Becoming Taller with chondrocyte apoptosis

We know that chondrocyte apoptosis plays an active role in endochondral ossification.  When chondrocytes undergo apoptosis TGF-Beta may be released into earlier segments of the growth plate maintaining balance.  Apoptosis is an important part of height growth and in fact in fusing growth plates there were no signs found of apoptosis.  Chondrocyte apoptosis may also play a role in exerting force on the growth by water release from apoptotic cells.  When osteoclasts reabsorb the cartilage ECM, the swelling chondrocytes open.  The ECM contains a lot of water.  When the ECM is absorbed(the ECM is hydrophillic so it contains a lot of water) the reduction in water content outside the cell reduces leading to water being released out of the cell to restore equilibrium.  The force of this water being released may be what leads to bone deformation and you becoming taller.

Since fusing growth plates do not show signs of apoptosis and apoptosis may play a key role in the water release in order to make you taller it is clear that chondrocyte apoptosis is important to height growth.  Lack of chondrocyte apoptosis may be signs of senescence as chondrocyte apoptosis may release TGF-Beta in order to keep the height growth train chugging along.

Influence of stress magnitude on water loss and chondrocyte viability in impacted articular cartilage.

"Mature bovine cartilage explants were impacted with peak stresses ranging from 10 to 60 MPa at a stress rate of 350 MPa/s{This is huge, it's hard to get even 1 MPa with a method like LSJL}. Water loss, matrix axial deformation, dynamic impact modulus (DIM), and cell viability were measured immediately after impaction. The water loss through the articular surface (AS) was small and ranged from 1% to 6% with increasing peak stress{this is for the whole cartilage unit and not individual cells}. The corresponding axial strains ranged from 2.5% to 25%, respectively, while the DIM was 455.9 +/- 111.9 MPa. Chondrocyte death started at the articular surface and increased in depth to a maximum of 6% (70 microns) of the cartilage thickness at the highest stress{the deeper depth means more hydrostatic pressure since chondrocyte death stopped occurring at a depth of 6%, the chondrocyte death was likely not due to hydrostatic pressure}. The volumetric (axial) strain was more than twice the amount of water loss at the highest peak stress. Specimens impacted such that the interstitial water was forced through the deep zone (DZ) had less water loss, a higher DIM, and no cell death{So the cell damage could be due to the actual impact force and not to hydrostatic pressure as the region with the most hydrostatic had no cell death}. Matrix compaction in the superficial region [cause] higher compressive strains to occur at the surface rather than in the deeper zones."

Therefore, it is likely that very high impact force causes cell death rather than hydrostatic pressure.  Low levels of repeated impact may induce hydrostatic pressure via oscillatory interstitial fluid flow.  Hydrostatic pressure is likely to just increase the amount of water uptake by the chondrocytes leading to more force generated by chondrocyte apoptosis.

Increasing the osmolarity of joint irrigation solutions may avoid injury to cartilage: a pilot study.

"Saline (0.9%, 285 mOsm) and Hartmann's solution (255 mOsm) are two commonly used joint irrigation solutions that alter the extracellular osmolarity of in situ chondrocytes during articular surgery. [Does] varying the osmolarity of these solutions influences in situ chondrocyte death in mechanically injured articular cartilage{a higher osmolarity means more concentration of the substance per unit of water, so the higher the osmolarity the more water being released by the chondrocytes}? We initially exposed osteochondral tissue harvested from the metacarpophalangeal joints of 3-year-old cows to solutions of 0.9% saline and Hartmann's solution of different osmolarity (100-600 mOsm) for 2 minutes to allow in situ chondrocytes to respond to the altered osmotic environment. The full thickness of articular cartilage then was "injured" with a fresh scalpel. Using confocal laser scanning microscopy, in situ chondrocyte death at the injured cartilage edge was quantified spatially as a function of osmolarity at 2.5 hours. Increasing the osmolarity of 0.9% saline and Hartmann's solution to 600 mOsm decreased in situ chondrocyte death in the superficial zone of injured cartilage{so when more water was released from chondrocytes there was less chondrocyte death}. Compared with 0.9% saline, Hartmann's solution was associated with greater chondrocyte death in the superficial zone of injured cartilage, but not when the osmolarity of both solutions was increased to 600 mOsm."

So as the relative water outside the cell decreases, there is less chondrocyte apoptosis which is bad for the growth plates.

"The extracellular osmolarity experienced by in situ chondrocytes has not been measured directly. Cartilage (with high fixed negative charges) contains a high concentration of free cations (mainly Na+) and a low concentration of free anions (mainly Cl−) compared with surrounding synovial fluid, and its interstitial osmolarity is greater with precise values determined by the local proteoglycan concentrations and the Gibbs-Donnan equilibrium conditions"<-proteoglycan concentrations determine the osmolarity of the environment.  Growth plate chondrocytes don't have synovial fluid but neither do the chondrocytes in this study.

