Growth Plate Height Research Articles

CCR7 depletion alleviates bony growth imbalance following physeal injury in mice

Growth plates are the frequent sites of skeletal injury in children, leading to skeletal growth imbalances. Chemokines, including the receptor CCR7, play a crucial role in stem cell recruitment and cartilage homeostasis, with previous studies linking CCR7 to osteoarthritis progression. However, its role in growth plate cartilage remains unclear. We analyzed the role of CCR7 in the physeal cartilage repair process in mice model. Physeal injury was created in the proximal tibia in 3-week-old C57BL/6 mice (WT) and CCR7-knockout mice (CCR7−/−). Tibial length was measured macroscopically and sections of the physeal injury were analyzed histologically and immunohistochemically. Height and bone volume of the tibial growth plate and bone mineral density (BMD) of the subchondral area were measured by micro-CT. Mesenchymal stem cells (MSCs) were harvested and gene expression after osteogenic differentiation was analyzed using qRT-PCR. At 1, 3 and 5 weeks postoperatively, injured tibiae of CCR7−/− mice were less shortened than those of WT mice. Bone volume of the physeal bridge was significantly lower in CCR7−/− mice than in WT mice. In contrast, BMD of the subchondral area was comparable between CCR−/− and WT mice, and between sham and operated tibiae. In osteogenic differentiation, CCR7−/− mice showed significantly lowered expression of osteogenic markers such as Osterix, Runx2 and Type X collagen. We demonstrated CCR7 depletion in mice inhibited physeal bridge formation and ameliorated growth imbalances after physeal injury.

9267 Targeting IGF-I to Growth Plate Cartilage Augments Therapeutic Effects on Skeletal Growth and Reduces Hypoglycemic Effects in Mice

Abstract Disclosure: K. Tailor: None. T. Stowe: None. J. Ree: None. B. Ventura: None. J. Deursen: None. R. O'Brien: None. J. Baron: None. J. Lui: None. Recombinant human growth hormone (GH) is commonly used to treat short stature in children but has limited efficacy in severe, non-GH-deficient conditions, such as skeletal dysplasias, and has adverse off-target effects. Because short stature is the result of reduced growth plate chondrogenesis, we hypothesized that targeting chondrogenic growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing adverse effects on non-target tissues. Toward this end, we previously developed fusion proteins conjugating insulin-like growth factor-1 (IGF-1) with a single-chain human monoclonal antibody fragment (ScFv) targeting matrillin-3 (1), a protein specifically expressed in cartilage matrix, and demonstrated increased on-target therapeutic efficacy at the growth plate and decreased off-target effects in a GH-deficient (lit) mouse model (2). In the current study, we established a second GH-deficiency model by administration of a GH antagonist, pegvisomant (PEG), in C57BL/6 wild-type male mice. Alternate-day injection of pegvisomant at 40 mg/kg for 7 days reduced body weight, serum IGF-1 levels, and growth plate height in mice. Using this model, we found that once-daily SQ administration of cartilage-targeted IGF-I (named CV1574-1) for five days increased body weight and growth plate height greater than an equimolar dose of IGF-I itself administered twice daily. Hypoglycemia is an adverse effect of recombinant IGF-1 treatment. Therefore, we also monitored blood glucose levels after a single SQ injection of CV1574-1 and found that this fusion protein showed a reduced hypoglycemic effect compared to injection of IGF-I itself. Lastly, to gain mechanistic insight into the role of matrillin-3 targeting on therapeutic efficacy, we assessed the ability of CV1574-1 to induce pAKT signaling in vitro. Transient exposure to CV1574-1 caused more prolonged induction of pAKT signal in wild-type rat chondrosarcoma (RCS) cells compared to matrillin-3-knockout RCS cells, suggesting that matrillin-3 targeting extends the duration of IGF-1 receptor activation. To summarize, the current findings demonstrate that CV1574-1, a fusion protein that targets IGF-1 to cartilage, improves efficacy at the growth plate, reduces hypoglycemic effects, and, in vitro, prolongs receptor activation. Our study provides further evidence that cartilage-targeted therapy has the potential to provide new pharmacological approaches for the treatment of childhood linear growth disorders, including skeletal dysplasias and growth failure due to systemic disease. Reference:(1) Cheung et al. Pharma Res 2015, 32(7):2439-49.(2) Lui et al. Molecular Therapy 2019, 27(3):673-680 Presentation: 6/3/2024

