Growth Of Metatarsal Bones Research Articles

Genome-wide association study of growth curve parameters reveals novel genomic regions and candidate genes associated with metatarsal bone traits in chickens

The growth and development of chicken bones have an enormous impact on the health and production performance of chickens. However, the development pattern and genetic regulation of the chicken skeleton are poorly understood. This study aimed to evaluate metatarsal bone growth and development patterns in chickens via non-linear models, and to identify the genetic determinants of metatarsal bone traits using a genome-wide association study (GWAS) based on growth curve parameters. Data on metatarsal length (MeL) and metatarsal circumference (MeC) were obtained from 471 F2 chickens (generated by crossing broiler sires, derived from a line selected for high abdominal fat, with Baier layer dams) at 4, 6, 8, 10, and 12 weeks of age. Four non-linear models (Gompertz, Logistic, von Bertalanffy, and Brody) were used to fit the MeL and MeC growth curves. Subsequently, the estimated growth curve parameters of the mature MeL or MeC (A), time-scale parameter (b), and maturity rate (K) from the non-linear models were utilized as substitutes for the original bone data in GWAS. The Logistic and Brody models displayed the best goodness-of-fit for MeL and MeC, respectively. Single-trait and multi-trait GWASs based on the growth curve parameters of the Logistic and Brody models revealed 4 618 significant single nucleotide polymorphisms (SNPs), annotated to 332 genes, associated with metatarsal bone traits. The majority of these significant SNPs were located on Gallus gallus chromosome (GGA) 1 (167.433–176.318 Mb), GGA2 (96.791–103.543 Mb), GGA4 (65.003–83.104 Mb) and GGA6 (64.685–95.285 Mb). Notably, we identified 12 novel GWAS loci associated with chicken metatarsal bone traits, encompassing 35 candidate genes. In summary, the combination of single-trait and multi-trait GWASs based on growth curve parameters uncovered numerous genomic regions and candidate genes associated with chicken bone traits. The findings benefit an in-depth understanding of the genetic architecture underlying metatarsal growth and development in chickens.

Humanin Treatment Protects Against Venetoclax-Induced Bone Growth Retardation in Ex Vivo Cultured Rat Bones.

Recent preclinical studies reported that the BCL-2 inhibitor venetoclax can impair bone growth. A strategy to prevent such a side effect of this promising anticancer drug is highly desired. Earlier in vitro and in vivo studies suggested that the mitochondrial peptide humanin has the potential to prevent drug-induced growth impairment. We hypothesized that co-treatment with the humanin analog HNG may prevent venetoclax-induced bone growth impairment. Ex vivo studies were performed in fetal rat metatarsal bones and human growth plate samples cultured for 12 and 2 days, respectively, while in vivo studies were performed in young neuroblastoma mice being treated daily for 14 days. The treatment groups included venetoclax, HNG, venetoclax plus HNG, or vehicle. Bone growth was continuously monitored and at the end point, histomorphometric and immunohistochemical analyses were performed in fixed tissues. Venetoclax suppressed metatarsal bone growth and when combined with HNG, bone growth was rescued and all histological parameters affected by venetoclax monotherapy were normalized. Mechanistic studies showed that HNG downregulated the pro-apoptotic proteins Bax and p53 in cultured metatarsals and human growth plate tissues, respectively. The study in a neuroblastoma mouse model confirmed a growth-suppressive effect of venetoclax treatment. In this short-term in vivo study, no significant bone growth-rescuing effect could be verified when testing HNG at a single dose. We conclude that humanin dose-dependently protects ex vivo cultured metatarsal bones from venetoclax-induced bone growth impairment by restoring the growth plate microstructure.