This study however shows a case where higher osmolarity levels resulted in more apoptosis.

Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes.

"Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. Osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. [We] investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. A highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. A 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture."

It could be the different properties of the fluids used in the two stuides that cause different results for example Saline contains a lot of positively charge ions.  Hartmann's solution is considered to be isotonic.  So at higher concentrations those two compounds may have had the effect of balancing the osmolarity rather than creating a hypo- or hyper- osmolarity.

"The interstitial osmolarity of cartilage ranges between 350 and 450 mOsm"

"Apoptosis leads to plasma membrane asymmetry and the externalization of phophatidylserine residues, which bind annexin V with high affinity. In the early stages of apoptosis, cells typically have an intact cell membrane. Thus, they do not stain with propidium iodide, whereas externalization of phophatidylserine can be detected by annexin V. In the late phase of apoptosis, cells stain with both dyes. The control group had more than 90% of intact, living cells and only less than 10% of cells in the early and late phases of apoptosis"

"The 400 mOsm-treated group showed no significant difference in the percentage of living and apoptotic cells compared to the control group. A marked, approximately 10-fold increase of apoptotic cells was observed in the 600 mOsm-treated group with a parallel decrease of viable cells"<-So an increase in osmolarity increases apoptosis.  Remember this study only occurs on chondrocytes so we don't know if there's more MSC differentiation into chondrocytes to compensate.  This is likely possible due to the release of TGF-Beta by apoptotic chondrocytes.

"Hyperosmotic stress leads mainly to apoptotic cell death that involves changes in the apoptotic signaling molecules in a primary chondrocyte cell culture. Hyperosmotic environment induced the activation of proapoptotic signaling factors such as JNK, p38 and caspase-3 and also led to an increased level of ERK1/2"

"Increasing osmotic pressure leads to a slight, transient change in cell growth, rate of protein synthesis and amino acid transport" 

The chondrocyte: a cell under pressure.

"The composition of cartilage reflects the net response of the chondrocytes to the prevailing [mechanical] loading pattern, with cartilage proteoglycan content highest in heavily loaded regions and removal of load leading to cartilage thinning and proteoglycan loss. Chondrocytes react to cartilage deformation and to the changes in hydrostatic pressure, extracellular ionic composition and streaming potentials induced by the load."

"When load is applied to cartilage, the tissue deforms as does the cell, leading to a rise in the hydrostatic pressure of the matrix within.  If the load is removed immediately, cartilage returns to its original conformation and pressure falls. However, cartilage is an osmotic system, and application of load disturbs the osmotic balance. Thus if load is maintained for any length of time, fluid is expressed in an attempt to restore osmotic equilibrium"

"Load deforms the matrix and chondrocyte, hydrostatic pressure rises, fluid is expressed increasing the extracellular concentration of proteoglycans and hence of cations."  Cell volume of the chondrocyte decreases.  Note we are trying to do this with the bone marrow and stem cells.  However, deformation of stem cells and bone marrow may lead to an increase in proteoglycans and differentiation into chondrocytes thus inducing height growth.  However, LSJL may delay chondrocyte apoptosis due to a reduction in cell volume when the growth plates are present.  However, it also increases extracellular concentration of proteoglycans leading to an eventual greater amount of water force expelled when the chondrocytes do undergo apoptosis.

Osmolarity affects chondrocyte apoptosis.  A high extracellular osmolarity may delay cellular apoptosis but may make the force of when apoptosis finally occurs stronger.

Some studies say that chondrocyte apoptosis is not necessary and chondrocytes can differentiate into pre-osteoblastic cells.  But either chondrocyte apoptosis is still important either to prevent it or to maximize the amount of water absorbed before apoptosis occurs.

Apoptosis may also be delayed by IGF-1 which is unusual considering IGF-1 increases chondrocyte hypertrophy(and other cells as well).  IGF-1 may delay apoptosis until hypertrophy is maximized.

Effect of exogenous IGF-1 on chondrocyte apoptosis in a rabbit intraarticular osteotomy model.