7237 Targeting IGF-I to Growth Plate Cartilage Augments Therapeutic Effects on Skeletal Growth and Reduces Hypoglycemic Effects in Mice

Abstract Disclosure: K. Tailor: None. T. Stowe: None. J. Ree: None. B. Ventura: None. J. Deursen: None. R. O'Brien: None. J. Baron: None. J. Lui: None. Recombinant human growth hormone (GH) is commonly used to treat short stature in children but has limited efficacy in severe, non-GH-deficient conditions, such as skeletal dysplasias, and has adverse off-target effects. Because short stature is the result of reduced growth plate chondrogenesis, we hypothesized that targeting chondrogenic growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing adverse effects on non-target tissues. Toward this end, we previously developed fusion proteins conjugating insulin-like growth factor-1 (IGF-1) with a single-chain human monoclonal antibody fragment (ScFv) targeting matrillin-3 (1), a protein specifically expressed in cartilage matrix, and demonstrated increased on-target therapeutic efficacy at the growth plate and decreased off-target effects in a GH-deficient (lit) mouse model (2). In the current study, we established a second GH-deficiency model by administration of a GH antagonist, pegvisomant (PEG), in C57BL/6 wild-type male mice. Alternate-day injection of pegvisomant at 40 mg/kg for 7 days reduced body weight, serum IGF-1 levels, and growth plate height in mice. Using this model, we found that once-daily SQ administration of cartilage-targeted IGF-I (named CV1574-1) for five days increased body weight and growth plate height greater than an equimolar dose of IGF-I itself administered twice daily. Hypoglycemia is an adverse effect of recombinant IGF-1 treatment. Therefore, we also monitored blood glucose levels after a single SQ injection of CV1574-1 and found that this fusion protein showed a reduced hypoglycemic effect compared to injection of IGF-I itself. Lastly, to gain mechanistic insight into the role of matrillin-3 targeting on therapeutic efficacy, we assessed the ability of CV1574-1 to induce pAKT signaling in vitro. Transient exposure to CV1574-1 caused more prolonged induction of pAKT signal in wild-type rat chondrosarcoma (RCS) cells compared to matrillin-3-knockout RCS cells, suggesting that matrillin-3 targeting extends the duration of IGF-1 receptor activation. To summarize, the current findings demonstrate that CV1574-1, a fusion protein that targets IGF-1 to cartilage, improves efficacy at the growth plate, reduces hypoglycemic effects, and, in vitro, prolongs receptor activation. Our study provides further evidence that cartilage-targeted therapy has the potential to provide new pharmacological approaches for the treatment of childhood linear growth disorders, including skeletal dysplasias and growth failure due to systemic disease. Reference:(1) Cheung et al. Pharma Res 2015, 32(7):2439-49.(2) Lui et al. Molecular Therapy 2019, 27(3):673-680 Presentation: 6/3/2024

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes bone length, regulates cortical and trabecular bone mass, and maintains growth plate architecture and width in a sex- and site-specific manner in mice

Tartrate-resistant acid phosphatase (TRAP) serum levels reflect osteoclast number, bone remodeling activity, and fracture risk. Deletion or loss of function of TRAP results in short stature in mice and man. Yet, the impact and mechanisms of TRAP for the site- and sex-specific development of bone and cartilage is not well understood. Here, we use a global TRAP knockout (TRAPKO) and wildtype littermate control (WT) mice of both sexes to investigate TRAP as a possible sex- and site-specific regulator of bone and growth plate development. TRAPKO mice of both sexes weighed less and had shorter tibial length than their WT, features that were more accentuated in male than female TRAPKO mice. These changes were not associated with a general reduction in growth as not all organs displayed a proportionally lower mass, and serum IGF-1 was unchanged. Using μCT and site-specificity analysis of the cortical bone revealed wider proximal tibia, a higher trabecular thickness, and lower trabecular separation in male TRAPKO compared to WT mice, an effect not seen in female mice. Histomorphometric analysis revealed that the growth plate height as well as height of terminal hypertrophic chondrocytes were markedly increased, and the number of columns was decreased in TRAPKO mice of both sexes. These effects were more accentuated in female mice. Proliferation and differentiation of bone marrow derived macrophages into osteoclasts, as well as C-terminal cross links were normal in TRAPKO mice of both sexes. Collectively, our results show that TRAP regulates bone and cartilage development in a sex-and site-specific manner in mice.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury

