Manipulating ECM stiffness for height increase

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.

2 thoughts on “Manipulating ECM stiffness for height increase

  1. Lucky7

    So is EMC stiffness to do with mineralized state and Lsjl increases stiffness or lowers it, would be good if you could investigate further things on this field, it is of great benefit to the method

    Reply

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