A recent post by Michael stated that one of the obstacles to LSJL success is that hydroxyapatite crystals have sharp edges and that could result in the damage to the cells of any microgrowth plate possibly induced by LSJL.

First, Shear Strain and Fluid Flow induced by LSJL may disrupt the crystals.  However, let’s look at the science of Hydroxyapatite crystals and cell death to chondrocytes.

Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation.

“Basic calcium phosphate (BCP) crystals, including octacalcium phosphate (OCP), carbonated-apatite (CA) and hydroxyapatite (HA) crystals are associated with destructive forms of osteoarthritis.  We assessed the ability of BCP to induce changes in intracellular calcium (iCa(2+)) content and oscillations and the role of iCa(2+) in BCP-induced cartilage degradation.

Bovine articular chondrocytes (BACs) and bovine cartilage explants (BCEs) were stimulated with BCP or monosodium urate (MSU) crystals. iCa(2+) levels were determined. mRNA expression of matrix metalloproteinase 3 (MMP-3), a disintegrin and metalloprotease with thrombospondin-like motifs 4 (ADAMTS-4) and ADAMTS-5 was assessed. Glycosaminoglycan (GAG) release was measured in the supernatants of BCE cultures.

All three BCP crystals significantly increased iCa(2+) content. OCP also induced iCa(2+) oscillations. Rate of BACs displaying iCa(2+) oscillations increased over time, with a peak after 20 min of stimulation. OCP-induced iCa(2+) oscillations involved both extracellular Ca(2+) (eCa(2+)) influx and iCa(2+) stores. Indeed, OCP-induced iCa(2+) oscillations decreased rapidly in Ca(2+)-free medium. Both voltage- and non-voltage-dependent Ca(2+) channels were involved in eCa(2+) influx. BCP crystal-induced variation in iCa(2+) content was associated with BCP crystal-induced cartilage matrix degradation. iCa²(+) was not associated with OCP crystal-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5.

BCP crystals can induce variation in iCa(2+) content and oscillations in articular chondrocytes. BCP crystal-induced changes in iCa(2+) content play a pivotal role in BCP catabolic effects on articular cartilage{and potentially possibly LSJL induced micro-growth plates}.”

“BCP crystal deposition in knee articular cartilage is associated with cartilage destruction, more severe clinical symptoms, and chondrocyte phenotype changes towards hypertrophy as suggested by increased expression of type X collagen and greater ability to produce BCPs in vitro”<-So the BCP crystals within the bone will encourage the chondrocytes to hypertrophy.

“BCP crystals may stimulate articular cells through two mechanisms. They can first activate cells as endocytosed or phagocytosed particles leading to intralysosomal crystal dissolution with subsequent elevation of intracellular Ca2+ levels and release of inflammatory cytokines. The other mechanism of cell activation by Ca2+ crystals involves a direct crystal–cell membrane interaction”

“In articular chondrocytes, BCP crystals induce increased DNA synthesis and cell division{this is anabolic}, IL-1β mRNA overexpression, nitric oxide and MMP-13 production, increased caspase-3 activity and chondrocyte apoptosis”

“iCa2+ was involved in OCP-induced proteoglycan degradation but not OCP-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5.”

This next study relates to HA crystals and causing apoptosis in another type of cell in bone osteoblasts.

Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts.

“Hydroxyapatite nanoparticles (nano-HAP) have been reported to cause inflammatory reactions. Here, we aimed to compare the effects of four types of nano-HAP with different nanocrystal morphologies (short rod-like, long rod-like, spherical or needle-shaped crystals) and sizes (10-20, 10-30 or 20-40 nm) on growth inhibition and apoptosis in primary cultured rat osteoblasts. The osteoblasts was treated with the four types of nano-HAP at various concentrations (20, 40, 60, 80 or 100 mg/l).  All four types of nano-HAP inhibited the growth of osteoblasts in a dose-dependent manner. These nano-HAP significantly induced apoptosis in osteoblasts. Nano-HAP with smaller specific surface areas induced lower apoptosis rates. The needle-shaped and the short rod-like particles induced greater cellular injury than the spherical and long rod-like particles, respectively. The increased apoptosis rates were accompanied by increased p53 and cytochrome c expression. nano-HAP inhibit the activity of osteoblasts and also induce the apoptosis of osteoblasts in vitro. The nano-HAP-induced apoptotic pathway is mediated by a mitochondrial-dependent pathway. Moreover, the sizes, morphologies and concentrations of nano-HAP have significant effects on the apoptotic level.”

