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.