BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3−/− mice.

Evaluation of a Diagnostic-Therapeutic Algorithm for Finger Epiphyseal Growth Plate Stress Injuries in Adolescent Climbers

Background: Finger epiphyseal growth plate stress injuries are the most frequent sport-specific injuries in adolescent climbers. Definitive diagnostic and therapeutic guidelines are pending. Purpose: To evaluate a diagnostic-therapeutic algorithm for finger epiphyseal growth plate stress injuries in adolescent climbers. Study Design: Case series; Level of evidence, 4. Methods: On the basis of previous work on diagnostics and treatment of finger epiphyseal growth plate stress injuries (EGPIs) in adolescent climbers, we developed a new algorithm for management of these injuries, which was implemented into our clinical work. During a 4-year period, we performed a prospective multicentered analysis of our patients treated according to the algorithm. Climbing-specific background was evaluated (training years, climbing level, training methods, etc); injuries were analyzed (Salter-Harris classification and UIAA MedCom score [Union Internationale des Associations d’Alpinisme]); and treatments and outcomes were recorded: union, time to return to climbing, VAS (visual analog scale), QuickDASH (shortened version of the Disabilities of the Arm, Shoulder, and Hand), and a climbing-specific outcome score. Results: Within the observation period, 27 patients with 37 independent EGPIs of the fingers were recorded (mean ± SD age, 14.7 ± 1.5 years; 19 male, 8 female; 66.7% competitive athletes). Regarding maturity at time of injury, the mean age at injury did not differ by sex. Average UIAA climbing level was 9.5 ± 0.8, with 6 ± 4.6 years of climbing or bouldering and 14 ± 9.1 hours of weekly climbing-specific training volume. Among the 37 injuries there were 7 epiphyseal strains, 2 Salter-Harris I fractures, and 28 Salter-Harris III fractures (UIAA 1, n = 7; UIAA 2, n = 30). Thirty-six injuries developed through repetitive stress, while 1 had an acute onset. Twenty-eight injuries were treated nonoperatively and 9 surgically. Osseous union was achieved in all cases, and there were no recurrences. The time between the start of treatment and the return to sport was 40.1 ± 65.2 days. The climbing-specific outcome score was excellent in 34 patients and good in 3. VAS decreased from 2.3 ± 0.6 to 0.1 ± 0.4 after treatment and QuickDASH from 48.1 ± 7.9 to 28.5 ± 3.3. Conclusion: The proposed management algorithm led to osseous union in all cases. Effective treatment of EGPIs of the fingers may include nonsurgical or surgical intervention, depending on the time course and severity of the injury. Further awareness of EGPI is important to help prevent these injuries in the future.

Effects of bisphosphonates on different zones of the epiphyseal growth plate of rats

Bisphosphonates (BIS) are indicated for several clinical disorders (e.g., osteoporosis). However, BIS has been associated with osteonecrosis and alterations in osteoclastogenesis and skeletal development. This study aimed to evaluate the effects of BIS (zoledronic acid - ZA and alendronate sodium - AS) on zones of the growth plate of rat femur. Animals (Wistar rats, n = 19) were divided into groups: 1) AS Group: animals received alendronate sodium orally (3 mg/kg per day); 2) ZA Group: ZA was administered intraperitoneally (0.2 mg/kg per week); and 3) Control Group (CG): a vehicle was administered. Animals were euthanized 21 days after the treatment, and femurs were collected for histological analysis. The images of all zones (resting, proliferative, hypertrophic, and calcified) were processed by the Qcapture® software providing a 40 and 400-fold increase. ZA decreased epiphyseal growth plate cell zones (ZA Group vs. CG) in most cases. Likewise, AS diminished the proliferative zone (AS Group vs. CG). Furthermore, ZA increased the calcified zone (ZA Group vs. CG). Previous works demonstrated that BIS decrease the epiphyseal disc. This reduction is probably due to the shortening of the cellular zones that undergoes calcification/ossification. The present results suggest that BIS should be carefully indicated because these drugs might accelerate epiphyseal closure.

