Long Bone Development Research Articles

Transcriptome-based comparison reveals key genes regulating allometry growth of forelimb and hindlimb bone in duck embryos

Allometric growth of the forelimb and hindlimb is a widespread phenomenon observed in vertebrates. As a typical precocial bird, ducks exhibit more advanced development of their hindlimbs compared to their forelimbs, enabling them to walk shortly after hatching. This phenomenon is closely associated with the development of long bones in the embryonic stage. However, the molecular mechanism governing the allometric growth of duck forelimb and hindlimb bones is remains elusive. In this study, we employed phenotypic, histological, and gene expression analyses to investigate developmental differences between the humerus (forelimb bone) and tibia/femur (hindlimb bones) in duck embryos. Our results revealed a gradual increase in weight and length disparity between the tibia and humerus from E12 to E28 (embryo age). At E12, endochondral ossification was observed solely in the tibia but not in the humerus. The number of differentially expressed genes (DEGs) gradually increased at H12 vs. T12, H20 vs. T20, and H28 vs. T28 stages consistent with phenotypic variations. A total of 38 DEGs were found across all 3 stages. Protein-protein interaction network analysis demonstrated strong interactions among members of HOXD gene family (HOXD3/8/9/10/11/12), HOXB gene family (HOXB8/9), TBX gene family (TBX4/5/20), HOXA11, SHOX2, and MEIS2. Gene expression profiling indicated higher expression levels for all HOXD genes in the humerus compared to tibia while opposite trends were observed for HOXA/HOXB genes with low or no expression detected in the humerus. These findings suggest distinct roles played by different clusters within HOX gene family during skeletal development regulation of duck embryo's forelimbs versus hind limbs. Notably, TBX4 exhibited high expression levels specifically in tibia whereas TBX5 showed similar patterns exclusively within humerus as seen previously across other species' studies. In summary, this study identified key regulatory genes involved in allometric growth of duck forelimb and hindlimb bones during embryonic development. Skeletal development is a complex physiological process, and further research is needed to elucidate the regulatory role of candidate genes in endochondral ossification.

Effects of pseudoephedrine on rat fetal bone development: evaluation by three different methods.

Pseudoephedrine (PSE) is an agent that is contained in common cold medications. The agent, which is used to treat cold and cough, is the fourth most prescribed drug group in some countries. During pregnancy, expectant mothers use PSE for colds and other reasons. One out of every four expectant mothers use PSE alone or in combination with other medicines for various reasons. This study was aimed to investigate effects of PSE on long bones development in rat during fetal growth. Pregnant rats were divided into five groups: control and four experimental groups (25mg/kg, 50mg/kg, 100mg/kg, 200mg/kg PSE). Between 1 and 20days of pregnancy, PSE was given to them by gavage. Weights and heights of fetuses isolated by cesarean on the 21st day were measured. Ossification of femur and humerus was examined by three different methods mentioned earlier. Depending on the dose increase, all morphometric data, ossification rate and bone length of the fetuses were decreased. Besides, it was determined that the amount of Calcium in the bone tissue decreased in the analyzes made with SEM-EDX Analysis. The data obtained from this study reveal that the use of PSE during pregnancy disrupts the existing balance in the bone and negatively affects ossification due to the dose increase. In conclusion, we present descriptive and novel data on the effects of PSE use during pregnancy on the bone development of rat fetal long bones.

Is mandible derived mesenchymal stromal cells superior in proliferation and regeneration to long bone-derived mesenchymal stromal cells?

Mesenchymal stromal cells (MSCs) are cells with the characteristic ability of self-renewal along with the ability to exhibit multilineage differentiation. Bone marrow (BM) is the first tissue in which MSCs were identified and BM-MSCs are most commonly used among various MSCs in clinical settings. MSCs can stimulate and promote osseous regeneration. Due to the difference in the development of long bones and craniofacial bones, the mandibular-derived MSCs (M-MSCs) have distinct differentiation characteristics as compared to that of long bones. Both mandibular and long bone-derived MSCs are positive for MSC-associated markers such as CD-73, -105, and -106, stage-specific embryonic antigen 4 and Octamer-4, and negative for hematopoietic markers such as CD-14, -34, and -45. As the M-MSCs are derived from neural crest cells, they have embryogenic cells which promote bone repair and high osteogenic potential. In vitro and in vivo animal-based studies demonstrate a higher rate of proliferation and high osteogenic potential for M-MSCs as compared to long-bones MSCs, but in vivo studies in human subjects are lacking. The BM-MSCs have their advantages and limitations. M-MSCs may be utilized as an alternative source of MSCs which can be utilized for tissue engineering and promoting the regeneration of bone. M-MSCs may have potential advantages in the repair of craniofacial or orofacial defects. Considering the utility of M-MSCs in the field of orthopaedics, we have discussed various unresolved questions, which need to be explored for their better utility in clinical practice.