SAT190 The Effect Of Mechanical Loading On Bone Growth Ex Vivo

Abstract Disclosure: T. Aeppli: None. Z. Zhang: None. F. Zaman: None. L.S. Savendahl: None. Background: Bone growth is a multi-step process that involves proliferation and differentiation of chondrocytes within the growth plate. Apart from local regulation by various signaling pathways, chondrocytes respond also to external cues such as nutrition, inflammation and mechanical load that may increase or stunt growth. The Hueuter and Volkmann law describes the effect of mechanical loading on bone growth stating that bone growth is inhibited by increased mechanical compression and is promoted by reduced loading. To this date, no data is available investigating the local effects of mechanical loading on bone growth in the absence of systemic growth factors in small embryonic bones. Aim: The aim was to study the effects of mechanical loading on bone growth in an ex vivo bone culture model. Methods: Cultured femur and metatarsal bones from rats (E19.5) were studied and local/direct effects of mechanical loading on bone growth were investigated. A range of different mechanical forces was applied to femur and metatarsal bones every 2-3 days. To monitor bone growth, digital pictures were taken and bone length was measured every 2-3 days. The loaded bones were compared to control bones (sham-loaded). Results: When applying different mechanical load to metatarsal bones, bone growth was suppressed in a dose-dependent manner. On day 5, after applying mechanical loading (0.4 N) twice on day 0 and 2, metatarsal bone growth was significantly suppressed when compared to unloaded control bones (p<0.05). In contrast, when mechanical loading was applied repetitively to femur bones, bone growth was significantly stimulated (p<0.001) when compared to unloaded control bones. Conclusion: In conclusion, depending on the type of bone (embryonic rat metatarsal bone and the larger rat femur bone) mechanical loading exerts opposite effects on bone growth in an ex vivo culture model. The effect of mechanical loading on bone growth requires further in vivo investigations to increase our understanding on the role of mechanical loading in different bones and bone growth disorders. Presentation: Saturday, June 17, 2023

Effects of the selective GPER1 agonist G1 on bone growth.

Estrogens may affect bone growth locally or systemically via the known estrogen receptors ESR1, ESR2 and G protein-coupled estrogen receptor 1 (GPER1). Mouse and human growth plate chondrocytes have been demonstrated to express GPER1 and ablation of this receptor increased bone length in mice. Therefore, GPER1 is an attractive target for therapeutic modulation of bone growth, which has never been explored. To investigate the effects of activated GPER1 on the growth plate, we locally exposed mouse metatarsal bones to different concentrations of the selective GPER1 agonist G1 for 14 days ex vivo. The results showed that none of the concentrations of G1 had any direct effect on metatarsal bone growth when compared to control. To evaluate if GPER1 stimulation may systemically modulate bone growth, ovariectomized C57BL/6 mice were treated with G1 or β-estradiol (E2). Similarly, G1 did not influence tibia and femur growth in treated mice. As expected, E2 treatment suppressed bone growth in vivo. We conclude that ligand stimulation of GPER1 does not influence bone growth in mice.

OR17-3 Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy particularly in severe, non-GH deficient conditions such as chondrodysplasias, and has potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we previously developed a cartilage-targeting single-chain human antibody fragment (CaAb) (1), aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. In the current study, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor-I (IGF-1), an endocrine/paracrine factor which positively regulates chondrogenesis. We first used ELISA to assess the ability of the CaAb-IGF1 fusion proteins to bind tissue lysates from different mouse organs and found that the fusion proteins bound to cartilage with high tissue specificity. We also found that these fusion proteins retain potent IGF-1 biological activity, as demonstrated by their ability to stimulate AKT and ERK phosphorylation in cell culture and stimulate metatarsal bone growth in organ culture. To assess in vivo efficacy, we administered daily subcutaneous injections to growth hormone-deficient (lit) mice for 5 days. In this model, twice-daily IGF-1 increased growth plate height but daily IGF-1 did not. In contrast, daily equimolar injections of the targeted fusion proteins did increase growth plate height whereas a fusion protein lacking the cartilage-targeting domain did not (P<0.01 targeted vs non-targeted or saline). Even alternate-day injections of the targeted fusion proteins were sufficient to produce a therapeutic effect (P<0.01) similar to daily injection. To assess off-target effects, we measured proliferation in kidney cortical cells by BrdU incorporation. Daily injections of the targeted fusion proteins did not increase kidney cell proliferation whereas twice-daily IGF-1 itself (the minimum IGF-1 regimen that stimulated the growth plate) did stimulate kidney proliferation (P<0.01). In summary, the fusion proteins, unlike IGF-I itself, were able to stimulate growth plate activity without significant off-target effects on kidney. Our findings therefore provide proof-of-principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders. Reference: (1) Cheung et al. Pharma Res 2015 Jul;32(7):2439-49. Sources of Funding: This work was supported by the Intramural Research Programs of NICHD and NCI, National Institutes of Health. Additional funding through Grant for Growth Innovation from EMD Serono, Inc. a biopharmaceutical business of Merck KGaA, Germany, and Endocrine Scholars Award in Growth Hormone Research from the Endocrine Society.

