Sunday, September 12, 2010

Can heterotopic ossification be used to increase height?

Heterotopic ossification is defined as to where bone is formed outside the skeleton.  Now this can be dangerous if bone forms in say the heart.  However, studying this process can provide insight into how to cause new bone growth even without a hyaline cartilage growth plate line.  Heterotropic ossification is usually caused by a fracture.  The heterotropic ossification may be caused by fracture releasing stem cells followed by those stem cells proceeding to differentiate into osteoblasts. 

Inherited human diseases of heterotopic bone formation. 

"Human disorders of hereditary and nonhereditary heterotopic ossification are conditions in which osteogenesis occurs outside of the skeleton, within soft tissues of the body. The resulting extraskeletal bone is normal. The aberration lies within the mechanisms that regulate cell-fate determination, directing the inappropriate formation of cartilage or bone, or both, in tissues such as skeletal muscle and adipose tissue. Specific gene mutations have been identified in two rare inherited disorders that are clinically characterized by extensive and progressive extraskeletal bone formation-fibrodysplasia ossificans progressiva and progressive osseous heteroplasia. In fibrodysplasia ossificans progressiva, activating mutations in activin receptor type-1, a bone morphogenetic protein type I receptor, induce heterotopic endochondral ossification, which results in the development of a functional bone organ system that includes skeletal-like bone and bone marrow. In progressive osseous heteroplasia, the heterotopic ossification leads to the formation of mainly intramembranous bone tissue in response to inactivating mutations in the GNAS {downregulated by LSJL} gene. Patients with these diseases variably show malformation of normal skeletal elements, identifying the causative genes and their associated signaling pathways as key mediators of skeletal development in addition to regulating cell-fate decisions by adult stem cells." 

"FOP is caused by a mutation in ACVR1, which encodes a bone morphogenetic protein type I receptor; POH is caused by a mutation in the GNAS locus"

"The underlying mutation in ACVR1 in FOP alters bone formation during embryonic skeletal development in addition to inducing heterotopic bone formation postnatally"

Note that activin receptor type-1 induces endochondral ossification.  Perhaps if that mutation occurred within the bone then a new hyaline cartilage growth plate line could be formed.  Activin receptor type-1 is most likely involved in the mechanotransduction mechanism of bone morphonogenic protein-2. 

The genetics of heterotopic ossification: insight into the bone remodeling pathway 

"Three SNPs[Single nucleotide polymorphisms] (beta2-adrenergic receptor, toll-like receptor 4, complement factor H) were identified that were associated with an increased or decreased frequency of HO. The less common polymorphism of the beta2-adrenergic receptor gene was associated with increased risk of HO. For toll-like receptor 4 and complement factor H, the less common polymorphism was associated with a decreased risk of HO.  
The SNPs identified as predictors of HO formation are representative of the adrenergic system[the adrenaline system], immune system, and the alternative complement system[a part of the immune system]. This represents the interplay of multiple pathways that affect bone remodeling, aberrations of which may be found in the genome." 

"high mobility group protein A2 is one of the proteins expressed with TLR4 activation by lipopolysaccharide. High mobility group protein A2 is one of the high mobility group family of transcriptional factors that have been associated with growth regulation. knockout mice deficient in TLR4 have higher bone mass than the wild-type phenotype."

So it seems like heterotropic ossification requires for the immune system to be malfunctioning.  So perhaps if you increased expression of activin receptor type-1 within the bone and made sure that the immune system was functioning properly you wouldn't be at risk for heterotopic ossification and could re-induce endochondral ossification within the bone.  

We'll need to study activin receptor type-1 and see if there are any chemical or mechanical ways to modify it.

Mandibular coronoid process in parathyroid hormone-related protein-deficient mice shows ectopic cartilage formation accompanied by abnormal bone modeling.

"Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller [due to an acceleration of differentiation or a change in direction of differentiation], this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller."

Heterotopic ossification induced by Achilles tenotomy via endochondral bone formation: expression of bone and cartilage related genes.