“nano-HAP with diameters less than 100 nm can cause inflammatory reactions, especially when the particles are needle-shaped”

nano-HAP increases caspase 3 and 9 and increases Bax levels while decreasing Bcl2.  The first three being pro-apoptotic proteins and the last being anti-apoptotic.

If HA crystals affect osteoblasts it’s likely they would affect chondrocytes too.  However, osteoblasts manage to survive in bone tissue despite the existence of HA crystals.

Annexin 5 overexpression increased articular chondrocyte apoptosis induced by basic calcium phosphate crystals.

“Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis. In advanced osteoarthritis, cartilage shows chondrocyte apoptosis, overexpression of annexin 5 (A5) and BCP crystal deposition within matrix vesicles.

Apoptosis was induced by BCP crystals, tumour necrosis factor (TNF)-alpha (20 ng/ml) and Fas ligand (20 ng/ml) in normal articular chondrocytes (control) and in A5 overexpressed chondrocytes, performed by adenovirus infection. Apoptosis was assessed by caspase 3 (Cas3) activity, and DNA fragmentation.

All BCP crystals, TNF-alpha and Fas ligand induced chondrocyte apoptosis as demonstrated by decreased cell viability and increased Cas3 activity and DNA fragmentation. TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling)-positive staining chondrocytes were increased by OCP (12.4 (5.2)%), CA (9.6 (2.6)%) and HA (9.2 (3.0)%) crystals and TNF-alpha (9.6 (2.4)%) stimulation compared with control (3.1 (1.9)%). BCP crystals increased Cas3 activity in a dose-dependent fashion. BCP-crystal-induced chondrocyte apoptosis was independent from TNF-alpha and interleukin-1beta pathways but required cell-crystal contact and intralysosomal crystal dissolution. Indeed, preincubation with ammonium chloride, a lysosomal inhibitor of BCP crystal dissolution[dissolved], significantly decreased BCP-crystal-induced Cas3 activity. Finally, overexpression of A5 enhanced BCP crystal- and TNF-alpha-induced chondrocyte apoptosis.

Overexpression of A5 and the presence of BCP crystals observed in advanced osteoarthritis contributed to chondrocyte apoptosis.”

So, Chondrocyte Apoptosis doesn’t occur unless the crystal is dissolved.  But it’s possible this may occur as a result of shear strain due to LSJL.    However, the apoptosis induced is not complete and wouldn’t totally inhibit micro-growth plate formation due to LSJL.

“Chondrocytes undergo apoptosis after exposure to NO or Fas ligand (Fas-L).”

“Annexins are ubiquitous proteins that can interact with acid phospholipids, membranes and cytoskeleton constituents in the presence of Ca2+. They are involved in regulating intracellular and extracellular activities such as endocytosis and exocytosis and Ca2+ fluxes. Chondrocytes produce annexins 2, 5 and 6 (A2, A5 and A6), whose levels are increased in OA cartilage. A2, A5 and A6 have been identified on matrix vesicles. A5 can form voltage-gated Ca2+ channels and mediates Ca2+ influx into matrix vesicles, which initiates extracellular mineralisation, and into cellular cytoplasm, which induces apoptosis of growth-plate chondrocytes”

” chondrocyte apoptosis induced by BCP crystals was independent from elevations in extracellular calcium and/or phosphate concentrations but required direct cell-crystal contact.”

