The Synthesis, Characterization, and Cell Seeding of a Collagen/Hydroxyapatite Porous Scaffold for Treatment of Osseous Defects
Date of Award
Master of Science (MS)
The need for finding a bone graft substitute stems from the fact that approximately 200,000 bone grafting procedures are performed annually in the U.S. alone. Although biological grafting options (i.e. autografts & allografts) exist, they do suffer from inherent problems. These include limited resources, costly processing, and potential for pathogen transfer. Thus, investigators have sought synthetic alternatives.
The objective of this research was to utilize hydroxyapatite (HA) and collagen, both analogues to the major constituents of bone, to fabricate an optimal synthetic osteoconductive/osteoinductive bone scaffold. Current attempts by other investigators to combine the two materials have been met with some difficulties, resulting in free or loosely bound particulate HA within the collagen matrix. This may ultimately result in a foreign body response to the disseminated crystals. Yet, this study revealed that through manipulation of the collagen's collodial chemistry, it could be made into a more effective carrier medium for particulate HA. It was found that treating the collagen with either IOOmM NaOH or hydrochloric acid followed by titration with 1 N NaOH to a basic pH ( approximately 11 . 8) would yield an adhesive, paste-like matrix capable of incorporating the HA, following lyophilization of the matrix. Composites made under acidic, neutral, and conditions correlating to pH values below 11 .8 were found to possess free HA particulates and loose, friable collagen fibers.
The composites were also frozen at two different temperatures to study their effects on microporosity formation. Porosity is an essential characteristic given that it serves as 111 pathways by which vasculature is established, cellular elements may infiltrate; and by which nutrients are supplied to the graft. It was found that porosity of the composite could be controlled by regulating the ice crystal formation prior to lyophilization of the material. Micron size pores were achieved by freezing the composite to -l 5°C and controlling the amount of particulate HA added to the system.
TGF-β 1 was also studied for its efficacy in serving as an osteoinductive catalyst with the collagen/HA composite. It was chosen due to its ability to modulate the growth and differentiation of osteoblast precursor cells. Utilizing 1125 as a tracer, release kinetics of the adsorbed polypeptide from the composite were evaluated. Elution was rapid, with approximately 56 ± 3.5% of the theoretical load release following 24 hours incubation. Improved methods by which to prolong the release of the growth factor may be needed in order to provide for optimal inductive properties.
Last, the cytocompatibility of the composite was evaluated. Primary cultures of adult, rabbit and fetal bovine osteoblasts were seeded into two formulations of the collagen/HA composite. Following three days of culture, an acid-to-base titrated composite formulation grafted with fetal bovine cells was the only combination which exhibited the presence of adherent cells. The lack of fetal cells upon a base-only treated collagen/HA composite was speculated to be a result of modifications to the collagen due to processing. Seeding technique and cell donor age were also suggested as possible reasons for the absence of cells utilizing adult rabbit cells on either composite formulation.
Together, the findings suggest that the composite could serve as a suitable, porous bone scaffold. Due to its compressive strength values, it would best be reserved for non-load-bearing applications, such as an osseous filler material used, for example, in IV conjunction with plated fractures. Improved TGF-β1 incorporation, in-depth in vitro studies, as well as an in vivo model will need to be assessed to determine the true effectiveness of the composite to replace existing biological sources.
Patterson, Kim Rachel, "The Synthesis, Characterization, and Cell Seeding of a Collagen/Hydroxyapatite Porous Scaffold for Treatment of Osseous Defects" (1997). All Theses. 3905.