Features of the skeleton of the girdle of the pelvic limb of the sable of the Black Pushkin breed in the age aspect

The study of the main morphophysiological parameters of mammalian loco- motor apparatus organs in the age aspect is an important and urgent problem for modern biology and morphology. The development and formation of the pelvic limb skeleton are determined by the living conditions of animals. Fur-bearing animals of natural populations have progressive and harmonious growth and development of long tubular bones in propor- tion to age, but the conditions of the cellular regime make significant adjustments to the morphogenetic transformations of the studied somatic system. The purpose of the study is to study the features of the skeleton of the pelvic limb belt bones of the sable of the black Pushkin breed in the age aspect and to establish the morphometric characteristics of this area. The study was conducted at the Department of Animal Anatomy of the St. Petersburg State University of Veterinary Medicine. Cadaver material for the study was delivered to the Department of Animal Anatomy of the St. Petersburg State University of Veterinary Medicine from the fur farming of the Leningrad region. The objects for this study were the corpses of the black Pushkin sable breed of two age groups: 15-20 months from birth (physi- ological maturity) and 36-40 months from birth (puberty) in the amount of eight pieces in each group. To achieve this task, a complex of traditional anatomical research methods was used: fine anatomical dissection, maceration, photographing and morphometry. When describing anatomical terms, the International Veterinary Anatomical Nomenclature (fifth edition) was used. The measurement was carried out with an electronic vernier caliper Orbit OT-INM02 with a division scale of 0.01 mm, manufactured in Russia. The features of the skeleton of the pelvic limb girdle in the age aspect and morphometric characteristics of the bones of this area in the sable of the black Pushkin breed are established

Endostructural and periosteal growth of the human humerus.

The growth and development of long bones are of considerable interests in the fields of comparative anatomy and palaeoanthropology, as evolutionary changes and adaptations to specific physical activity patterns are expected to be revealed during bone ontogeny. Traditionally, the cross-sectional geometry of long bones has been examined at discrete locations usually placed at set intervals or fixed percentage distances along the midline axis of the bone shaft. More recently, the technique of morphometric mapping has enabled the continuous analysis of shape variation along the shaft. Here we extend this technique to the full sequence of late fetal and postnatal development of the humeral shaft in a modern human population sample, with the aim of establishing the shape changes during growth and their relationship with the development of the arm musculature and activity patterns. A sample of modern human humeri from individuals of age ranging from 24 weeks in utero to 18 years was imaged using microtomography at multiple resolutions and custom Matlab scripts. Standard biomechanical properties, cortical thickness, surface curvature, and pseudo-landmarks were extracted along radial vectors spaced at intervals of 1° at each 0.5% longitudinal increment measured along the shaft axis. Heat maps were also generated for cortical thickness and surface curvature. The results demonstrate that a whole bone approach to analysis of cross-sectional geometry is more desirable where possible, as there is a continuous pattern of variation along the shaft. It is also possible to discriminate very young individuals and adolescents from other groups by relative cortical thickness, and also by periosteal surface curvature.