IntroductionGrowth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair.MethodsChondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT.ResultsGlycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks.ConclusionThis study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Age-related changes in densitometry and histomorphometry of long bone during the pubertal growth spurt in male rats.

Because experimental studies to determine the developmental toxicity of exposure to various substances in children are impossible, many studies use immature male rats. This study aimed to provide normative data for longitudinal bone growth with age during the puberty in male rats. In order to evaluate long bone growth and mineralization we examined bone size and bone density by dual-energy X-ray absorptiometry, analyzed histomorphometry of the growth plate, and serum hormone levels relevant to bone growth from postnatal day (PD)20 to PD60. The length and weight of long bones increased strongly by PD40, and no further increase was observed after PD50. On the other hand, tibial growth plate height decreased sharply after PD50 along with a reduction in the number of cells and columns, which was probably responsible for the absence of further lengthening of long bones. Parameters related to bone formation such as bone area ratio, and the thickness and number of trabeculae, also increased significantly between PD40 and PD50. Furthermore, serum levels of IGF-1 peaked at PD30 and testosterone increased rapidly on and after PD40, when IGF-1 levels were going down. These changes may participate in the parallel increase in mineral acquisition, as well as lengthening of long bones. Our findings provide comprehensive data for changes in bone density, histomorphometry of long bones, and hormone levels relevant to bone growth during the growth spurt. This will be useful for planning animal toxicological studies, particularly for deciding on the appropriate age of animals to use in given experiments.

Rheb1 is required for limb growth through regulating chondrogenesis in growth plate

Ras homology enriched in the brain (Rheb) is well established as a critical regulator of cell proliferation and differentiation in response to growth factors and nutrients. However, the role of Rheb1 in limb development remains unknown. Here, we found that Rheb1 was dynamically expressed during the proliferation and differentiation of chondrocytes in the growth plate. Given that Prrx1+ limb-bud-like mesenchymal cells are the source of limb chondrocytes and are essential for endochondral ossification, we conditionally deleted Rheb1 using Prrx1-Cre and found a limb dwarfism in Prrx1-Cre; Rheb1fl/fl mice. Normalized to growth plate height, the conditional knockout (cKO) mice exhibited a significant decrease in column count of proliferative zones which was increased in hypertrophic zones resulting in decreased growth plate size, indicating abnormal endochondral ossification. Interestingly, although Rheb1 deletion profoundly inhibited the transcription factor Sox9 in limb cartilage; levels of runx2 and collagen type 2 were both increased. These novel findings highlight the essential role of Rheb1 in limb growth and indicate a complex regulation of Rheb1 in chondrocyte proliferation and differentiation.

Pharmacological inhibition of BCL-2 with the FDA-approved drug venetoclax impairs longitudinal bone growth

Treatment-related skeletal complications are common in childhood cancer patients and survivors. Venetoclax is a BCL-2 inhibitor that has shown efficacy in hematological malignancies in adults and is being investigated in pediatric cancer clinical trials as a promising therapeutic modality. Venetoclax triggers cell death in cancer cells, but whether it exerts similar effects in normal bone cells, is unknown. Chondrogenic ATDC5 cells, E20 fetal rat metatarsal bones, and human growth plate biopsies were treated with different concentrations of venetoclax. Female NMRI nu/nu mice were treated with venetoclax or vehicle for 15 days. Mice were X-rayed at baseline and at the end of the experiment to assess longitudinal bone growth and body weight was monitored throughout the study. Histomorphometric and immunohistochemical analyses were performed to evaluate treatment effects on the growth plate cartilage. Venetoclax decreased the viability of chondrocytes and impaired the growth of ex vivo cultured metatarsals while reducing the height of the resting/proliferative zone and the hypertrophic cell size. When tested in vivo, venetoclax suppressed bone growth and reduced growth plate height. Our experimental data suggest that venetoclax directly targets growth plate chondrocytes suppressing bone growth and we, therefore, encourage careful monitoring of longitudinal bone growth if treating growing children with venetoclax.