“[BCP induced chondrocyte apoptosis requires] cell-crystal contact, crystal endocytosis and intralysosomal crystal dissolution responsible for intracellular Ca2+ elevation.

Hydroxapatite crystals require too many things to go wrong to induce apoptosis in cells to likely occur in a normal physiological environment.  And if they did they would cause apoptosis to a variety of cells not just chondrocytes so you would want to eliminate them regardless.

Thus, I do not believe that HA Crystals or BCP crystals are a hindrance of micro-growth plate formation.

Unfortunately, as of now there is no clear direction on how to manipulate the circadian clock to alter longitudinal bone growth.

Prolonged bioluminescence monitoring in mouse ex vivo bone culture revealed persistent circadian rhythms in articular cartilages and growth plates.

“we revealed a robust and extremely long-lasting circadian rhythm in ex vivo culture maintained for over six months from the femoral bone of a PERIOD2(Luciferase) mouse{How do we manipulate or re-initiate this circadian cycle for height growth?}. Furthermore, we also identified robust circadian clocks in flat bones. High- or low-magnification real-time bioluminescence microscopic imaging revealed that the robust circadian rhythms emanated from the articular cartilage and the epiphyseal cartilage within the growth plate of juvenile animals. Stimulation by forskolin or dexamethasone treatment caused type 0 phase resetting, indicating canonical entraining properties of the bone clock. Together, our findings from long-term ex vivo culture revealed that “tissue-autonomous” circadian rhythm in the articular cartilage and the growth plate of femoral bone functions for several months even in an organ culture condition, and provided a useful in vitro assay system investigating the role of the biological clock in bone formation or development.”

“the plasma PTH rhythm persisted under “constant routine” conditions where subjects were deprived of any exogenous time information, indicating that the rhythm is driven by the intrinsic circadian clock”

“At the molecular level, the circadian clock is composed of a set of clock genes forming cell-autonomous transcription/translation feedback loops; the molecular oscillators in turn drive the expression of output genes governing a variety of clock-controlled physiological processes. Specifically, two transcription factors, BMAL1 and CLOCK, heterodimerize and transactivate core clock genes such as Period genes (Per1 and Per2), Cryptochome1 (Cry1), and Rev-Erb genes (Rev-Erbα and Rev-Erbβ). Expressions of these genes (Bmal1, Per1, Per2, Cry1, Cry2, RevErbα and RevErbβ) show clear circadian rhythms with distinct peak times”

“circadian bioluminescence rhythms from long bones (proximal femoral ends and radiuses) and flat bones (calvariae and scapulae) showed similar period lengths”<-so the circadian rhythm may not be something that distinguishes between long and flat bones.

“both epiphyseal cartilage and articular cartilage in femoral trochlea  showed clear circadian rhythms for 4 days”

“expression of certain clock genes has been shown in growth plates”

” local circadian clocks in the epiphyseal cartilage may affect bone growth in juvenile animals.”

“Exogenous time cues (e.g. light) can reset internal clocks.”

“[The] phase of the bone clock [that] was strongly altered by forskolin, functions to increase intracellular cAMP levels through adenylate cyclase activation. Therefore, it is possible that in vivo circadian phase can be reset by endogenous substance(s) via the cAMP pathway in the bone. The hormone PTH increases intracellular cAMP levels via the PTH/PTHrP receptor, consequently inducing Per1 and Per2 expression through the cAMP–PKA–CREB pathway. In addition, sympathetic signaling stimulated by leptin has been shown to regulate bone remodeling in part through a β-adrenergic receptor (β-AR). The β-AR agonist isoproterenol also enhances intracellular cAMP levels and up-regulates Per1/2 and Bmal1 expression in primary mouse osteoblasts. In accordance, isoproterenol has been reported to stimulate the circadian rhythmic expression of Per1/2/3 and Bmal1 in human SaM-1 osteoblastic cells”

