Effect of Bone Marrow Derived Exosomes Loaded with Estradiol on Bone Defect

Abstract

Fractures occur under strong impact and pressure and with the reduction of the bone's ability to bear the pressure, which is more common in the elderly and people with Background diseases. In many cases, common treatment methods have favorable results, but in 10% of cases, complications such as infection, swelling, and even lack of fusion occur, which cause permanent physical problems. Today, researches to solve these problems continue in the field of biological scaffolds, cells and their derivatives. Hydrogel scaffolds provide a suitable substrate for cell growth. Exosomes, as cell-derived membrane bags containing cellular regulatory macromolecules, have shown remarkable results in various studies, including the bone field. Also, estradiol is one of the estrogenic hormones, which has the effects of stimulating bone formation, inhibiting bone resorption, and anti-inflammatory effects. Therefore, in this study, a hydrogel scaffold loaded with exosomes of bone marrow cells loaded with estradiol was used to repair a bone defect model. Materials and Methods: Extraction of exosome from bone marrow cells of New Zealand rabbit was done by ultracentrifugation method. The morphology and size of exosomes were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). By western blot analysis, CD63 and CD9 proteins were investigated as exosome markers. In order to estradiol loading in exosomes, the incubation method was used, and drug loading was confirmed by HPLC and TEM methods. Rheological evaluation, pressure coefficient and bio-ability of hydrogel scaffold were measured by FTIR, TGA and SEM methods. In order to create bone defect models with dimensions of 0.5×0.5 in the tibia bone of New Zealand rabbits, surgery was performed using a dental bur. This study was conducted in 5 different groups of animals, where scaffolds, exosomes and estradiol were placed in these five groups in a distinct and combined manner in the bone defect area, and then the surgical site was completely closed. After two months, the animals were sacrificed and the tibia bones of the rabbits were removed and subjected to Real Time-PCR, immunohistochemistry (IHC), H&E staining and Micro-CT imaging to evaluate the repair. Results: The extracted exosomes had round and two-layered morphology by TEM method. In terms of size and zeta potential, they were in the range of 120-30 nm and the approximate electric charge was -10 mv. The loading rate of estradiol in exosomes was 59.77 ng by HPLC method. Scaffold quality tests included: TGA and structural toxicity, which showed appropriate thermal resistance and non-toxicity and high bioavailability of the hydrogel. After the treatment period, the results of Real Time-PCR, IHC, H&E tissue staining and Micro-CT imaging showed that the expression of bone factors, the production of new bone tissue in the last group (scaffold-exosome-estradiol) were significantly (P<0.0001) higher than other study groups.