Spinal nociceptive transmission by mechanical stimulation of bone marrow.

“in addition to the periosteum, many unmyelinated calcitonin gene-related peptide-labeled fibers innervate bone marrow.”

“nociceptors in bone marrow are likely to be excited by increased pressure in bone marrow, possibly resulting in activation of pain pathways including the spinal dorsal horn (SDH)”

“mechanical stimulation to bone marrow, which induces an increase in intraosseous pressure, elicits nociceptors located in bone marrow.”

“both electrical stimulation and increase in pressure within bone marrow generate a blood pressure increase that may be indicative of nociceptive activation.”

“An increase in intraosseous pressure has been shown to activate fine-diameter afferent nerve fibers arising from bone marrow, as do irritant and inflammatory agents such as H+and K+ ions and histamine and bradykinin.”

Recreational runners with patellofemoral pain exhibit elevated patella water content.

“Increased bone water content resulting from repetitive patellofemoral joint overloading has been suggested to be a possible mechanism underlying patellofemoral pain (PFP). To date, it remains unknown whether persons with PFP exhibit elevated bone water content. The purpose of this study was to determine whether recreational runners with PFP exhibit elevated patella water content when compared to pain-free controls. Ten female recreational runners with a diagnosis of PFP (22 to 39years of age) and 10 gender, age, weight, height, and activity matched controls underwent chemical-shift-encoded water-fat magnetic resonance imaging (MRI) to quantify patella water content (i.e., water-signal fraction). Differences in bone water content of the total patella, lateral aspect of the patella, and medial aspect of the patella were compared between groups using independent t tests. Compared with the control group, the PFP group demonstrated significantly greater total patella bone water content (15.4±3.5% vs. 10.3±2.1%; P=0.001), lateral patella water content (17.2±4.2% vs. 11.5±2.5%; P=0.002), and medial patella water content (13.2±2.7% vs. 8.4±2.3%; P<0.001). The higher patella water content observed in female runners with PFP is suggestive of venous engorgement and elevated extracellular fluid. In turn, this may lead to an increase in intraosseous pressure and pain.”

It would be interesting if running caused greater patella(kneecap) size and this increase in patella size was intraosseous pressure related.  But I couldn’t find evidence of running increasing patella size.  The change in water content is low so it’s possible that that increase in hydrostatic pressure was not significant enough.  All it would take is one example of running increasing patella size though.

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes?

“Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three-dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind{Perhaps we can find a way to recreate cartilage canals?}. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation. We conclude that cartilage canals contribute to both perichondral and endochondral bone formation and that osteoblasts have the same origin in both processes.”

“Cartilage canals are tubes of vascularized mesenchyme that are present in bones of vertebrates.”

“cartilage canals regress with age in the distal femur of pigs”

“Cartilage canals originate from the perichondrium (p) and penetrate into the reserve zone (rz) of the cartilage matrix.”

“In the chicken femur cartilage canals contain arterioles, venules, capillaries and mesenchymal cells which are embedded in the canal matrix”

“the matrix of the canals contains no type II collagen, whereas the surrounding cartilage matrix of the reserve and the proliferative zone is rich in this type of collagen. Thus, a demarcation between the canals and the surrounding cartilage matrix appears and ultrastructural data clearly show that no epithelium is elaborated around the canals.”

“the perichondrium is composed of an inner layer that has the characteristics of osteoprogenitor cells and an outer fibroblastic layer. Both layers can provide their mesenchymal stem cells, which migrate into and along the cartilage canals. Several cartilage canals penetrate into the SOC and multinucleated chondroclasts resorb the calcified cartilage matrix and hence open the lacunae of hypertrophic chondrocytes.”

Being able to induce chondrogenesis directly by stem cell implantation would be a huge breakthrough as there are stem cell sources available in breast milk for instance or umbillical cords.

Developmental-like Bone Regeneration By Human Embryonic Stem Cell-derived Mesenchymal Cells.

“The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice{This is a problem in extrapolating results to humans as humans are not immunodeficient!  The human immune system may reject stem cells}. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After six weeks, x-ray, microCT and histological analysis showed that the hESC-MCs had consistently formed highly vascularized new bone that bridged the bone defect and integrated seamlessly with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without pre-implantation differentiation to osteo- or chondro-progenitors.”

It’s not quite true that the hESCs were implanted as is into the bone as the hESCs were first differentiated into MSC-like cells which requires for instance silencing or activating some genes.

“direct transplantation of undifferentiated hESCs induces uncontrollable spontaneous differentiation and teratoma formation instead of the desired healthy, functional tissue”<-a teratoma is a tumor made of ectopic tissue.

The hESCs were more epithelial cell types whereas the hESCs-MCs were more fibroblastic cell type.

“The hESC-MCs have a fibroblastic morphology resembling adult hMSCs“<-So they were like adult MSCs but with some epigenetic modifications.

