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Notes for U781 seminars – Celine Colnot in Valerie Cormier-Daire's group
Treatment – BMP2/7 to induce bone growth, grafts from iliac crest or other allografting, other therapies as best as possible.
Intramembranous ossification bone direct from mesenchyme, not via perichondium and cartilage. Similarities between development and repair.
Renal capsule transplants to test. Cf. Dev Biol 2004 (269:55-69) and Development 2005 (132:1057-67.
Rosa26 mice where WT graft in Rosa26 renal capsule and contrary – the blood and vasculature do not contribute osteoblasts, and to refine the cartilage graft, only the perichondrium is necessary.
Repair recapitulate development? For endochondral ossification, when not stabilized (aligned) get large callus out of cartilage that is replaced by bone. Reactivation of gene expression. Cf. other articles, Mmp9/13 get too much hypertrophic cartilage in callus or normally in these mutant mice, get delayed bone formation. However, source of cells and the environment is quite different in repair vs development.
Searching for origin of stem/progenitor cells in callus during bone repair ? Not necessarily systemically but also periosteum, local bone marrow. Lineage analyses approaches. Eg lethally irradiated WT mice and bone marrow from GFP mice – cells at surface of callus but not integrated into the bone matrix. However, if do same with Rosa26 donor, saw more carefully that only the osteoclasts come from the donors. So bone marrow not a major contributor – at a distance anyhow. Or other blood-circulating cells.
Idea that mesenchymal stem cells can reconstitute bone but not really strong evidence for their contribution.
Their positive effect may be more on the inflammatory reaction at the fracture site. Cf Granero-Molto Stem Cells 2009.
Grafting of bone more direct. Rosa26 donor into cortical bone of WT mice – subgrafted periosteum , endosteum or endosteum + marrow. They give rise to osteocytes + chondrocytes, or osteocytes respectively, or local osteocyte and marrow, nothing much at a distance.
No migration much away from the grafts in any of the cases.
When fractures are stabilized nearly no cartilage formation, get an intramembranous-type direct ossification (is it just the stage at which she examines, and that healing is faster with stabilization?)
Cell fate decisions (at least, presence of cartilage precursors) happen during the inflammatory stage of repair. Microarrays to compare stable vs non-stable fractures. Of course lots of immune and chemokine upregulation in non-stable fractures, but this is maintained in stable and not in non-stable? Yes, that is right.
MMP9 in coordination with the inflammation might regulate the cell differentiation. Bone marrow transplantation can rescue the Mmp9-/- phenotype, where bone formation is delayed, by reintroducing inflammatory cells, and inducing endochrondral ossification.
Bone graft approach in Mmp9 -/- mice, to see if there is endochondral ossification when periosteum is transplanted.
Growth factors in the fracture environment?
BMP members and pSmad in non-stable fractures – activated in periosteum, not endosteum, and leads to endochondral ossification. BMP2 can act directly on periosteum to activate cartilage formation. BMPs can be made by the inflammatory cells, but also released into the environment in case of a fracture.
Avenir project proposal (accepted): origins and functions of skeletal stem cells
No good markers for progenitors – cf Sox9 and Runx2 in the mesenchymal condensation, remain in low Wnt environment, keeps Sox9 and downreg of Runx2 (endochondral ossification) followed subsequently by Runx2 increase to get the actual ossification. For intramembranous ossification, high Wnt environment, directly ossification.
Limb bud – Sox9+ cells give rise to osteochondroprogenitors, where Prx1 labels ALL the limb bud mesenchyme.
Prx1-Cre;R26R and Sox9-Cre;R26R mice to look at lineage contributions. Also have Prx1-CreER and Sox9-CreER (what is ER?)
Presumably ER is to label only during the adult stages – inducible perhaps with tamoxifen (estrogen-responsive?)
Injury in bone transplantations into the renal capsule assay, host vs graft in callus?
Prx1-Cre;ZEG is a GFP-producing reporter line and do renal capsule transplantations.
Do embryonic osteochondroprogenitors give rise to mesenchymal stem cells?
Will also examine systemic recruitment when injury is sustained over time/ chronic.
Can get bone that normally is intramembranous ossification to make cartilage by using a floxed Catn allele combined with dermo1-Cre. Will similarly play with Wnt levels in the Prx1/Sox9 Cre mice.
Grafts of muscle in case of traumatic fractures but not sure if this is beneficial because the tissue acts directly on bone, or because help revascularize.