"Thirty rats underwent bilateral midpoint Achilles tenotomy through a posterior approach under aseptic condition. At 3, 5 and 10 weeks post-operation, X-ray and histological examinations were carried out to investigate the formation of HO. At different phases of HO formation, mRNA levels of transforming growth factor (TGF)-beta1, TGF-beta2, TGF-beta 3, bone morphogenetic proteins (BMP)-2, BMP-4, BMP-7, hypoxia inducible factor (HIF)-1 alpha, Sox9, Runx2, vascular endothelial growth factor (VEGF), aggrecan and collagen type I, II and X were evaluated by real-time RT-PCR. Protein levels of TGF-beta1, TGF-beta2, TGF-beta 3, BMP-2, BMP-4, BMP-7, HIF-1 alpha, Sox9 and Runx2 were also examined by immunohistochemical staining. During the chondrogenic phase, the expressions of HIF-1 alpha and Sox9 were significantly up-regulated. Runx2 expression was significantly up-regulated during osteogenic phase, while HIF-1 alpha and Sox9 expression was significant decreased. TGF-beta1 mRNA levels were rather constant, and the mRNA levels of TGF-beta2, TGF-beta 3 and BMPs were changed throughout HO formation. The presences of the proteins of HIF-1 alpha, Sox9, Runx2, TGF-betas and BMPs within the HO tissues were confirmed by immunohistochemical staining. Our study indicates that HO induced by Achilles tenotomy is by endochondral bone formation, and HIF-1 alpha activation plays an important role during chondrogenesis in this model."

"in an age-matched study that the content of TGF-β1 was 6.8 times higher in HO than in normal bone"

"BMP-4 is found in primitive mesenchymal and chondrocytic cells, the cambium layer of the periosteum, the marrow cavity and the muscles adjacent to the fracture site"

Inhibition of ectopic bone formation by a selective retinoic acid receptor alpha-agonist: a new therapy for heterotopic ossification?

"Because chondrogenesis requires a decrease of nuclear retinoic acid receptor alpha (RARalpha) action, we reasoned that pharmacologic activation of this receptor pathway should inhibit HO. Thus, we selected the synthetic retinoid NRX195183, a potent and highly selective RARalpha-agonist, and found that it did inhibit chondrogenesis in mouse limb micromass cultures. We established a mouse HO model consisting of subcutaneous implantation of Matrigel mixed with rhBMP-2. Control mice receiving daily oral doses of vehicle (peanut oil) or retinol (a natural nonactive retinoid precursor) developed large HO-like masses by days 9-12 that displayed abundant cartilage, endochondral bone, vessels, and marrow. In contrast, formation of HO-like masses was markedly reduced in companion mice receiving daily oral doses of alpha-agonist. These ectopic masses contained sharply reduced amounts of cartilage and bone, blood vessels, and TRAP-positive osteoclasts, and expressed markedly lower levels of master chondrogenic genes including Sox9, cartilage genes such as collagen XI and X, and osteogenic genes including Runx2."

Genes Upregulated by RARA agonist(activator) also upregulated by LSJL:


Genes downregulated by RAR agonist also downregulated by LSJL:


Heterotopic ossification in complex orthopaedic combat wounds: quantification and characterization of osteogenic precursor cell activity in traumatized muscle.

"Heterotopic ossification frequently develops following high-energy blast injuries sustained in modern warfare. We hypothesized that differences in the population of progenitor cells present in a wound would correlate with the subsequent formation of heterotopic ossification.
We obtained muscle biopsy specimens from military service members who had sustained high-energy wartime injuries and from patients undergoing harvest of a hamstring tendon autograft. Plastic-adherent cells were isolated in single-cell suspension and plated to assess the prevalence of colony-forming cells. Phenotypic characteristics were assessed with use of flow cytometry. Individual colony-forming units were counted after an incubation period of seven to ten days, and replicate cultures were incubated in lineage-specific induction media. Immunohistochemical staining was then performed to determine the percentage of colonies that had differentiated along an osteogenic lineage. Quantitative real-time reverse-transcription polymerase chain reaction was used to identify changes in osteogenic gene expression.
Injured patients had significantly higher numbers of muscle-derived connective-tissue progenitor cells per gram of tissue (p < 0.0001; 95% confidence interval [CI], 129,930 to 253,333), and those who developed heterotopic ossification had higher numbers of assayable osteogenic colonies (p < 0.016; 95% CI, 12,249 to 106,065). In the injured group, quantitative real-time reverse-transcription polymerase chain reaction performed on the in vitro expanded progeny of connective-tissue progenitors demonstrated upregulation of COL10A1{up in LSJL}, COL4A3, COMP, FGFR2, FLT1, IGF2, ITGAM, MMP9, PHEX, SCARB1, SOX9{up}, and VEGFA in the patients with heterotopic ossification as compared with those without heterotopic ossification.
Our study suggests that the number of connective-tissue progenitor cells is increased in traumatized tissue. Furthermore, wounds in which heterotopic ossification eventually forms have a higher percentage of connective-tissue progenitor cells committed to osteogenic differentiation than do wounds in which heterotopic ossification does not form."