TNF overexpression and dexamethasone treatment impair chondrogenesis and bone growth in an additive manner

Children with chronic inflammation are often treated with glucocorticoids (GCs) and many of them experience growth retardation. It is poorly understood how GCs interact with inflammatory cytokines causing growth failure as earlier experimental studies have been performed in healthy animals. To address this gap of knowledge, we used a transgenic mouse model where human TNF is overexpressed (huTNFTg) leading to chronic polyarthritis starting from the first week of age. Our results showed that femur bone length and growth plate height were significantly decreased in huTNFTg mice compared to wild type animals. In the growth plates of huTNFTg mice, increased apoptosis, suppressed Indian hedgehog, decreased hypertrophy, and disorganized chondrocyte columns were observed. Interestingly, the GC dexamethasone further impaired bone growth, accelerated chondrocyte apoptosis and reduced the number of chondrocyte columns in huTNFTg mice. We conclude that TNF and dexamethasone separately suppress chondrogenesis and bone growth when studied in an animal model of chronic inflammation. Our data give a possible mechanistic explanation to the commonly observed growth retardation in children with chronic inflammatory diseases treated with GCs.

Magnetic resonance and diffusion tensor imaging of the adolescent rabbit growth plate of the knee.

To assess the ability of MRI-DTI to evaluate growth plate morphology and activity compared with that of histomorphometry and micro-CT in rabbits. The hind limbs of female rabbits aged 16, 20, and 24 wk (n=4 per age group) were studied using a 9.4T MRI scanner with a multi-gradient echo 3D sequence and DTI in 14 directions (b-value=984 s/mm2 ). After MRI, the right and left hind limb were processed for histological analysis and micro-CT, respectively. The Wilcoxon signed-rank test was used to evaluate the height and volume of the growth plate. Intraclass correlation and Pearson correlation coefficient were used to evaluate the association between DTI metrics and age. The growth plate height and volume were similar for all modalities at each time point and age. Age was correlated with all tractography and DTI metrics in both the femur and tibia. A correlation was also observed between all the metrics at both sites. Tract number and volume declined with age; however, tract length did not show any changes. The fractional anisotropy color map showed lateral diffusion centrally in the growth plate and perpendicular diffusion in the hypertrophic zone, as verified by histology and micro-CT. MRI-DTI may be useful for evaluating the growth plates.

Blocking circadian clock factor Rev-erbα inhibits growth plate chondrogenesis via up-regulating MAPK-ERK1/2 pathway

ABSTRACT Emerging evidence indicated circadian clock gene Rev-erbα was involved in cartilage metabolism, however the contribution of Rev-erbα to growth plate chondrogenesis remains unknown. Here, we found that Rev-erbα exhibited the spatiotemporal expression model in growth plate. Moreover, Rev-erbα antagonist SR8278 inhibited longitudinal elongation of metatarsal bone ex vivo. And morphological analysis exhibited SR8278 led to the reduced height of growth plate and hypertrophic zone. Furthermore, blocking Rev-erbα suppressed the proliferation and hypertrophic differentiation of chondrocytes in growth plate. Similarly, knock-down Rev-erbα inhibited the proliferation and differentiation of primary chondrocytes in vitro. The mechanistic study indicated that knock-down Rev-erbα up-regulated MAPK-ERK1/2 pathway in chondrocytes. However, restraint of MAPK-ERK1/2 pathway alleviated partially SR8278-inhibited longitudinal elongation of metatarsal bone and growth plate development. Therefore, our results provide evidence of the vital role of Rev-erbα on growth plate chondrogenesis.