“Flow cytometry analysis of the hESC-MCs for markers of adult MSCs demonstrated they were positive for CD73 (99.9%), CD90 (85.4%), CD105 (100%), CD146 (99.6%), and CD166 (100%), and were negative for the hematopoietic markers CD34 and CD45. These values were nearly identical to those obtained for the control adult hMSCs, except that the hESC-MCs had lower Stro-1 expression than adult hMSCs (0.3% vs 11%).”

“The percent of cells positive for SSEA-4 was 60% for hESC-MCs vs 35.5 % for adult hMSCs. For Oct4 the percent of hESC-MC cells expressing the marker was 85.8 vs 94.1 % for adult hMSCs, for Nanog: 67.8% positive in hESC-MCs vs 63.7% in adult hMSCs, and lastly 100% of hESC-MCs were positive for Sox 2 vs 99.9% for adult hMSCs.”

Osteogenic differentiation was three times higher for adult MSCs than for ESC-MCs.  Endochondral ossification was observed in the bone defect healing for ESC-MCs but not for the adult MSCs.

“adult bone marrow-MSCs are an adult tissue resident stem cell whose normal function is small scale tissue repair to maintain homeostasis and its own self-renewal. When extracted and cultured, adult bone marrow-MSCs will have a higher tissue specific gene expression because of their developmental lineage in that tissue. However, this also potentially limits their capacity for large-scale tissue regeneration, perhaps because of inherent functionality or even limited proliferation.”

“adult hMSCs from bone marrow are capable of tissue repair, while hESC-MC are capable of induced developmental tissue generation.”

“the hESC-MC cell morphology is similar to that of adult MSCs, although adult MSCs have more elongated filopodia[slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells].”

So the breakthrough isn’t that you can grow taller by eating umbillical cords as these cells were pre-differentiated into mesenchymal cells.  The breakthrough is that the limitation on height growth after puberty is based on the characteristics on the cells themselves.  The presence or absence of the growth plate or bone mineralization may not be the limiting factor but rather the cells themselves.

That means that any height increase modality such as LSJL should be ensured to have an effect on the cells themselves to induce them to a more developmental stem cell type.  Now stimuli induced by LSJL like hydrostatic pressure, interstitial fluid flow, and dynamic compression have all been shown to induce changes in cellular gene expression.  MSCs and hESC-MCs were largely similar between pluripotency markers Oct4, SSEA4, Sox2, and Nanog.  Differences lied mainly in the expression of Stro-1 was lower in hESC-MCs than adult MSCs.  Which is odd as Stro-1 positive MSCs tend to decline with age.

The hESC-MCs were also implanted into a defect with a scaffold so it’s unclear whether these implanted cells could generate endochondral ossification on their old without a defect nor scaffold.

Here’s some stuides on how mechanical stimulation can alter the genetic expression of mesenchymal stem cells so we can see whether LSJL does in fact prime adult MSCs to be more chondrogenic.

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement.

“[We] investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. Methods: Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB{also upregulated by LSJL}. There were changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process.

Mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation.”

Ability of the MSCs to differentiate into chondrocytes was not tested.  This type of loading increased ERK1/2 phosphorylation but not Akt phosphorylation whereas LSJL increased both levels.

“the cytoskeletal organization of the cells displayed alterations, with MSCs taking a more rounded shape when loaded for 2 h, while cells appeared more flattened with a more prominent filamentous actin network when rested for 22 h.”

Comparison of genes altered to LSJL was not done but no genes altered seemed to be involved in chondrogenesis.

Intermittent traction stretch promotes the osteoblastic differentiation of bone mesenchymal stem cells by the ERK1/2-activated Cbfa1 pathway.

“We investigated the osteoblastic differentiation of bone mesenchymal stem cells (BMSCs) affected by intermittent traction stretch at different time points and explored the mechanism of osteoblastic differentiation under this special mechanical stimulation. The BMSCs and C3H10T1/2 cells were subjected to 10% elongation for 1-7 days using a Flexcell Strain Unit, and then the mRNA levels of osteoblastic genes and the expression of core-binding factor a1 (Cbfa1) were examined. Furthermore, we focused specifically on the role of the extracellular signal-regulated kinases 1/2 (ERK1/2) and Cbfa1 in the osteogenesis of BMSCs stimulated by the stretch. The results of these experiments showed that the stretch induces a time-dependent increase in the expression of osteoblastic genes. The synthesis of osteoblastic genes was downregulated after the knockdown of Cbfa1 expression by short-interfering RNA. Furthermore, the stress-induced increase in the expression of Cbfa1 mRNA and osteoblastic genes was inhibited by U0126, an ERK1/2 inhibitor. These results indicate that long periods of intermittent traction stretch promote osteoblastic differentiation of BMSCs through the ERK1/2-activated Cbfa1 signaling pathway.”<-couldn’t get full study.

