Next steps for proving LSJL

Finally, I got access to the LSJL gene expression data of genes upregulated over 2.0 fold.  Based on information about Interstitial Fluid Flow, Dynamic Compression, and Hydrostatic Pressure stimulus we knew that LSJL could likely induce chondrogenesis because all of those stimuli have been associated with chondroinduction and LSJL likely induces all those stimuli but the LSJL chondroinduction was confirmed by the statistically significant increases in Sox9, COL2A1, and Agc1.  The chondroinducer CCL2 was upregulated 3.691 fold. As a bonus, MATN3 was upregulated as well which is only expressed in cartilagenous tissues.  In addition, HMGA2 and Lin28B were expressed which are associated with overgrowth.  However, Growth Hormone expression decressed.  GH levels went down by half and SOCS3 expression increased. The only thing missing was Sox5 & Sox6 expression but they could have been expressed below 2 fold.  Also, H19 increased in expression which is associated with IGF2.  LSJL also upregulates ITGBL1 which may be involved in mesenchymal condensation(Beta-1Integrin is involved in the integrin if ITGBL is like Beta-1integrin as suggested in the name it may induce condensation as well. According to Identification of two novel chromosome regions associated with isolated growth hormone deficiency., ITGBL1 homozygous deletion causes GH deficiency.  ITGBL1 homozygous deletion also reduced height.

The genes were all analyzed for statistical significance and fold changes under 2 were not included thus there is low probability that the change expression is not statistically significant to LSJL.

The mouse did have open growth plates, they were female, and were 14 weeks of age.  However, the genes were determined by bone samples in 4 mouse groups.  The bones were entirely grounded.

In all 4 Control Samples, Sox9 expression was below 1.  However, in 3 of the 4 Sox9 expression increased to between 2 and 4.  In only one of the samples did expression decrease(Cluster 1).  Similar for COL2A1, expression was below 1 for 1 through 4 and increased above 1 for 2 to 4.  Same change for Agc1.  However, for MATN3 Clusters 1,3,4 were below 1 and all increase above 1, however cluster 2 was above 1 and decreased below 1.

A level of 1 means that it's mRNA is synthesized greater than GADPH.  A level less than 1 means it's synthesized less than GADPH.  GADPH is a control molecule that is stably expressed in most tissues and cells at high levels.  However, GADPH expression can alter in different cells in some conditions so that could have altered the data.

It's not likely that 3 out of 4 bone samples all contained growth plates indicating that Sox9 increased in expression in periosteum cells, Osteoblasts, Osteoclasts, fibroblasts, nerve cells, or Stem Cells.

According to Evolution of the osteoblast: skeletogenesis in gar and zebrafish., Sox9 can be expressed in osteoblasts.  However, I haven't seen any data that states that Col2A1 or Agc1 can be expressed in osteoblast(or osteoclast) cells.  Fibroblasts are the most likely cells that could be associated with those genes but there was still the increase in MATN3 expression which is solely associated with chondrogenesis.

So we have solid evidence that LSJL can induce new chondrogenesis.

LSJL upregulates several genes associated with neurons.  Serotonin Receptors were upregulated 10 fold.  There is very likely to be a conditioning mechanism.  That is why I am now altering 4 days LSJL on legs/4 days LSJL on arms.  Although gene expression continued to elevate on selected genes for 1 week but was almost eliminated on 2 weeks.  Another one of CH Turners studies showed different genes started to be expressed after 4 days.  I am now loading for 4 minutes.

Since now we have proven that LSJL can induce chondrogenesis in ectopic areas, lack of results could be either ineffective loading(a clamp is not a pizeoelectric mechanical loader) or due to adaptation which can be fixed by deconditioning periods?  Although several people have performed irregular loading which would allow for decondition time.