Cartilage to bone transitions in health and disease.

“The apical ectodermal ridge and the zone of polarising activity control proximo-distal and anterior-posterior patterning in the growing limb bud. These two centres are regulated by signalling pathways including Indian hedgehog (Ihh) and Wnt/beta-catenin”

“planar cell polarity (PCP), a non-canonical Wnt pathway involving the cadherins Fat and Dachsous (Fat/Dchs), is also important in embryonic skeletal development. Gradients of Dchs expression appear to regulate cell shape and directional movement during limb morphogenesis and growth”

“movement-induced mechanical bone loading regulates longitudinal growth of skeletal elements [via epigenetic alteration]”

“Endochondral ossification is initiated by embryonic mesenchymal cells migrating to form pre-cartilage condensations, which then undergo differentiation into chondrocytes and secrete an extracellular matrix rich in collagen type II and aggrecan. The chondrocytes of these cartilage condensations undergo an ordered and highly regulated process involving  predominant marginal proliferation and central maturation, hypertrophy and cell death”

Other events are described until eventually a secondray ossifaction center is formed called the epiphyseal plate.  Maybe the formation of the secondary ossification center can give insights into height growth.

Chondrocytes maintain fixed positions while undergoing their various differentiation states.

“The primary zone of the growth plate, often known as the ‘resting’ or ‘germinal’ zone, consists of undifferentiated chondrocyte progenitors. Unlike the remainder of the growth plate, the chondrocytes of the resting zone are distributed sporadically and have a low rate of proliferation”<-Our idea is that any stem cell can function as a chondrocyte progenitor to form a new growth plate.

“As chondrocytes progress from the resting zone, they gain a proliferative phenotype and adopt a flattened, oblate shape, arranging themselves into longitudinal columns. It is proposed that the creation of these highly organised columns is directed by the chondrocytes in the resting zone which have been postulated to produce a growth plate-orientating factor”<-Conceivably a mesenchymal stem cell could acquire the same phenotype.

WISP3 regulates IGF-1 control of chondrocyte hypertrophy which affects longitudinal growth.

The ECM matrix is mineralized during the hypertrophic stage to facilitate vascular invasion.  Since ECM matrix mineralization affects the degree of interstitial growth possible, this could be a key stage for affecting height growth.

“the formation of mineralised tethers[cords] between epiphyseal and diaphyseal bone [promote] the fusion of the primary and secondary ossification centres”<-The authors suggest that longitudinal bone growth cessation occurs at the end of sexual maturity but there is large reason to believe this is not the case.

“The chondrocytes of the growth plate reach a state of senescence as they exhaust their proliferative potential, and longitudinal bone growth is ceased. In humans, oestrogen mediates these effects in both males and females and the processes controlling fusion are relevant to understanding the ‘permanent’ loss of this transient chondrocyte phenotype”

Differences between type IIA and Type IIB collagen characterize the differences between articular and growth cartilage.

“As joint development progresses, the interzone differentiates into three recognisable layers; two chondrogenic layers which cover the articular surfaces of the developing opposed skeletal elements and an intermediate layer which separates them. There is evidence to suggest that the cells derived from this intermediate layer differentiate to become articular chondrocytes,  while the outer layer chondrocytes are incorporated into the growing epiphysis”<-Maybe some outer layer cells are retained that can be used for neo growth plate formation?

“Interzones first appear as densely cellular, homogenous regions with GDF-5, Wnt9a, autotaxin and chordin being known interzone markers”<-These four elements are linked to c-Jun by Wnt9a and c-Jun is upregulated by LSJL.  Maybe LSJL can form these interzones?

Articular chondrocytes tend to be smaller than GP chondrocytes and they express Tenascin C.  Chondrocyte progenitors are present in the superficial zone in mature cartilage.  Perhaps articular chondrocytes can become GP chondrocytes and induce longitudinal growth at the joints?