In vivo and in vitro evidence for growth hormone-like bioactivity of Rhizoma Anemarrhenae extract

Certain herbs used in traditional Chinese medicine may produce a growth-enhancing effect by promoting the secretion of growth hormone (GH) by the pituitary gland or mimicking the function of GH. In this study, we aimed to identify herbs that could serve as GH alternatives. A reporter gene assay for GH was developed, and 100 different herbal extracts were assayed. We found that Rhizoma Anemarrhenae (RA) water extracts exhibited transactivation activities that stimulate the activation of signal transducer and activator of transcription 5 (STAT5). The growth-promoting effect of RA in NB2–11 cells was inhibited by co-treatment with GH receptor (GHR)-Fc fusion protein. Unlike GH, RA extracts did not enhance the growth of B16F10 melanoma cells. The activation of the Janus kinase 2-STAT5 signaling pathway was confirmed in both NB2–11 cells and WI-38 human normal lung fibroblasts; the activation was inhibited by co-treatment with GHR-Fc fusion protein. Docking analysis of the active ingredients of RA, including mangiferin, neomangiferin, isomangiferin, anemarsaponin E, 7-O-methylmangiferin, officinalisinin I, timosaponin BII, timosaponin AI, and timosaponin AIII, using SWISSDOCK indicated a direct interaction of these compounds with GHR. The growth-promoting effects and activation of STAT5 were also confirmed. Moreover, we found that RA extract significantly increased the height of the tibial growth plate and stimulated the production of insulin-like growth factor 1 in the serum, liver, and muscle tissues. Our findings provide evidence that herbal extracts, particularly, RA extracts, can promote growth by mimicking GH bioactivity.

Juvenile mice exhibit inverse catch‐up growth but retain disorganized growth plates following a high‐fat to low‐fat diet intervention

Obesity can negatively impact the skeleton. We previously reported that a high‐fat diet causes rapid and dysregulated bone elongation. Our goal was to determine whether a diet intervention could mitigate or even reverse some of these effects on skeletal growth plates. We tested theHYPOTHESISthat rapid and dysregulated growth caused by a high‐fat diet will be mitigated by a low‐fat diet intervention. We expected to see a reduction in growth rate and changes in growth plate morphology that matched controls after switching to a low‐fat diet.METHODS3‐week old C57BL/6 mice (N=96 mixed sex) were fed control (10% kcal fat) or high‐fat (60% kcal fat) diets for one week. Some high‐fat diet mice were switched to control diet and examined at 5‐, 6‐, and 8‐weeks age (at least N=5/diet/age). Sex by diet interactions were tested at 5‐weeks. Body mass was recorded. Tibial elongation rate was measured from oxytetracycline labeled (7.5 mg/kg IP) slab sections. Growth plate height and columnar orientation (angle of proliferative column relative to long axis) were quantified in ImageJ. Statistical significance (p<0.05) was determined in SPSS by ANOVA with post‐hoc tests.RESULTSThere were no sex by diet interactions, so females were analyzed separately to discriminate diet effects. High‐fat and intervention groups were both heavier than controls at 4‐weeks before the intervention (F=7.7, p<.001). Body mass declined sharply after the diet switch and intervention mice were similar to controls at 5‐, 6‐, and 8‐weeks, while high‐fat diet mice remained heavier (p<0.001). Tibial elongation rate was faster in high‐fat diet mice (F=12.2, p<0.001), but slower in the intervention group compared to controls at both 5‐ (p<.001) and 6‐weeks age (p<0.01). Relative to controls, the most significant changes in growth plate height (F=10.7, p<0.001) were enlarged growth plates in high‐fat diet mice at 5‐weeks (p<0.001) and smaller growth plates in intervention mice at 6‐weeks (p=0.003). Chondrocyte columns in controls aligned within 2 degrees of the long axis at 5 weeks, while columns in both high‐fat and intervention mice deviated over 10 degrees (F=17.3, p<0.001). Columns remained disorganized in high‐fat and intervention groups at 6‐ and 8‐weeks (p<0.01).DISCUSSIONAfter switching from high‐ to low‐fat diet, intervention mice exhibited a rapid decrease in body mass, tibial elongation rate, and growth plate height. While we hypothesized that diet intervention would attenuate these to control levels, we surprisingly found that tibial elongation rate in the intervention group was actually slower than that of controls, resembling “inverse catch‐up growth” to achieve a potential target growth rate. However, chondrocyte columns remained disorganized, suggesting a possible lasting consequence of high‐fat diet.SIGNIFICANCEThese results demonstrate that the skeleton is exquisitely sensitive to diet in the earliest stages of postnatal bone elongation and are relevant for establishing interventions to prevent childhood obesity and its negative effects on bone structure.

