azad University of Research Sciences who supported the preparation of this research.

CONFLICT OF INTEREST 
The authors declare that they have no conflict of interest.

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