Improvement of bone allograft recolonization by adipose stem cells: impact of bone graft demineralization

Bone allografts combined with mesenchymal stem cells are
proposed for bone tissue engineering but remains limited by a
low degree of stem cells spreading and cellular recolonization.
We postulate to demineralize bone allografts in view to improve
adipose stem cells (ASCs) colonization and the bioactivity of
the graft.
Bone allografts (n=16) were treated for decellularization (4
groups of demineralization time: 0, 4, 8, 12 hours). The implants
were compared in terms of residual calcium, mineral density
and bioactivity (for BMP-2/VEGF contents). Each implant was
scanned by microtomography to analyze macroporosity and
open porosity. Helium pycnometry and Hg porosimetry were
performed to assess the absolute density and microporosity.
Bone surface analysis was assessed by X-Ray photoelectron
spectroscopy and SEM. The bone graft recolonization by ASCs
was studied in vitro by SEM, histology and DNA extraction at
24 hours/day 15 post-cellular seeding. Finally, ASCs combined
with non-/demineralized bone matrix were implanted into
the lumbar muscles of 10 nude rats (in comparison to bone
grafts w/o cells) to study the osteoinductivity/angiogenicity by
imagery/histology 29 days after implantation.
A significant reduction of the calcium concentration (>-90%)
was found in demineralized bone in comparison to native grafts
as revealed by ionometry (0.27 vs. 4.1 g/L) and pQCT (0 vs. 0.39 g/
cm?). Demineralization significantly increase the macroporosity
(>100μm by +13%) and the open porosity (>4 cm?/g vs. 2.1±1.0
cm?/g in comparison to the native graft,p<0.05). A significant
increase of microporosity (>10 μm by +158% and <100 nm
by 558%) was also found after demineralization. Helium
pycnometry confirmed the correlation between the decrease
of absolute density and demineralization of the bone graft
(R?=0.81). A positive linear correlation between the decrease of
calcium/increase of nitrogen atoms (at the bone surface) and
the time of demineralization was found (R?=0.99,p<0.001). At
day 15 post-incubation, a significant higher ASCs colonization
of the bone graft was found for tissue demineralized during 12
hours (p<0.05).
In conclusion, the demineralization of cancellous bones
significantly improves the colonization by ASCs in view to
return the bioactivity for bone regeneration.