“The calcified cartilage layer is semipermeable and whilst it acts as a physical barrier for vascular invasion of the overlying articular cartilage, it does permit the passage of small molecules from the underlying subchondral bone”

“there is increasing evidence implicating the re-initiation of the transient chondrocyte phenotype in osteoarthritic aetiology and pathology”

A BMP receptor ALK2 has been implicated in heterotopic ossification.

“in repair of fractured bone tissue in which there is a deliberate re-initiation of the endochondral processes”<-since re-initiation of endochondral ossification can occur in any fractured bone tissue it is likely that any set of mesenchymal cells can be induced to form an ectopic growth plate as well.

Edit 8/20/13 by Michael: The acronym ECM stands for Extracellular Matrix. More specifically, we are talking about the extracellular matrix that is inside the bone tissues, between the bone cells, which are composed of both living and non living components, and organic as well as non-organic compounds.

The obstacle to stretching adult bones is ECM stiffness and why bones do not grow longer as adults.  If the ECM is too stiff then interstitial growth, which is the mechanism by which bones grow longer, is not possible.  The cartilagenous growth plate has a less stiff ECM than the typical bone.  If LSJL can induce micro-growth plates such that the whole stiffness of the entire bone ECM is decreased than interstitial and in turn longitudinal growth should be possible.

Given that mechanical loading tends to increase matrix stiffness it is a must that LSJL induce micro-growth plates to decrease the overall matrix stiffness.  According to Effect of high hydrostatic pressure on biological properties of extracellular bone matrix proteins., hydrostatic pressure increased adhesion of osteoblasts to the ECM proteins Col 1, VN, and Fibronectin.

In order to insure that LSJL forms micro-growth plates we must insure that stem cells adhere to a demineralized bone matrix within the epiphyseal bone marrow.  That will insure that the LSJL method decreases matrix stiffness by neo-growth plate formation and does not cause adhesion of osteoblasts to the bone ECM.

Active Manipulation of Uniaxial ECM Stiffness by Magnetic Anchoring of Bio-Conjugated Beads

“by embedding magnetic beads in a ECM through bio-conjugation between the Streptavidin-coated beads and the collagen fibers, the stiffness of the ECM can be actively manipulated by the application of an external magnetic field”

The magnetic field had no effect on ECM stiffness without the presence of the beads.

“embedding 0.1 mg/ml of beads in the pure ECM reduces the difference in stiffness between pure collagen and magnetic bead embedded collagen” 0.5mg/ml on the other hand increased stiffness.

How matrix properties control the self-assembly and maintenance of tissues.

“Tissue formation is regulated, in part, by a balance between cell-cell cohesion and cell-extracellular matrix (ECM) adhesion. Decreasing cell-matrix adhesion by either reducing matrix stiffness or matrix ligand density induces the self-assembly of endothelial cells into network-like structures. These structures are stabilized by the polymerization of the extracellular matrix protein fibronectin. When fibronectin polymerization is inhibited, network formation does not occur. Interestingly, this interplay between substrate mechanics, ECM assembly, and tissue self-assembly is not limited to endothelial cells and has been observed in other cell types as well.”

“Substrates have been made as compliant as 50 Pa and as stiff as 100 kPa moduli which span a large range of physiological mechanical properties. ”

“Pairs of endothelial cells interacting on compliant substrates (E = 500 Pa) tend to remain in contact, while cells on stiffer substrates tend to separate and migrate away from each other. “<-More compliant substrates are likely more pro-chondrogenic.

“If cells are unable to adhere well to a substrate, then cell–cell adhesion is enhanced to enable the cells to assemble their cytoskeleton and spread.”

“fibronectin polymerization stabilizes endothelial cell–cell connections. ”

Influence of stress on extracellular matrix and integrin biology.

“non-lethal stress favors ECM stiffness, integrin activation and enhanced survival.”

