Apatite content of collagen materials dose-dependently increases pre-osteoblastic cell deposition of a cement line-like matrix.

Reference

Perrier A, Dumas V, Linossier MT, Fournier C, Jurdic P, Rattner A, Vico L, Guignandon A. Apatite content of collagen materials dose-dependently increases pre-osteoblastic cell deposition of a cement line-like matrix. Bone 2010.

Summary

The authors showed the increase in osteoblast activity during bone remodeling process is attributed to pre-osteoblastic secretion of VEGF-A and expression of RGD-containing proteins, osteopontin and fibronectin. Bone breakdown exposes varying degree of mineralization (dom) within a collagen I matrix; pre-osteoblast cells encounter such structure and release the above factors to promote cell viability and adhere to the collagen I-hydroxyapatite composite. The researchers evaluated this by observing cell morphology, mRNA production, adhesion rate, and cell migration. With increasing dom, the cells took on a more unique morphology, produced more mRNA for VEGF-A and RGD-containing proteins, displayed higher adherence rate, and migrated larger distances.

Results

The Apatite Collagen Complex (ACC) was made in 3 groups: 0%, 25%, and 50% (as the human bone is max 42%) apatite degree of mineralization. The ACC was first characterized as a substrate under light microscopy to quantify (via ImageJ) the amount of collagen and degree of mineralization was easily distinguishable. The ACC substrates were then tested for biocompatibility by seeding MC3T3-E1 cells; the light microscopy images are shown below. As the dom increases, the cells take on a more elongated, ciliated, stretched morphology showing adhesion and migration.

mRNA of VEGF-A was quantified using ELISA which as the figure below indicates, the 25% and 50% show marked, dose-dependent increases from 0% to 25% to 50% both in soluble VEGF-A and matrix-bound. The RGD-containing proteins OPN & FN were also shown to increase in mRNA content by RT-PCR; OPN increased 50-fold for 25% & 40-fold for 50% whereas FN increased by 7-fold for 25% and 8.5-fold for 50%. B3 integrin, the RGD-containing protein cell surface receptors, were also upregulated by 3-fold to 4-fold 25%-50%, respectively.

From protein expression, the authors moved on to the manifestation of increased expression by mechanically testing cell adhesion. A vertical sinusoidal vibration was applied to the cells; collagen I indeed did facilitate better adhesion and as the dom increased, the adhesion also increased dramatically: 4.59+0.82% cells resisted (TCPS), 29.00+5.77% (ACC0), 56.17+11.84% (ACC25) and 75.14+6.44 (ACC50). Finally, cell migration was observed using time-lapse microscopy in which the cells’ migration was recorded to move greater distances with increasing dom; it was also found that cells in unmineralized substrates (TCPS & ACC0) had low speed profile while those in 25% and 50% had alternating low and high speed profiles.

Shortfalls

Overall, this paper presents relatively minute shortfalls.

Although the paper used several methods to prove the changing profile of VEGF and OPN/FN, the most basic information of experimental setup was omitted: the authors did not provide a total cell count in the 4 categories they were comparing. Hence, there is no way to tell if the results are statistically significant except relative to each other.

The ACC substrate system was imported from those used in previous osteoclast experiments; osteoclasts, though derived from the same precursor, have ruffled border morphology to aid in adhesion and bone breakdown. The ACC was thus prepared in a way to aid adhesion which can contribute to the increasing cell adhesion and morphological changes to osteoblast with increasing dom, thus presenting a confounding factor.

A type of protein quantification for the RGD-containing proteins OPN, FN, and B3 integrin would have complemented the mRNA results as they had done for VEGF-A. Finally, the choice of 32hr post seeding to conduct experiments seem arbitrary given a doubling time of 27 hours for MC3T3-E1 cells; an scientific rationale would have strengthen the results.