Investigating the Effects of Vaping and Nicotine’s Block of Kir2.1 on Humerus and Digital Development in Embryonic Mice

Kir2.1 is an ion channel that is important for skeletal development. Nicotine, a common ingredient in electronic cigarettes, blocks Kir2.1. However, little research has been done to investigate how vaping nicotine during pregnancy affects embryonic development. We examined how vaping nicotine during pregnancy would affect embryonic skeletal development in mice. Mice pregnant with embryos of three genotypes—wild‐type, Kir2.1+/KO, or Kir2.1KO/KO—received either no vaping treatment, a 2.4% freebase nicotine vaping treatment, or a 3.6% nicotine salt vaping treatment throughout pregnancy. The embryos were dissected at E18.5, and their skeletons were extracted and stained for imaging. The lengths of the humerus bones and number of digital bones were calculated to quantify differences in bone development between groups. We found significant differences in humerus length between the wild‐type no vape and wild‐type 2.4% freebase nicotine groups, as well as the Kir2.1+/KO no vape and Kir2.1+/KO 2.4% freebase nicotine groups. However, no significant differences were found between wild‐type no vape and wild‐type 3.6% nicotine salt groups, as well as Kir2.1+/KO no vape and Kir2.1+/KO 3.6% nicotine salt groups. We suspect that nicotine salts are metabolized faster than freebase nicotine and thus inhibit Kir2.1’s function for a shorter amount of time, but further research needs to be conducted to test this hypothesis. We conclude that fetal exposure to vaped free base nicotine impacts the development of long bones and reduces long bone length.

Bone Mass and Osteoblast Activity Are Sex-Dependent in Mice Lacking the Estrogen Receptor α in Chondrocytes and Osteoblast Progenitor Cells.

While estrogen receptor alpha (ERα) is known to be important for bone development and homeostasis, its exact function during osteoblast differentiation remains unclear. Conditional deletion of ERα during specific stages of osteoblast differentiation revealed different bone phenotypes, which were also shown to be sex-dependent. Since hypertrophic chondrocytes can transdifferentiate into osteoblasts and substantially contribute to long-bone development, we aimed to investigate the effects of ERα deletion in both osteoblast and chondrocytes on bone development and structure. Therefore, we generated mice in which the ERα gene was inactivated via a Runx2-driven cyclic recombinase (ERαfl/fl; Runx2Cre). We analyzed the bones of 3-month-old ERαfl/fl; Runx2Cre mice by biomechanical testing, micro-computed tomography, and cellular parameters by histology. Male ERαfl/fl; Runx2Cre mice displayed a significantly increased cortical bone mass and flexural rigidity of the femurs compared to age-matched controls with no active Cre-transgene (ERαfl/fl). By contrast, female ERαfl/fl; Runx2Cre mice exhibited significant trabecular bone loss, whereas in cortical bone periosteal and endosteal diameters were reduced. Our results indicate that the ERα in osteoblast progenitors and hypertrophic chondrocytes differentially contributes to bone mass regulation in male and female mice and improves our understanding of ERα signaling in bone cells in vivo.

Effects of high-dose all-trans retinoic acid on longitudinal bone growth of young rats

ObjectiveThe signaling axis consisting of GH-IGF1-IGFBP3 is the primary signal taht acts prepubertally to influence height development. Growth plate thinning and even premature closure have been reported in children with tumors treated with retinoid chemotherapy, resulting in long bone dysplasia. Growth failure may occur despite received GH treatment, but the reason is unknown. This study investigate the effect of high-dose all-trans retinoic acid (ATRA) on the development of long bones in growing SD rats. MethodsA total of 20 three-week-old male SD rats were randomly divided into a control group and an experimental group (n = 10). Rats were treated by gavage with or without high-dose ATRA for 10 days. The body weights of the rats were recorded daily. At the end of the experiment, we measured the length of nose-tail and tibia, stained the tibia and liver for pathological tissue and RT-PCR reaction, and measured the levels of serum GH, IGF1 and IGFBP3, and so on. ResultsCompared with controls, experimental rats exhibited reduced body weight and shortened nasal-tail and radial tibial length. Cyp26b1 enzyme activity in the liver was elevated, and histopathological staining revealed that the cartilaginous epiphyseal plate was narrowed, the medullary cavity of trabecular bone was sparse, the number of trabecular bones was decreased, trabecular separation was increased, bone marrow mineralization was enhanced, osteoclastic activity was increased, and circulating GH-IGF1-IGFBP3 levels were decreased. However, RT-PCR reaction results of localized proximal tibiae showed upregulation of IGF1 and downregulation of IGFBP3. ConclusionsHigh-dose ATRA intake over a short period of time can reduce GH-IGF1-IGFBP3 levels, affect cartilage and bone homeostasis, and inhibit bone growth in developing animals.

Implications of leg length for metabolic health and fitness.