"Heterotopic ossification most commonly occurs as a sequel of head trauma, blunt elbow or acetabular trauma, and burns and following total hip arthroplasty"

Downregulated genes in connective tissue progenitors:

Other upregulated genes upregulated between 2 and 4 fold:

Only Col4a3 was statistically significant p < 0.05.

"Progeny cells composing primary muscle-derived connective-tissue progenitor colony-forming units were neither hematopoietic cells (i.e., were negative for CD45 and HLA-DR) nor endothelial cells (were negative for CD34 and CD31) and were positive for CD29, CD44, CD73, and HLA-ABC."

"Post hoc analysis revealed that the 1.56-fold increase in the average number of osteogenic colony-forming (connective-tissue progenitor-O) colonies per gram of tissue in the injured patients who developed heterotopic ossification, as compared with the average number in those who did not, was significant (p < 0.016; 95% CI, 12,249 to 106,065). There was also a significant, 1.34-fold increase in the average number of connective-tissue progenitor colonies per gram of tissue in the injured patients who developed heterotopic ossification compared with the average number in those who did not (p < 0.048; 95% CI, 409 to 111,687). Many of the connective-tissue progenitors, especially those isolated from normal tissue (∼75%), failed to differentiate into any of the mesenchymal lineages tested (bone, cartilage, or fat) and thus were considered to be fibroblasts (connective-tissue progenitor-F)."

Fibrodysplasia ossificans progressiva: mechanisms and models of skeletal metamorphosis.

"Fibrodysplasia ossificans progressiva (FOP) is a debilitating genetic disorder of connective tissue metamorphosis. It is characterized by malformation of the great (big) toes during embryonic skeletal development and by progressive heterotopic endochondral ossification (HEO) postnatally, which leads to the formation of a second skeleton of heterotopic bone. Individuals with these classic clinical features of FOP have the identical heterozygous activating mutation (c.617G>A; R206H) in the gene encoding ACVR1 (also known as ALK2), a bone morphogenetic protein (BMP) type I receptor."

"During the first decade of life, children with FOP develop inflammatory soft tissue swellings (known as flare-ups) that appear suddenly and expand rapidly. A flare-up is the first clinical indication of the tissue metamorphosis, which involves the catabolism of soft connective tissues (including aponeuroses, fascia, ligaments, tendons and skeletal muscles) and their replacement by extraskeletal bone through HEO"

"Trauma induced by operative removal of heterotopic bone also leads to new bone formation"

"Flare-up: an episodic activation of FOP characterized by edema (swelling), pain and decreased range of motion"

"Osteochondroma: benign endochondral neoplasms or developmental lesions that have a cartilage cap and arise from the underlying bone"

Heterotopic Bone Formation About the Hip Undergoes Endochondral Ossification: A Rabbit Model.

"We used a rabbit model that included intramedullary instrumentation of the upper femur and ischemic crush injury of the gluteal muscle. Bilateral surgical induction procedures were performed on 30 animals with the intention of inciting the process of HO; no supplemental osteogenic stimulants were used. Three animals were sacrificed at each of 10 predetermined times between 1 day and 26 weeks postoperatively and the progression of tissue maturation was graded histologically using a five-item scale.
Heterotopic bone reliably formed de novo and consistently followed a pathway of endochondral ossification. Chondroid elements were found in juxtaposition with immature woven bone in all sections that contained mature osseous elements."

Chondroblasts were observed at day 7.  Chondrocytes at day 10 and mineralization occurred at day 21.

Here's what an ectopic growth plate looks like:
"A high-magnification photomicrograph shows skeletal muscle 4 weeks after the induction procedure. Dense fibrous tissue can be seen with collagen deposition in continuity with palisading chondrocytes, evolving through stages of calcification and ossification reminiscent of the growth plate"
"An intermediate-magnification photomicrograph shows skeletal muscle 12 weeks after the induction procedure. Typical endochondral ossification is apparent, with capillary ingrowth of cartilaginous matrix around hypertrophic chondrocytes and ongoing matrix mineralization"

"Heterotopic bone that arose directly from the femur was derived via endochondral ossification that originated from a periosteal proliferation of chondrocytes"

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