Effect of a high crude protein content diet during energy restriction and re-alimentation on animal performance, skeletal growth and metabolism of bone tissue in two genotypes of cattle.

The objective of this study was to investigate the effect of diet crude protein (CP) content and metabolisable energy (ME) intake on skeletal growth and associated parameters of growing steers prior to and during compensatory growth in weight and catch-up growth in skeletal elongation. The experiment was a factorial design with two cattle genotypes [Brahman crossbred (BX, 178 ± 6 kg) and Holstein-Friesian (HF, 230 ± 34 kg)] and three nutritional treatments; high CP content and high ME intake (HCP-HME), high CP content and low ME intake (HCP-LME) and low CP content and low ME intake (LCP-LME) with the ME intake of HCP-LME matched to that of LCP-LME. Nutritional treatments were imposed over a 103 d period (Phase 1), and after this, all steers were offered ad libitum access to the HCP-HME nutritional treatment for 100 d (Phase 2). Steers fed the high CP content treatment with a low ME intake, showed higher hip height gain (P = 0.04), larger terminal hypertrophic chondrocytes (P = 0.02) and a higher concentration of total triiodothyronine in plasma (P = 0.01) than steers with the same ME intake of the low CP content treatment. In addition, the low CP treatment resulted in significant decreases in bone volume (P = 0.03), bone surface area (P = 0.03) and the concentration of bone-specific alkaline phosphatase in plasma (P < 0.001) compared to steers fed the HCP-HME treatment. A significant interaction between genotype and nutritional treatment existed for the concentration of thyroxine (T4) in plasma where HF steers fed LCP-LME had a lower T4 concentration in plasma (P = 0.05) than BX steers. All steers with a restricted ME intake during Phase 1 demonstrated compensatory growth during Phase 2. However, HF steers fed the LCP treatment during Phase 1 showed a tendency (P = 0.07) for a greater LWG during Phase 2 without any increase in dry matter intake. Results observed at the growth plate and hip height growth suggest that catch-up growth in cattle may also be explained by the growth plate senescence hypothesis. Contrary to our initial hypothesis, the results demonstrate that greater CP intake during ME restriction does not increase compensatory gain in cattle during re-alimentation.

Part 2. Review and meta-analysis of studies on modulation of longitudinal bone growth and growth plate activity: A micro-scale perspective.

Macro-scale changes in longitudinal bone growth resulting from mechanical loading were shown in Part 1 of this review to depend on load magnitude, anatomical location, and species. While no significant effect on longitudinal growth was observed by varying frequency and amplitude of cyclic loading, such variations, in addition to loading duration and species, were shown to affect the morphology, viability, and gene and protein expression within the growth plate. Intermittent compression regimens were shown to preserve or increase growth plate height while stimulating increased chondrocyte presence in the hypertrophic zone relative to persistent and static loading regimens. Gene and protein expressions related to matrix synthesis and degradation, as well as regulation of chondrocyte apoptosis were shown to exhibit magnitude-, frequency-, and duration-dependent responses to loading regimen. Chondrocyte viability was shown to be largely preserved within physiological bounds of magnitude, frequency, amplitude, and duration. Persistent static loading was shown to be associated with overall growth plate height in tension only, reducing it in compression, while affecting growth plate zone heights differently across species and encouraging mineralization relative to intermittent cyclic loading. Lateral loading of the growth plate, as well as microfluidic approaches are relatively understudied, and age, anatomical location, and species effects within these approaches are undefined. Understanding the micro-scale effects of varied loading regimes can assist in the development of growth modulation methods and device designs optimized for growth plate viability preservation or mineralization stimulation based on patient age and anatomical location.

A Mixture of Humulus japonicus Increases Longitudinal Bone Growth Rate in Sprague Dawley Rats.