Lengthening of radius in patients with congenital radial club hand, type II

Background. Congenital radial club hand (CRCH) is characterized by longitudinal underdevelopment of the forearm and hand on the radial surface. Underdevelopment can range from hypoplasia to aplasia of the radius. More than 50 methods to correct the forearm deformities, depending on the degree of radius underdevelopment, have been proposed.&#x0D; Aim. We evaluated the results of CRCH treatment using microsurgical technique and external fixation.&#x0D; Methods. We analyzed 16 patients (age, 4.6 0.9 years) with CRCH type II, according to the classification of Bayne and Klug, treated between 1994 and 2017. The patients were divided into two groups: Group 1 were patients undergoing microsurgical autotransplants of the epimetaphyseal second metatarsal bone with growth plate to the position of the radius defect and group 2 were patients treated by lengthening of the radius with external fixation. We analyzed the types of deformities, size of the radius defects, and range of motion in upper limb joints before the stage of the lengthening. External fixation index and number of complications also were determined. The type and number of recurrent deformities and timing of their detection were analyzed.&#x0D; Results. The observation period ranged from 12 months to 10 years (average, 3.8 years). In group 1, good results were obtained in 62.5% of cases. After transplantation of the metatarsal bone growth plate, the work of the growth plate continued, characterized by increasing radius length in the later observation period. In group 2, good results were obtained in 50% of cases. Clinical and X-ray examinations showed recurrent hand deviation and radius shortening, which required repeated radius lengthening.&#x0D; Conclusion. Microsurgical transplantation of the second metatarsal bone with growth plate is accepted more in reconstruction of the radial bone in patients with CRCH type II due to creation of a growth zone in the distal part of the radius. Radius lengthening via external fixation is applicable while maintaining the distal epimetaphysis and normal transverse dimensions of the radial bone.

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes.

Congenic mice provide in vivo evidence for a genetic locus that modulates intrinsic transforming growth factor β1–mediated signaling and bone acquisition

Osteoporosis, the most common skeletal disorder, is characterized by low bone mineral density (BMD) and an increased risk of fragility fractures. BMD is the best clinical predictor of future osteoporotic fracture risk, but is a complex trait controlled by multiple environmental and genetic determinants with individually modest effects. Quantitative trait locus (QTL) mapping is a powerful method for identifying chromosomal regions encompassing genes involved in shaping complex phenotypes, such as BMD. Here we have applied QTL analysis to male and female genetically-heterogeneous F(2) mice derived from a cross between C57BL/6 and DBA/2 strains, and have identified 11 loci contributing to femoral BMD. Further analysis of a QTL on mouse chromosome 7 following the generation of reciprocal congenic strains has allowed us to determine that the high BMD trait, which tracks with the DBA/2 chromosome and exerts equivalent effects on male and female mice, is manifested by enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and by increased growth of metatarsal bones in short-term primary culture. An insertion/deletion DNA polymorphism in Ltbp4 exon 12 that causes the in-frame removal of 12 codons in the DBA/2-derived gene maps within 0.6 Mb of the marker most tightly linked to the QTL. LTBP4, one of four paralogous mouse proteins that modify the bioavailability of the transforming growth factor β (TGF-β) family of growth factors, is expressed in differentiating MSC-derived osteoblasts and in long bones, and reduced responsiveness to TGF-β1 is observed in MSCs of mice homozygous for the DBA/2 chromosome 7. Taken together, our results identify a potential genetic and biochemical relationship between decreased TGF-β1-mediated signaling and enhanced femoral BMD that may be regulated by a variant LTBP4 molecule.

Androgen Receptor Modulation Does Not Affect Longitudinal Growth of Cultured Fetal Rat Metatarsal Bones

Background: Systemic administration of the nonaromatizable androgen oxandrolone stimulates growth in girls with Turner syndrome and boys with a constitutional delay of growth and puberty. It is unknown if oxandrolone acts locally at the growth plate level to stimulate longitudinal bone growth. Methods: Metatarsal bones from female and male rat fetuses (day E20) were cultured for 14 days in the presence of oxandrolone, testosterone or the androgen receptor (AR) antagonist flutamide with/without insulin-like growth-factor-I (IGF-I) or charcoal-treated serum. Results: The AR was found to be expressed in both male and female fetal rat metatarsal bones. Neither oxandrolone nor testosterone had any effect on metatarsal bone growth when tested at a wide concentration range (1 nM to 10 μM), not even in the presence of IGF-I (100 ng/ml) or charcoal-treated serum (10%). Bone growth was also unaffected when the AR was blocked by flutamide. Control experiments confirmed that metatarsal bone growth was significantly stimulated by IGF-I (p < 0.001). Conclusion: Modulation of AR activity in the fetal rat growth plate does not affect linear bone growth. Extrapolating from these in vitro data, it could be speculated that oxandrolone stimulates longitudinal bone growth in treated children by acting indirectly rather than directly through AR activation in growth plate chondrocytes.

