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Bioinspired Hydrogel Formulations for Bone Regeneration- Fabrication, Characterization, and In Vivo Efficacy Evaluation in Mice and Rabbits

Patel, Akhil P (2019) Bioinspired Hydrogel Formulations for Bone Regeneration- Fabrication, Characterization, and In Vivo Efficacy Evaluation in Mice and Rabbits. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Constant increase in aging population, bone diseases incidence, and traumatic/ accidental injuries have led to millions of orthopedic surgeries worldwide. In the USA, 280K hip, 700K vertebral, and 250K wrist fractures are treated every year. Most clinical interventions, like autografts and allografts, face challenges of rejection, significant clinical morbidity, prolonged hospitalization, delayed rehabilitation, and surgical complications. Relatively new approaches employ growth factors such as bone morphogenetic proteins (BMPs) to exert potent bone regenerative activity; however, they are limited due to serious side effects such as uncontrolled bone growth, prohibitive costs, and stability issues in the clinical setting.
In the process of natural bone development, collagen acts as an organic template to guide mineral deposition in the bone extracellular matrix (ECM) and apatite minerals act as inorganic phase, both of which interact at molecular level forming hierarchical nanocomposites. However, most of the currently explored materials are just physical blends of organic and inorganic phases. Therefore, these materials show limited potential to recapitulate bioactivity to regenerate bone without added growth factors.
This dissertation focuses on our progress towards the development of bioinspired hydrogel scaffolds using self-assembly of oppositely charged polysaccharides to regenerate critical-sized bone defects without the use of growth factors. We summarized the results from our preclinical studies to assess its efficacy using mice and rabbits.
First, we optimized process parameters such as concentration and temperature to facilitate interfacial polyionic complexation (IPC) between cationic (chitosan (CHT)) and one of the three anionic polysaccharides, namely, gellan gum (GG), alginate (ALG), and kappa-carrageenan (KCA). This resulted in three variants of hydrogel scaffolds: CHT-GG, CHT-ALG, and CHT-KCA. We characterized these three variants of hydrogel scaffolds for their collagen-mimetic multi-scale hierarchy. We further demonstrated the versatility of hydrogel scaffolds to load small molecule or metal nanoparticles. Further, we developed automated method to produce, collect and orient IPC fibers to make into a bilayer scaffold.
We then assessed ability of hydrogel scaffolds to sequester bone-like minerals from simulated body fluid in vitro. All three variants of hydrogel scaffolds promoted biomimetic mineralization in vitro both on the surface and inside the scaffolds.
We further assessed the efficacy of all three variants of hydrogel scaffolds, processed as films, in non-load bearing, critical-sized (5 mm diameter) mouse calvaria defects. Non-mineralized CHT-KCA showed significantly higher calvaria regeneration and defect closure compared to the empty defect control group; however, the defect was still far from closure. More interestingly, implantation of in vitro mineralized CHT-KCA further enhanced bone regeneration significantly compared to the empty defect. Additionally, preservation of minerals by lyophilization of the mineralized scaffold condition prior to implantation significantly increased bone regeneration as compared to (hydrated) mineralized CHT-KCA.
We further tested efficacy of CHT-KCA in semi-load bearing ulna defect. Here, we first processed hydrogel scaffolds as 1.5 cm long cylindrical scaffolds, which were implanted in a critical-sized rabbit ulna defect. Cylindrical hydrogel scaffolds showed an onset of bone regeneration as early as 4 weeks. Moreover, mineralized CHT-KCA significantly enhanced bone regeneration and improved functional mechanical properties of the regenerated bone compared to empty defect after 12 weeks. Histological assessment revealed complete bone healing of the mineralized CHT-KCA through endochondral ossification.
Collectively, these studies suggest that hydrogel scaffolds can be considered as a versatile platform technology and they can be processed in any shape and sizes; pre-fabricated in the lyophilized, hydrated or even injectable form. Hydrogel scaffolds can conform to and maintain the shape of the defect. Hydrogel scaffolds demonstrated great potential to regenerate bone in mice calvaria defect and rabbit ulna defect.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Patel, Akhil Pakp38@pitt.eduakp380000-0001-8915-8995
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSant, Shilpashs149@pitt.edushs149@pitt.edu
Committee MemberRohan, Lisalrohan@mwri.magee.edulrohan@mwri.magee.edu
Committee MemberBeniash, Eliaebeniash@pitt.eduebeniash@pitt.edu
Committee MemberLi, Songsol4@pitt.edusol4@pitt.edu
Committee MemberFeinberg, Adamfeinberg@andrew.cmu.edu>feinberg@andrew.cmu.edu
Date: 20 November 2019
Date Type: Publication
Defense Date: 17 October 2019
Approval Date: 20 November 2019
Submission Date: 19 November 2019
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 185
Institution: University of Pittsburgh
Schools and Programs: School of Pharmacy > Pharmaceutical Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Chitosan; gellan gum, carrageenan, alginic acid, interfacial polytonic complexation, microfluidic, self-assembly, bone graft, osteoblast, nanoparticle, hydrogel, fiber, biocompatible, osteoinduction, mouse calvaria defect, rabbit ulna defect, hydroxyapatite, biomineralization, osteointegration, bone healing, bone regeneration.
Date Deposited: 20 Nov 2019 13:42
Last Modified: 20 Nov 2019 13:42
URI: http://d-scholarship.pitt.edu/id/eprint/37833

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