Growth Plates: They don't fuse

Note:  I am still a little sick.  I will start with pictures again this Thursday.  Virus is a confounding variable as the body may with-hold anabolic factors to stop the virus.  I'm still working out and performing LSJL but I'm waiting until I'm healthier to start measuring.  Also this page is inaccurate as the hyaline cartilage growth plate line does eventually fuse into bone but that does not inhibit height growth by new growth plate formation. 

Look at this page.  It's a pretty interesting explanation of all the ways that growth occurs but I want you to take note of one sentence:

6). The epiphysis ossify.

*Hyaline cartilage remains on the growth plate and the articulating surface.

The hyaline cartilage is not what's hypertrophying(differentiating) and then being ossified.  During primary ossification, stem cells turn into new chondrocytes(differentiate into chondrocytes) and then those chondrocytes are fused into bone.  The hyaline cartilage does not fuse into bone because there is already an extracellular matrix.  Stem cells within or transported to the hyaline cartilage by methods such as LSJL differentiate into chondrocytes.  Those chondrocytes hypertrophy and those chondrocytes are ossified into bone.  The chondrocytes within the hyaline cartilage do hypertrophy and eventually ossify.

Bone formed at the primary and secondary ossification centers push the articular(hyaline) cartilage away.  Some being at the end of the bone and others at the growth plate line.  The reason that growth doesn't occur indefinitely is a lack of stem cells in the hyaline cartilage growth plate line which is what LSJL tries to rectify.  The hyaline cartilage environment has genetic markers that shape the stem cells into hypertrophying and differentiating chondrocytes.  Methods like IGF-1 increase height growth by increasing stem cell proliferation thereby increasing growth.

Now, the reason that a growth plate line does not remain after fracture healing like in limb lengthening surgery is that in fracture healing endochondral ossification occurs as a result of periosteal progenitor cells which differentiate into chondrocytes and so on.  There's never any hyaline cartilage matrix so the entirety of the homeless chondrocytes are ossified.

So you see growth plates don't fuse.  New chondrocytes are created from stem cells and those chondrocytes are ossified.  Not the ones in the growth plate.

Mechanisms of Growth Plate Maturation and Epiphyseal Fusion

"Longitudinal growth occurs within the long bones at the growth plate. During childhood, the growth plate matures, its total width decreases and eventually it disappears at the end of puberty with complete replacement by bone along with cessation of longitudinal growth. The exact mechanism of epiphyseal fusion is still not completely understood and experimental studies are complicated by the fact that there is a species difference between humans and rabbits that do fuse their growth plates and rodents that do not. This mini review summarizes hypotheses and theories postulated in the literature regarding growth plate maturation and epiphyseal fusion. Growth factors, local regulators and hormones involved in growth plate maturation are described as well as four postulated hypotheses and theories regarding the final steps in epiphyseal fusion: apoptosis, autophagy, transdifferentiation and hypoxia. A better insight into the mechanisms of epiphyseal fusion may ultimately help to develop new strategies for the treatment of cartilage and growth disorders. "

Remember that growth plates have to do more than merely transform into bone otherwise they would never increase in length they have to deform the bone into a longer shape which is why I think apoptosis is the most logical final step as that could generate force deforming the bone.

"Senescence is a term for the structural and functional changes over time in the growth plate, such as a gradual decline in the overall growth plate height, proliferative zone height, hypertrophic zone height, size of hypertrophic chondrocytes and column density"<-senescence is what we try to prevent not fusion

"A new hypothesis is that proliferation is influenced by a multiorgan genetic program and that proliferation declines when this genetic program has reached a critical point"<-although this would not explain individuals of different proportions

"A most widely held hypothesis is that at the chondro-osseous junction site of the growth plate, terminally hypertrophic chondrocytes die by undergoing apoptosis leaving behind a scaffold of cartilage matrix for osteoblasts that invade and lay down bone[this apoptosis also releases water that generates force to induce bone deformation]. It is assumed that the same mechanism eventually also results in epiphyseal fusion. Studies in the rat showed that apoptosis-regulating proteins (the so-called caspases, which are cysteine proteases) are expressed in the growth plate and that there is an increased expression of proapoptotic factors with age. Typical morphological changes when cells undergo apoptosis include cell shrinkage with intact organelles and integrity of membranes, pyknotic nuclei by aggregation of chromatin, fragmented DNA, partitioning of the cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies) and absence of an inflammatory response. Interestingly, several recent studies failed to demonstrate a typical apoptotic appearance in the terminal hypertrophic chondrocytes and, therefore, these studies have questioned whether apoptosis is the final mechanism through which chondrocytes die in the terminal hypertrophic zone. Furthermore, we recently analyzed a unique piece of fusing human growth plate tissue during epiphyseal fusion and were not able to find signs of classical apoptosis"<-Note that if apoptosis is a critical role during endochondral ossification than it is logical not to find signs of apoptosis during fusion as this stage of growth may release factors to encourage further growth.  It may be at time of senescence apoptosis ceases and other forms of cell death occur.

