Skip to main content

Table 1 In vitro assessment of osseointegration

From: Electrical stimulation to promote osseointegration of bone anchoring implants: a topical review

 

Assessment

Assessment description

Outcome measures

Cell proliferation

Cell viability

Cell adhesion

Cell morphology

Bone markers*

Colorimetric assay

MTT1

MTT is reduced into fluorescent purple formazan crystals by living cells, which determines mitochondrial activity with a spectrophotometer [32]

X

X

   

WST-12

WST-1 is cleaved to soluble fluorescent formazan by a complex cellular mechanism by living cells, which determines living cell activity with a spectrophotometer [32, 33]

X

X

   

Staining

LIVE/DEAD

Fluorescent staining of cells using calcein-AM (viable cells), propidium iodide (dead cells) and Hoechst 33,342 (total cells) [34]

 

X

   

Trypan blue

Staining elimination test where viable cells do not take up the dye, but dead cells are permeable to it [34]

 

X

   

Imaging

SEM3

Electron microscope that enables high-resolution imaging and generates specimen images by scanning of the surface using a focused beam of electrodes [31]

  

X

X

 

Fluorescence microscopy

Optical imaging method used to study cell physiology by using fluorescence [35]

  

X

X

 

CLSM4

Optical imaging method used for enhancing optical resolution and contrasting a micrograph by usage of a spatial pinhole to stop out-of-focus light in image formation [36]

   

X

 

PCR methods

RT-PCR6

A variation of the standard PCR5 method where cDNA7 is made from RNA8 via reverse transcription, which allows amplification of specific mRNA transcripts from small biological specimens [37]

    

X

qPCR9

Another variation of standard PCR where two elements are added to the standard procedure: fluorescent dye and fluorometer. Widely used for quantifying RNA transcript levels in cells and tissues [37]

    

X

Immunoassays

ELISA10

An immunoassay used for quantification which utilises an antibody labelled with an enzyme marker where either the enzyme or antibody is bound to an immunosorbent substrate. The change in enzyme activity is a result of the enzyme-antibody-antigen reaction which is proportional to the antigen concentration [38]

    

X

RIA11

An immunoassay for quantification of antigen–antibody reaction by usage of a radioactively labelled substance either directly or indirectly to quantify the binding of the unlabelled substance to a specific antibody [39]

    

X

Western blotting

Identification assay used to identify proteins or peptides that have been electrophoretically separated via blot relocating from electrophoresis gel into strips of nitrocellulose paper followed by labelling with antibody probes [40]

    

X

  1. *Common bone markers include alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2), collagen type 1 (Col 1), procollagen type 1, osteoprotegerin (OPG), osteocalcin (OC), and vascular endothelial growth factor (VEGF)
  2. 1MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 2WST-1, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; 3SEM, scanning electron microscopy; 4CLSM, confocal laser scanning microscopy; 5PCR, polymerase chain reaction; 6RT-PCR, reverse transcription PCR; 7cDNA, complementary DNA; 8RNA, ribonucleic acid; 9qPCR, quantitative PCR; 10ELISA, enzyme-linked immunosorbent assay; 11RIA, radioimmunoassay