More LSJL studies on longitudinal growth!

Since I have bumped this several times I have highlighted the important new relevant information with (*NEW*).  You can control-F to find it if you have not read this post I recommend you do because it has a lot of promising LSJL information. For the first time, using the same 16 week old C57/BL/6 rats a length increase was given for the LSJL study but not in the axial loading study.  Thus providing further evidence that LSJL is a novel height increasing loading mechanism.

Here's a study that shows that LSJL increases height in adult rats!  This is evidence that LSJL could possibly induce adult height growth.

Knee loading promotes longitudinal bone growth in both young and adult mice

"21 young (8 weeks of age) and 15 adult (16 weeks of age) C57/BL/6 female mice were used{rodents retain a cartilagenous template post growth plate cessation however if LSJL still involves differentiation of stem cells into chondrocytes then it can still work in human adult mice it'll just be harder as mice already have a cartilagenous matrix to work with.  Also, adult female mice tend to stop growing at 6 months as opposed to the four month old mice used in the study}. The left hindlimb was given 5-min loading bouts (0.5 N at 5 Hz) for 10 days (young mice) and 20 days (adult mice), while the right hindlimb was used as sham loading control. Mice were sacrificed 2 weeks after the last loading
Knee loading lengthened tibiae in both young and adult mice. Compared to sham loading control, the longitudinal tibia length was elevated by 2.3% from 16.68 ± 0.06 mm (control) to 17.06 ± 0.05 mm (loading) in young mice and 1.5% from 17.82 ± 0.04 mm (control) to 18.09 ± 0.03 mm (loading) in adult mice{so the growth plates were not senescent in adult mice which means that it is not proof that LSJL induces chondrogenic differentiation}. The tibia weight was increased by 10.4% in young mice and by 6.0% in adult mice. Loads elevated BMD and BMC. In young mice, for instance, BMD was increased from 0.0409 ± 0.0003 g/cm2 (control) to 0.0427 ± 0.0003 g/cm2 (loading) and BMC was elevated from 0.0136 ± 0.0002 g (control) to 0.0145 ± 0.0002 g (loading).

Knee loading enhances longitudinal bone growth in both young and adult mice [with stronger effects in young mice]. Joint loading may have a potential usage in development of load-driven therapies for limb length discrepancy and short stature."

The adjunct LSJL scientist Stuart J. Warden did an axial loading study also on 16 week old female C57/BL/6:

Cortical and trabecular bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model.

"Adult (16weeks old) female C57BL/6 mice were randomly divided into three load magnitude groups (5, 7 and 9N), and had their right tibia axially loaded using a continuous 2-Hz haversine waveform for 360cycles/day, 3days/week for 4 consecutive weeks."  The Newtons used was lower in LSJL than Warden's study.  The frequency was 5Hz in LSJL versus 2Hz here.  20 days were used in the LSJL study versus 28 days here which means that the axial loading rats had more time to grow.

"Bone strain on the medial surface of the midshaft tibia demonstrated a linear increase in response to incremental externally applied loads (R2 = 0.99), with 9 N inducing a tensile strain of 1,833 με "

"The ultimate finding of the study was that a load of 9 N (engendering a tensile strain of 1,833 με on medial surface of the midshaft tibia) was able to simultaneously induce lamellar cortical and trabecular bone adaptation when using the mouse tibial axial compression loading model in 16 week old female C57BL/6 mice."

Since the scientists took measurements related to "Effect of loading and load magnitude on percent difference in polar moment of inertia (IP) between loaded and nonloaded tibiae at 1 mm increments along the bone length."  They would have had to measure the bone length.

These images serve to contrast against the images in the LSJL study here. The female mice in that study were 14 weeks old.  In the LSJL study you can clearly see the degradation of bone in the non-microfracture mice.  Thus, in contrast to axial loading, LSJL induces bone degradation which may allow for new growth plate formation.

Absence of proof is not proof of absence so it's possible that there was a length increase in the mice but it was not mentioned so I will be investigating.  If I don't get an answer due perhaps to my reputation I will need your help to find out what the length change is.  Specifics would be very helpful even if the results are not statistically significant to compare to the LSJL study.

(*NEW*)

Here's a patent related to LSJL:

"The methods and apparatus provide a mechanical load applied to the epiphysis of a bone associated with a joint{just like we are trying to do with LSJL}. The magnitude of the mechanical load is oscillated and is applied periodically for a short durations of time{this oscillation could be important to LSJL as both fluid flow and compression are important for inducing chondrogenesis, the current method of LSJL may not induce enough fluid flow}. The applied mechanical force induces formation of trabecular and cortical bones. Embodiments of the apparatus include passively and actively actuated bands which are positioned around at least a portion of a joint and provide mechanical loading on the joint{so loading the joint capsule is important for LSJL in addition to loading the bone epiphysis}, for example, on the epiphysis of a bone and oriented traverse to the longitudinal axis of the bone{so essentially lateral loading}."<-We are not really oscillating the loads but I suppose we could with the clamp.  Rotate the clamp tighter than loosen a little, then a little tighter than before and so on...

Some difficulties for an LSJL like device:

"Generating strain in the diaphysis in limb bones requires a loading device that is large enough to span the entire length of a long bone, for example, a femur or tibia{this device however was designed to stimulate bone formation in the entire bone whereas we are only looking for chondrogenic differentiation in the epiphysis}. Accordingly, the portability and ease of use of such devices are detrimentally affected. In addition, it may be difficult to determine the appropriate loading magnitude for particular individuals; therefore, operation of such a device may cause unwanted bone fractures{<-no reported bone fractures have been reported with LSJL however}. Furthermore, enhancing bone formation by generating a strain in the diaphysis may require that all bones be treated independently{bones have different shapes and differences in other bones present at joints}. Moreover, the use of such devices can require dedicated exercise time which may not be easily incorporated into daily activities[this is an issue people have been having with LSJL]."

Here's some methods described to perform LSJL. They are not specifically described to generate height increase but they are all designed to increase intramedullary pressure and fluid flow which is our goal as to generate chondrogenic differentiation:

"The method of enhancing bone formation in a mammal can include applying a mechanical load to a joint of the mammal so that viscoelastic joint tissues of the joint are deformed{this is what we do with the clamp or dumbell}. In one embodiment the method includes oscillating the amplitude of the mechanical load, for example at about 2 Hz, so that fluid flow is generated in the bones forming the joint. The fluid flow can be interstitial cellular fluid flow and include the diaphysis of a bone. The method can also include varying the amplitude of the mechanical load to generate a streaming potential measured on the periosteal surface of the diaphysis of the bones forming the joint, for example so that the peak-to-peak amplitude of the mechanical load is about 0.5 N. The joint can be, but is not limited to, one of the knee, ankle, hip, shoulder, elbow, and wrist, and the mechanical load in one embodiment is applied laterally to the joint to cause viscoelastic deformation of joint tissues. The method can also include applying a circumferential band around at least a portion of the joint and exercising the joint to vary the load applied to the joint by the band{basically like putting a ring on the finger or something like a ring on your leg.  I think it's basically describing tying off the joint and then exercising to induce fluid flow basically like the cuff used to measure blood pressure only over the epiphysis}. The method can also include positioning a wall of a bladder in contact with the joint and advancing a fluid into the bladder{a bladder is a hydration system like the platypus, the wall would be one side of the bag that holds the fluid, so you would put the side of the bladder onto the joint and then put fluid into the bladder(something dense would work best)}. Alternatively, applying the mechanical load to the joint can include positioning a band around at least a portion of the joint and tightening the band.

In another embodiment the method of enhancing bone formation in a mammal having a joint connecting a first bone and a second bone includes applying a mechanical load to the epiphysis of at least one of the first bone and the second bone. The magnitude of the mechanical load can be oscillated and the mechanical load can be applied laterally to the joint such that fluid flow is generated in at least one of the first bone and the second bone. The orientation of the mechanical load can be in a direction that is about transverse to the longitudinal axis of the bone and can be applied to the joint tissue connecting the first bone and the second bone{so you would stack your joints one on top of each other and then load the top joint with a dumbell and hope that you get both joints}."

