Evaluation of Recombinant Amelogenin as a Potential Biomaterial Coating

Abstract

Amelogenin, a protein that takes part in enamel development, has some interesting physicochemical properties such as the formation of nanospherical aggregates. Furthermore, its derivatives have shown regenerative properties and have already been commercialized for that purpose. Therefore, it was of interest for this project to evaluate mammalian cell attachment and proliferation on engineered variants of the amelogenin protein in order to evaluate its potential as a biomaterial coating. Seven different amelogenin constructs were evaluated in this project by growing cells on coatings of the constructs. However, the number was soon narrowed down and the focus put on a construct that had fused RGD cell adhesion peptide (termed 108) and a construct with added residues (termed 121), to increase the coating's stability.

Cell proliferation was measured using the PrestoBlue assay. The assay results indicated that the cells were proliferating on all construct coatings. However, when cells were grown on a petri-dish with several discs of amelogenin protein coatings, where cells could choose where to grow, the cells seemed to avoid attachment to the wild-type amelogenin (rH174) and construct 121. On construct 108, the cells attach and spread more quickly compared to the other constructs. When it comes to cell attachment, a combination of constructs 121 and 108 showed the highest degree of attachment compared to 108 alone and rH174. This could, however, be caused by the stability/solubility of the coatings rather than their cell attachment properties, and the washing steps could have washed away parts of the coatings and consequently the attached cells. Furthermore, in the case of construct 107 (amino acid sequence with positively charged amino acids), the cell adhesion was greatly improved when on a coating formed with nanospherical protein.

Popular Abstract

You wouldn't believe how many different proteins there are in the body. Each of these proteins has a special, and often very specific, role when it comes to structure, function and regulation of our bodies. One of these proteins is amelogenin. The role of amelogenin in the body is to regulate the formation and growth of teeth enamel which happens during fetal development, a process called amelogenesis.

Since amelogenin is not the most accessible, the gene that codes for the protein can be placed into the genome of a bacteria (in this case E.coli). The bacteria will then be able to produce the protein. The protein is then purified from the bacterial culture for further use.

The use of biomaterials in medical purposes can be traced back as far as to the Egyptians. Biomaterials have been described as: “any substance (other than a drug) or combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or as a part of a system which treats, augments, or replaces any tissue, organ, or function of the body”. At first, biomaterials were used as support materials that were completely bioinert to avoid immune response, and thereby rejection, of its host. But recently biomaterials have been evolving towards, so-called, “biomimetic” biomaterials, made of naturally occurring materials such as proteins. Those materials are designed in a way that allows the surface to interact with its surrounding tissue and to act as a scaffold for tissue regeneration. However, natural materials often lack the rigidity of the formerly used synthetic materials, and that is where bioactive coatings of inert materials come in to play.

In this study, amelogenin’s potential as a biomaterial coating was assessed. Amelogenin was of interest since the protein has a characteristic solubility profile and an ability to self-assemble to form nanosized aggregates. Furthermore, it has been shown to have tissue regenerative properties. Two products made out of amelogenin derivatives, that is a mix of amelogenin proteins, have already been commercialized and have successfully been used to treat both dental defects and hard to heal leg ulcers.

Proteins, like amelogenin, are composed of a chain of amino acids, which all have different chemical properties. It is therefore possible to manipulate the function of a protein by changing its amino acid sequence or fuse a sequence with desirable properties to the existing protein, using molecular methods. This process is often referred to as protein engineering.

In this study, the amelogenin protein (termed the wild-type protein) and six engineered variants of it were studied. The purpose was to assess if the wild-type, or any of its variants, would make a surface suitable for mouse cells to grow on. The modifications made on the protein were e.g. removal of a critical part of the protein, addition of a cell adhesion domain, addition of a positively charged cell penetration domain, addition of a single amino acid.

Coatings were made of the different proteins by allowing a solution of the proteins evaporate so only the protein would remain. The coatings were then seeded with mouse cells, so-called fibroblasts that are cells involved in connective tissue and wound healing. The interaction and proliferation of the cells on the surfaces was monitored through a microscope with regular time intervals and by using a special assay to determines the relative number of active cells. The results indicated that the wild-type amelogenin was not suitable on its own for cell adhesion, however, addition of a cell binding domain to amelogenin made it a feasible surface for the cells to grow on.

The stability of that coating could be increased by mixing the amelogenin, without compromising the cell adhesive properties. Furthermore, the cells would also grow on the amelogenin with added cell penetration domain, but only if the protein would be allowed to self-assemble beforehand. Amelogenin self-assembly is induced by simply increasing the pH of the protein solution. When the protein self-assembles it forms so-called, nanospheres, round structures that will expose the ends of the proteins where the additions were made. This will increase the amount of positive charges on the surface compared to when the amelogenin is not assembled, and since the cell membrane is negatively charged the surface aids cell adhesion.

• author: Smáradóttir, Björk
• supervisor: Johan Svensson Bonde
• organization: Pure and Applied Biochemistry
• course: KBKM01 20191
• year: 2019
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Amelogenin, Recombinant protein, Biomaterials, Applied biochemistry
• language: English
• id: 8985451
• date added to LUP: 2022-08-01 12:28:17
• date last changed: 2022-08-01 12:28:17