Gene expression is the most fundamental level at which the
genotype gives rise to phenotype. The development of human salivary
exosomes has become one of the promising researches to improve cell-based
tissue engineering but their functions in human periodontal ligament fibroblast
(HPdLF) cells are not well studied. To study the effect of human salivary
derived exosomes on the gene expression of HPdLF cells. In vitro, HPdLF
cells were cultured for 24 hours with 10 µg/ml of human salivary exosomes.
Determination of gene expression levels of basic fibroblast growth factor
(bFGF) and collagen type I (COL1) in the presence and absence of human
salivary exosomes in HPdLF culture was performed using quantitative reverse
transcriptase polymerase chain reaction (RT-qPCR). Human salivary
exosomes significantly upregulated bFGF gene expression but not COL1
gene in HPdLF cells after 24 hours of culture. Human salivary exosomes are
able to upregulate bFGF gene in HPdLF cells. Thus, they might have potential
to be used as an alternative biomaterial in tissue engineering for periodontal
regeneration.
Abstract—The functions displayed by exosomes derived from saliva and
other body fluids have been established. This paper studied the stability of
human salivary exosome beginning from the collection mode, storage, and its
preservation methods. Unstimulated saliva samples were collected from
healthy subjects. Protease inhibitor was added into each samples and stored
under different temperatures and at varying periods of time. The exosomes
were isolated by ultracentrifugation and confirmed by using Western Blot.
Exosome morphology was inspected by Scanning Electron Microscope (SEM)
and the protein concentration was determined using the Protein (Bradford)
Assay. The exosome particle size distribution and concentration were
calculated using Nanoparticle Tracking Analysis (NTA). The protein assay
showed no significant differences in the exosome protein concentration values
for all conditions. Western Blot analysis also showed no differences in the
presence of exosome and all the samples were positive for protein CD63.
SEM analysis showed the fine shape of exosome which is round, in vesicle
form with the size ranging between 10 nm and 100 nm. NTA determined the
individual mean and the clumping exosome size was 203 nm. Human salivary
exosomes remained intact in the absence of protease inhibitor and in different
storage temperatures.