The hydration system would be interesting to try. A platypus hydration system is available for about 30$. We'd have to find the best liquid to put in the platypus. We'd want the most dense liquid but that's still save and affordable(not something like mercury). For the circumferential band, you could tie off two exercise bands at the end of two joints and then go running.

"In yet another embodiment, the apparatus for enhancing bone formation includes a band configured to fit around at least a portion of a joint of a mammal, a bladder coupled to the band, and a pump in fluid communication with the bladder and operable to advance a fluid, for example water or air, into the bladder. The pump can have a controller for oscillating the volume of fluid in the bladder. The band can form a loop having an adjustable circumference. The band can also be a belt that defines a loop having a circumference configurable to fit around a joint and an electric motor operatively coupled to the belt and capable of varying the circumference of the loop. The band can alternatively include an electro-chemical material, for example, a polymer that undergoes dimensional changes upon exposure to an electric field, such as polypyrrole or polythiophene. Oscillation of the electric field can be used to change the circumference of the loop, thereby varying the mechanical load on the joint.

In another exemplary embodiment, the apparatus for enhancing bone formation includes a band configured to be positioned around a joint and an element coupled to that band that is configured to apply lateral pressure to the joint. The band can include an elastic wrap. The element can also include a pad, for example a fluid filled bladder. In one embodiment two pads are positioned on opposite sides of the joint."

"One exemplary method of the present invention uses exemplary apparatus to provide brief periods of periodic mechanical loads for example for three minutes per day. Additionally, mechanical loads may be oscillated in magnitude, for example sinusoidally between 2 Hz and 30 Hz with a peak to peak load sufficient to induce bone formation enhancing interstitial fluid flow, for example, about 100 N"

"That may be driven by air pressure approximately 40 kPa (5.8 psi) was needed to provide 0.5 N to murine elbows in the second exemplary study discussed below. Assuming that 100 N is used to press a lateral wall of a human knee joint, 51 kPa (7.4 psi) is required for a 50 mm in diameter bladder"<-So we can figure out that 40 kPa produces 0.5N is for mice. So humans need 11more kPa of pressure to generate 0.5N of force given a 50mm in diameter bladder. So for humans 51 kPa per 0.5N of force given a 50mm bladder.

"a micro air pump driven at 6-24 NDC with 180 g weight (available from Sensidyne, of Clearwater, FL)."

"ctuators can be formed in any shape, for example, belts, and it can easily generate strain of 10 % and stress above 20 MPa with a small amount of electricity. For example, a conductive polypyrrole polymer (available from EAMEX Co., of Osaka, Japan) with a cross-sectional area of 50 mm (width) x 3 mm (thickness) can generate 189 Ν"

We can combine clamping and a bladder hydration system. We could clamp the sides of the bone while putting the platypus hydration system on top using just water at this time. Anyone have ideas for substances more dense than water?

Elbow loading promotes longitudinal bone growth of the ulna and the humerus.

"Mechanical stimulation plays a critical role in bone development and growth. In view of recently recognized anabolic responses promoted by a joint-loading modality, we examined the effects of elbow loading on longitudinal growth of the ulna and the humerus. Using a custom-made piezoelectric loader, the left elbow of growing C57/BL/6 female mice was given daily 5-min bouts of dynamic loading for 10 days. The right forelimbs of those mice served as contralateral controls, and the limbs of non-treated mice were used as age-matched controls. The effects of elbow loading were evaluated through measurement of bone length, weight, bone mineral density (BMD), and bone mineral content (BMC), as well as mRNA expression levels of load-sensitive transcription factors such as c-fos, egr1, and atf3. The results revealed that the humerus was elongated by 1.2% compared to the contralateral and age-matched controls, while the ulna had become longer than the contralateral control (1.7%) and the age-match control (3.4%)[We can't be sure if these actually increased final height or merely accelerated height growth until we actually identify how lateral joint loading increases growth]. Bone lengthening was associated with increases in bone weight, BMD and BMC. Furthermore, the mRNA levels of the selected transcription factors were elevated in the loaded ulna and humerus. Interestingly, the increase was observed not only at the elbow but also at the wrist and shoulder in the loaded limb{loading at one area likely changes hydrostatic pressure throughout the bone and the periosteum of one bone is connected to another}."

"Loads were 0.5 N at 5 Hz and given for 5 min per day for 10 days . The lateral

and the medial sides of the ulna and humerus were in contact with the loading rod and the stator, respectively. We chose a forelimb configuration that made the right angle (90) between the ulna and the humerus, since in this position the forelimb was relaxed and stably immobilized. To position the elbow properly for loading, the lower end of the loading rod and the upper end of the

supporter (nylon screw) were designed to form a pair of semi-spherical cups. The olecranon process and coronoid process of the ulna together with the ulnar tuberosity, and medial and lateral epicondyles of the humerus were confined in the cups. The tip of the loader had a contact surface of 3 mm in diameter. To avoid a local stress concentration between the elbow and the loader, both the loading surface

and supporter were covered with silicon rubber. The right elbow was used as a sham loading control (contralateral control), which was placed under the loader with no dynamic loading. In the age-matched control group, the same procedure was applied without application of lateral

loads."

In contrast to the other LSJL lengthening study, the contralateral humerus did not increase in length.  However, the contralateral ulna did increase in length.  If this was a periosteal signaling or a fluid flow related reasons than you'd expect the humerus to increase in length but not the ulna as the ulna's are closer to each other than the humerus'. The length increase was larger in the ulna than humerus so there may be a threshold of adaptation that occurs before the other limb begins to lengthen to compensate.  Messenger RNA expression was much higher in the ula than humerus which could be another reason.

"During the loading experiment no bruising or other damage was detected at the loading site"

"We did not observe any load-driven damage in the subchondral bone and the articular cartilage in the humerus and ulna."

A statement from Hiroki Yokota:  "We did not conduct histological analysis.  I do not know likelihood but it is possible that microdamages are induced in the subchondral bone."

"the load-drive fold change was 4.5 (c-fos), 4.0 (egr-1), and 6.0 (atf3) in the proximal ulna, and 2.9 (c-fos), 3.1 (egr-1), and 3.8 (atf3) in the distal humerus."

"elbow loading (1,500 daily cycles for 10 days) stimulates longitudinal bone growth in the humerus and the ulna. Compared to the contralateral controls, longitudinal length was increased on average by 1.2% (humerus) and 1.7% (ulna). Compared to the age-matched controls, these increases were 1.2% (humerus) and 3.4% (ulna)."

"In response to lateral loads applied to the knee ex vivo, the maximum strain at the loading site was in the order of a few millistrains[10^-3] and the strain at the midshaft cortical bone was in the order of 10 microstrains[10^-6]{So the strain is much higher on the loading site than in the middle of the bone which is good because the loading site is near the epiphysis which means that LSJL may induce more than the 1500 microstrain required to induce bone adaptation}. The longitudinal strain along the bone length was positive, indicating that tensile force acts in the growth plate. As a biophysical mechanism for induction of bone formation with joint loading, it is proposed that alterations in intramedullary pressure are induced and interstitial molecular transport is activated by a dynamic pressure gradient"

According to A comprehensive characterization study of human bone marrow mscs with an emphasis on molecular and ultrastructural properties, c-Fos is a neurogenic marker.  Fos- and Jun-related transcription factors are involved in the signal transduction pathway of mechanical loading in condylar chondrocytes states that overexpression of c-Fos inhibits chondrogenic differentiation.  LSJL did upregulate C-Fos along with ATF3 and egr1.  According to Gene expression profiling of primary human articular chondrocytes in high-density micromasses reveals patterns of recovery, maintenance, re- and dedifferentiation, egr1 is associated with the chondrocyte differentiation process.