“DEX, synthetic glucocorticoids (GCs), was found to reset the bone circadian rhythm. GCs are secreted from the adrenal gland in a circadian manner. Although the SCN is not reset by GCs due to a lack of glucocorticoid receptor (GR) expression, circadian clocks in peripheral organs such as the liver, kidney, and heart are highly responsive to GCs. GCs are considered as internal time-cues which relay timing information within the body and synchronize the peripheral clocks, including bones, as shown here. At the molecular level, GCs bind to GR and regulate target gene expression via glucocorticoid response elements. Previous studies have identified Per1, Per2, and E4bp4 as direct targets of GRs in mice”

Forskolin alters Ca2+ and cAMP levels, I haven’t seen any studies regarding the effects of Forskolin on longitudinal bone growth.  Dexamethasone is widely known to inhibit longitudinal bone growth.  However, Dexamethasone is also known to help induce chondrogenic differentiation.

The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis.

“SOX9 is a transcriptional activator required for chondrogenesis, and SOX5 and SOX6 are closely related DNA-binding proteins that critically enhance its function. We use here genome-wide approaches to gain novel insights into the full spectrum of the target genes and modes of action of this chondrogenic trio. Using the RCS cell line as a faithful model for proliferating/early prehypertrophic growth plate chondrocytes, we uncover that SOX6 and SOX9 bind thousands of genomic sites, frequently and most efficiently near each other. SOX9 recognizes pairs of inverted SOX motifs, whereas SOX6 favors pairs of tandem SOX motifs. The SOX proteins primarily target enhancers. While binding to a small fraction of typical enhancers, they bind multiple sites on almost all super-enhancers (SEs) present in RCS cells. These SEs are predominantly linked to cartilage-specific genes. The SOX proteins effectively work together to activate these SEs and are required for in vivo expression of their associated genes. These genes encode key regulatory factors, including the SOX trio proteins, and all essential cartilage extracellular matrix components. Chst11, Fgfr3, Runx2 and Runx3 are among many other newly identified SOX trio targets. SOX9 and SOX5/SOX6 thus cooperate genome-wide, primarily through SEs, to implement the growth plate chondrocyte differentiation program.”

“Once settled in skeletogenic sites, progenitor cells coalesce into precartilaginous condensations and activate the chondrocyte differentiation program. Early-stage chondrocytes proliferate and build an abundant, cartilage-specific extracellular matrix. They express such genes as Col2a1 (encoding collagen type II) and Acan (proteoglycan aggrecan). Elongation of cartilage anlagen occurs in growth plates, structures in which chondrocytes proceed through terminal maturation steps in a precise spatial and temporal manner. They proliferate and produce cartilage matrix while aligning into longitudinal columns. They then cease proliferation, become prehypertrophic and express novel markers, such as Ihh (Indian hedgehog). As they become hypertrophic, they turn off most early markers and activate unique ones, including Col10a1 (collagen type X). They eventually die or switch to the osteoblast fate to participate in endochondral ossification. Chondrocytes forming permanent cartilage never proceed through these growth plate maturation steps. They keep expressing pancartilaginous early-chondrocyte markers and also express tissue-specific markers. For instance, superficial articular chondrocytes distinctively express Prg4 (lubricin).”

“Expression of the three SOX genes culminates in growth plate proliferating and prehypertrophic chondrocytes, and is abruptly turned off when chondrocytes undergo hypertrophy. SOX9 is required for chondrogenesis.”

“SOX9 is required to turn on and maintain chondrocyte-specific genes and that SOX5/6 strikingly augment SOX9’s transcriptional activity by securing SOX9 binding to DNA”

“The phenotype of RCS[rat chondrosarcoma] cells thus faithfully matches that of growth plate chondrocytes at the proliferating/early prehypertrophic stage. “<-For more on this read the study.

“RCS cells are bona fide growth plate proliferating/prehypertrophic chondrocytes “<-Since chondrosarcoma can occur in older individuals this is a very promising statement for the creation of neo-growth plates.