Substance P impact on endochondral ossification

All this may have an impact on LSJL but I’m not sure what it is yet.  However, nerves are something that you can feel.  However, different nerves have different effects so it’d be hard to gauge what the effect is.  But as alluded to in the study reduced pain sensitivity is a sign of reduced substance P and SNF.  So if you have reduced pain sensitivity due to LSJL you may have less SP and SNF.  The reduced mechanical stability may be beneficial to neo-growth plates also.  However, mesenchymal stem cells and chondrocytes stained heavily for SP so enhanced pain sensation may be more indicative of the success of LSJL.

Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification.

substance P pdf<-Read the full study.

“Sensory and sympathetic nerve fibers innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of absence of sensory neurotransmitter substance P (SP) and removal of sympathetic nerve fibers (SNF) on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation.
In order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1-/-) mice (no SP) and animals without sympathetic nerve fibers. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and β2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2 and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all three groups for mechanical testing and μCT-analysis.
Callus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained β2- adrenoceptor and TH-negative. Absence of SP and SNF did not change general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5days after fracture{so more sympathetic nerve fibers are pro-differentiation?}. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1-/- mice and mice without SNF, a lower number of osteoblasts in Tac1-/- mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of BMM, osteoclasts and osteoblasts isolated from Tac1-/- and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21days after fracture, revealed an overall reduced mechanical bone quality in Tac1-/- mice and mice without SNF. μCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF.
The absence of SP and SNF reduces pain sensitivity and mechanical stability of bone in general. The micro-architecture of bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, absence of SP reduces bone formation rate whereas absence of SNF induces bone resorption rate{maybe this could be beneficial as bone resorption would leave room for neo-growth plates}. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.”

“Under rigid, stable fixation regimen, bone regenerates with no or only minor callus formation”

“When applying more flexible fixation regimens, bone healing occurs in consecutive stages which involve intense callus formation. Firstly, an acute inflammatory response and recruitment of mesenchymal stem cells (mesenchymal callus) occur in order to subsequently generate a primary cartilaginous callus populated mostly with chondrocytes (soft callus). Later, this cartilaginous callus undergoes revascularization and calcification (calcified hard callus) and is finally remodeled to fully restore a normal bony structure and architecture”<-The idea with LSJL is that perhaps the accute inflammatory respose and recruitment of mesenchymal stem cells can occur without a fracture.

“SP plays a role in pain transmission; tibial fractures cause an early and strong induction of sensory nerve regeneration and growth into the site of injury (sensory sprouting). The presence of NK1 receptors was demonstrated on bone cells”  SP can also affect proliferation in mesenchymal stem cells.

“skeletal growth or activity of bone tissue might be regulated by SNF”

“At day 5 after fracture, when chondrogenic differentiation starts, a substantial number of mesenchymal and chondrocyte-like cells stained positive for NK1R and some cells double-stained for SP. At day 9 after fracture, when most of the callus matrix has adopted a cartilaginous phenotype (soft callus), nearly all of the callus chondrocytes were SP- and NK1R-positive. At 13 days after fracture, when remodeling of the callus progressed toward tissue mineralization and the bony, hard callus was about to be formed, number of SP-positive callus cells appeared to be reduced compared to day 9 but NK1R staining seems to be unaltered in hypertrophic chondrocytes. SP- and NK1R staining pattern in sympathectomized mice was similar to WT”

“mesenchymal callus cells and periosteum stained positive for α1d adrenergic receptor 5 days after fracture whereas only few chondrocyte-like cells were α1d-positive”

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