Generation and evaluation of recombinant amelogenin

Abstract

Amelogenin is a major constituent of enamel matrix protein that is responsible for the development of tooth enamel. Due to its self-assembly and physicochemical properties, amelogenin has been widely used as one of the natural biomaterials. Amelogenin, along with the other enamel matrix proteins have been known for its regenerative properties. The purpose of this project is to evaluate the engineered amelogenin constructs to be used as a biomaterial coating and to assess its compatibility with the fibroblast cells lines. Six engineered amelogenin proteins were evaluated for their solubility characteristics by forming protein coatings in two different formulations. Apart from the six, mixes of two different proteins were also prepared in different ratios to enhance the stability of the protein coatings. Fibroblast cells were grown on these coating materials to evaluate cell attachment and proliferation.
Fibroblast cells, when grown on these proteins, displayed various morphology such as elongated, spherical and clumping of cells growing on top of each other. The cells were grown on the coatings, using different protein concentrations to determine the attachment and proliferation over time. The cells were found to grow very well on the protein coating containing the RGD sequence whereas some protein coatings were unfavourable for the cell attachment. For such constructs, the cells preferred to grow more on the plate surface than on the coatings. The protein mixes made in different ratios showed good stability and promoted cell growth.

Popular Abstract

Biomaterials are substances that are used for making implants, to support or replace a biological structure, is used for various treatments like bone fracture, tooth replacement, and for many other complications. The implants are made of inert materials to have biocompatibility and also to promote regeneration and wound healing at the site of injury. To increase the longevity, and limit the corrosion, the biomaterials are subjected to surface modification by mechanical, physical and chemical means. Coatings are an important class of surface modification that enhances the compatibility of the biomaterials. Studies have shown that coatings prepared from biological substances, such as collagen and other extracellular matrix derivates promotes the mammalian cell interaction and increases the pace of healing process.
This project involves the use of one such biological molecule, called amelogenin, to be used as a coating material for biomaterials. Amelogenin is a tooth enamel protein that involves in the formation and structural organization of hydroxyapatite crystals to form enamel rods. The characteristic property of amelogenin is to self-assemble and form nanospheres and nanoribbons under physiological conditions. Thus, it was of interest to use amelogenin and its recombinant variants to evaluate if the proteins can act as a potential coating agent for biomaterials.
Recombinant amelogenin variants were produced by genetically engineering the amelogenin gene and subsequently expressing the gene in a host system (E.coli BL21(DE3)) for protein production. The proteins were purified and the coatings were formulated into dried and precipitated forms. The proteins were coated on to a 96-well microtiter plate (surface modification) to evaluate its solubility and cell attachment property. The solubility of the proteins was determined by analyzing the protein release in phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle medium (DMEM) at various time points. The compatibility of the coating material with the mammalian cells was determined by cultivating fibroblast cells on the coatings and their attachment and proliferation were monitored over the period of time. Also, the cell attachment on different concentrations of amelogenin coatings on a Petri dish was observed after 2 and 24 hours of cell seeding. The cells were then stained with fluorescent dyes to visualize the nucleus and actin filaments of the attached cells on the coatings. Two proteins were mixed either to promote cell attachment or to decrease the solubility of the proteins. All the protein mixes complemented each other so well that they exhibited both the characteristics of the protein. Thus further studies have to be conducted such as quantifying the number of cells, cytotoxicity studies etc., for a better understanding of how the coating material affects the cell attachment and proliferation.

• author: Velayuthan, Lok Priya
• 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: Applied biochemistry, amelogenin, tooth enamel, biomaterial coating
• language: English
• id: 8997188
• date added to LUP: 2022-08-01 12:29:38
• date last changed: 2022-08-01 12:29:38