Amelogenin as a Biomaterial : Cultivating osteoblasts on assembled amelogenin

Abstract

The exact workings of amelogenin (AMG) as a key-regulator in the formation of enamel is still to be determined. It has been observed however, that AMG promotes tissue-regrowth and has promotive effect on e.g., osteoblastic cells and could therefore be of interest for medical applications. This project aimed to utilize AMG’s self-assembly properties to fabricate a protein-based biomaterial for cultivating osteoblasts on and examining possible effects on the cells.

Recombinant AMG was produced in E. coli and allowed to self-assemble by controlling the concentration of calcium, phosphate, and the pH. Microscope analysis with Thioflavin T staining was used to analyze AMG aggregates, which included fibrillar structures as well as irregular clusters. To analyze the samples after cell seeding fluorescence microscopy was used.

Protein-films created from AMG aggregates were only transparent enough to allow visualization of seeded osteoblasts after 190+ hours of incubation. It was hypothesized that this allowed all protein to form fibrillar structures, together producing an intricate, interwoven net with sufficient transparency to make osteoblast observation possible. Osteoblasts seemingly growing along the fibrillar structures could be observed, indicating a preference for attachment to the protein. Attempts to determine if they indeed grew on the material, or through the gaps of it, remained inconclusive, making it uncertain if the cells preferred it over the normal plastic. Nonetheless, the osteoblasts' strong inclination for contact was evident.

Popular Abstract

Film of the Year: Evaluating films made from teeth-protein for cultivation of skeletal-cells

The main character of this project is amelogenin, a protein that can self-organize into complex structures and direct the growth of enamel, the body's hardest tissue. As amelogenin also has bone-regenerating properties it was here used as a material for growing skeletal-cells on.

The initial results of this research were promising, showing the protein self-organizing rapidly into long, yarn-like structures in the microscope, which were then successfully made into protein-film prototypes. An issue arose however when skeletal cells were to be grown on it. In the microscope, the amelogenin-film was too opaque to see the translucent cells through. Serendipitously, one sample that was forgotten over a weekend came to the rescue. It turned out, upon examining it in the microscope, that the protein had to have longer time to self-organize completely, than initially thought, into the longer string-structures. This particular film looked like an intricate, see-through network of strings where skeletal cells visibly nestled within. They could be seen stretched along the protein net and seemed to favor anchoring to it, especially to a material with an engineered version of amelogenin with improvements made for cell attachment. Unfortunately, it could not be proven with the existing methods that cells did grow on the actual film and not through the gaps of it. Nonetheless, the findings show that skeletal cells prefer to grip onto the protein and associate with it.

Normally, growing cells in a lab is done on plastic. The surfaces of these plastics are often treated so the cells can grip it and they better reflect the body's conditions. Amelogenin will form string-like microscopic structures on its own in specific condition which, by simply air-drying a droplet of, turns into a thin film consisting only of the protein. Letting cells grow on a layer of this biological material instead of the plastic could prove to be a novel, maybe favorable option that is cheap and easy to produce. Amelogenin is already used as a component of several medical treatments, one being Emdogain® which is used to treat gum-disease and prevent tooth-loss. Amelogenin has had proven positive effects on regrowth of bone which opens up the possibilities of using it as a biological coating for an implant or bonding agent inserted to heal bone fractures. Although this project could not confirm direct skeletal cell-growth on the films that were tested, the observed preference to attach to it suggests a potential for new ways of using amelogenin in the field of regenerative medicine.

• author: Gabrielson, Caroline
• supervisor: Johan Svensson Bonde
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Amelogenin, biomaterial, molecular biotechnology, applied biochemistry
• language: English
• id: 9141962
• date added to LUP: 2023-12-18 08:30:42
• date last changed: 2023-12-18 08:30:42