Date of Award
5-1-2014
Degree Name
Doctor of Philosophy
Department
Molecular Biology, Microbiology and Biochemistry
First Advisor
El-Amin III, Saadiq
Second Advisor
Cao, Deliang
Abstract
Introduction: Bone defects and non-unions caused by trauma, tumor resection, pathological degeneration, or congenital deformity pose a great challenge in the field of orthopedics. Traditionally, these defects have been repaired by using autografts and allografts. Autografts have set the gold standard for clinical bone repair because of their osteoconductivity, osteoinductivity and osteogenicity. Nevertheless, the application of autografts is limited because of donor availability and donor site morbidity. Allografts have the advantage that the tissues are readily available and can be easily applied, especially when large segments of bone are to be reconstructed. However, their use is also limited by the risk of disease transfer and immune rejection. To circumvent these limitations tissue engineering has evolved as a means to develop viable bone grafts. An ideal bone graft should be both osteoconductive and osteoinductive, biomechanically strong, minimally antigenic, and eliminates donor site morbidity and quantity issues. The biodegradable polymer, Poly lactic-co-glycolic acid (PLAGA) was chosen because of its commercial availability, biocompatibility, non-immunogenicity, controlled degradation rate, and its ability to promote optimal cell growth. To improve the mechanical properties of PLAGA, Single Walled Carbon Nanotubes (SWCNT) were used as a reinforcing material to fabricate composite scaffolds. The overall goal of this project is to develop a Single Walled Carbon Nanotube composite (SWCNT/PLAGA) for bone regeneration and to examine the interaction of MC3T3-E1 cells (mouse fibroblasts) and hBMSCs (human bone marrow derived stem cells) with the SWCNT/PLAGA composite via focusing on extracellular matrix production and mineralization; and to evaluate its toxicity and bio-compatibility in-vivo in a rat subcutaneous implant model. We hypothesize that reinforcement of PLAGA with SWCNT to fabricate SWCNT/PLAGA composites increases both the mechanical strength of the composites as well as the cell proliferation rate on the surface of the composites while expressing osteoblasts phenotypic, differentiation and mineralization markers; and SWCNT/PLAGA composites are biocompatible and non-toxic, and are ideal candidates for bone tissue engineering. Methods: PLAGA and SWCNT/PLAGA composites were fabricated with various amounts of SWCNT (5, 10, 20, 40 and 100mg), characterized and degradation studies were performed. PLAGA (poly lactic-co-glycolic acid) and SWCNT/PLAGA microspheres and composites were fabricated; characterized and mechanical testing was performed. Cells were seeded and cell adhesion/morphology, growth/survival, proliferation and gene expression analysis were performed to evaluate biocompatibility. Sprague-Dawley rats were implanted subcutaneously with Sham, poly lactic-co-glycolic acid (PLAGA) and SWCNT/PLAGA composites, and sacrificed at 2, 4, 8 and 12 week post-implantation. The animals were observed for signs of morbidity, overt toxicity, weight gain, food consumption, hematological and urinalysis parameters, and histopathology. Results: Imaging studies demonstrated uniform incorporation of SWCNT into the PLAGA matrix and addition of SWCNT did not affect the degradation rate. Composites with 10mg SWCNT resulted in highest rate of cell proliferation (p<0.05) among all composites. Imaging studies demonstrated microspheres with uniform shape and smooth surfaces, and uniform incorporation of SWCNT into PLAGA matrix. The microspheres bonded in a random packing manner while maintaining spacing, thus resembling trabeculae of cancellous bone. Addition of 10mg SWCNT led to greater compressive modulus and ultimate compressive strength. Imaging studies revealed that MC3T3-E1 cells adhered, grew/survived, and exhibited normal, non-stressed morphology on the composites. SWCNT/PLAGA composites exhibited higher cell proliferation rate and gene expression compared to PLAGA. No mortality and clinical signs were observed. All the groups showed consistent weight gain and rate-of-gain for each group was similar. All the groups exhibited similar pattern for food consumption. No difference in urinalysis parameters, hematological parameters; and absolute and relative organ weight was observed. A mild to moderate summary toxicity (sumtox) score was observed for animals treated with the PLAGA and SWCNT/PLAGA whereas the sham animals did not show any response. At all the time intervals both PLAGA and SWCNT/PLAGA showed a significantly higher sumtox score compared to the Sham group. However, there was no significant difference between PLAGA and SWCNT/PLAGA groups. Conclusion: Our SWCNT/PLAGA composites, which possess high mechanical strength and mimic the microstructure of human trabecular bone, displayed tissue compatibility similar to PLAGA, a well known biocompatible polymer over the 12 week study. Thus, the results obtained demonstrate the potential of SWCNT/PLAGA composites for application in BTE and musculoskeletal regeneration. Future studies will be designed to evaluate the efficacy of SWCNT/PLAGA composites in bone regeneration in a non-union ulnar bone defect rabbit model. As interest in carbon nanotube technology increases, studies must be performed to fully evaluate these novel materials at a nonclinical level to assess their safety. The ability to produce composites capable of promoting bone growth will have a significant impact on tissue regeneration and will allow greater functional recovery in injured patients.
Access
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