” ECM is [composed of] collagens (27 members), glycoproteins (fibronectin, laminin, vitronectin, tenascin, thrombospondin, SPARC for secreted protein acidic and rich in cysteine), proteoglycans (aggrecan, decorin, perlecan, syndecan and versican) and elastin.”

“ECM composition notably influences its mechanical properties such as compliance, which, at least in part, regulates integrin biology. For example, collagen, especially when polymerized, increases the stiffness of the matrix support, compared with fibronectin.”<-Depolymerize collagen to enable bone stretching to grow taller?

“Cells interact physically and functionally with ECM through transmembrane proteins termed integrins, which connect ECM to cell cytoskeleton”

Hypoxia alters the ECM and affects integrin signaling.  It does so to favor ECM-cell and cell-cell contacts.  Mechanical stimulation tends to increase cellular adhesion.  Ultraviolet light also affects ECM.

Elucidating the role of matrix stiffness in 3D cell migration and remodeling.

“in matrices with low stiffness, single cells can overcome the resistance of the matrix by engaging in a degradation-independent three-dimensional migration mode”

“Cells in soft gels quickly adopted a spindle-shaped morphology. With increasing stiffness the morphology became less elongated and reticulate filopodia were formed. In the stiff gels, the cells generally remained round with frayed filopodia.”<-The stiffness of the bone may inhibit hypertrophy which is a key stage for bone elongation.

“the overall mobility of cells entrapped in the stiffest gels was dramatically reduced compared to the intermediate and soft gels”

“With increasing stiffness, the density of these cellular networks decreased, as cells were increasingly hindered from proliferating and penetrating the matrix.”<-This may be way too stiff an ECM inhibits interstitial growth.

Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration.

Conversely, removal of hydroxyapatite may reduce stiffness.

“e investigated how the addition of discrete quantities of HA affected scaffold porosity, interconnectivity, mechanical properties, in vitro mineralisation and in vivo bone healing potential. The results show that the addition of HA[hydroxyapatite] up to a 200 weight percentage (wt%) relative to collagen content led to significantly increased scaffold stiffness and pore interconnectivity (approximately 10 fold) while achieving a scaffold porosity of 99%. In addition, this biomimetic collagen-HA scaffold exhibited significantly improved bioactivity, in vitro mineralisation after 28 days in culture, and in vivo healing of a critical-sized bone defect.”

Imaging articular cartilage tissue using atomic force microscopy (AFM).

“Cartilage is a complex avascular tissue composed of cells (“chondrocytes”) embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends.  mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety.”

New insights into adhesion signaling in bone formation.

“The bone matrix is deposited in a cyclic fashion during homeostasis and integrates several environmental cues. These include diffusible elements that would include estrogen or growth factors and physicochemical parameters such as bone matrix composition, stiffness, and mechanical stress.”

Couldn’t get full study.

Matrix mechanics and fluid shear stress control stem cells fate in three dimensional microenvironment.

“matrix mechanics that control stem cells (primarily mesenchymal stem cells (MSCs)) fate in 3D environment, including matrix stiffness and extracellular matrix (ECM) stiffness.”<-couldn’t get full study.

Elaengus angustfolia is also known as silver berry or wild olives.

Toxic effects of Elaeagnus angustifolia fruit extract on chondrogenesis and osteogenesis in mouse limb buds.

“We determined the effect of Elaeagnus angustifolia extract on chondrogenesis and osteogenesis in mouse embryo limb buds in vitro and in vivo. Limb bud mesenchyme from day 12.5 embryos were used for high-density micromass cultures. Water/alcohol extract was added to culture media at 10, 100, 1000 and 10000 μg/L. For in vivo experiments, 40 pregnant mice were given 0.5, 5.0 or 50.0 mg/kg of the extract between days 8 and 18 of gestation.