EXTH-70. ESTABLISHMENT OF INTRAVENTRICULAR SHH INHIBITION AS A THERAPEUTIC OPTION IN YOUNG PATIENTS WITH MEDULLOBLASTOMA

Medulloblastoma is the most common malignant brain tumor in children. The embryonal tumor arises in the posterior fossa and disseminates via the cerebrospinal fluid. Medulloblastoma divides in four molecular subgroups, one of which is characterized by mutations in the sonic-hedgehog (SHH) -pathway. SHH inhibition provides an elegant way of targeted therapy. The small molecule Vismodegib allosterically inhibits Smoothened (SMO), an upstream activator of SHH. Unfortunately, Vismodegib has shown to cause irreversible premature epiphyseal growth plate fusions in preclinical studies and clinical trials, preventing its systemic application in children (Kimura et al. 2008; Robinson et al. 2017). We established an intraventricular therapy with Vismodegib, combining the benefits of targeted drug delivery and minimizing systemic side effects. In a mouse model for SHH medulloblastoma, we compare intraventricular, oral and placebo treatment regarding effects on survival, tumor biology, and bone morphology. Math1-cre::Ptch1 Fl/Fl mice show a homozygous loss of PTCH1 in Math1-positive cells, resulting in SHH-pathway overactivation and development of SHH medulloblastomas. At postnatal day 11-13, Math1-cre::Ptch1 Fl/Fl mice were randomized in four treatment arms: group A (n= 9) received placebo intrathecally, B (n= 9) received Vismodegib 200 mg/kd/d orally, C (n= 19) received Vismodegib 0.2 mg/kg/d intrathecally, and D (n= 8) received Vismodegib 1.6 mg/kg/d intrathecally. Kaplan-Meier survival analysis showed a significant survival benefit for 1.6 mg/kg/d intraventricular Vismodegib compared to placebo (p= 0.012). Bone histology and X-ray analysis of intraventricular treated mice showed intact femoral and tibial growth plates, in contrast to orally treated mice that developed skeletal malformations. Further analyses such as DNA sequencing, gene expression analysis, and histological evaluation are ongoing and will add to the picture of the anti-tumor effects of intraventricular SHH-inhibition. Based on the preliminary experimental results, we conclude that intrathecal application of a SMO-inhibitor might evolve as a promising new way of targeted treatment of SHH medulloblastomas.

Diazinon exposure reduces bone mineral density in adult and immature rats: A histomorphometric and radiographic study

Diazinon has been widely used as a domestic and agricultural pesticide. This study examined the effects of diazinon on bone mineral density (BMD) of mature and immature rats. For this purpose, 24 adult Wistar rats (male; 8 weeks old) were initially divided into four groups ( n = 6). Corn oil was used as the control while diazinon at 15, 30, and 45 mg/kg in corn oil was given to mature rats via gavage per day. Since these dosages were lethal for the immature rats, 12 immature Wistar rats (male; 4 weeks old) ( n = 6) were gavaged with corn oil as control and 5 mg/kg of diazinon in corn oil. The animals were sacrificed on day 28 with their left femur bones removed for histomorphometric studies. BMD was measured in the right femur, using standardized radiographs in the femoral head, femoral neck, greater trochanter, and shaft. The Image J Program was used for measuring the bone lamellae and epiphyseal growth plates. The results of this study for the first time revealed that diazinon reduced BMD in both adults and immature rats. Diazinon exposure was associated with diminished trabecular and cortical bone density. Correspondingly, our results indicated that in immature rats, DZN led to the reduction in the epiphyseal growth plate width, both in the proliferation and hypertrophic zones. These results suggested that diazinon might be associated with impaired bone longitudinal growth as well as bone metabolism in adults.

IRON DEPOSITION IN THE GROWTH PLATE OF LONG BONES OF THE OFFSPRING WHEN GIVEN DURING PREGNANCY IN RAT MODEL

Objective: To evaluate histologically the deposition of iron in the epiphyseal cartilage of offspring’s of dams given iron supplementation during pregnancy and lactation in rat model. Study Design: Laboratory based experimental study. Place and Duration of Study: Department of Anatomy, Army Medical College, Rawalpindi and National Institute of Health (NIH) Islamabad, from Mar to Nov 2016. Methodology: In this study, 16 female and 4 male adult rats were chosen for breading. After confirmation of pregnancy, pregnant rats were separated in two groups. One group was given oral iron supplementation for four weeks till delivery and half of the pups fed by mothers who were given iron during lactation. The other group was kept on normal lab diet. Deposition of iron in the epiphyseal cartilage of newborn rats after four weeks was evaluated histologically in pups. Results: Iron deposition was maximum in group C i.e. 1.30 ± 0.48; in group B it was 0.20 ± 0.44. Statistically significant iron deposition (p<0.001) was observed in the growth plate of off springs when mothers were given iron supplements during pregnancy and lactation. Conclusion: Present study proves that injudicious iron supplementation through pregnancy results in deposition of iron in epiphyseal growth plate of the fetus and it can have damaging effects on bones of fetus.

Associations of Obesity with Linear Growth and Puberty

<b><i>Background:</i></b> The prevalence of obesity in childhood has increased dramatically in recent decades with increased risk of developing cardiometabolic and other comorbidities. Childhood adiposity may also influence processes of growth and puberty. <b><i>Summary:</i></b> Growth patterns of obesity during childhood have been shown to be associated with increased linear growth in early childhood, leading to accelerated epiphyseal growth plate (EGP) maturation. Several hormones secreted by the adipose tissue may affect linear growth in the context of obesity, both via the growth hormone IGF-1 axis and via a direct effect on the EGP. The observation that children with obesity tend to mature earlier than lean children has led to the assumption that the degree of body fatness may trigger the neuroendocrine events that lead to pubertal onset. The most probable link between obesity and puberty is leptin and its interaction with the kisspeptin system, which is an important regulator of puberty. However, peripheral action of adipose tissue could also be involved in changes in the onset of puberty. In addition, nutritional factors, epigenetics, and endocrine-disrupting chemicals are potential mediators linking pubertal onset to obesity. In this review, we focused on interactions of obesity with linear growth and pubertal processes, based on basic research and clinical data in humans. <b><i>Key Message:</i></b> Children with obesity are subject to accelerated linear growth with risk of impaired adult height and early puberty, with its psychological consequences. The data highlight another important objective in combatting childhood obesity, for the prevention of abnormal growth and pubertal patterns.

