"To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, which is a potential regulator of cell fate decisions during fracture repair. Mmp9-/- mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification[So to grow taller being MMP knockout may be better]. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9-/- compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9-/- mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9-/- stroma and periosteum. Following transplantation, Mmp9-/- mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Mmp9-/- periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9-/- hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9-/- mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation."
The mice were 12-16 weeks old. Mast cells(connective tissue cell) typically produce MMP9. Fractures were produced by three point bending loading.
"Inflammatory mediators such as tumor necrosis factor-α (TNFα) are required for bone formation but can also impair later stages of repair by stimulating cartilage degradation"
"The role of Cox2 is counterbalanced by 5-lipoxygenase (5-LO) and leukotriene inhibitors can stimulate cartilage and bone formation in the early phase of repair through direct actions on chondrocytes"<-Grow taller with leukotriene inhibitors? Pharmacological inhibition of 5-lipoxygenase accelerates and enhances fracture-healing. states that 5-LO inhibition via AA-861 increased COL2, COX2, COL10, and osteopontin. Celecoxib[COX2 inhibitor] also increased COL2.
"MMP9−/− but not wild type skeletal progenitors differentiate into chondrocytes in both non-stabilized and stabilized fractures"
"the presence of Mmp9−/− inflammatory cells alone was not sufficient to induce chondrogenic cell differentiation at the fracture site. "
"Osteoclast recruitment was impaired in Mmp9−/− fracture calluses and bone marrow transplants rescued the cartilage remodeling defects in non-stabilized fractures via providing MMP9-expressing osteoclasts. This result was in concordance with the rescue of the growth plate phenotype in Mmp9−/− developing long bones"
"the recruitment of T cells and macrophages was affected [in] Mmp9−/− fracture calluses"
"This indirect effect of MMP9 on periosteal cell fate was via the regulation of macrophage localization. Only macrophages were located within the periosteum to locally influence chondrogenic and osteogenic cell differentiation. CD4 + T cells were found away from the periosteum, and so are unlikely to have an effect on chondrogenesis and osteogenesis."
"MMP9 acts indirectly at the level of the periosteum"
"Ectopic localization of macrophages in the periosteum of Mmp9−/− stabilized fractures may be responsible for the induction of endochondral ossification as we found an increase in BMP2 expression."
"MMP9 indirectly regulates angiogenesis by regulating the bioavailability of VEGF"
"the lack of MMP9 in both the periosteum and inflammatory cells was responsible for the cartilage phenotype."
Genes upregulated in Unstablized versus Stabilized(MMP9 knockout) at Day 7 that were also upregulated(or -down) by LSJL:
All below are signal transduction related molecules:
COL10A1
C1QTNF3
COL12A1
COL11A1
COL6A1
PTN
MOXD1
ASPN
EDIL3
NOV
LRIG3(down)
TNN
COL16A1
EMILIN3
DKK3
COL1A1
PTGS2
MATN4
HTRA1
Col9A1
MATN3
Col9A2
SMOC2
THBS3
CSPG4
THBS2
CCL21C
COL2A1
KERA
Glycoprotein Related:
TNMD
COL13A1
PRSS35
EPHA3
GPR88(down)
Car12
HAPLN1
Cell Differentiation:
Sox9
COL13A1
ALDH1A3(down)[an enzyme produced by retinoic acid]
NR4A2
SLITRK6
OTHER:
WNT2
LSJL was highly homologous to the most chondrogenic stimulation group in the study MMP9-/- unstabilized fractgure.
Genes upregulated in stabilized versus unstabilized MMP9 KO mice that were also altered in LSJL
So these are essentially downregulated by unstabilized mice:
CCL2(up)
CXCL5
CXCL1(up)
IL6(up)
CD14(up)
Unstabilized non-MMP9 knockout on day 7 versus pre-fracture gene upregulation that were also upregulated(or -downregulated) by LSJL:
Saa3
CCL2
CXCL1
IL6
SERPINA3N
CXCL5(down)
CCL7
CD276
CFI
ANXA8
At day 2 these are the genes that are also upregulated by LSJL that were not previously reported above:
IL1RL1
LSJL produces a similar gene expression profile to MMP9-/- and Unstabilized fracture mice which were pro-chondrogenic thus providing further evidence that LSJL is pro-chondrogenic. No change in MMP9 expression was reported due to LSJL.