In limb bud cultures 10 μg/mL of extract reduced chondrogenesis but not osteogenesis. Higher concentrations had no effect on chondrogenesis or osteogenesis. In pregnant mice 50 mg/kg of the extract significantly increased fetal femur and ossified zone length, but significantly decreased bone and cartilage volumes{How can something increase femur length but decrease bone and cartilage volume?}.

The extract had no favorable effects on chodrification or ossification and appeared to reduce chondrogenesis. This is in apparent contradiction to its empirical effects in human adults.”

“Elaeagnus angustifolia (Russian olive, Russian silverberry, Oleander), [is] a plant native to Western Asia”  How the authors prepared the extract can be gotten from the Extract Preparation section in the full study.

The authors hypothesize that the effect of the extract is due to flavinoids and antioxidants.

Both 0.5mg/kg and 50mg/kg reduced cartilage volume but only 5mg/kg and 50mg/kg increased embryo femoral bone length.  5mg/kg actually increased cartilage volume in contrast to 0.5mg/kg, 50mg/kg, and control.

“1000 µg/mL of the extract reduced cell survival. High cell density can induce the mesenchymal cells to differentiate into the chondroblasts. At 1000 µg/mL, cell density decreased; therefore the number of differentiated nodules was reduced. Lower concentrations of the extract led to a decrease in the number and the area of the nodules. Stereological study confirmed that bone and cartilage volumes were reduced by feeding the animals with a high dose of the extract. The difference in the effective concentrations in vitro and in vivo may be attributed to differences in the bioavailability of the extract components”

“the extract may exert an effect on the chondrogenic potential of mesenchymal cells through GPR30.”<-Royal jelly is also associated with GPR30.  Also, GPR30KO is also associated with longer femur length.

Wild Olives wouldn’t be the first supplement to inhibit chondrogenesis and yet increase bone length.  It would be nice to compare the ingredients to Royal Jelly to see if there are any similarities and then the direct height increasing materials could be identified.

Given the chondroinhibitory effects, it would only have potential to increase height during development and not post growth plate cessation.

This post is a note on resolving an issue I realized I had created almost a few minutes after I had pressed the Publish Button on the most recent post I wrote about using a pulse massager to possibly let the growth plate in still growing adolescences increase in their longitudinal growth. The post was entitled Using An Electrical Pulse Massager Physiotherapy Device To Increase Longitudinal Growth In Vivo For Open Growth Plates (Big Breakthrough)

I realized that I had made a huge mistake in not realizing that when the term frequency, or wavelength, or Herts, or Amplitude, or Intensity, there is actually almost two meanings for these things, since what I was talking about and what the Patent on Non-Invasive In Vivo Stimulation of the Growth Plates Using Capacitative Electrical Fields was talking about were two different things.

The Abstract of the Patent…

Epiphyseal growth plate stimulation in the bone of a living body is achieved by applying electrodes non-invasively to a body and supplying to said electrodes an AC signal in the range of about 2.5 to 15 volts peak-to-peak at a frequency of about 20-100 KHz.

What I realized was that what Brighton was suggesting to do was release an AC signal, and that is not what the TENS or EMS Pulse massager was doing. First I reference the Wikipedia article on Pulsed Electromagnetic Field Theory.

The Pulse Massagers that I was talking about seemed to be not releasing an AC electrical signal, but a DC signal but in a specific pattern which mimicked what a impulse function would look like. I was definitely wrong before.

There is a picture below comparing the differences between an AC Current, a DC Current, and a Pulsed Electrical Current…

Note how while the AC (or alternating current) is moving in a sinusoidal way, the Pulsating Current does not, but moves in a repeating pattern which does not drop below the Applied Current at 0.

This is my way of trying to explain the problem with my logic in the last post using an Electrical Engineering perspective. The x-y graph on the left represent the applied current over time where the x axis represents time and the y axis if for current.

The reason that the current can even go into the negatives is because when you are supplying AC Current, the electrons that are flowing down the conducting material, something like copper or iron, actually switches directions.

