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Concept: Endochondral ossification

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Composite biological and synthetic grafts with progenitor cells offer an alternative approach to auto- or allografts for fracture repair. This study aimed to evaluate osteogenesis of autologous serum-derived albumin (ASA) scaffolds seeded with canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) in a canine segmental bone defect model. ASA scaffold was prepared with canine serum using cross-linking and freeze-drying procedures. Beta-tricalcium phosphate (β-TCP) was mixed at the cross-linking stage. Ad-MSCs were seeded into the scaffold and incubated for a day before implantation. After 16 weeks, the grafts were harvested for histological analysis. The dogs were divided into five groups: control, ASA scaffolds with and without Ad-MSCs, and ASA scaffolds including β-TCP with and without Ad-MSCs. ASA scaffolds with Ad-MSCs had significantly larger area of increased opacity at the proximal and distal host cortex-implant interfaces in radiographs 16 weeks after implantation compared to the groups with β-TCP (p < 0.05). Histomorphometric analysis showed that ASA scaffolds with Ad-MSCs had significantly more new bone formation than other groups (p < 0.05). These results suggest that Ad-MSCs seeded into ASA scaffolds enhanced osteogenesis in the bone defect model but β-TCP in the ASA scaffold might prevent penetration of the cells required for bone healing.

Concepts: Stem cell, Mesenchymal stem cell, Bone marrow, Stem cells, Adipocyte, Endochondral ossification

136

Both stem cell therapy and physical treatments have been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli for large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low-power laser irradiation (LPLI) and human adipose-derived stem cell (ADSC) treatments during fracture repair using a comparative rat calvarial defect model. We evaluated the viability of human ADSCs, which were cultured on a porous PLGA scaffold using an MTS assay. The critical-sized calvarial bone defect rats were divided into 4 groups: control group, LPLI group, ADSC group, and ADSC+LPLI group. Bone formation was evaluated using micro-CT. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. Our data showed that PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses revealed that both the LPLI and ADSC groups showed improved calvarial bone defect healing compared to the control group. In addition, the ADSC+LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC+LPLI group. There were significant differences between the groups. In addition, both ADSC and ADSC+LPLI groups showed strong signals of vWF expression. ADSC and LPLI treatments improved fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and LPLI further enhances the bone healing process.

Concepts: Cell, Bone fracture, Developmental biology, Stem cell, Stem cells, Endochondral ossification, Bone healing, Stem cell treatments

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The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFβ sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction.

Concepts: Bone, Stem cell, Mesenchymal stem cell, Bone marrow, Skeletal system, Cartilage, Endochondral ossification, Intramembranous ossification

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Fibrodysplasia Ossificans Progressiva (FOP) is a rare and as yet untreatable, genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment and premature death. Most FOP patients carry an activating mutation in a BMP type I receptor gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and in turn HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist Palovarotene effectively inhibited HO in injury-induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional-on knock-in mouse line carrying the human ACVR1(R206H) mutation for classic FOP. In addition, Palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, Palovarotene maintained joint, limb and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. This article is protected by copyright. All rights reserved.

Concepts: Gene, Genetics, Skeletal system, Cartilage, Endochondral ossification, Intramembranous ossification, Fibrodysplasia ossificans progressiva, Retinoic acid receptor

28

Skeletal maturation is divisible to three main components; the time of appearance of an ossification center, its change in morphology and time of fusion to a primary ossification center. With regard to the knee, the intermediate period between appearance and fusion of the ossification centers extends over a period of greater than 10 years. This study aims to investigate radiographically the age at which morphological changes of the epiphyses at the knee occur in a modern Irish population. Radiographs of 221 subjects (137 males; 84 females) aged 9-19 years were examined. Seven nonmetric indicators of maturity were assessed using criteria modified from the Roche, Wainer, and Thissen method and Pyle and Hoerr’s atlas of the knee. Reference charts are presented which display the timeline for each of the grades of development of the seven indicators. Mean age was found to increase significantly with successive grades of development of each of the seven indicators. A significant difference was noted between males and females at the same grade of development for six of the seven indicators. The narrowest age range reported for a single grade of development was 2.2 years for Grade 2 of development of the tibial tuberosity for males. The information on changing morphology of the epiphyses at the knee in the present study may provide an adjunct to methods used for evaluation of skeletal maturity before surgery for orthopedic disorders or to evaluate skeletal age in clinical scenarios where either delayed or precocious skeletal maturation is suspected. Clin. Anat. 26:755-767, 2013. © 2012 Wiley Periodicals, Inc.