"The oldest hypothesis is that at the chondro-osseous junction site of the growth plate terminal hypertrophic chondrocytes can transdifferentiate into osteoblasts. This theory is based on mostly organ and cell culture models, like for example chondrocytes in mice and murine metatarsal bone cultures that were able to transdifferentiate into osteoblasts producing bone matrix. Adams and Shapiro discussed that evidence in support of transdifferentiation is mostly circumstantial. It is based on microscopic examination of chondrocyte and osteoblast populations at the chondro-osseous junction and results from different studies are inconsistent. Although direct evidence is lacking, others speculate that transdifferentiation is present at the chondro-osseous junction because terminally differentiated cells are producing collagen type 1 together with extracellular matrix factors. In addition, to our knowledge human studies on transdifferentiation at the chondro-osseous junction in the growth plate have not been described."<-some chondrocytes may transdifferentiate into osteoblasts whereas others may undergo apoptosis.  Transdifferentiation is unlikely to induce bone deformation.

"In a unique human growth plate tissue specimen in the process of undergoing epiphyseal fusion, we observed a dense border of thick bone surrounding growth plate remnants at the site where normally the growth plate is located[just because apoptosis is not involved during fusion does not mean that it may not be involved in earlier stages of development]. In addition, signs of hypoxia and early necrosis were found. We postulated that the border of dense bone might function as a physical barrier for oxygen and nutrients to reach the fusing growth plate resulting in hypoxia and eventually cell death in a nonclassical apoptotic way through necrosis or a mixture of apoptosis and necrosis[if this is the case than osteoclasts can help continue endochondral ossification by degrading this dense bone]. In line with this new hypothesis, White et al recently demonstrated bridging bone in the center of a distal human tibial growth plate obtained from a 12.9-year-old girl, which might be an early sign of this shelling process. Signs of a hypoxia-related process were also reported by Stewart et al who observed an upregulated expression of hypoxia-inducible factor 2α mRNA during chick and murine chondrocyte differentiation in vitro. Hypoxia-inducible factor 2α knockout mice are small, which might indicate that this gene has an important role in the growth plate and subsequently in the regulation of longitudinal growth. Thus, epiphyseal fusion might be a hypoxia-related process leading eventually to cell death of growth plate chondrocytes."

It is likely that all four methods are involved with apoptosis being the most important but apoptosis is likely not present near fusion.

Growing Pains: The Need for Engineered Platforms to Study Growth Plate Biology

"Growth plates, or physis, are highly specialized cartilage tissues responsible for longitudinal bone growth in children and adolescents. Chondrocytes that reside in growth plates are organized into three distinct zones essential for proper function. Modeling key features of growth plates may provide an avenue to develop advanced tissue engineering strategies and perspectives for cartilage and bone regenerative medicine applications and a platform to study processes linked to disease progression. In this review, a brief introduction of the growth plates and their role in skeletal development is first provided. Injuries and diseases of the growth plates as well as physiological and pathological mechanisms associated with remodeling and disease progression are discussed. Growth plate biology, namely, its architecture and extracellular matrix organization, resident cell types, and growth factor signaling are then focused. Next, opportunities and challenges for developing 3D biomaterial models to study aspects of growth plate biology and disease in vitro are discussed. Finally, opportunities for increasingly sophisticated in vitro biomaterial models of the growth plate to study spatiotemporal aspects of growth plate remodeling, to investigate multicellular signaling underlying growth plate biology, and to develop platforms that address key roadblocks to in vivo musculoskeletal tissue engineering applications are described."

"the osteochondral interface is a transitional zone tissue that is composed of articular cartilage (90%), a calcified cartilage transitional tissue (5%), and subchondral bone (5%)."

"The calcified cartilage transitional zone is a permanent ≈20–250 µm thick layer that joins articular cartilage to subchondral bone in joints. This calcified cartilage is 10–100 times stiffer than articular cartilage but less stiff than subchondral bone when there is the same mineral content, and serves to reduce stress between mechanically dissimilar articular cartilage and bone. Another example of a transitional tissue is the enthesis, a permanent fibrocartilage tissue that joins connective tissue (e.g., tendons and ligaments) to bone to facilitate joint motion."

