All ETDs from UAB

Advisory Committee Chair

Susan L Bellis

Advisory Committee Members

Amjad Javed

Selvarangan Ponnazhagan

Michael S Reddy

Timothy M Wick

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) School of Engineering

Abstract

The “gold standard” of bone grafting materials is autograft. Unfortunately, autogenous bone must first be harvested from the patient, leading to complications such as donor site morbidity. Because of this, many surgeons rely on non-autogenous bone grafting materials. While these grafts are more available than autogenous bone, they require extensive processing which significantly reduces their osteoinductivity. Therefore, there is substantial interest in finding ways to add osteoinductive molecules to these grafts. Previously, our lab has focused on utilizing molecules derived from bone morphogenetic protein 2 (BMP2) with an attached hydroxyapatite-binding domain (polyglutamate) to anchor them onto bone grafting materials. Previous work by our lab found a peptide derived from rBMP2, BMP2pep, with an added E7 domain (E7- BMP2pep) to be effective at stimulating osteoblastic activity in a rat defect model. To further the understanding of this peptide’s cell signaling capabilities, we investigated the early signaling capabilities of BMP2pep and compared it to rBMP2. We found BMP2pep is capable of stimulating early BMP2 signaling such as pSMAD 1/5 induction, but at a lower level than rBMP2. We introduced activating mutations into these peptides, but they did not enhance the efficacy of BMP2pep. These studies found BMP2pep is a relatively weak osteoinducer compared to rBMP2. Knowing this, we hypothesized that the addition of a polyglutamate domain to rBMP2 may have clinical significance since many of the deleterious side effects associated with rBMP2 are due to dissemination of rBMP2 from the graft. Therefore, we used recombinant protein engineering to create rBMP2 with a polyglutamate domain, rBMP2-E8, and expressed this new protein in E. coli. We subsequently found this protein tightly anchors onto hydroxyapatite, in stark contrast to commercial rBMP2. Importantly, rBMP2-E8 stimulated pSMAD 1/5 signaling, indicating the addition of the hydroxyapatite-binding domain did not eliminate the protein’s signaling potential. These were substantial findings considering the addition of an anchoring domain to the full-length BMP2 molecule is largely unexplored. In summary, BMP2pep and its mutants were found to be weak osteoinducers when used to treat cells, but rBMP2-E8 not only tightly anchored onto hydroxyapatite, but also stimulated BMP2 signaling.

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