Bone tissue morphology of rat offspring lactationally exposed to polychlorinated biphenyl 169 and 155

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent, organic pollutants also considered endocrine-disrupting chemicals. Our study examined the effects of lactational exposure to nondioxin-like PCB-155 and/or dioxin-like PCB-169 on longitudinal femur growth at the distal epiphyseal growth plate (EGP) in young rats at three different ages [postnatal days (PNDs) 9, 22, and 42]. After delivery, lactating rats were divided into four groups (PCB-169, PCB-155, PCB-155 + 169, and control) and administered PCBs intraperitoneally. The femurs of offspring were used to estimate growth rate (µm/day), and histomorphometric analysis on the distal femur included the thickness of the EGP and zones of proliferation and hypertrophy with calcification. Stereometry was used to determine trabecular bone volume density. In the PCB-169 and PCB-155 + 169 groups, PCB-169 affected longitudinal bone growth in the early postnatal period by interfering with chondrocytes in the EGP zone of proliferation and, to a lesser extent, the zone of hypertrophy. Morphometric alterations in EGP structure diminished until puberty. A slow growth rate persisted in the PCB-169 group until PND 42, while in the PCB-155 group, a fast growth rate between PNDs 9 to 22 was significantly reduced between PNDs 22 to 42. Sterometric assessment showed decreased trabecular bone volume in the PCB-155 + 169 group compared with that in the control on PND 9 and increased in the PCB-169 group compared with that in the PCB-155 group on PND 42. To summarize, studied PCB congeners exerted congener- and age-dependent effects on femur growth rate and its histomorphometric characteristics.

Forensic age assessment of the knee: proposal of a new classification system using two-dimensional ultrasound volumes and comparison to MRI

Objectives To assess epiphyseal growth plate closure of the knee for forensic age estimation using an ultrasound (US)-based method and to compare the findings with MRI. Methods Thirty-three healthy male individuals (age, 14.4–19.3 years) were prospectively evaluated for epiphyseal growth plate closure of the right knee by recordings of two-dimensional US volumes and a high-resolution T1-weighted MRI sequence. The degree of epiphyseal growth plate closure was rated independently by two readers for each method using a modality specific three-point scale that differentiates between an open physis (S1), a partially closed physis (S2), and a closed physis (S3). Results The inter-rater agreement was high for the US (Cohen’s kappa (CK): femur 95.2%, tibia 81.3%, fibula 86.3%) and the MRI method (CK: femur 70.2%, tibia 90.8%, fibula 79.8%). The degree of growth plate closure associated positively with advancing age. The US system showed a clearer separation of median ages with lower overlap than the MRI system. Open growth plates on minors (< S3 on femur and tibia) were identified by US with higher sensitivity (1.0 vs. 0.7) and slightly lower specificity (0.7 vs. 0.85) compared with MRI. The examination time was substantially shorter on US than on MRI (2.65 ± 0.91 min vs. 24.72 ± 2.72 min; p < 0.001). Conclusions The US method for evaluation of growth plate closure of the knee can reliably assign male individuals to different ossification stages and identifies minors with high accuracy. More studies with larger numbers are needed to further evaluate this method. Key Points • US is feasible to determine the degree of epiphyseal growth plate closure of the knee, shows a high degree of reliability, and is comparable to MRI. • US of the knee can detect open growth plates on male minors with high accuracy. • US of the knee may be used as a fast, non-invasive imaging tool for forensic age estimation to identify male minors.

Selective Chemonucleolysis With Condoliase in Cynomolgus Monkeys

Selective chemonucleolytic effects of condoliase, a glycosaminoglycan degrading enzyme, was investigated histopathologically in cynomolgus monkeys. Condoliase was administered once into the lumber intervertebral disc (IVD), and as a comparative control, chymopapain, a proteolytic enzyme, was administered in a similar manner. Histopathological changes of the IVD and the adjacent vertebral body (VB) were examined at 1 to 26 weeks after administration. Major changes induced by condoliase in the IVD were degenerative and necrotic changes in the nucleus pulposus, annulus fibrosus, cartilaginous endplate (CEP), and epiphyseal growth plate (EGP); focal disappearance of the EGP; and neovascularization and ossification of the CEP. Decreased/necrosis of bone marrow cells with new bone formation was observed in the VB. Cellular regeneration in the IVD was observed as a recovery changes on and after week 4. The changes in the IVD and VB subsided at week 26. Chymopapain induced qualitatively similar but more widely extended changes. The degrees of the changes in the IVD and VB were more severe than those of condoliase, and the changes were exacerbated even at week 26. These results indicated that histopathological changes caused by condoliase were less severe and more selective than those by chymopapain.