Concepts: Time, Bone, Human, Grade, The Age, Change, Endochondral ossification, Irish language

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HT042, a new herbal prescription consisting of Astragalus membranaceus, Phlomis umbrosa and Eleutherococcus senticosus, is used in traditional Korean medicine to stimulate growth in children. This study was conducted to investigate the effects of HT042 on skeletal growth, insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) levels, and oestrogenic activity in female rats. Female Sprague-Dawley rats were divided into control, recombinant human growth hormone (rhGH; 20 µg/kg/day), and HT042 (100 mg/kg/day) groups and treated for 3 weeks. Axial skeletal growth, femur length, and growth plate length were measured every 3 weeks. The serum IGF-1 and IGFBP-3 levels were analysed. Moreover, the oestrogenic activity of the herbal extracts in the immature and ovariectomized rats was tested. The nose-anus, nose-tail, femur and growth-plate lengths were increased significantly in the HT042 group. Both IGF-1 and IGFBP-3 were highly expressed in the hypertrophic zone of the growth plate. The serum IGF-1 levels were increased. Moreover, HT042 had no uterotrophic effects in the rats. Consequently, HT042 promoted longitudinal bone growth by stimulating cell proliferation in the epiphyseal plate and inducing the expression of IGF-1 without an oestrogenic response. HT042 may be helpful in stimulating growth in children with short stature. Copyright © 2012 John Wiley & Sons, Ltd.

Concepts: Immune system, Insulin-like growth factor 1, Growth hormone, Growth factors, Endochondral ossification, Insulin-like growth factor, Idiopathic short stature, Long bone

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We studied the effect of iron saturation level on the osteogenic activity of lactoferrin (LF) in vitro and in vivo. Different iron saturation levels of LF (1.0, 9.0, 38, 58, and 96%) were prepared as the following samples: apo-LF, LF-9, LF-38, LF-58, and holo-LF. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we observed that the stimulating osteoblast proliferation activity of LF in vitro decreased with increasing iron saturation level at 100 and 1,000 μg/mL. In vivo, 4-wk-old ICR Swiss male mice were randomly divided into 4 groups: blank control (physiological saline), negative control (BSA), apo-LF, and holo-LF. Four groups of mice were injected subcutaneously with physiological saline, BSA, apo-LF, or holo-LF over the calvarial surface twice a day for 5 consecutive days at a dose of 4 mg/kg per day. Bone histomorphometry showed that new bone formation (assessed using tetracycline-HCl labels) tended to be stronger with apo-LF than with holo-LF. Using fluorescence spectroscopy and circular dichroism measurements, we found that exposure of tryptophan increased, α-helix content increased, but β-structure content decreased with increasing iron saturation level. These findings indicated that the osteogenic activity of LF decreases with increasing iron saturation level in vitro and in vivo, which may be related to conformational changes in LF.

Concepts: Spectroscopy, Bone, Skeletal system, In vivo, Cartilage, In vitro, Endochondral ossification, Intramembranous ossification

