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In vitro and in vivo study using chitosan microparticles with growth factors and antibiotics for bone tissue regeneration

Mantripragada, Venkata Prasanna R

Abstract Details

2014, Doctor of Philosophy, University of Toledo, Bioengineering.
The objective of this study is to fabricate chitosan microparticles under mild environmental conditions. These microparticles will serve as a better carrier for relevant growth factors and facilitate the repair and regeneration of critical-sized defects in bones. Chitosan is structurally similar with glucosaminoglycans, and this makes it particularly attractive as a biomaterial for bone defects, as glucosaminoglycans are usually the interacting molecules with osteoblasts. Chitosan and tri-ppolyphosphate (TPP) microparticles were employed because this system can be engineered quantitatively to obtain a scaffold with desired physical and biological properties. Coacervation technique was employed to cross-link amine groups in chitosan with phosphate groups in TPP. Temperatures below room temperature were maintained during the cross-linking process to ensure maximum encapsulation efficiency of the growth factors. Physical and morphological characteristics of the microparticles were determined using scanning electron microscopy; the microparticles have a diameter in the range of 400-700 µm, and the surface was found to be groovy and rough. Degradation study conducted in vitro indicated that the microparticles remained unaltered for 30 weeks when suspended in phosphate buffered saline (PBS) of pH 7.4 containing lysozyme (10 mg/L), but when the microparticles were suspended in PBS solution of pH 5.1 containing lysozyme enzyme, the microparticles degraded within 15 weeks. In order to evaluate the harms of organic solvents like hexane and acetone on growth factor encapsulation, cumulative release profiles of insulin-like growth factor 1 (IGF-1) was compared between particles prepared using the emulsification and coacervation techniques. These results indicated a significant decrease (p<0.05) in the encapsulation efficiency, which in turn also decreased the cumulative release from the emulsification microparticles in comparison with coacervation technique during the two-week period. In order to optimize various parameters that control the release kinetics of growth factor from microparticles, IGF-1 was used and it was found that microparticles with IGF-1 encapsulated in it and prepared at below room temperatures using 50% TPP, exhibited a better controlled release profile. With these optimized parameters, microparticles were fabricated incorporating bone morphogenetic protein 7 (BMP-7), which is the protein of interest in this study and in vitro experiments were conducted. BMP-7 was incorporated in two ways: encapsulation and coating. The release study results suggest that BMP-7 encapsulated microparticles exhibited a controlled release profile, while a burst release was observed in BMP-7 coated microparticles. Cell proliferation study conducted using DNA assay indicated a statistical significant increase (p<0.05) in the amount of DNA obtained from BMP-7 encapsulated and coated microparticles in comparison with microparticles without any growth factors. Cell differentiation study conducted by real time RT-PCR demonstrated a significant up-regulation in the expression of transcription factors - runx 2 and osx as well as late osteoblast markers - OCN, OPN and BSP, which lead to increased mineralization. These results indicate that chitosan microparticles obtained by coacervation method are biocompatible and help in improving the encapsulation efficiency of BMP-7. The local application and controlled release of growth factors and antibiotics is beneficial to stimulate bone healing and prevent infection at the same time. Therefore, dual release of growth factor bone morphogenetic protein-7 (BMP-7) along with antibiotics- vancomycin and cefazolin respectively was also studied. In both the release study experiments, only about 50% of the total encapsulated drug is released and it was observed that cefazolin could inhibit bacterial growth to a greater extent (~85%) in comparison with vancomycin (~80%). BMP-7 release profile in the presence of antibiotics remained unaltered, indicating that the drugs do not affect the stability of the protein. It is also important to consider the amount of drugs incorporated into the microparticles, as it is cytotoxic for osteoblasts. 50 µg/ml and 100 µg/ml of cefazolin were found to positively influence osteoblasts cell proliferation. In the complete duration of our release study, the concentration of cefazolin remained <70 µg/ml. Interestingly, in cefazolin and BMP-7 group, a significant increase in osteoblasts cell proliferation was observed in comparison with BMP-7 only, indicating that cefazolin also may play a role in osteoblast proliferation. On the other hand, 1000 µg/ml of vancomycin was found to significantly reduce cell proliferation, indicating it is toxic for osteoblasts. Therefore, the amount of vancomycin to be incorporated was chosen accordingly. Microparticles with and without BMP-7 were finally tested in vivo to determine the biological response in bone tissue in rats. Healthy bone growth was observed adhered to the microparticles. Histology images indicated minor inflammatory response observed around the microparticles at 6 weeks, which reduced by 12 weeks. Bone volume fraction was found to be significantly less in comparison with controls, which can be explained by the presence of microparticles occupying the defect site. µ-CT analysis of bone surface density and porosity was found to be significantly more (p<0.05) for microparticles containing groups, which include microparticles without growth factors, microparticles with BMP-7 encapsulated and BMP-7 coated microparticles in comparison with controls, which suggests that the new bone formed in the presence of microparticles is more interconnected and porous. Collagen fibrils analysis conducted using multiphoton microscopy indicated a significant improvement in the formation of bundled collagen area (%) in microparticles containing groups in comparison with controls, from which we understand that the collagen fibril diameter is higher and therefore the cross-linking between the fibrils is higher. The effect of BMP-7 was not effectively observed and this can be hypothesized to be due to (i) insufficient concentration of BMP-7 incorporated (ii) formation of fibrous capsule by macrophages around the microparticles, which inhibit growth factor release and (iii) highly acidic conditions created by macrophages at the surface of the microparticles may degrade the growth factor being released. This study therefore proves that even though the microparticles with growth factors significantly improved proliferation and differentiation of osteoblasts in vitro, in vivo conditions are completely different, and all the observations from this study should be considered for fabricating chitosan microparticles in future for bone tissue engineering applications.
Ambalangodage Jayasuriya (Committee Chair)
Beata Lecka Czernik (Committee Member)
Mark Wooten (Committee Member)
Patricia Relue (Committee Member)
Stephen Callaway (Committee Member)
243 p.

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Citations

  • Mantripragada, V. P. R. (2014). In vitro and in vivo study using chitosan microparticles with growth factors and antibiotics for bone tissue regeneration [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1398861353

    APA Style (7th edition)

  • Mantripragada, Venkata Prasanna. In vitro and in vivo study using chitosan microparticles with growth factors and antibiotics for bone tissue regeneration. 2014. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1398861353.

    MLA Style (8th edition)

  • Mantripragada, Venkata Prasanna. "In vitro and in vivo study using chitosan microparticles with growth factors and antibiotics for bone tissue regeneration." Doctoral dissertation, University of Toledo, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1398861353

    Chicago Manual of Style (17th edition)