Based on mathematical conventions, the flow of electrons/electrical charge (aka Current) is defined by a mathematical term known as a vector, which has both a magnitude and a direction. Since the concept of current has to be defined by not only a magnitude, but also direction, when you see a ‘negative current’ that means that the direction of the flow of electrons (aka Current) is going in the opposite direction.

However I wanted to make some extremely accurate distinctions between 1. E&M Fields and Waves, 2. what Brighton is actually proposing in the patent, and 3. what I had proposed which was a mistake.

1. Elementary Electrodynamics Theory

Our bodies when broken down as much as possible turns out to be a bunch of molecules. Those molecules broken down even further through snapping the covalent, ionic, and hydrogen bones turns into atoms. Almost all atoms have at least 1 electron, and the reason that any type of chemical reaction is even possible is due to the exchange of electrons between element atoms which either have too much or too little electrons. Based on Quantum Theory the electron itself has both the properties of a particle and a wave. We can actually use mathematical tools used to describe waves to describe the behavior and movements of electrons.

What this implies is that if we really wanted to, we could say that everything has a sort of sinusoidal electrical wave of field that is generated from it. We could say that every single object does emit some type of alternating electro-magnetic signal, which means that every single object would have some type of electrical signal going through it already.

The way that all electromagnetic waves move is in a sinusoidal way, from the ones with the lowest frequency and biggest wavelength like the radio waves to gamma rays. Like the electromagnetic waves, our bodies themselves emit electromagnetic waves is a sinusoidal, alternating pattern.

However that doesn’t help us on differentiating what Brighton is talking about and what I was talking about.

2. What Brighton is actually proposing in the patent

Brighton is talking about using a function generator to send a electrical current that is of the alternating current type through the metal electrode pad.

When he talks about frequency, he is referring to how many times the flow of electrical charges or electrons reverse themselves and them reverse themselves back. The device that he reference in the patent was a

From the patent he writes the following under the section entitled DESCRIPTION OF THE PREFERRED EMBODIMENT….

FIG. 1 shows a signal frequency generator 10 which generates an AC stimulation signal having a frequency within the range of about 20-100 KHz. The waveform of the AC stimulation signal is preferably an unmodulated symmetrical sine wave having a peak-to-peak amplitude within the range of about 2.5-15 volts peak-to-peak and more preferably within the range of about 5-10 volts peak-to-peak. The frequency generator 10 can be a Wave Tech Model 148 Function Generator

3. What I had proposed which was a mistake. 

I was proposing that we use one of those TENS Pulse Massagers with the electrode pads to do the same job as the patent. They are not the same. The electrical signal that is being sent is different.

Pulse electrical signals are also measured in intensity and frequency, but the frequency is the number of impulse signals that the electrical signal sees every second. When a TENS device says that it can get upwards of 100 Hz it means that within a 1 second time frame, the electrical current was increased to a certain intensity 100 times and then decreased as well.

When you press the button to increase or decrease the Intensity, you are telling the device to increase the amplitude of the impulse signal of the electric current. The signal becomes stronger, and when graphed out in terms of current vs. time, the amplitude of the current function will multiply by a certain factor.

So I was talking about pulsed electrical signals and he was talking about alternating electrical signals, two completely different things.

Currently I have no idea what the effect of using one of those pulse massagers would be if it was applied to the skin close to the growth plate of kids who are still growing.

Would the pulse electrical signals have a similar effect as the AC signals proposed by brighton? I am not sure

Me as the amateur researcher made the mistake in thinking that a pulse massager and a function generator was the same thing.

The actual device that the Ivy League Ph. D & M.D. Orthopaedic Surgeon is suggesting is actually a Function Generator, and his proposed one was one from back in the mid 1980s. It was called a Wave Tech Model 148 Function Generator. For more information on Wave Tech, (aka Wave Tek) the company and what happened, Click Here.