The aim of this study was to investigate the effects of administration of a mixture of Humulus japonicus (MH) on longitudinal bone growth in normal Sprague Dawley (SD) rats. We measured the femur and tibia length, growth plate area, proliferation of chondrocytes, and expression of insulin-like growth factor-1 (IGF-I) and IGF binding protein-3 (IGFBP-3), and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) phosphorylation after dietary administration of MH in SD rats for four weeks. The nose–tail length gain and length of femur and tibia increased significantly in the group that received MH for a period of four weeks. We performed H&E staining and Bromodeoxyuridine/5-Bromo-2′-Deoxyuridine (BrdU) staining to examine the effect of dietary administration of MH on the growth plate and the proliferation of chondrocytes and found that MH stimulated the proliferation of chondrocytes and contributed to increased growth plate height during the process of longitudinal bone growth. In addition, serum levels of IGF-1 and IGFBP-3 and expression of IGF-1 and IGFBP-3 mRNAs in the liver and bone were increased, and phosphorylation of JAK2/STAT5 in the liver was increased in the MH groups. Based on these results, we suggest that the effect of MH on longitudinal bone growth is mediated by increased JAK2/STAT5-induced IGF-1 production.

High‐fat diet increases IGF‐I activity in skeletal growth plates before the onset of obesity

Childhood obesity can be one of the greatest perils to the growing skeleton because its effects are often hidden until a child is disabled by pain. Obesity is characterized by accelerated linear growth and skeletal maturation, which often leads to slipped epiphyses and fractures. It is unclear what causes this growth acceleration. IGF‐I, the major growth‐promoting hormone, is paradoxically normal in obesity. Equally puzzling, our lab found that a high‐fat diet accelerates bone lengthening in mice before they become obese. Our goal here was to determine whether a high‐fat diet alters local IGF‐I signaling and structure of the growth plate. We tested the HYPOTHESIS: that a high‐fat diet increases IGF‐I activity in growth plates before the onset of obesity. If diet‐induced changes in IGF‐I signaling underlie the growth acceleration, we expected to find dysregulated IGF‐I activity and concomitant changes in growth plate structure without changes in body mass.METHODS3‐week old C57BL/6 mice (N=8/diet/group) were fed control (10% kcal fat) or high‐fat (60% kcal fat) diets for 14 days. Immunostaining was performed on proximal tibial histology sections using antibodies against the phosphorylated (activated) IGF‐I receptor and IGFBP‐4. Growth plate height (total and individual zones), protein expression (percent cells positively stained) and columnar orientation (angle of a line drawn through a column of at least 5 proliferative cells relative to long axis of the bone) were quantified in ImageJ. Statistical significance (p&lt;0.05) was determined in SPSS using t‐tests.RESULTSTibial elongation rate was over 10% greater in the high‐fat diet group (t=2.1, p&lt;0.05) with no difference in body mass (t=0.5, p=0.62). Growth plate height was also greater in high‐fat diet mice (t=8.6, p&lt;0.001), with marked increases in height of hypertrophic (t=7.3, p&lt;0.001) and proliferative (t=6.5, p&lt;0.001) zones. Columnar orientation significantly differed between diets (t=7.1, p&lt;0.001). High‐fat diet mice exhibited columns that deviated nearly 8 degrees from the longitudinal axis of the bone, while columns in the control mice aligned within 2 degrees of the long axis. Immunostaining revealed a significant increase in IGF‐I activity in high‐fat diet relative to control mice, assessed by the percentage of chondrocytes expressing the phosphorylated IGF‐I receptor (t=2.4, p&lt;0.05), but a decrease in IGFBP‐4 expression (t=5.9, p&lt;0.001).DISCUSSIONOur results support the hypothesis that a high‐fat diet increases IGF‐I activity in growth plates before the onset of obesity. Key findings that suggest IGF‐I signaling is dysregulated by a high‐fat diet include: increased IGF‐I receptor activation, decreased inhibitory binding proteins, increased growth plate height and decreased columnar organization.SIGNIFICANCEThese results highlight the fundamental role of diet in the earliest stages of developing obesity‐related skeletal complications and are relevant for establishing early interventions to prevent childhood obesity and its damaging effects.Support or Funding InformationSupported by the National Institute of General Medical Sciences (1P20GM121299‐01) and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH (1R15AR067451‐01).

Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans.

The purposes of this study are to evaluate which growth plate parameters are associated with bone growth in mice and to compare the mouse results with those in humans. The sagittal sections of the proximal growth plate of the mouse tibia from neonate to young adult stages were subjected to histomorphometric and functional analyses. The radiographic images of tibias of human patients until puberty were analyzed to obtain the tibia length and the proximal growth plate height. It was found that a linear correlation best modeled the relationship between the growth plate variables with the tibia growth rate and length. In mice, total height, resting zone height, combined height of the proliferation and prehypertrophic zones, proliferation activity, and the total width of tibia growth plate showed high linear correlation with tibia bone length and bone growth rate, but the hypertrophic zone height and the growth plate area did not. In both mice and humans, the total growth plate width of tibia was found to have the strongest correlation with tibia length and growth rate. The results validated that growth plate total height, the height of the resting zone and cell proliferation activity are appropriate parameters to evaluate the balance between growth plate activity and bone growth in mice, consistent with previous reports. The study also provided a new growth plate parameter candidate, growth plate width for growth plate activity evaluation in both mouse and human tibia bone.

Indirubin-3\u2032-oxime stimulates chondrocyte maturation and longitudinal bone growth via activation of the Wnt/\u03b2-catenin pathway

Researchers have shown increased interest in determining what stimulates height. Currently, many children undergo precocious puberty, resulting in short stature due to premature closure of the growth plate. However, the current approach for height enhancement is limited to growth hormone treatment, which often results in side effects and clinical failure and is costly. Although recent studies have indicated the importance of paracrine signals in the growth plate for longitudinal bone growth, height-stimulating agents targeting the signaling pathways involved in growth plate maturation remain unavailable in the clinic. The Wnt/β-catenin pathway plays a major role in the maturation of growth plate chondrocytes. In this study, by using an ex vivo tibial culture system, we identified indirubin-3′-oxime (I3O) as a compound capable of enhancing longitudinal bone growth. I3O promoted chondrocyte proliferation and differentiation via activation of the Wnt/β-catenin pathway in vitro. Intraperitoneal injection of I3O in adolescent mice increased growth plate height along with incremental chondrocyte maturation. I3O promoted tibial growth without significant adverse effects on bone thickness and articular cartilage. Therefore, I3O could be a potential therapeutic agent for increasing height in children with growth retardation.

Status of growth plates can be monitored by MRI.

Growth plate injuries and disorders cause premature closure, resulting in shortened or deformed limbs. Quantitative assessment by MRI might monitor the status of the growth plate and may assist in the prediction of these deformations. To investigate whether the status of the growth plate can be monitored by quantitative evaluation using MRI of the noninjured region of the growth plate in a physeal injury model. Prospective, longitudinal. A 3.0-mm drill was used to create an injury to the central region of the right proximal tibial growth plate in 5-week-old male Japanese white rabbits (N = 18). The left tibia served as the control. 7.04T, T2 -weighted imaging, diffusion-weighted imaging. Eight of 18 rabbits underwent MRI, proton density-weighted imaging, and T2 -weighted and diffusion-weighted imaging. T2 and apparent diffusion coefficient (ADC) maps were generated for each image. The growth plate height and the T2 and ADC values of the noninjured region were measured. Two rabbits were sacrificed at 2, 4, 6, 8, and 10 weeks postinjury. Proximal tibial bones were evaluated using microcomputed tomography, histological, and immunohistological methods. Data were compared using repeated-measures analysis of variance followed by Tukey post-hoc multiple comparison. Growth plate height decreased at 10 weeks postinjury (P = 0.018) on the injured side. T2 values were greater at 2 weeks postinjury (P = 0.0478) and decreased at 8 and 10 weeks (P = 0.0226, P = 0.0470, respectively) on the injured side. ADC values increased at 6 weeks on the lateral side (P = 0.0304) and decreased at 8 weeks and 10 weeks postinjury (P < 0.01) on the medial and injured sides, respectively. Quantitative MRI can help monitor the status of the growth plate and capture its changes early. 1 Technical Efficacy Stage: 3 J. Magn. Reson. Imaging 2020;51:133-143.