Background and objectivesSeveral studies have linked longer legs with favorable adult metabolic health outcomes and greater offspring birth weight. A recent Mendelian randomization study suggested a causal link between height and cardiometabolic risk; however, the underlying reasons remain poorly understood.MethodologyUsing a cross-sectional design, we tested in a convenience sample of 70 healthy young women whether birth weight and tibia length as markers of early-life conditions associated more strongly with metabolically beneficial traits like organ size and skeletal muscle mass (SMM) than a statistically derived height-residual variable indexing later, more canalized growth.ResultsConsistent with the ‘developmental origins of health and disease’ hypothesis, we found relatively strong associations of tibia length—but not birth weight—with adult organ size, brain size, SMM and resting energy expenditure measured by magnetic resonance imaging (MRI), dual-energy X-ray absorptiometry and indirect calorimetry, respectively.Conclusions and implicationsBuilding on prior work, these results suggest that leg length is a sensitive marker of traits directly impacting metabolic and reproductive health. Alongside findings in the same sample relating tibia length and height-residual to MRI-measured pelvic dimensions, we suggest there may exist a degree of coordination in the development of long bone, lean mass and pelvic traits, possibly centered on early, pre-pubertal growth periods. Such phenotypic coordination has important implications for fitness, serving to benefit both adult health and the health of offspring in subsequent generations.

An Avascular Niche Created by Axitinib-Loaded PCL/Collagen Nanofibrous Membrane Stabilized Subcutaneous Chondrogenesis of Mesenchymal Stromal Cells.

Engineered cartilage derived from mesenchymal stromal cells (MSCs) always fails to maintain the cartilaginous phenotype in the subcutaneous environment due to the ossification tendency. Vascular invasion is a prerequisite for endochondral ossification during the development of long bone. As an oral antitumor medicine, Inlyta (axitinib) possesses pronounced antiangiogenic activity, owing to the inactivation of the vascular endothelial growth factor (VEGF) signaling pathway. In this study, axitinib‐loaded poly(ε‐caprolactone) (PCL)/collagen nanofibrous membranes are fabricated by electrospinning for the first time. Rabbit‐derived MSCs‐engineered cartilage is encapsulated in the axitinib‐loaded nanofibrous membrane and subcutaneously implanted into nude mice. The sustained and localized release of axitinib successfully inhibits vascular invasion, stabilizes cartilaginous phenotype, and helps cartilage maturation. RNA sequence further reveals that axitinib creates an avascular, hypoxic, and low immune response niche. Timp1 is remarkably upregulated in this niche, which probably plays a functional role in inhibiting the activity of matrix metalloproteinases and stabilizing the engineered cartilage. This study provides a novel strategy for stable subcutaneous chondrogenesis of mesenchymal stromal cells, which is also suitable for other medical applications, such as arthritis treatment, local treatment of tumors, and regeneration of other avascular tissues (cornea and tendon).

The Effect of Complex Feed Supplements on the Growth and Development of Long Bones in Broilers

Poultry farming as one of the most intensive branches of animal husbandry, and it uses a large amount of various feed supplements in order to obtain the maximum production volume. This article presents the results of a study on the effect of feed supplements used for feeding broiler chickens. Until the age of 42 days, the chickens received a phytase-containing enzyme supplement, mineral supplements Mn and Ca in a citrate form and a vitamin supplement of 1,25-dioxycholecalciferol (vitamin D3, calcitriol). The live weight of the chickens was measured at the age of 1, 4, 7, 14, 21, 28, 35 and 42 days. The femur development of the chickens was studied through morphological methods (morphometry and histological examination). The influence of various supplements (from the combined to the mutually absorbing ones) on the growth and development of the long bones of broilers was identified.
 Keywords: poultry, broilers, feed supplements, vitamin D, mineral supplements, enzyme supplements, histology, bones

The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression.

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3′ untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.

Evaluation of impaired growth plate development of long bones in skeletally immature mice by antirheumatic agents.