You can find more information about the cost of function generators on Amazon by Click Here. Some of the models are expensive but any of the devices that can just get 60 KHz and in the range of 5-10 Volts would be good enough possibly stimulate the epiphyseal growth plates.

The White spots within bone marrow are adipose tissue.  There are four above the arrow for instance.

Bone size and bone strength are increased in obese male adolescents.

“We recruited 51 male ObAs (10-19 years) at the entry of a residential weight-loss program and 51 healthy age-matched and 51 bone-age-matched controls. vBMD and geometric bone parameters, as well as muscle and fat area were studied at the forearm and lower leg by peripheral quantitative computed tomography. Muscle force was studied by jumping mechanography. In addition to an advanced bone maturation, differences in trabecular bone parameters (higher vBMD and larger trabecular area) and cortical bone geometry (larger cortical area and periosteal and endosteal circumference) were observed in ObAs both at the radius and tibia at different pubertal stages. After matching for bone age, all differences at the tibia, but only the difference in trabecular vBMD at the radius, remained significant. Larger muscle area and higher maximal force were found in ObAs compared with controls, as well as higher circulating free estrogen, but similar free testosterone and IGF-I levels. ObAs have larger and stronger bones at both the forearm and lower leg. The observed differences in bone parameters can be explained by a combination of advanced bone maturation, higher estrogen exposure, and greater mechanical loading resulting from a higher muscle mass and strength. ”

Obese individuals have a higher bone age than non-obese individuals until age 16.  Obese individuals had taller height than age matched controls but shorter height than bone age matched controls.

“higher values of trabecular vBMD, trabecular area, periosteal circumference, and cortical area at the different pubertal stages in the obese group. ”  So bodyfat causes increase in bone parameters outside of the growth plate.  This includes the bone age matched group and not just the age matched group.

Obese individuals had increased estrogen and leptin but similar levels of free testosterone.

What would be interesting if the fat itself did not increase various hormones and genes to cause bone growth or some kind of loading effect.  But if the fat within the bone itself caused an expansion of bone parameters.

The main difference between a growth plate and adipose tissue is that adipose tissue is disorganized as you can see in the image of the bone marrow however adipose tissue cells are huge.  So is it possible that there could be enough adipose tissue cells to cause an expansion of the bone even if they are not coordinated like a growth plate.

The effect of weight on the femur: a cross-sectional analysis.

“if stresses associated with biomechanical modifications of the obese surpass the strain threshold of a bone or bony location, it is possible that discernible differences in long-bone morphology could be observed between different weight categories as a direct result of long-term, abnormal mechanical compensation.”

“The Pearson’s product-moment correlation coefficient results show no correlation between weight and stature. “<-Since only very large amounts of weight would influence stature it’s possible that effect of extreme weights are overlooked.

Mediolateral dimensions of the bone at the midshaft at the bone increased at 4 out of 5 of the sites measured in the bone.  It’s possible that other parameters were increased but not statistically significant.  Anteriorposterior dimensions were increased only at the midshaft.

Is it the adipose tissue cells themselves that increase the bone dimensions or is it a weight loading effect increasing the dimenions.  The question is why would the bone increase in size in only one dimension.  The increase in bone size being mainly in one axis is consistent with it being a weight loaded effect and not a result of internal forces from adipose tissue cells.

“research has shown elongation of the proximal ML dimension of the femur in pregnant women”

“As ML diameter measures resistance to ML bending, these results suggest that as weight increases, alterations to the femoral angle result in greater ML pressures, forcing the femur to adapt or risk failure.”

Reduced size-independent mechanical properties of cortical bone in high-fat diet-induced obesity.