"The enthesis is a stratified fibrocartilaginous tissue ≈250–500 µm thick that contains gradients in cell phenotype, biochemical cues, mineral content, as well as matrix composition and alignment. Bone is 100 times stiffer than tendon and ligament, and the gradient within the enthesis protects it from failure by minimizing the strain concentrations between mechanically dissimilar tissues."

"Growth plates, or physis, are highly organized cartilage tissues ≈800–1000 µm thick that lie between the epiphysis and metaphysis at the ends of most long bones. However, unlike the osteochondral interface and tendon-bone or ligament-bone entheses which are permanent transitional tissues, growth plates are temporary cartilage tissues involved in development and growth that disappear at skeletal maturity in humans."

" growth plates are avascular and aneural—relying on the vascular systems of the adjacent bone tissue to maintain chondrocyte proliferation{manipulating the vascular system may be a way to grow atller for longer}—and are composed of chondrocytes embedded in an extracellular matrix"

"Cranial vault sutures differ from the growth plates of the long bones in that they do not facilitate bone growth via a cartilage intermediate (i.e., endochondral ossification). Rather, cranial vault sutures facilitate bone growth via a process of intramembranous ossification where bone growth occurs directly by mesenchymal stem cell differentiation in response to extrinsic growth signals caused by the expanding brain. Growth plates in the long bones facilitate bone growth via endochondral ossification and a cartilage intermediate, and possess intrinsic growth potential (i.e., no external signal is required)."

"Compositionally, bone is composed of inorganic calcium phosphate mineral (50–70%), organic matrix (i.e., collagen, 20–40%), water (5–10%), and lipids (<3%)."<-so calcium has the power to powerfully change bone.

" Long bones receive blood supply from arteries in the metaphysis and epiphysis, periosteal arteries, and the central nutrient artery. A dense capillary system within bone tissue is responsible for distributing oxygen and nutrients to bone resident cells: osteoblast, osteocytes, and osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and they deposit collagen proteins that compose the bone extracellular matrix and are responsible for mineralization of these proteins. Osteoblasts terminally differentiate into osteocytes, bone cells that are embedded in the extracellular matrix and sense mechanical strain. Osteoclasts derive from mononuclear monocyte-macrophage precursor cells, and they are responsible for resorption of the mineralized bone matrix. Bone is constantly remodeled and maintained by reciprocal interactions mediated by osteoblasts and osteoclasts that are mediated through growth factor signaling. Notably, fibroblast growth factors (FGFs), transforming growth factor beta (TGFB), bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), and platelet-derived growth factor (PDGF) are all critical for bone homeostasis"

"Skeletal stem cells have been identified in the growth plates, and subpopulations of these cells can generate chondrocytes and osteocytes, but not adipocytes"

"he molecular mechanism of bony bridge formation, and their work suggests bony bridge formation begins immediately following injury. Namely, they identified pro-osteoblastic factors following growth plate injury including interleukin-6, bone morphogenic protein 2, and osteoprotegerin. The premature mineralization of the extracellular matrix and early osteogenic differentiation of mesenchymal and preosteoblast cells in the growth plate cartilage following a fracture leads to improper skeletal development"

"The resting zone cartilage is important for orienting the developing growth plates by producing a “growth plate-orienting factor” that may direct cells to align parallel to the long-axis of the bone. Additionally, vasculature originating in the epiphysis provides nutrients to chondrocytes in this zone."

"The middle layer of the growth plate is called the proliferative zone, and it primarily functions as a site of matrix production and cellular proliferation.  In the proliferative zone, chondrocytes appear flattened, begin to divide, and are arranged in columnar structures parallel to the long bones.  The rearrangement of chondrocytes into columns requires cell–cell adhesion and that chondrocytes elongate to take up space, and this is indicative of cell spreading event.  [In the proliferative zone there's no] cell separation, cell body contraction, or a leading edge which would have been an indication of cell migration.  Thus, they assert that chondrocyte rearrangement into columnar structures is more like cell spreading than cell migration.  The columnar architecture in the proliferative zone specifies the primary direction of growth in long bones. The long axis of the flattened chondrocytes is perpendicular to the long axis of the bone, and this columnar structure is maintained via cell–cell adhesion."