Restriction of physical growth and development is a known problem in patients with juvenile idiopathic arthritis (JIA). However, the effect of medical treatment for JIA on skeletal growth in affected children has not been properly investigated. We, therefore, hypothesize that naproxen and methotrexate (MTX) affect endochondral ossification and will lead to reduced skeletal development. Treatment of ATDC5 cells, an in vitro model for endochondral ossification, with naproxen or MTX resulted in increased chondrogenic but decreased hypertrophic differentiation. In vivo, healthy growing C57BL/6 mice were treated with naproxen, MTX, or placebo for 10 weeks. At 15 weeks postnatal, both the length of the tibia and the length of the femur were significantly reduced in the naproxen‐ and MTX‐treated mice compared to their controls. Growth plate analysis revealed a significantly thicker proliferative zone, while the hypertrophic zone was significantly thinner in both experimental groups compared to their controls, comparable to the in vitro results. Micro‐computed tomography analysis of the subchondral bone region directly below the growth disc showed significantly altered bone microarchitecture in naproxen and MTX groups. In addition, the involvement of the PTHrP‐Ihh loop in naproxen‐ and MTX‐treated cells was shown. Overall, these results demonstrate that naproxen and MTX have a profound effect on endochondral ossification during growth plate development, abnormal subchondral bone morphology, and reduced bone length. A better understanding of how medication influences the development of the growth plate will improve understanding of endochondral ossification and reveal possibilities to improve the treatment of pediatric patients.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

Integrating levels of bone growth control: From stem cells to body proportions.

The study of the mechanisms controlling organ size during development and regeneration is critical to understanding how complex life arises from cooperating single cells. Long bones are powerful models in this regard, as their size depends on a scaffold made from another tissue (cartilage, composed of chondrocytes), and both tissues interact during the growth period. Investigating long bone growth offers a valuable window into the processes that integrate internal and external cues to yield finely controlled size of organs. Within the cellular and molecular pathways that control bone growth, the regulation of stem-cell renewal, along with amplification and differentiation of their progeny, are key to understanding normal and perturbed long-bone development. The phenomenon of "catch-up" growth-where cellular hyperproliferation occurs following injury to restore a normal growth trajectory-reveals key aspects of this regulation, such as the fact that bone growth is target-seeking. The control mechanisms that lead to this behavior are either bottom-up or top-down, and the interaction between these modes is likely critical to achieve a highly nuanced, yet flexible, degree of control. The role of cartilage-intrinsic mechanisms has been well studied, establishing a very solid groundwork for this field. However, addressing the unanswered questions of bone growth arguably requires new hypotheses and approaches. Future research could for example address to what extent extrinsic signals and cells, as well as communication with other tissues, modulate intra-limb and inter-organ growth coordination. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Vertebrate Organogenesis > Musculoskeletal and Vascular.

Dysbacteriosis-Derived Lipopolysaccharide Causes Embryonic Osteopenia through Retinoic-Acid-Regulated DLX5 Expression.

Growing evidence suggests an adverse impact of gut microbiota dysbiosis on human health. However, it remains unclear whether embryonic osteogenesis is affected by maternal gut dysbacteriosis. In this study, we observed that elevated lipopolysaccharide (LPS) levels led to skeletal developmental retardation in an established mouse model of gut microbiota dysbiosis. Using chick embryos exposed to dysbacteriosis-derived LPS, we found restriction in the development of long bones as demonstrated by Alcian blue and alizarin red staining. Micro-CT and histological analysis exhibited decreased trabecular volume, bone mineral density, and collagen production, as well as suppressed osteoblastic gene expression (Ocn, Runx2, Osx, and Dlx5) in chick embryonic phalanges following LPS treatment. Atomic force microscopy manifested decreased roughness of MC3T3-E1 cells and poorly developed matrix vesicles (MVs) in presence of LPS. The expression of the aforementioned osteoblastic genes was suppressed in MC3T3-E1 cells as well. High-throughput RNA sequencing indicated that retinoic acid (RA) may play an important role in LPS-induced osteopenia. The addition of RA suppressed Dlx5 expression in MC3T3-E1 cells, as was also seen when exposed to LPS. Quantitative PCR, Western blot, and immunofluorescent staining showed that retinoic acid receptor α (RARα) was upregulated by LPS or RA treatment, while the expression of DLX5 was downregulated. CYP1B1 expression was increased by LPS treatment in MC3T3-E1 cells, which might be attributed to the increased inflammatory factors and subsequently activated NF-κB signaling. Eventually, blocking RA signals with AGN193109 successfully restored LPS-inhibited osteoblastic gene expression. Taken together, our data reveals that maternal gut microbiota dysbiosis can interfere with bone ossification, in which Dlx5 expression regulated by RA signaling plays an important role.