According to Egr-1 mediates transcriptional repression of COL2A1 promoter activity by interleukin-1β, egr-1 is an inhibitor of COL2A1 via interleukin-1Beta.

"In the hypertrophic zone of the growth plate in the proximal tibia, the number of chondrocytes and their cellular height were elevated{increase in number of chondrocytes indicates possibility of new stem cells differentiating into chondrocytes thus bypassing proliferative capacity, also increase in cellular height means more height growth per chondrocyte thus also likely leading to a resultant final adult height rather than more acceleration}. Thus, joint loading is potentially useful to lengthen long bones in the hindlimb and the growth plate at the loading site exhibits morphological alterations; however, no studies have been conducted for the forelimb."

"mice were mask-anesthetized using 1.5% isoflurane and received loads to the left elbow in the lateral-medial direction with the custom-made piezoelectric mechanical loader"<-the mice were anesthetized meaning no muscle contraction but that doesn't mean that muscle contraction can't increase hydrostatic pressure.

"Loads were 0.5 N at 5 Hz and given for 5 min per day for 10 days"<-5 bouts of 1/2 the force of gravity per second. Hard to convert this to our purposes but the load seems pretty low. I am now loading at 100 seconds. Perhaps lower loading is needed for LSJL and longer duration. This study uses electricity but a pizeoelectric current is generated by the bone in response to deformation due to loading thus the same effect is achieved due to loading.

"To avoid a local stress concentration between the elbow and the loader, both the loading surface and supporter were covered with silicon rubber"<-thus supporting the usage of socks and clothing to eliminate some of the irritation of LSJL.

"The longitudinal strain along the bone length was positive, indicating that tensile force acts in the growth plate[so the growth plate was stretched but previous growth plate stretching methods have had a lack of success]. As a biophysical mechanism for induction of bone formation with joint loading, it is proposed that alterations in intramedullary pressure[Hydrostatic pressure is likely a subset of intramedullary pressure thus inducing chondrocyte differentiation] are induced and interstitial molecular transport is activated by a dynamic pressure gradient"

"dynamic tensile stress along the length of the ulna and the humerus as well as alterations in intramedullary pressure [may contribute to the bone lengthening]. We observed differential sensitivity to elbow loading in the humerus and ulna. A potential factor for the observed difference may originate from anatomical dimensions and geometries, micrometric and nanometric structures in the lacunocanalicular network, and populations of osteocytes and osteoblasts. In the present study, the loading frequency is 5 Hz. Depending on loading frequencies, it might be possible that the humerus becomes more responsive to elbow loading that the ulna."

"Load-induced longitudinal growth suppression has been reported to be proportional to load magnitude in the growing rat ulna[thus perhaps growth stimulation by lateral joint loading may be proportional to load magnitude as well], but it is not known whether the change in growth rate is also proportional to the number of loading bouts per day or the number of loading days. It is conceivable that the rate of bone lengthening is dependent on various loading conditions such as load magnitude, frequency in Hz, number of bouts per day, and loading duration in days as well as animal age. In summary, the current study demonstrates that elbow loading is an effective means to promote longitudinal bone growth in the mouse humerus and ulna. Joint loading may therefore be potentially useful for the development of load-driven therapies for limb length discrepancy and short stature."

According to Artificial ants deposit pheromone to search for regulatory DNA elements., egr-1 has a strong involvement in chondrogenesis.  Other genes of note are E47, CREB, AP-1, AP-2 and Erg-1.

There's also been a review study recently that Hiroki Yokota is a part of that mentions LSJL and may provide some insight:

Mechanical intervention for maintenance of cartilage and bone.

"Moderate loads to the synovial joint suppress the expression levels of matrix metallproteinases (MMPs), while loads above a threshold tend to increase their destructive activities{although some catabolic effects of MMPs may be good for height growth, MMPs may degrade bone allowing for cartilage growth}."

"Moderate shear stress(2–5 dyn/cm2) reduced MMP expression levels, while high shear stress (10–20 dyn/cm2) increased them. Similarly, moderate hydrostatic pressure (1–5 MPa) suppressed MMP-1 expression, while higher loads (10 MPa) elevated it."<-you're likely not to get above 5MPa of hydrostatic pressure even with very large loads so that is not really an issue with LSJL.

"The required magnitude of loads for joint loading is in general smaller than that for axial loading (e.g. 0.5 N for elbow loading and 2–3 N for ulna axial loading in mice). Bone is less stiff in a lateral direction than an axial direction."<-Note that more than 0.5N(100N is mentioned) is likely required for humans. 0.5N is what was used in the mouse arm lengthening study.

"It has been proposed that joint loading periodically alters the pressure in the medullary cavity and activates molecular transport in a lacunocanalicular network in cortical bone."<-It is our hypothesis that this increase in pressure in the medullary cavity induces chondrogenic differentiation.  The medullary cavity is continuous into the spaces of the spongy bone of the epiphysis.  It is these spaces where we aim to induce chondrogenic differentiation and thus induce endochondral ossification to grow taller.

"That is, a pressure gradient in the medullary cavity generates oscillatory fluid flow in the porous bone cortex."<-and fluid flow into the spongy bone spaces of the epiphysis.

"Modulation of the intramedullary pressure with knee loading is exerted throughout the length of the tibia and the femur."<-the epiphysis is part of the entire length thus knee loading like by LSJL alters pressure in the epiphysis.

"Proinflammatory cytokines such as IL-1β upregulate the expression and activity of MMP-1 and MMP-13. It has been shown using cultured chondrocytes that mechanical stimulation, given in a form of fluid flow shear stress, can suppress the IL-1β-induced upregulation of MMP-1 and MMP-13. In accordance with those in vitro results, joint motion in vivo is able to reduce inflammatory responses in a murine collagen-induced arthritis model. Additionally, in an antigen-induced arthritis model in rabbits, continuous passive motion suppressed transcription of IL-1β and synthesis of inflammatory mediator COX-2 and MMP-1. These mechanical signals also induced IL-10 synthesis, suggesting that moderate joint loading can generate anti-inflammatory signals."<-there are good and bad MMPs for height growth.  MMP-1 and MMP-13 seem to be bad for height growth.

"When knee loading was applied to one leg, the loaded tibia and femur were reported to be longer than the non-loaded contralateral bones. In response to knee loading, the number of cells in the growth plate of the proximal tibia increased and their cellular shape was altered."<-If LSJL increases the number of cells in the growth plate by differentiation of stem cells into chondrocytes than LSJL will work in adults as well.  It's possible that during knee loading only chondrocyte proliferation was increased but chondrocytes have a finite proliferative capacity and an increase in chondrocyte proliferation without increasing stem cell differentiation into chondrocytes should accelerate the the transition of proliferating chondrocytes into hypertrophic chondrocytes and not the number of cells in the growth plate.

"Homeostasis of the articular cartilage is affected through interactions with the subchondral bone underneath the cartilage.  Both MMPs and ADAMTS need to be post-translationally activated, and this activation process is regulated by many factors including MMPs themselves and many proteoglycans."<-Thus loading of the articular cartilage may itself play a role in the height gain by triggering a response in the subchondral bone in response to the stimulation of the articular cartilage.

Here's another study involving Hiroki Yokota that mentions that lateral joint loading modality:

Mechanical Loading: Bone Remodeling and Cartilage Maintenance 

"Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other[So an increase in TGF-Beta expression in bone can affect cartilage growth]. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway[loading the cartilage may also have an impact on the bone enabling the stimulation of cartilage growth within bone].."

Here's the section that mentions LSJL:

"Joint Loading applies moderate lateral loads to bone as well as joint tissues including articular cartilage and synovium. In the knee loading modality, loads are transmitted not only to the distal femur and the proximal tibia but also to the articular cartilage of the femur and tibia. [LSJL] is capable of stimulating bone remodeling throughout the lengths of the femur and the tibia, as well as suppressing the expression and activities of matrix metalloproteinases (MMPs) in the articular cartilage. A joint loading induces a periodic alteration in the pressure in the medullary cavity[it's this alteration in pressure in the epiphysis that we hope induces chondrogenic differentiation] and activates a Wnt signaling pathway in bone."