Hydrostatic pressure has been shown to induce more chondrogenic expression in MSCs when co-cultured with chondrocytes.

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture.

“Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.”<-couldn’t get the full study but this one seems to suggest that adult MSCs can upregulated chondrogenic genes by mechanical stimulation.  The details of the mechanical stimulation are left absent in the abstract unfortunately.

A combination of dynamic and shear stress has been used to induce chondrogenic differentiation in adult MSCs.

One tensile strain study at 3000 microstrain found that it upregulated both chondrogenic and osteogenic genes.

Forces induced by LSJL such as tensile strain, dynamic and shear stress, and hydrostatic pressure can induce chondroinduction of MSCs.  Whether these stimuli induce osteo- or chondro-(the ideal) induction may depend on various concentrations of growth factors in the serum(altered by supplements) and properties of the bone itself.

Sometimes I am amazed as what I see when I manage to focus all of my attention on any type of issue related to height, even if the connection is very small. This is what happened yesterday, while I was walking through the famous COEX Mall that is located off of the Samseong Subway Station in Seoul, South Korea. I was having a rough day and I took my GF to the mall to walk around and as we walked around the a mall the sight of a young boy in crutches really caught my eye.

This young boy, who was very young was walking by himself on crutches and had one of his lower legs wearing one of the external fixators which I am so familiar with. The sight was so extraordinary, and when my GF say the metal device that had its metal spoke coming out of his skin, she expressed shock over the fact that this boy was walking around in public which his external fixator. For her, the sight was too disturbing. I felt a little bit of sadness over what this boy was going through. Personally I probably had more of an understanding of why he needed to get this procedure done.

It was clear that this boy was still very young, who was still growing. He was not using the method of distraction osteogenesis to make his legs longer, and look taller. He had a serious medical condition. Most of the procedures of distraction osteogenesis done is to correct for a difference in the length of a certain bone region between the left and right side of a person. You can do callotasis on more than just the long bones, but also the mandibular bone. One of his legs was shorter than the other. To walk with a more normal gait, his leg was being lengthened. I understood that there was a lot of pain associated with the process, but I have never experienced it myself.

This is the first time I have ever seen anyone were an external fixator out in public. However I am not that surprised to see it in a place like Seoul, South Korea. Seoul has been also the same place where I saw a case of a korean women who suffered from gigantism, multiple people suffering from albinism, one case of a person who suffered from Osteogensis Imperfecta (Refer to Sean Stephenson), and various types of achondroplasia and dwarfism. For a country that seems to pride itself on being so homogeneous and image conscious, there are many people who just don’t look ‘average’ whatever that means.

After see the young boy in the crutched, we actually went to donate blood. Apparently blood donating is a popular thing to do these days in Korea. Everyone there in the donation clinic seemed to know what their blood type is, except me. I was once told that my blood type is the most common type there is. The Asian fascination with blood type is something I probably won’t understand very well. It would turn out that my GF’s blood’s plasma is low on Calcium and that her blood did not have the requirements to make her a potential doner. This concerned me greatly, since she has been complaining about how her knees have been hurting for years, the symptom of cold knees, and the desire to be taller. Immediately I realized that there was a clear connection between her lack of calcium, the knee pain, and the cold knees.

How could a women desire to be taller and not even be able to have a sufficient level of Calcium and Vitamin D in her bones and system?

I would immediately take her to the GNC in the mall and tried to stock up on Calcium & Vitamin D 1500 MG as well as Glucosamine Sulphate & Chondroitin 1500 mg. Her resistance on spending $50 on these supplements which would help at least a little with the bone mineral density, combined with her strong desire to become taller, her choice on diet to avoid protein, and her symptom of having intense knee discomfort and cold knees is actually making me realize that she is asking for things in life which are in direct disagreement with each other. She doesn’t know about this website. She doesn’t realize that I have been dedicating for over a year of my life towards this goal, in not just figuring out how to increase height, but also in treating bone and orthopedic disorders. I know how to help her, but she just won’t listen to my advice. Maybe she thinks she is helping me save a little bit of money, but she is risking her own health, and her height by not fixing this problem.

I wrote about the issue of having low Calcium levels leading to severe height loss later in mid age due to menopause in the post The Connection Between Bone Loss From Osteoporosis And Decreases In Height In East Asian Females. The study Usefulness of Estimated Height Loss for Detection of Osteoporosis in Women showed that while a lose of 2-4 cm in height is normal, any more than 4 cms of height loss due to bone density decreases means that osteoporosis has set in. My biggest worry is that not only will she not increase in height, which she desperately desires, due to refusing to listen to my insistence on taking certain supplements to help improve the health of the knees, she is also going to risk getting osteoporosis due to diet choices causing the exact opposite effect for her. 

If she only knew what I know, and saw what I see.

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.