Akt promotes BMP2-mediated osteoblast differentiation and bone development

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is crucial for normal skeletal development and for bone remodeling. Osteogenesis is primarily regulated by bone morphogenetic proteins (BMPs), which activate gene expression programs driven by bone-specific transcription factors. In a mesenchymal stem cell model of osteoblast commitment and differentiation controlled by BMP2, we show that an inhibitor of PI3-kinase or a dominant-negative Akt were as potent in preventing osteoblast differentiation as the IGF binding protein IGFBP5, whereas a Mek inhibitor was ineffective. Conversely, an adenovirus encoding an inducible-active Akt was able to overcome the blockade of differentiation caused by IGFBP5 or the PI3-kinase inhibitor, and could restore normal osteogenesis. Inhibition of PI3-kinase or Akt did not block BMP2-mediated signaling, because the Smad-responsive genes Sox9 and JunB were induced normally under all experimental conditions. When activated during different stages of osteoblast maturation, dominant-negative Akt prevented accumulation of bone-specific alkaline phosphatase and reduced mineralization, and more significantly inhibited the longitudinal growth of metatarsal bones in primary culture by interfering with both chondrocyte and osteoblast development and function. We conclude that an intact IGF-induced PI3-kinase-Akt signaling cascade is essential for BMP2-activated osteoblast differentiation and maturation, bone development and growth, and suggest that manipulation of this pathway could facilitate bone remodeling and fracture repair.

Effects of alendronate and pamidronate on cultured rat metatarsal bones: Failure to prevent dexamethasone-induced growth retardation

Bisphosphonates are widely used anti-resorptive drugs in the adult population. In children, their use has mainly been limited to patients with osteogenesis imperfecta. However, the powerful effects of bisphosphonates on bone turnover have raised concern about their long-term effects on the growing skeleton. We aimed to study the effects of two commonly used bisphosphonates, alendronate (Aln) and pamidronate (Pam) on normal bone growth as well as their potential to prevent glucocorticoid-induced growth retardation. Effects on bone growth were studied in fetal rat metatarsal bones (day E20) that were cultured for 5–47 days and measured every 2–7 days. Cellular mechanisms were investigated in metatarsal bones and also in the human chondrocytic cell line HCS-2/8. Chondrocyte viability (WST-1), proliferation (BrdU incorporation), differentiation (collagen type X immunohistochemistry) and apoptosis (TUNEL and Cell Death ELISA) were determined. At a clinically relevant concentration of bisphosphonates (1 μM), metatarsal bone growth was stimulated by both Aln ( p < 0.001 for length and p < 0.05 for width) and Pam ( p < 0.05 for both length and width) from day 19 of culture. The growth-stimulatory effect was associated with increased chondrocyte proliferation (+ 21% with Aln and + 24% with Pam), while cell differentiation and apoptosis were not affected. Despite the finding that both Aln and Pam (1 μM) rescued HCS-2/8 cells from undergoing dexamethasone-induced apoptosis, neither of them was able to prevent dexamethasone-induced growth retardation of fetal rat metatarsal bones. Aln and Pam have the capacity to stimulate the growth of cultured fetal rat metatarsal bones; an effect associated with increased proliferation of growth plate chondrocytes. Our experimental data suggest that bisphosphonates are ineffective in preventing glucocorticoid-induced growth retardation. Nevertheless, based on our in vitro data, both Aln and Pam appear safe to use in growing children, at least with regard to their effects on linear bone growth.

Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17beta-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 +/- 5.3 fmol/3 days/10(6) cells). The aromatase inhibitor letrozole (1 microM) and the pure estrogen receptor antagonist ICI 182,780 (10 microM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P < 0.01) and almost 50% (P < 0.001), respectively. Treatment with ICI 182,780 (10 microM) increased apoptosis by 228% (P < 0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P < 0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 microM) and letrozole (1 microM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P < 0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.

The developmental skeletal growth in the rat foot is reduced after denervation.

It has long been known that bone is innervated. In recent years it has been suggested that the local nerves may influence the growth and metabolism of bone by way of neuropeptides. The transient local presence of nerve-containing cartilage canals just before formation of secondary ossification centres in rat knee epiphyses seems to support that view. The purpose of the present study was to see if denervation affects the developmental growth of metatarsal bones in the rat hindfoot. We made sciatic and femoral neurectomies in 7-day-old rat pups and examined the hindfeet at various times after surgery. Immunohistochemical analysis showed that denervation was complete. Radiographic examination revealed that the metatarsal bones were significantly shorter in denervated hindfeet 30 days after denervation (average relative shortening 9.9 +/- 2.3%). Measurements of total foot length showed that denervated feet were subnormally sized already five days postoperatively, before the onset of secondary ossification. The timing of the latter was not affected by denervation. Control rats subjected to tenotomies exhibited normal metatarsal bone lengths. On the basis of these results we suggest that the local nerves may influence the growth of immature bones but do not affect secondary ossification.