"Moderate loading of the articular cartilage generates mechanical signals that increase the synthetic activities of the chondrocyte while suppressing its catabolic actions"<-moderate loading is prochondrogenic so it'll make it easier for stem cell in the marrow to differentiate into chondrocytes.  So you should definitely do some form of moderate loading in addition to LSJL.

Note that joint rotation may increase VEGF which is important for the formation of cartilage canals.  Joint rotation may help augment the effects of LSJL.

"Mechanical stimulation also suppresses cell death through signaling pathways including tumor necrosis factor-α (TNF-α) and Wnt"<-normal mechanical stimulation may help you grow taller through normal height development by suppressing TNF-alpha.

"CITED2 expression is increased by moderate flow shear (5 dyn/cm²), intermittent hydrostatic pressure (1–5 MPa)[thus CITED2 may be stimulated by LSJL], and joint motion. The induction of CITED2 in vivo by joint motion loading was correlated with the downregulation of MMP-1 and the maintenance of cartilage matrix integrity"

"CITED2 suppresses MMP-1 expression by preventing MMP transactivator Ets-1 from recruiting limiting amounts of co-activator p300 to the MMP-1 promoter"
 
"Moderate loading can block interleukin (IL)-1β–induced transcriptional activity of nuclear factor-κB (NF-κB) by interfering with multiple steps in the NF-κB signaling cascade. High-amplitude loading activates NF-κB, which regulates expression of proinflammatory cytokines and mediators such as nitric oxide synthase (NOS-2), cyclooxygenase 2 (COX-2), MMPs, TNF-α, and IL-1β. NF-κB also controls the differentiation or activity of other skeletal cell types, including osteoclasts, osteoblasts, and osteocytes"<-NF-kappaB stimulates chondrogenesis so maybe high-amplitude loading is a good thing.

"Mechanical overloading also stimulates expression of vascular endothelial growth factor (VEGF), which appears to be involved in the induction of MMP-1, −3, and −13 expressions"<-Note that LSJL does upregulate MMP-3. So it's possible that LSJL does stimulate VEGF expression.

"Quickly after osteocyte stimulation, there is an increase in intracellular Ca2+ concentrations and the release of adenosine triphosphate. These events are followed by the participation of secondary messengers prostaglandin and nitric oxide. Further downstream are elements of the Wnt pathway, which regulate bone formation and bone remodeling by promoting osteoblast proliferation and differentiation"<-Note that this release in adenosine triphosphate(ATP) can stimulate chondrocytes as well.

Wnt activation may reduce COL2A1 levels.

Here's a study not directly written by H. Yokota but was cited as being highly influential on the previous study:

Mechanical loading, cartilage degradation, and arthritis

"Moderate mechanical loading maintains the integrity of articular cartilage; however, both disuse and overuse can result in cartilage degradation. In instances of cartilage breakdown, inflammatory cytokines such as interleukin-1 beta and tumor necrosis factor-alpha stimulate the production of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTSs), enzymes that can degrade components of the cartilage extracellular matrix. In order to prevent cartilage destruction, tremendous effort has been expended to design inhibitors of MMP/ADAMTS activity and/or synthesis. Accumulating evidence suggests that physiologic joint loading helps maintain cartilage integrity."

"There appears to be a critical threshold of 15–20 megapascals (MPa) for cell death and collagen damage due to a single impact load in bovine cartilage explants"<-15 MPa is incredibly high.  I think we'd be lucky to get 2 MPa at most with LSJL style loading.  In one study they found that 25lbs of loading increased Hydrostatic pressure by 12-14mmHg.  There are 7500mmHg in one MPa.  The smallest MPa listed as damaging is 5 MPa in the article.

"The range of nonphysiological load intensities in vivo should be greater than those reported in these in vitro studies."<-Due to distribution of force in a living organism thus the threshold for a damaging amount of pressure should be higher than 5MPa.

"The MMP family consists of the collagenases, (MMPs 1, 8, and 13) which degrade collagens types I, II, and III, the gelatinases (MMPs 2 and 9), which target denatured collagen, the stromelysins (MMPs 3, 7, 10, and 11), which degrade several ECM proteins and are involved in proenzyme posttranslational activation, the membrane-type MMPs (MT-MMP 1–4), and a diverse subgroup including MMPs 12, 20, and 23."<-LSJL downregulates MMP-1 which is good as you don't want degradation of type II collagen. You may want degradation of type I collagen however to make room for chondrogenesis. LSJL upregulates MMP-3 although whether this is beneficial is unknown.

"LIPUS promotes synthesis of several matrix components in chondrocytes in vitro, including type II collagen, type X collagen, and aggrecan. LIPUS also increased production of type II collagen in an experimental OA rat model and ameliorated histological cartilage damage when compared to untreated groups."<-Since LIPUS stimulates Type II Collagen and Aggrecan it too can help stimulate chondrogenesis of mesenchymal stem cells.

"Mechanical overloading stimulates expression of VEGF, which appears to be necessary for mechanically induced MMP-1, -3, and -13 expressions"<-Since LSJL upregulates MMP-3 and VEGF is necessary to the mechanically induced upregulation of MMP-3, it is logical that LSJL increases VEGF fashion possibly in a non-estrogen mediated fashion.

"Epigenetic regulation includes the activation of normally silent genes through DNA hypomethylation, which allows for an open chromatin structure, or silencing of normally expressed genes through DNA hypermethylation, which prevents access of transcription factors to their promoter."<-DNA hypomethylation could be used to activate tall genes in normal individuals?  It is hard to control specific gene methylation however.

"Gene transcription can also be regulated by deacetylation through histone deacetylases (HDACs)."

"There is evidence in adult endothelial cells that shear stress regulates gene expression. Shear stress at 10 dyn/cm2/sec modifies core histones H3 and H4 in human umbilical vein endothelial cells."<-Shear stress modifies gene expression. It's not bone or cartilage cells but it shows that shear stress can modify gene acetylation. LSJL causes shear stress.

Here's a study involving Sun HB who seems to be working with Hiroki Yokota on LSJL related issues.

Mechanotransduction and cartilage integrity.

"Chondrocytes are able to sense and react to mechanically induced changes within the cartilage matrix[this is important for people who still have growth plates]. Chondrocyte mechanotransduction is initiated at the interface between the cell membrane and extracellular matrix, and the processing of these mechanical signals involves mechanoreceptors such as ion channels and integrins. Membrane stretch, a condition that chondrocytes experience during compression or during hypo-osmotic conditions that cause swelling, activates potassium channels. The function of ion channels in chondrocyte membranes is not clear, but they may be involved in chondrocyte functions such as cell proliferation and matrix secretion[so ion channels are involved in anabolic therefore height increasing functions]. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits and interact with cytoskeletal proteins such as fibronectin, vitronectin, and osteopontin.  Mechanical stimulation of human chondrocytes increases expression of aggrecan and decreases MMP-3{upregulated in LSJL} gene expression in a pathway involving the α5β1 integrin and IL-4 release. However, this response to mechanical stimulation is absent in chondrocytes derived from OA cartilage, suggesting abnormal chondrocyte signaling may be involved in OA disease progression[this may be one of the reasons why osteoarthritis doesn't help make you taller]"

So growth plates are capable of anabolic responses in response to mechanical load. OA however involves endochondral ossification so maybe mechanical stimulation doesn't effect chondrocytes undergoing endochondral ossification such as growth plate chondrocytes.