"n the hypertrophic zone, chondrocytes begin to separate from one another, become rounded, and expand in size. Chondrocytes in this zone enlarge to four times the size of chondrocytes in the proliferative zone, stop proliferating, and undergo apoptosis. Terminal differentiation of chondrocytes is associated with an increase in cell volume, intracellular calcium concentration, alkaline phosphatase enzyme activity, and synthesis and secretion of Collagen X.  Hypertrophic chondrocytes direct mineralization of the surrounding matrix and attract blood vessels and chondroclasts from the metaphysis. Blood vessels from the metaphysis invade the cartilage of the hypertrophic zone, form new vascular channels via angiogenesis, and provide access for other cell types involved in bone formation. Differentiating osteoblasts and osteoclasts invade the hypertrophic region via the perichondrium and remodel the cartilage tissue into bone tissue. This remodeling leads to new bone formation underneath the growth plate and thus bone elongation. There is some evidence indicating chondrocytes trans-differentiate into osteoblasts during vascular invasion of this zone. The hypertrophic zone is the weakest and is most likely to have a cleavage plane pass through leading to growth plate injury."

"compressive loading is detrimental [to the growth plate] when applied statically or dynamically; although, dynamic loading appears to be less detrimental than static loading [but loading is needed for proper growth plate development in general]"

"The primary extracellular matrix proteins in the growth plate are aggrecan, collagen, and hyaluronic acid"

"matrix vesicles containing mineral are deposited in the hypertrophic zone and serve as nodes for mineralization and subsequent bone formation. Aggrecan, a chondroitin sulfate proteoglycan, is the primary proteoglycan in growth plate cartilage and plays a structural role in the extracellular matrix. Aggrecan expression is upregulated during chondrocyte differentiation and its presence is important for chondrocyte-matrix and chondrocyte-chondrocyte interactions."

"Aggrecan plays an important role in growth factor regulation and signaling and is essential for chondrocyte organization, morphology, and survival during limb development."

"The glycosaminoglycans contained within aggrecan create a negatively charged molecule that binds growth factors required for chondrocyte maturation such as Indian hedgehog and this negative charge enables hydration of cartilage tissue."

"Aggrecan and hyaluronic interactions are stabilized by a link protein that interacts with both molecules, with hyaluronic acid necessary for chondrocyte-matrix stabilization. Free hyaluronic acid, synthesized by hypertrophic chondrocytes, is present in higher concentrations in the hypertrophic zone. Free hyaluronic acid is present between chondrocytes and their immediate extracellular matrix (i.e., sulfated proteoglycans, aggrecan), though the amount of free hyaluronic acid in the pericellular space increases as chondrocytes mature."

"CCN2 stimulates the proliferation and differentiation of growth plate chondrocytes.  CCN2 promotes endochondral ossification by acting on chondrocytes, osteoblasts, and osteoclasts."

"estrogen-resistant patients (i.e., patients with mutations in the estrogen receptor gene) grow into adulthood and lack proper growth plate fusion"

"estrogen can stimulate growth hormone and insulin-like growth hormone expression to further stimulate longitudinal bone growth."

"FGF receptor 3 (FGFR3) inhibits growth plate chondrocyte proliferation and limits the lengthening of the long bones"

"Growth plates are avascular and hypoxic, thus hypoxia-inducible factors are likely critical for survival, maintenance, and differentiation of growth plate chondrocytes. HIF1α is expressed in the hypoxic regions of the growth plates: the center portion of the proliferative zone and the upper portion of the hypertrophic zone. HIF1α enhances the expression of chondrocyte marker Sox9 and extracellular matrix components Collagen II and Aggrecan. HIF1α is also required for differentiation of hypoxic prechondrogenic cells. The primary hypothesis in cartilage biology is that hypoxia modulates differentiation and condensation of mesenchymal stem cells (MSCs) into chondrocytes by activating HIF1α. A downstream target of HIF1α is vascular endothelial growth factor, which is discussed in a later section. HIF1α is another excellent target for incorporation into specific zones where it is most relevant"

"Runt-related transcription factor 2 (RUNX2) drives proliferative chondrocytes to differentiate into hypertrophic chondrocytes.  RUNX2 is expressed in the proliferative and hypertrophic zones of the growth plate, and it is needed for osteoblast differentiation and chondrocyte maturation in osteogenesis. RUNX2 expression is regulated by PTHrP and induces Ihh signaling. RUNX2 is highly expressed in perichondral cells and in osteoblasts. RUNX2 also regulates chondrocyte proliferation via the upregulation of Anthrax toxin receptor 1 (ANTXR1).  Endochondral ossification proceeds normally in ANTXR1 negative mice, but they have shorter femur and tibia bones compared to wild-type mice."