Editorial Type:
Article Category: Research Article
 | 
Online Publication Date: 01 Apr 2016

Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface

DMD, MS,
PhD,
PhD, and
DMD
Page Range: 138 – 144
DOI: 10.1563/aaid-joi-D-14-00199
Save
Download PDF

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

<bold>
  <sc>Figure</sc>
  1
</bold>
Figure 1

Reverse transcription-polymerase chain reaction agarose gel electrophoresis of MMP expression in control vs stretched cells after 48 hrs showing upregulation of MMP-9 expression with stretching (box). 1kb (1 kilobase DNA ladder standard); gd (GDPH, internal cellular RNA control); 2 (MMP-2, gelatinase A); 3 (MMP-3, stromelysin 1); 9 (MMP-9, gelatinase B); 10 (MMP-10, stromelysin 2); h4 (H4 Histone, internal proliferation control); t2 (TIMP-2, tissue inhibitor of MMP-2); t3 (TIMP-3, tissue inhibitor of MMP-3).


<bold>
  <sc>Figure</sc>
  2
</bold>
Figure 2

Increased expression of MMP-9, mRNA after application of mechanical force for specific time intervals of 0.5, 2, 6, 24, 48, and 96 hours vs controls. The increase was variable over time but was observed at the first time point, as early as 30 minutes.


<bold>
  <sc>Figure</sc>
  3
</bold>
Figure 3

Increased MMP-9 RNA expression in response to mechanical stretching. Digitized densitometry of polymerase chain reaction product comparing control groups to stretched groups at each time point (means of three replicates).


<bold>
  <sc>Figure</sc>
  4
</bold>
Figure 4

Change in MMP-2 (72k) and MMP-9 (92k) activity with cell stretching. MMP-2 pro-form and MMP-9 band decrease when cells are stretched.


Contributor Notes

Corresponding author, e-mail jborke@westernu.edu
  • Download PDF