"One transcriptional regulator that appears to play a crucial role in cartilage homeostasis is CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2). CITED2 is a transcriptional coregulator that does not bind DNA directly. It positively regulates transcription by recruiting CBP (cAMP-responsive element-binding protein) and p300 to interact with other DNA-binding transcription factors such as Lhx2, PPARα, PPARγ, Smad 2, and TFAP2. CITED2 also negatively regulates target genes by competing for CBP/p300 binding with transcription factors including Ets-1, NF-κB, HIF-1α, STAT2, and p53. Through these mechanisms, CITED2 is able to regulate many cellular processes such as embryonic development, cell proliferation, inflammation, and matrix turnover."

"With regard to cartilage integrity, CITED2 expression in chondrocytes in vitro is increased by moderate intensities of flow shear and intermittent hydrostatic pressure[LSJL induces both these things but does flow shear and hydrostatic pressure stimulate CITED2 release in stem cells?] (IHP) and in chondrocytes in vivo by joint motion. Increased CITED2 expression in vivo correlated with the maintenance of cartilage integrity and the suppression of collagenase MMP-1, suggesting the anticatabolic effects of physiologic joint loading were mediated by CITED2. CITED2 suppresses MMP-1 transcription by competing with MMP transactivator Ets-1 for binding to its coactivator p300. In addition to MMP-1, Ets-1 binds to the promoter regions of other MMPs including MMP-2, -3, -8, -9, and -13. Therefore, it is likely CITED2 may regulate additional MMPs through a similar manner.

Upstream of CITED2, moderate IHP loading phosphorylated p38δ, which was required for the transactivation of CITED2[stem cells have p38 meaning that hydrostatic pressure likely can stimulate CITED2 activation in stem cells too]. p38 belongs to the MAP kinase family, which is activated in response to mechanical stresses. While moderate loading activated p38δ and CITED2, high levels of IHP phorphorylated p38α and MMP-1, but not CITED2. This may explain why CITED2 is specifically activated by moderate loading and also suggests that p38α is involved in the upregulation of MMP-1[MMP-1 is catabolic however very high levels of hydrostatic pressure has been shown to be anabolic in inducing chondrocyte differentiation]. Different members of the p38 family may act as a “mechanosensitive switch” in chondrocytes, which act to upregulate or downregulate MMP expression based on the mechanical loading regimes. The evidence that CITED2 is inducible by IL-4 and may interact with components of NF-κB, suggests a potential role of CITED2 as a central mediator in these mechanotransduction pathways involved in maintaining cartilage integrity"

So CITED2 competes with MMP-1 a catabolic protein.  So moderate levels of hydrostatic pressure may be better with existing growth plates to activate CITED2 and not MMP-1 but high levels of hydrostatic pressure may be better to induce new chondrocyte differentiation where growth plates are absent.

Here's a study that shows that CITED2 may be inhibitory towards new cartilage and bone growth:

Identification of CITED2 as a negative regulator of fracture healing.

"The transcription regulator CITED2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is known to suppress genes mediating angiogenesis and extracellular matrix (ECM) remodeling. We tested the hypothesis that CITED2 functions in bone fracture healing by suppressing the expression of genes critical to ECM remodeling, angiogenesis and osteogenesis, importantly the matrix metalloproteinases (MMPs). Three hours following mandibular osteotomy[removal of bone] or sham surgery of adult rats, osteotomy fronts were harvested and the expression of CITED2 and genes associated with fracture healing was ascertained by quantitative PCR. In parallel, gain-of-function studies examined the effect of overexpressing CITED2 on the expression and activity of several MMPs. In the fractured mandible, CITED2 expression was inversely related to the expression of MMP-2, -3, -9, -13, VEGF, HIF-1alpha, M-CSF, RANK-L, and OPG[Hypoxia inducible Factor-1 and CSF are highly anabolic factors for endochondral ossification]. Consistent with this, the over-expression of CITED2 in osteoblasts inhibited the expression and activity of MMP-2, -3, -9, and -13."

Since fracture healing often involves endochondral ossification, perhaps CITED2 inhibits new endochondral ossification from occurring. However, fracture healing usually requires catabolic activity to remodel bone. New height growth requires catabolic activity to make way for the new growth plates. However, CITED2 may also be important to discourage catabolism of chondrogenic components formed in the bone.

"Early fracture healing is characterized by the initial formation of cartilage tissue in the callus, which is then resorbed by MMPs to allow for vascular invasion with the eventual replacement of cartilage with osseous tissue[so perhaps allowing for CITED2 may allow grow plates to stay active longer]. MMPs 9 and 13 are critical to normal skeletal development; most notably, MMP-9 deficiency delays fracture healing with poor cartilage resorption and impaired capillary and chondroclast invasion. MMP-2, in contrast, participates in cartilage degradation, while MMP-3 activates pro-MMP-9 during wound repair"

So inhibiting CITED2 may aid in the formation of new growth plates but CITED2 may help preserve existing growth plates.

Confirmation that the LSJL scientists believed it can cause height growth

Here's a grant submitted by Ping Zhang that was proposed to do research on whether LSJL could cause adult height growth!

Load-Driven Bone Lengthening

"The long-term objective of the proposed project is to understand the mechanism of load-driven bone lengthening. Although distraction osteogenesis is effective to treat the patients with limb length discrepancy, this invasive procedure occasionally generates problems such as premature consolidation, delayed union, and infection. In order to investigate a possibility of load-driven non-invasive therapy[like LSJL], we will focus on knee loading a form of joint loading modalities. We address a set of questions:
(1) Does knee loading enhance proliferation of chondrocytes in the growth plate and lengthen the proliferative and hypertrophic zones in the distal femur and the proximal tibia[if knee loading can cause differentiation of stem cells into chondrocytes it can cause new height growth]?
(2) Is the lengthening effect dependent on ages[will it work on adults?]?
(3) Does injection of insulin growth factor-2 (IGF-2) into the growth plate enhance the loading effects?
We hypothesize
 (a) Lateral loads, applied to the knee with appropriate loading conditions, can lengthen both the femur and the tibia through stimulating proliferation of chondrocytes in the growth plate;
(b) Efficacy of load-driven bone lengthening is stronger in the youth than the elderly;
(c) The lengthening effect can be augmented in the elderly by injecting IGF-1 in the growth plate[so LSJL will work even in aged individuals!!!!  However, aged individuals do not have growth plates so maybe he means injecting IGF-1 into the bone marrow?  I will concede that based on some communications with Ping Zhang that he does not have excellent english skills so perhaps he used the wrong terminology].

In order to examine the above hypotheses, three specific aims are proposed using a hindlimb of C57BL/6 mice as a model system.

Evaluation of load-driven bone lengthening under varying mechanical conditions
Comparison of the loading effects among mice with varying ages
Effects of local administration of IGF-2 into the growth plate with and without knee loading.

We will use a custom-made piezoelectric mechanical loader, and conduct bone histomorphometric and gene expression analyses using varying imaging modalities. The proposed study is expected to contribute to developing a non-invasive physical therapy for treatment of patients with limb length discrepancy.

The proposed project will contribute to examining a possibility of non-invasive physical therapy for treatment of patients with limb length discrepancy"

Here's the grant name to track it: Grant 1R03AR055322-01A1.

Here's a link with more info on the project.  It should be noted that a Salubrinal study is included as part of the grant.  So maybe Salubrinal does have an undisclosed effect on lengthening.

Here's Ping Zhang's research information.

Can LSJL work on adults?

LSJL was tested on 14 week old male rats.  Many chondrogenic genes were upregulated and studies have shown that Adult Humans BMMSC's are capable of chondroinduction.  However, more evidence would be helpful.

The next step would be to test LSJL on adult female mouse ERalphaAF-1 knockout mice which experience growth plate fusion as described in the study The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice.  Then see if there's any length increase when LSJL is applied.  You can't apply LSJL to another species because the Yokota lab pizeoelectric loader is custom built for rats and LSJL rat growth plates don't fuse which may alter gene expression.  So by forcing fusion using ERa AF-1 knockout mice, we can test how LSJL would work on results.  Gene expression data would be interesting but length increase would be what is needed.