"Insulin-like growth factor-1 (IGF-1) [protects] chondrocytes from apoptosis in vitro. IGF-1 expression may also assist in maintaining a fully differentiated chondrocyte phenotype{Good for LSJL if we can find supplements to increase IGF-1, IGF-1 increases p-Akt which also inhibits apoptosis}. Administration of IGF-1 after fracture inhibits apoptosis in vivo. Twenty-four mature female New Zealand white rabbits were randomized to control and IGF-1 groups. All subjects underwent standardized medial femoral condyle fracture and repair. Fibrin clot was administered in all subjects, with 25 mcg/ml IGF-1 in the clot in half the subjects. Half of the animals in each group were sacrificed at 2 weeks and half at 4 weeks. Two-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects (21 +/- 6 vs. 12 +/- 6). Likewise, 4-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects{although we don't know if the IGF-1 subjects would eventually catch up or the IGF-1 hypertrophic cells differentiate directly into bone cells rather than undergoing apoptosis, in this study the 4 week IGF-1 don't show more apoptosis than 2 weeks but we don't know if this applies to the growth plates as this study involved an injury} (23 +/- 7 vs. 10 +/- 2). There was no significant administration difference between 2-week control and 4-week control subjects, or between 2-week IGF-1 and 4-week IGF-1 subjects. Intraarticular IGF-1 at the time of fracture repair appears to inhibit chondrocyte apoptosis in vivo, as judged by TUNEL staining, in this animal model. Administration of IGF-1 [may] inhibit human chondrocyte apoptosis in vivo"

"[IGF-1 stimulates] the addition of sulfated glycosaminoglycans (GAGs) to aggrecan molecules that will ultimately form the proteoglycans that make up much of the matrix of articular cartilage[and growth plate]. IGF-1 acts throughout skeletal immaturity to stimulate the proliferation of epiphyseal chondrocytes[this likely occurs indirectly through IGF-1's ECM stimulating properties] and thereby direct the linear growth of bones"<-IGF-1 stimulates ECM, ECM stimulates cellular proliferation and slows down chondrocyte apoptosis.

"During adulthood IGF-1 continues to regulate the homeostasis of articular cartilage by stimulating chondrocytes to produce GAGs and type II collagen. IGF-1 counteracts matrix degradation and chondrocyte apoptosis"<-this is true for growth plate cartilage too.

"Exogenous IGF-1 administration in an equine osteochondral defect model stimulates a response in chondrocytes implying that exogenous exposure of IGF-1 may extend and amplify endogenous IGF-1 response to articular injury"<-IGF-1 administration or indirect ways of increasing IGF-1 will amplify IGF-1 levels.

Since this study involves an injury a complete relationship cannot be drawn to growth plate chondrocytes but we know that IGF-1 inhibits(or delays) apoptosis.

Once chondrocytes have undergone autophagy or apoptosis they cannot dedifferentiate from bone into chondrocytes as they no longer exist.  This must be a key regulatory mechanism to prevent ectopic growth plate formation.

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate.

"The terminally differentiated [hypertrophic chondrocyte] cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis{if some cells of the cartilage plate undergo transdifferentiation then we can use those transdifferentiated cells that should maintain some of the epigenetic characteristics of epiphyseal chondrocytes to form new growth plates}. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens.  Hypertrophic cells [may] die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis."

"Pi uptake is required for activation of chondrocyte apoptosis, and specific Na-Pi transporters were identified in growth plate chondrocytes. When these transporters were inhibited, apoptosis was blocked. Ca2+ [is] a key cofactor in the induction of chondrocyte apoptosis"

The fate of the terminally differentiated chondrocyte: evidence for microenvironmental regulation of chondrocyte apoptosis.

"Chondrocytes contained within the epiphyseal growth plate promote rapid bone growth. To achieve growth, cells activate a maturation program that results in an increase in chondrocyte number and volume and elaboration of a mineralized matrix; subsequently, the matrix is resorbed and the terminally differentiated cells are deleted from the bone. The terminally differentiated epiphyseal cells are deleted from the cartilage by apoptosis. Indeed, morphological, biochemical, and end-labeling techniques confirm that death is through the apoptotic pathway. Since the induction of apoptosis is spatially and temporally linked to the removal of the cartilage matrix, current studies have examined the apoptogenic activity of Ca(2+)-, Pi-, and RGD-containing peptides of extracellular matrix proteins. All of these molecules are powerful apoptogens. With respect to the molecular mechanism of apoptosis, Pi [the] apoptogen anion is transported into the cytosol via a Na(+/)Pi transporter. Subsequently, there is activation of caspases, generation of NO, and a decrease in the thiol reserve. Specific microenvironments exist in cartilage that can serve to direct chondrocyte apoptosis."

