טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKhoury Nizar
SubjectThe Combined Use of Transforming Growth Factor-BETA Loaded
onto BETA-tri Calcium Phosphate Scaffold to
Enhance Bone Eneration
DepartmentDepartment of Medicine
Supervisors Professor Eli Machtei
Dr. Hadar Zigdon
Full Thesis textFull thesis text - English Version


Abstract

Transforming Growth Factor-β (TGF-β1) belongs to the TGF beta superfamily. It plays a pivotal role in osteogenesis mainly by enhancing Mesenchymal Stem Cells (MSCs) differentiation into osteoblasts. The aim of the study is to enhance bone regeneration in rat calvaria model by combining TGF-β1 loaded onto β-Tri Calcium Phosphate (β-TCP) synthetic scaffold.

Methods: in-vitro experiments were performed in order to evaluate the release kinetics of TGF-β1 from β-TCP scaffold and to follow the effect of TGF-β1 on various MSCs functions.  To this end, TGF-β1 in various concentrations (1-40 ng/ml) was loaded onto β-TCP scaffold, and the release kinetics was monitored by ELISA. Next, MSCs were isolated from rats, cultured in DMEM and characterized using FACS. The effect of TGF-β1 on the adhesion and proliferation of MSCs were assayed using Alamar Blue. Then the effect of TGF-β1 on MSCs differentiation was evaluated by Alizarin Red quantification assay. The seeding efficiency of MSCs on β-TCP was also examined and calculated. We performed an in-vivo study to evaluate bone augmentation following transplantation of TGF-β1 loaded on β-TCP in a rat calvaria model. 14 sprague-dawley rats were divided into two groups: GBRβ-TCP (control), GBR?β (40ng/ml) β-TCP (test). At 3 months, following their euthanasia, specimens were fixed and decalcified and then embedded in paraffin and sectioned. To examine bone quality and morphology the specimens were stained by: 1. Hematoxylin and Eosine (H&E) and 2. Masson's Trichrome.  Histomorphometric analysis was performed using Image-pro? software. 

Results: the release kinetics demonstrated that greater TGF-β1 was released from the 40ng/ml compared to other concentrations that were tested. Approximately 26% of initial TGF-β concentration was released in the first hour and remained relatively constant after two hours until the ninth hour.  FACS analysis revealed that more than 92% of the cells were CD90 positive and were negative for CD14, CD31 and CD34.  The adhesion of MSCs to plastic plate showed that neither the time nor the concentration of TGF-β significantly influenced cell adherence. Furthermore, after 24 hours we found a suppressive effect of TGF-β on the MSCs proliferation in a dose dependent manner. The maximum inhibition was obtained with 40ng/ml concentration, compared to the control group (p=0.028), also we found a positive effect on MSCs osteogenic differentiation. Alizarin Red (AR) staining were strongest in the osteiogenic medium TGF-β1 group, compared to a weak mineralization in the DMEM groups (p=0.021). In the seeding efficacy test it was shown that TGF-β1 didn't influence MSCs adherence to the scaffold. In-vivo, bone height and bone area fraction were similar with or without TGF-β1; however, blood vessel density and degradation of scaffold were significantly higher in the TGF-β1 group.

Summary and Conclusion: The present study demonstrated that β-TCP is a suitable carrier for TGF-β1. In-vitro, TGF-β1 increased MSCs differentiation. In-vivo, the addition of TGF-β1 to TCP scaffold led to faster degradation of the scaffold and increased blood vessel density. Taken together, these results may suggest that 40 ng/ml TGF-β1 may improve β-TCP scaffold performance in bone formation.