The transactivating function 1 of estrogen receptor α is dispensable for the vasculoprotective actions of 17β-estradiol explains how to generate ERa AF-1 knockout mice.

"ERα AF-1 was generated through a targeted deletion by using a knockin strategy, through which 441 nt of exon 1 were deleted. The truncated protein lacks the A domain and all three motifs constituting ERα AF-1 (AF-1 boxes 1–3) in the B domain, thus yielding a 451-aa-long, 49-kDa protein"

The cost of these mice is at least $5000 but it should cheaper from there on.  I'll try to do some research and see what can be done.  Then there's the confounding variable that the knockout may affect sensitivity of cells to the stimulus.

Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton.

"Age-related increases in osteocyte apoptosis is observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"there is a marked age-related increase in marrow adiposity"<-although adiposal stem cells are capable of differentiating into chondrocytes.

"aging is accompanied by a decline in periosteal lining cell numbers"

" Age-related increases in osteocyte apoptosis is also observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"attenuated biophysical stimulation could arise in part from decreases in the surface to volume ratio of bone mineral matrix and increased viscosity of interstitial fluids.  If these physical mechanisms were to occur in bone, the magnitudes of fluid flow induced secondary to skeletal loading could be reduced with age."<-since fluid flow is a vital part of the LSJL stimulus this could reduce LSJL based chondrogenesis.

"PGE2 release has been found to be substantially increased in cells derived from aged vs. young human donors. In contrast, the number of cells displaying spontaneous Ca2+ oscillations and fluid flow induced oscillations were found to be significantly lower in primary cells derived from aged animals, while the amplitudes of Ca2+ oscillations themselves were equivalent"<-Ca2+ oscillations may be critical to induce chondrogenesis.

"Activation of phosphatases and kinases downstream of second messenger signaling including Calcineurin, CAMK, and MAPK are decreased with age. Further downstream (of the phosphatases and kinases), activation and DNA binding by a variety of transcription factors such as NFAT, AP-1, and Wnt/β-catenin also display age-related deficits or suppression"<-LSJL involves both AP-1 and Wnt transcription factors.

"primary cells isolated from young and senescent mice reveals that relative increases in Cox-2 and c-fos gene expression levels in response to fluid flow (vs. no flow controls) were not altered with age "<-Cox-2 and c-fos are both upregulated by LSJL.

"The absolute Cox-2 but not c-fos levels displayed age-related declines in large part due to lower baseline levels of Cox-2 expression in cells derived from aged animals."

"aging markedly diminishes the periosteal response to mechanical stimuli"

"suppression of increased osteoclastic activity [is] associated with aging"

"Cyclosporin A (CsA)[immunosuppresent drug that is available with prescription only], at low-dosages, could be a candidate agent to address age-related deficits in NFAT (or AP-1) activation and transcription"

"senescent mice (22 months) were subjected to mechanical loading in conjunction with low-dose CsA treatment, the resulting periosteal bone formation was significantly increased compared to vehicle-treated aged matched controls subject to loading alone."

CsA inhibits p38, GSK-3beta, and Calcineurin.  Lithium is another GSK-3Beta inhibitor.

Other genes that may be differentially regulated due to aging in LSJL:

Turner's axial loading study used 20 week old mice versus 14 week old.  So we can also study whether genes were deferentially regulated in the two studies due to age rather than due to the different loading modalaties(axial vs. lateral).

MicroRNA-199a-3p, microRNA-193b, and microRNA-320c are correlated to aging and regulate human cartilage metabolism.

"The expression of two miRNAs (miR-199a-3p and miR-193b) was upregulated with age and that of one miRNA (miR-320c) was downregulated with age."

"Type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR-199a-3p mimic group[axial loading 20 week old mice upregulated COL2A1 and acan but not Sox9, whereas all three were upregulated in LSJL thus the significant upregulation of Sox9 versus axial loading is likely due to the different modalaties and not aging] but [were] upregulated in the inhibitor group. Similar results were observed for miR-193b. By contrast, ADAMTS5 expression was downregulated in the miR-320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR-193b inhibitor group compared with the control group. miR-199a-3p and miR-193b are involved in the senescence of chondrocytes, and miR-320c is involved in the juvenile properties of chondrocytes."

LSJL upregulates the anti aging genes Lin-28b and HMGA2.

Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells.

"chondrogenic potential did not change [with aging]"

The growth of adult(25-50) MSCs was only slightly than child MSCS(0-12) but old had significantly less growth of MSCs.

Thus LSJL inducible chondrogenesis may not change due to alterations in chondrogenic potential with aging but rather due to secondary signaling from osteocytes which do experience alterations in aging.

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats.

"protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age."  IRS1 was deferentially expressed in the two studies up in axial loading and down in LSJL despite the axial loading rats being older.  The tissues are different, muscle versus bone, but the insulin pathway has a general anabolic effect in all tissues.

Effect of aging on the basal expression of c-Fos, c-Jun, and Egr-1 proteins in the hippocampus. found that aging decreased egr1 expression in the hippocampus.  egr1 was downregulated in axial loading whereas it was upregulated in LSJL.  The differential expressions of egr1 in the two studies could be based on aging rather than loading modality.

Age-related changes in rat bone-marrow mesenchymal stem cell plasticity.

"The expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs{chondrogenic potential is not compromised in human BM-MSCs so maybe epiphyseal fusion adult female mice would not be the best model}. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs."

"Old BM-MSCs displayed spread out, flat enlarged morphology which is consistent with late passage and extensively cultured BM-MSCs"

"Human fetal bone marrow is known to contain approximately 1 in 10, 000 MSCs in comparison to 1 in 250, 000 in adults "

Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation.

"Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation[It's osteoblasts and not chondrocytes but it should be indicative of the ability for MSCs to respond to NO, ERK1/2, and PGE2]. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation."

"Calcium concentration also increased linearly in differentiating MSCs from both aged and mature rats over the time course, but it was consistently lower in cells from aged animals; the difference was statistically significant at day 28"<-This may be applicable to chondrogenic differentiation as calcium secretion may help trigger chondrogenesis.

Aged animals had increased expression of the pro-chondrogenic protein BMP-2.

"Differentiating cells from aged rats expressed higher p21 and p53 levels at day 7 therefore senescence could play a role in the age-related changes in mineralization and response to mechanical stimulation observed."

Time course of epiphyseal growth plate fusion in rat tibiae.

"there has been a question as to when or to what extent the rat growth plate fuses. To investigate this question, we used microcomputed X-ray tomography, at voxel resolutions ranging from (5.7 micro m)(3) to (11 micro m)(3), to image the proximal epiphyseal growth plates of both male (n = 19) and female (n = 15) rat tibiae, ranging in age from 2 to 25 months. The three-dimensional images were used to evaluate fusion of the epiphyseal growth plate by quantitating the amount of cancellous bone that has bridged across the growth plate. The results suggest that the time course of fusion of the epiphyseal growth plate follows a sigmoidal pattern, with 10% of the maximum number of bridges having formed by 3.9 months in the male tibiae and 5.8 months in the female tibiae, 50% of the maximum number of bridges having formed by 5.6 months in the male tibiae and 5.9 months in the female tibiae, and 90% of the total maximum of bridges have formed by 7.4 months for the males and 6.5 months for the females. The total volume of bridges per tibia at the age at which the maximum number of bridges per tibia has first formed is 0.99 mm(3)/tibia for the males and 0.40 mm(3)/tibia for the females. After the maximum number of bridges (-290 for females, -360 for males) have formed the total volume of bridges per tibia continues to increase for an additional 7.0 months in the males and 17.0 months for the females until they reach maximum values (-1.5 mm(3)/tibia for the males and -2.2 mm(3)/tibia for the females)."

No bony bridges were observed for the female rat until 5 months.