” femora from C57BL/6 mice fed either a HFD or standard laboratory chow (Chow) were evaluated for structural changes and tested for bending strength, bending stiffness and fracture toughness. Here, we find that in young, obese, high-fat fed mice, all geometric parameters of the femoral bone, except length, are increased, but strength, bending stiffness, and fracture toughness are all reduced. This increased bone size and reduced size-independent mechanical properties suggests that obesity leads to a general reduction in bone quality despite an increase in bone quantity; yield and maximum loads, however, remained unchanged, suggesting compensatory mechanisms. We conclude that diet-induced obesity increases bone size and reduces size-independent mechanical properties of cortical bone in mice.”

Mice were fed high fat diet over 19 weeks.  4 week old mice were used.

“the HFD group showed a 34% increase in serum IGF-I concentration compared to Chow”

Cellular dynamics and tissue interactions of the dura mater during head development

“Morphogenesis of the cranial bones and sutures is dependent on tissue interactions with the dura mater, which control the size and shape of bones as well as sutural patency. Development of the brain also involves interactions with dura mater: secretion of stromal derived factor 1 (SDF-1) is a critical event in directing migration of the external granular layer precursors of the cerebellar cortex and the Cajal-Retzius (CR) cells of the cerebral cortex. The dura mater is also required for growth of the hippocampal dentate gyrus. Wnt1Cre/R26R transgenic reporter mice were used to study the origin and fates of the cells of dura mater during head development. The dura mater of mammals is derived entirely from the cranial neural crest. Beginning around neonatal day 10 (N 10), the dura mater is infiltrated by cells derived from paraxial mesoderm, which later come to predominate. Over the course of infancy, the neural crest–derived cells of the dura mater become sequestered in niche-like distribution characteristic of stem cells. Simultaneously, dura mater cells underlying the sagittal suture migrate upward into the mesodermally-derived mesenchyme separating the parietal bones. Although initially the parietal bones are formed entirely from paraxial mesoderm, the cellular composition gradually becomes chimeric and is populated mainly by neural crest–derived cells by N 30. This occurs as a consequence of osteoblastic differentiation at the dura mater interface and intravasation of neural crest–derived osteoclastic and other hematopoietic precursors. The isolated cells of the dura mater are multipotent in vitro, giving rise to osteoblasts, neuronal cells and other derivatives characteristic of cranial neural crest, possibly reflecting the multipotent nature of dura mater cells in vivo.

” neural crest cells can be found throughout the intrafrontal suture. These cells give rise to fibroblast-like mesenchymal cells in the sutures, as well as chondrocytes, osteoblasts, and osteocytes in developing bones.”

“Mineralized bone is incapable of interstitial growth{this would explain why adipose tissue cells don’t cause interstitial growth, however is unmineralized bone capable of interstitial growth; unfortunately there is no citation}, and bones grow at the marginal growth sites—growth plates in long bones and sutures in the skull. ” I also couldn’t find any emails either so I can’t ask where they retrieved that conclusion from.

Further research shows that the possibility of interstitial growth is related to the the rigidity of the ECM.  So adipocytes may be capable of interstitial growth if the ECM is too rigid.

I write about the optimal stiffness of ECM for chondrocyte differentiation here.  However, the stiffness for ECM for chondrocyte differentiation may be different from that for interstital growth.  Here I mention, that the compounds that give the bone ECM it’s stiffness are Calcium, Phosphorus, and Vitmain D.  However, people with deficiencies in those three compounds do not grow taller.  Also, mentioned is that demineralized bone matrix is an effective scaffold for chondroinduction.

In conclusion, the reason that adipose cells do not cause interstitial growth in bone despite being enormous and potentially present in massive quantities is that the ECM of bone is too stiff due to the mineral content.  Although during longitudinal growth, the bone is stiff at the bony area between the top and bottom area of the growth plate.  Thus, a key factor for micro-growth plate success via induction by LSJL is to reduce bone ECM stiffness.  LSJL may do this itself by causing interstitial fluid flow and shear strain.

LSJL dincreasing interstitial fluid flow and shear strain can be supported by the histological slides presented here.  The pink area which represents the bone appears to be much less rigid(compare slides A and B).