Pharmacologic iron chelation reduces markers of chondrocyte hypertrophy and osteoarthritis-associated cartilage lesions in an animal model of idiopathic disease

Purpose: It has previously been documented that chondrocytes from joints afflicted with osteoarthritis (OA) undergo improper dedifferentiation into hypertrophic chondrocytes; thus, it is postulated that this process may contribute to the progression of OA. Relative to unaffected adult chondrocytes, hypertrophic chondrocytes mimic cellular processes for endochondral ossification observed during initial development of long bones. In particular, they express markers of hypertrophic differentiation such as runx family transcription factor 2 (runx2), collagen type x, and the matrix degrading enzyme matrix metalloproteinase-13 (MMP-13). In addition to these markers, hypertrophic chondrocytes exhibit increased apoptosis, decreased autophagy, and promote cartilage vascularization and mineralization of the extracellular matrix. One theory for why this hypertrophic state occurs is that oxidative stress from reactive oxygen species (ROS) contributes to this shift in chondrocyte phenotype. We had previously demonstrated the ability of systemic iron reduction, achieved by an iron deficient diet, to reduce ROS production and thereby decrease structural changes of OA in an animal model of idiopathic/spontaneous disease. Given the success of this study, we hypothesized that systemic iron chelation - achieved by administration of the pharmacologic iron chelator deferoxamine (DFO) - would reduce markers of chondrocyte hypertrophy and decrease the development of knee OA in this animal model. Methods: All procedures received University approval and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Three-month-old male Dunkin-Hartley guinea pigs were randomly assigned to receive either DFO or vehicle control (n=8 per group). Animals within the DFO group received 46 mg/kg of DFO injected subcutaneously twice daily while animals within the control group received an equivalent dose of saline. Animals were subjected to 10-minute sessions of open field behavior monitoring once per month for the duration of the study, with baseline activity levels collected prior to initiating treatment. The study was terminated when animals were 8-months-old, at which time body weights were recorded. Hind limbs were removed and the right tibia was measured using calipers. Articular cartilage was collected from the right knee joint for gene expression analysis using Nanostring nCounter technology. Femoral head cartilage and liver tissue were collected for iron quantification by atomic absorption spectroscopy. Left knee joints were formalin-fixed, paraffin-embedded, and stained with toluidine blue. For histologic analysis, all joint compartments were blindly scored by two assessors using OARSI published guidelines. Data were compared using non-parametric Mann-Whitney U tests, Welch’s t-tests, or unpaired student’s t-tests as appropriate given the normality and heteroscedasticity of the data. All statistical analyses were performed with Prism 8.0; statistical significance was set at P≤0.05. Results: No significant differences in body weight (p=0.576) or tibia length (p=0.628) were present between groups. As anticipated, animals treated with DFO had lower levels of iron within the liver (p=0.0218) and articular cartilage (p=0.0005) tissue than control animals. Histologic grading of knee joints confirmed that animals treated with DFO had lower total joint OA scores than control animals (p<0.0001). Gene transcript analysis demonstrated that, relative to controls, animals treated with DFO had lower expression of runx2 (p=0.0010), MMP-13 (p=0.0151), vascular endothelial growth factor (VEGF; p=0.0140), β- catenin (p<0.0001), wnt (p=0.0008), mammalian target of rapamycin (mTOR; p=0.0018), and the serine/threonine protein kinase AKT (p=0.0041). Animals treated with DFO also had increased expression of b cell lymphoma 2 (BCL-2; p<0.0001), a prototypical anti-apoptotic marker. There was no significant difference in the gene expression of collagen type x between groups (p=0.3163). Open field behavior monitoring indicated that DFO treated animals maintained their baseline activity level throughout the study, while the movement of control animals declined throughout the study. Conclusions: Administration of a pharmacologic iron chelator was successful in reducing iron levels both systemically and within articular cartilage tissue, which was associated with a reduced development of OA-associated cartilage lesions and maintained mobility in these animals. Gene transcript analysis of cartilage tissue revealed that DFO treated animals had a decreased expression of: select markers of chondrocyte hypertrophy (runx2 and MMP-13), negative regulators of autophagy (AKT and mTOR), the angiogenesis promoter VEGF, and genes associated with chondrocyte dedifferentiation (β- catenin and wnt); while displaying increased expression of the anti-apoptotic protein BCL-2. These results indicate that removal of excess iron may help prevent chondrocyte hypertrophy and development of OA-related changes within the cartilage of knee joints.