"the growth cartilage of female Sprague–Dawley rats to be bridged by bone between 6 and 18 months of age"

"Although the tibiae from the 6-month-old rats exhibited an increase in number and volume of bridges when compared to the younger rats, the growth plates still appeared to show proliferative and hypertrophic zonest hence, there is a local limited potential for further elongation. Conversely, there seemed to be an absence of all four of the distinct zones of an active growth plate (zones of resting cells, cell proliferation, cell maturation, and lacunar hypertrophy) in the 9-, 13-, and 24-month-old male growth plates and the 8-, 12-, and 25-month-old female growth plates, as demonstrated by conventional histological evaluation "<-So you can grow taller despite some briding.

16 weeks the oldest LSJL mice age is 4 months. Note that the C57BL/6J female used have delayed senescence.  Here's the growth rate for C57BL/6J mice according to Growth of C57Bl/6 mice and the material and mechanical  properties of cortical bone from the tibia:

Because of the loss of chondrogenic differentiation potential in aged rats but not adult humans it's necessary to have more adult humans test LSJL(read: you).  It's possible that the existing growth plates in the rats inhibited chondrogenic differentiation and thus if growth plates were removed in old rats this would not be the case.  This theory is supported by research on the tide mark.  However, the stem cells were cultured outside of the rat bone and differentiation was induced there where there would be no growth plate.

Thus, there is not likely to be an age related difference in LSJL, independent on the loss of the growth plates of course, in humans but there is in rats with respect to chondrocyte differentiation potential but there may be an indirect effect due to secondary osteocyte signaling and changes in fluid viscosity and bone shape.

Impact of aging on rat bone marrow-derived stem cell chondrogenesis.

"We measured the responses of rat bone marrow-derived mesenchymal stem cells (BMSCs) to chondrogenic induction in vitro. BMSCs from immature rats (1 week old), young adult rats (12 weeks old), and old adult rats (1 year old) were analyzed for cartilage extracellular matrix (ECM) production. Histologic analysis showed strong cartilage ECM formation by BMSCs from 1-week-old rats, but not by BMSCs from 12-week-old[LSJL has been proven effective on 8 and 16 week old mice. or 1-year-old rats. Age-related declines [were observed] in messenger RNA encoding type II collagen, aggrecan, and link protein, three major cartilage ECM components. [There were] significant age-related differences in the expression of genes that influence cartilage ECM formation. These findings support the hypothesis that the chondrogenic potential of mesenchymal stem cells declines with age."

"BMSC isolates contain large, slowly replicating cells and small, rapidly growing cells, which retain multipotential differentiation status longer than do larger cells"

"The level of c-Kit, a protein tyrosine kinase receptor for stem cell factor appeared to decrease with increasing age."

Type II collagen gene expression was decreased in 1 year old rats.  Aggrecan expression decreased in twelve week old rats.  Link protein decreased in one year old rats.  Sox9 expression decreased in 1 year old rats but was still greater than that of 1 week old rats.

The decrease in Aggrecan is significant but not the decrease in collagen.  Most genes related to chondrogenesis did not change significantly in the microarray data(with the noted exception of aggrecan) except that many chondrogenic genes increased in expression between 1 week old and 12 week old mice.

Increasing Aggrecan may be a way to enhance the chondrogenic potential of MSCs in adults.

Here's a grant related to the effects of aging on Mechanotransduction:

AGING-RELATED DEGRADATION IN BONE MECHANOTRANSDUCTION

"Mechanical loading and physical exercise hold promise for enhancing bone mass and morphology. [Previously], we examined how real-time Ca2+ signaling/NFAT pathway activation induced acutely by mechanical stimuli influences bone formation and the degraded response of bone at senescence. [Our] hypothesis [is] that age-related decline in bone formation induced by mechanical loading arises primarily via deficits in activation of the Ca2+/NFAT pathway{NFAT5 and NFATC3 are downregulated by LSJL}. We will quantify agerelated alterations in gene expression downstream of the Ca2+/NFAT pathway and their relation to deficits in cell function and bone formation. We will also demonstrate the requirement for this pathway in bone mechanotransduction by examining the impact of inhibiting (using NFATc1 knock-out mice and high dose Cyclosporine A; CsA) or enhancing its activation (using low-dose CsA). These experimental data will in-turn be used to develop multi-scale simulations for how modulating activation of the Ca2+/NFAT pathway influences the dynamics of cell function and bone formation in young adult and aged animals. In the final S. Aim, [we] will optimize activation of the Ca2+/NFAT pathway with the objective of restoring bone response to loading in the aged skeleton to levels observed in the young adult skeleton. Success of this project would clarify the Ca2+/NFAT pathway as a critical mechanism underlying the agerelated degradation in bone's ability to respond to physical stimuli, and will demonstrate the benefits that are anticipated via interventions in this pathway. Cyclosporine A (one of the proposed interventions) is currently approved for clinical use."

Here's a study detailing the properties of 17 week old female mice tibias(LSJL has induced length gain in female mice ages up to 16 weeks):

Intrinsic material properties of cortical bone.

"The G171V mutation (high bone mass, HBM) is autosomal dominant and is responsible for high bone mass in humans {The humans are transgenic for LRP5, being transgenic for LRP5 inhibits GSK-3Beta so it may help with height growth}. Transgenic HBM mice in which the human LRP5 G171V gene is inserted also show a similar phenotype with greater bone mass and biomechanical performance than wild-type mice, as determined by whole bone testing. Whole bone mechanics, however, depend jointly on bone mass, architecture, and intrinsic bone tissue mechanical properties. To determine whether the HBM mutation affects tissue-level biomechanical performance, we performed nano-indentation testing of unembedded cortical bone from HBM mice and their nontransgenic (NTG) littermates. Femora from 17-week-old mice (female, 8 mice/genotype) were subjected to nano-indentation using a Triboscope. For each femoral specimen, approximately 10 indentations were made on the midshaft anterior surface with a target force of either 3 or 9 mN{LSJL uses 0.5N of Force} at a constant loading rate of 400 mN/s. The load-displacement data from each test were used to calculate indentation modulus and hardness for bone tissue. The intrinsic material property that reflected the bone modulus was greater (48%) in the HBM as compared to the NTG mice. The greater intrinsic modulus in HBM reflects greater bone mineral content as compared to NTG (wild-type, WT) mice. The greater intrinsic property of cortical bone is derived from the greater bone mineral content and BMD, resulting in greater bone strength in HBM as compared to NTG (WT) mice."

"Elastic depth (nm, deformation) is elastic, that is, the deformation recovers when the load is removed, whereas plastic depth (nm, deformation) is plastic, or the deformation is permanent."<-plastic depth of the long bones in a longitudinal direction may way to growth taller.  And the plasticity and elasticity of the bone may effect the ability of bone to respond to deformation by newly induced growth plate.  And this plasticity and elasticity may be affected by age.

This is what a 17 week old bone looks like.  There seems to be lots of porous areas to induce new growth plates.

"the composition (mineral, collagen, etc.) of bone may be responsible for the differences in the elastic and plastic components of total deformation"<-We can alter this composition to get more permanent changes in bone growth.

Skeletal growth and the changing genetic landscape during childhood and adulthood.

"All processes of skeletal growth (longitudinal growth as well as gains and losses of bone mass) are subjected to environmental and genetic influences. These influences, and their relative contributions to the phenotype, can be asserted at any stage of life."

" The shape and content of bone will change throughout the life span with fluctuating contributions of [environmental and genetic] influences."

"in humans, long bones lengthen with age from birth to ∼18 years (or until skeletal maturity), with fluctuations in growth velocity occurring at various periods therein. Increases in total bone diameter and cortical thickness occurs from birth to approximately 30 years of age"

"Genetic influence on bone changes over the life course. The mechanisms for changes in genetic influence can include developmental timing of gene action, epigenetic modification of the bone-related gene product, or even variation in environmental factors influencing gene expression."

"Quantitative measures of bone size and shape were obtained from the second metacarpal from anteroposterior radiographs of the left hand."