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Until final completion of maturation processes at the age of approximately 18 years, determination of the skeletal age of the hand plays a central role in forensic age diagnostics in living persons in criminal proceedings. In this process, assessment of hand radiographs relies primarily on the stage of development of the epiphyseal nuclei, the increase in size of the individual bones and of the hand skeleton as a whole, changes in the shape of the various skeletal elements and ossification of the epiphyseal plates. To achieve this, there are a variety of methodological approaches based on two different fundamental principles. The methods proposed by Greulich and Pyle, Thiemann et al. and Gilsanz and Ratib rank among the so-called atlas techniques, whilst the methods proposed by Tanner et al. and Roche et al. are classified as so-called bone-specific techniques. In order to be applicable in the field of criminal procedure, the methods of estimating the skeletal age of the hand developed with clinical aspects in mind must satisfy the demands of a high degree of estimate accuracy and good reproducibility of the estimated results. In the course of the present study, a study population of 92 persons was used to compare the above-mentioned atlas and bone-specific techniques for determining hand skeleton age in view of these qualitative criteria. Estimate accuracy was studied using Pearson’s correlation coefficients, and weighted kappa coefficients were determined for studying the intra-and interobserver agreement of an estimate result. In the inter-method comparison, a basically good agreement was shown between the skeletal ages and the chronological age of the test persons on the one hand and the skeletal age diagnoses of one or of two examiners on the other. No general advantage of the methodological approach of the bone-specific technique was discernible in the course of comparison; in the female gender, particularly, the RUS2 and RUS3 score of the method of Tanner et al. proved unfavourable. For age estimation practice in criminal proceedings, the atlas methods of Greulich and Pyle and Thiemann et al. are particularly recommendable.

Concepts: Bone, Statistics, Pearson product-moment correlation coefficient, Approximation, Estimation, Endochondral ossification, Human skeleton, Legal burden of proof

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OBJECTIVE: To transect blood vessels within epiphyseal cartilage canals and observe whether this resulted in ischaemic chondronecrosis, an associated focal delay in enchondral ossification (osteochondrosis) and pathological cartilage fracture (osteochondrosis dissecans) in the distal femur of foals, with potential translational value to the pathogenesis of juvenile osteochondritis dissecans in children. METHOD: Ten Norwegian Fjord Pony foals were operated at the age of 13-15 days. Two vessels supplying the epiphyseal growth cartilage of the lateral trochlear ridge of the left distal femur were transected in each foal. Follow-up examination was carried out from 1-49 days post-operatively and included plain radiography, macroscopic and histological examination. RESULTS: Transection of blood vessels within epiphyseal cartilage canals resulted in necrosis of vessels and chondrocytes, i.e. ischaemic chondronecrosis, in foals. Areas of ischaemic chondronecrosis were associated with a focal delay in enchondral ossification (osteochondrosis) in foals examined 21 days or more after transection, and pathological cartilage fracture (osteochondrosis dissecans) in one foal examined 42 days after transection. CONCLUSION: The ischaemic hypothesis for the pathogenesis of osteochondrosis has been reproduced experimentally in foals. There are several similarities between osteochondrosis dissecans in animals and juvenile osteochondritis dissecans in children. It should be investigated whether juvenile osteochondritis dissecans also occurs due to a focal failure in the cartilage canal blood supply, followed by ischaemic chondronecrosis.

Concepts: Blood, Heart, Blood vessel, Cartilage, Necrosis, Endochondral ossification, Osteochondritis dissecans, Fjord horse

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Coronoid dysplasia (CD) or medial coronoid disease is part of canine elbow dysplasia and eventually results in osteoarthrosis. Although CD was originally attributed to disturbed endochondral ossification, more recent data point to the subchondral bone. The objective of this study was to assess dysplastic bone and cartilage of dogs that underwent unilateral or bilateral arthroscopic subtotal coronoidectomy for the treatment of CD. Arthroscopic findings and histopathology of bone and cartilage removed from elbow joints with CD were compared. The most common arthroscopic finding was fragmentation with softening of the subchondral bone of the central part of the medial coronoid process. In dogs without obvious fragmentation, CD was characterised by bone softening and chondromalacia. During arthroscopic intervention dysplastic bone and cartilage were collected for histopathological assessment. Forty-five slices of formalin-fixed, paraffin-embedded bone and cartilage samples were stained using haematoxylin and eosin and evaluated. Histopathological findings primarily consisted of osteonecrosis of subchondral bone with necrosis within the marrow spaces. Histopathological changes in the articular cartilage were characterised by fibrillation, chondrocyte clone formation, and focal cartilage necrosis. The pathology was found primarily in the subchondral bone and not in the articular cartilage. Vascular compromise may play a role in the pathogenesis of osteonecrosis in CD.

Concepts: Bone, Pathology, Skeletal system, Histopathology, Cartilage, Endochondral ossification, Chondrocyte, Ossification