Análisis bioarqueológico de un feto anencefálico del sitio arqueológico cueva de los niños (provincia de última esperanza, Región de Magallanes, Chile): A 29 años de su hallazgo

The re-evaluation of human bones from the Cueva de los Ninos site located in the province of Ultima Esperanza, Chile, is presented. This work focuses on deepening the analysis of the anencephalic fetus called Ultima Esperanza A, sustained by the availability of new methods for the analysis of bone material and by the presence of unstudied remains from the individual. After estimating the age using new regression equations and considering the alterations produced by fetal neurological defects in the development of long bones, we suggest the possibility that the age of the fetus is between one or two weeks less than that proposed previously. New bioanthropological data on the individual are provided, emphasizing its importance and the few documented cases of anencephalic fetuses found in archaeological contexts worldwide. Finally, the possibility that the individual Ultima Esperanza A and the other fetus found at the site (Ultima Esperanza B) were born from a multiple pregnancy is discussed, based on the possible lower age difference between them and the higher incidence of anencephaly in these cases.

CYTL1 regulates bone homeostasis in mice by modulating osteogenesis of mesenchymal stem cells and osteoclastogenesis of bone marrow-derived macrophages

We previously showed that mice with knockout of Cytl1, a functionally uncharacterized cytokine candidate, exhibit normal endochondral ossification and long-bone development. Here, we investigated the potential functions of CYTL1 in bone homeostasis. We found that Cytl1−/− mice exhibited higher bone mass than wild-type littermates and resisted ovariectomy-induced bone resorption. This led us to investigate the functions of CYTL1 in the osteogenesis and osteoclastogenesis of bone marrow-derived stem cells. CYTL1 was down-regulated during the osteogenesis of human mesenchymal stem cells (hMSCs). The osteogenesis of hMSCs was inhibited by overexpression or exogenous treatment of CYTL1, but enhanced by CYTL1 knockdown. CYTL1 decreased osteogenesis by inhibiting RUNX2 and promoted proliferation among undifferentiated hMSCs, but stimulated apoptosis among osteogenically differentiating cells. Finally, Cytl1−/− mice exhibited inhibition of osteoclast activity and the osteoclastogenesis of bone marrow-derived macrophages. Our results collectively suggest that CYTL1 negatively regulates the osteogenesis of MSCs and positively regulates osteoclastogenesis to modulate bone mass in mice.

RVis: a freely available modelling platform

This study investigated the mechanisms underlying the retarded development of long bone in fetus by prenatal nicotine exposure (PNE) which had been demonstrated by our previous work. Nicotine (2.0 mg/kg.d) or saline was injected subcutaneously into pregnant rats every morning from gestational day (GD) 9 to 20. Fetal femurs or tibias were harvested for analysis on GD 20. We found massive accumulation of hypertrophic chondrocytes and a delayed formation of primary ossification center (POC) in the fetal femur or tibia of rat fetus after PNE, which was accompanied by a decreased amount of osteoclasts in the POC and up-regulated expression of osteoprotegerin (OPG) but by no obvious change in the expression of receptor activator of NF-κB ligand (RANKL). In primary osteoblastic cells, both nicotine (0, 162, 1620, 16,200 ng/ml) and corticosterone (0, 50, 250, 1250 nM) promoted the mRNA expression of OPG but concentration-dependently suppressed that of RANKL. Furthermore, blocking α4β2-nicotinic acetylcholine receptor (α4β2-nAChR) or glucocorticoid receptor rescued the above effects of nicotine and corticosterone, respectively. In conclusion, retarded osteoclastogenesis may contribute to delayed endochondral ossification in long bone in fetal rats with PNE. The adverse effects of PNE may be mediated via the direct effect of nicotine and indirect effect of maternal corticosterone on osteoblastic cells.