"the genetic contribution to bone length remains high and is relatively consistent throughout the remainder of childhood and into young adulthood (average heritability of 0.92 from age of 7 to 29 years). In later adulthood, the genetic influence on metacarpal length again fluctuates, with heritabilities ranging between 0.64 and 0.88. "

Functional comparison of chronological and in vitro aging: differential role of the cytoskeleton and mitochondria in mesenchymal stromal cells.

"We established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages[a round of cell growth and proliferation in cell culture]. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics{so by reducing some of of these division related changes may enable stem cells to differentiate into chondrocytes once more}, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function{or loss of progenitor characteristics could be due to culture and it's possible similar changes may occur in aging by degeneration of the microenvironment}. Altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels [occurred] in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging."

"Cell roundness increases during long term culture of aMSCs and yMSCs"

Comparison of genes upregulated and downregulated by long term passaging to LSJL to be done.  A number of genes downregulated by passaging are focal adhesion proteins, regulation of actin cytoskeleton, mitochondrion, wnt signaling pathway, and TGF/BMP signaling.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

"We examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases.
All stem cell lines were expanded with fibroblast growth factor-2{upregulated by lsjl} and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels{all upregulated by LSJL}. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.
Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.
The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells."

GAG content of the chondrogenic medium decreased fairly dramatically by age and by age 90 was almost non-existent.    But is still quite significant in the ages of 20-40 in life.  Sox9 and Aggrecan were non-existant by age 70.   Whereas, Col2a1 was non-existent by age 65.  Osteogenic potential on the other hand increased with age.

Vascular disease decreased chondrogenic potential whereas it increased the osteogenic potential.  So the aging trends in osteogenic vs. chondrogenic differentiation may be related to vascular issues.

"The age-dependent decrease in SOX9 expression must limit the cartilage regeneration capability of MSCs."

Human fetal and adult bone marrow derived mesenchymal stem cells use different signalling pathways for the initiation of chondrogenesis.

"Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues including first trimester bone marrow and have demonstrated enhanced expansion and differentiation potential. However their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signalling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signalling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modelling chondrogenic differentiation and skeletal development studies."

"Fetal MSCs have enhanced plasticity, proliferation propensity and expansion potential compared to adult MSCs. They appear to form an intermediate cell type between adult MSCs and embryonic stem cells (ESCs) as they have active telomerase and express pluripotency markers, albeit at a considerably lower level than ESCs"

"Fetal MSCs also lack intracellular HLA class II and have lower HLA class I expression compared to adult MSCs which suggests that these cells may be immunologically inert"

"the bone morphogenetic proteins (BMPs) have been shown to have the ability to induce de novo ectopic cartilage formation in a system that recapitulates endochondral ossification during skeletogenesis. Specifically, BMP2 has been shown to promote condensation of the mesenchymal cells in the developing limb"

"Fetal and adult MSCs both induced strong phosphorylation of BMP-specific SMAD1 and SMAD5 with BMP2 treatment alone or in combination with TGFβ3. However, strikingly we detected TGFβ3-mediated phosphorylation of SMAD1/5 in adult MSCs as well as a marginal level in fetal MSCs. TGFβ-induced signalling is predominantly activated through TGFBR1 and TGFBR2 to phosphorylate SMAD2/3. However, TGFβ has also been shown to activate SMAD1/5 phosphorylation, traditionally activated by BMP signals in numerous cell types. By signalling through TGFBR1, activin A receptor type II-like 1 (ALK1) is recruited and in combination with TGFBR2, activates SMAD1/5 phosphorylation. This dual signalling results in the formation of mixed-receptor SMAD complexes, which may bind to BMP promoters and influence BMP-mediated transcriptional responses. The expression of TGFBR2 and upregulation of BMPR2 in adult MSCs stimulated with TGFβ3 indicates that TGFβ-mediated SMAD1/5 phosphorylation may signal through a receptor complex incorporating TGFBR2-BMPR2 receptors to mediate the initiation of chondrogenesis."

"phosphorylation of both SMAD2/3 and SMAD1/5/8 is essential at the onset of chondrogenic differentiation and these SMADs remain in an active state in differentiated MSCs, while only SMAD2/3 is present in native articular cartilage"

"the initiation of chondrogenic differentiation of MSCs may be mediated by either TGFβ or BMP, but can only be maintained through signalling associated with SMAD3. This may explain the need for TGFβ3 in addition to BMP2 for optimal cartilage tissue engineering by fetal MSCs even though BMP2 alone is sufficient for the initiation of chondrogenic differentiation"

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.

Lateral Joint Loading for the Jaw?

People have asked before if it's possible to do LSJL for the spine.  So far LSJL has only been done for the arms and legs and there's more research done on this field.  Here's a study on lateral loading for the jaw that predates the LSJL longitudinal growth studies that may provide some insight on how to grow taller via the spine or how to get a bigger jaw.

Influence of extraoral lateral force loading on the mandible in the mandibular development of growing rats.

"Thirty growing Wistar rats were divided into 3 groups: control, sham, and experimental. To determine longitudinal developmental changes, each animal was placed under anesthesia and immobilized in a custom-built body retainer. In the experimental group, a fixing device of aluminum was fitted to the zygomatic arch. Lateral force was then applied to the mandible with an open coil for 2 weeks. Absolute lengths and perpendicular heights from the baseline of the lower border of the mandible were measured.
Lateral force induced the mandible to shift toward the nonloaded side; absolute mandibular length at menton-condylion on the side where the load was applied was greater than that in the control group. No difference from the control group was noted on the nonloaded side, and there were no significant differences in perpendicular heights. Lateral loading on the mandible resulted in histopathologic changes: (1) on the side where the load was applied, the cartilaginous zone hypertrophied in the highest margin of the condylar head, the erosive zone expanded, and the width of the mandibular neck decreased; (2) the chondrocyte layer shifted to the medial side on the nonloaded side, and cartilaginous ossification occurred in the lateral direction immediately below the chondrocyte layer, which deformed the mandibular neck toward the medial side and caused asymmetric development of the mandible."

"A fixation device for loading lateral force onto the mandible was attached to the zygomatic arch. It was made of aluminum and was 7 mm wide, 50 mm long, and 0.5 mm thick. The device was fixed bilaterally to the cheek bones (horizontal to the Frankfort plane and immediately below the orbit) with a stainless steel wire with a diameter of 0.3 mm, so that the device passed under the lower border of the mandible. In the experimental group, a hole was made in the left mandibular incisor cervical area, and an open coil spring was ligated to the tooth by using a 0.25 mm stainless steel wire. The device and the coil spring were bound with a stainless screw. The force was loaded to displace the mandibular central incisor 2 mm to the right of the maxillary central incisor. The open coil spring, made of stainless steel containing nickel and chromium, measured 0.25 mm thick, 1.02 mm in diameter, and 7.0 mm long. In the load-shift curve obtained in a preliminary experiment with this coil spring, 2 mm of compression provided about a 15-g force. The direction of the force was assumed to be parallel to the Frankfort plane."<-The force used in the LSJL studies was 0.5N which is much less force.

"The lateral force loaded on the mandible was 15 g; at that level, the rats could move the mandible back to its normal position by themselves when awake. Force was loaded for 24 hours a day."<-LSJL loading is intermittent.

At 2 weeks the absolute mandibular length increased from 17 to 18.  That is huge.  There was no change in the control group.

B and D are loaded side and A and C are non-loaded.  It's hard to tell if there's any evidence of new mesenchymal chondrogenesis or articular cartilage endochondral ossification which would be what increases height.  However, the control group did not grow at all indicating that this is not merely an increase in growth rate.

So lateral loading of the mandible increases it's length and perhaps intermittent loading could provide this increase without the deformity.  You could also load both sides equally.  Growth can be stimulated in the mandible by lateral loading.  No clamp is likely needed and you can just use your hands.  Ultrasound has been tested on the condyle as well.