"The most superficial region, closest to the joint surface, is the reserve or resting cell zone. In this region, spherical chondroprogenitor cells, embedded in an extensive extracellular matrix, are present. Below the reserve cell zone is the proliferative layer. Here, the chondroprogenitors generate columns of chondrocytes (5-8 cells deep in mammalian plates; 15-30 deep in avian cartilage). These cells divide in the long axis of the plate so that flattened mother and daughter cells appear to lie on top and slightly to the side of each other. The shape of the cells suggests that they are under axial loading. After a limited number of rounds of proliferation, the cells change their shape and phenotype. These maturing chondrocytes become swollen and express type X collagen as well as type II collagen; in addition, they exhibit high alkaline phosphatase activity. From a functional viewpoint, these hypertrophic chondrocytes secrete membrane-limited vesicles (matrix vesicles) that serve to initiate mineralization of the extracellular matrix. As the cells become hypertrophic, they increase their volume five- to 12-fold and elevate three-fold the total amount of territorial extracellular matrix. When terminally differentiated, the increase in cell volume and the presence of a bulky extracellular matrix provide much of the space required for bone elongation. At the calcified end of the growth plate, trabeculae of woven bone are deposited on the calcified cartilage septa."

"Osteoblasts often occupy chondrocyte lacunae [thus providing some evidence for transdifferentiation]"

"Hypertrophic chondrocytes have been reported to continue to synthesize type I collagen, glycosaminoglycans, and glycolytic and oxidative enzymes and phosphatases"

"terminally differentiated hypertrophic chondrocytes expressed type II collagen, osteopontin, osteocalcin, osteonectin, and aggrecan core-binding protein mRNA."

"when immature sternal chondrocytes were challenged with an apoptogen, they were far more resistant to the induction of apoptosis than were hypertrophic cells"

"treatment of hypertrophic tibial chondrocytes with Pi induced death in a dose- and time-dependent manner. Within 48 hours, 3 mM Pi increased chondrocyte apoptosis by 30%; lower concentrations of Pi induced death after 48 hours. More recently, it was shown that this effect is in large part dependent on another apatitic ion, Ca2+. A modest increase in the Ca2+ concentration from 1.9 to 2.3 mM caused a dramatic increase in chondrocyte death. At a Ca2+ level of 2.8 mM, a small rise in the Pi concentration promoted rapid cell death. Since Ca2+ alone, even at concentrations of 2.8 mM, did not influence the rate of killing, it was concluded that the concentration of the ion pair served as a primary death signal."<-Since hypertophic chondrocytes are so sensative to apoptosis maybe inhibiting this apoptosis can enable these chondrocytes to hypertrophy for longer and enable you to grow taller.

"A null mutation in the MMP-9/gelatinase B gene caused an abnormal pattern of skeletal growth. While terminal differentiation appeared to be normal, apoptosis was delayed, resulting in an eight-fold lengthening of the growth plate."  It's unclear from the study whether this translated into increased adult bone length.

Hyperbaric oxygen treatment prevents nitric oxide-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

"Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on interleukin-1β (IL-1β)-induced NO production and apoptosis of rabbit chondrocytes and healing of articular cartilage defects. For the in vitro study, RT-PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL-1β-treated chondrocytes. To clarify that the HSP70 was necessary for anti-iNOS and anti-apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO-induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70."

Apoptosis of growth plate chondrocytes occurs through a mitochondrial pathway.

"Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated.

Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA."

"[Apoptotic] cells are characterized by shrinkage of their cytoplasm, condensation of chromatin, blebbing, and formation of vesicles containing the remnants of the cell (apoptotic bodies) that are engulfed by macrophages. During this highly regulated process, there is activation of proteases that lead to cleavage of enzymes and structural proteins and, ultimately, to DNA fragmentation, the hallmark of apoptosis"

"[Alterations occurred in] chondrocytes exposed to 5 mM Pi. Their mitochondria accumulated in the perinuclear region of the healthy cell. Chondrocytes maintained this appearance for the first 4 hours of Pi treatment. Approximately 8 hours after the initiation of treatment, chondrocytes presented signs of stress and began to loose attachment to the glass coverslips; numerous vesicles could be observed in their cytoplasm. While remaining functional, the mitochondria presented a different cellular distribution. After 24 hours of Pi exposure, chondrocytes had shrunk into smaller cellular masses without loosing their membrane integrity. Mitochondria lost their membrane potential. Some of the vesicles in the cytoplasm of these cells contain DNA, the result of endonuclease activity "

"When serum-free media was supplemented with 5 mM Pi, we observed a larger increase in cytochrome c in the cytoplasm fraction. As expected, cytochrome c content was greater in the cell fraction containing mitochondria."

"Release of cytochrome c into the cytosol results in caspase 3 activation and starts a cascade of protease activities (caspases and endonucleases) that ultimately leads to DNA degradation and death. Supplementing serum-free media with 5 mM Pi for 24 hours caused a significant increase in caspase 3 activity in chondrocytes. When the cells were treated with PFA (an inhibitor of Pi cellular transport) before exposure to Pi, caspase 3 activity remained low. "