Current Status of Next-Generation Sequencing in Bone Genetic Diseases.

The development of next-generation sequencing (NGS) has dramatically increased the speed and volume of genetic analysis. Furthermore, the range of applications of NGS is rapidly expanding to include genome, epigenome (such as DNA methylation), metagenome, and transcriptome analyses (such as RNA sequencing and single-cell RNA sequencing). NGS enables genetic research by offering various sequencing methods as well as combinations of methods. Bone tissue is the most important unit supporting the body and is a reservoir of calcium and phosphate ions, which are important for physical activity. Many genetic diseases affect bone tissues, possibly because metabolic mechanisms in bone tissue are complex. For instance, the presence of specialized immune cells called osteoclasts in the bone tissue, which absorb bone tissue and interact with osteoblasts in complex ways to support normal vital functions. Moreover, the many cell types in bones exhibit cell-specific proteins for their respective activities. Mutations in the genes encoding these proteins cause a variety of genetic disorders. The relationship between age-related bone tissue fragility (also called frailty) and genetic factors has recently attracted attention. Herein, we discuss the use of genomic, epigenomic, transcriptomic, and metagenomic analyses in bone genetic disorders.

Progress and Current Status in Hajdu-Cheney Syndrome with Focus on Novel Genetic Research.

Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant manifestation of a congenital genetic disorder caused by a mutation in the NOTCH2 gene. NOTCH signaling has variations from NOTCH 1 to 4 and maintains homeostasis by determining and regulating the proliferation and differentiation of various cells. In HCS, the over-accumulated NOTCH2 causes abnormal bone resorption due to its continuous excessive signaling. HCS is characterized by progressive bone destruction, has complex wide-range clinical manifestations, and significantly impacts the patient's quality of life. However, no effective treatment has been established for HCS to date. There are genetic variants of NOTCH2 that have been reported in the ClinVar database of the U.S. National Institutes of Health. In total, 26 mutant variants were detected based on the American College of Medical Genetics and Genomics (ACMC). To date, there has been no comprehensive compilation of HCS mutations. In this review, we provide the most comprehensive list possible of HCS variants, nucleotide changes, amino acid definitions, and molecular consequences reported to date, following the ACMC guidelines.

Multiscale characterization of pathological bone tissue.

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.

3D printable magnesium-based cements towards the preparation of bioceramics.

HYPOTHESIS: Among all the materials used so far to replace and repair damaged bone tissues, magnesium silicate bioceramics are one of the most promising, thanks to their biocompatibility, osteoinductive properties and good mechanical stability. EXPERIMENTS: Magnesium silicate cement pastes were prepared by hydration of MgO mixed with different SiO2 batches at different Mg/Si molar ratios. Pastes were either moulded or 3D printed to obtain set cements that were then calcined at 1000 °C to produce biologically relevant ceramic materials. Both cements and ceramics were characterized by means of X-ray diffraction, while two selected formulations were thoroughly characterized by means of injectability tests, Raman confocal microscopy, scanning electron microscopy, atomic force microscopy, gas porosimetry, X-ray microtomography and compressive tests. FINDINGS: The results show that bioceramic scaffolds, namely forsterite and clinoenstatite, can be effectively obtained by 3D printing MgO/SiO2 cement pastes, paving the way towards important advances in the field of bone tissue engineering.

Progress in Advanced Glycation End-Products Effects on Biomechanics of Bone Tissues / 医用生物力学

Advanced glycation end-products (AGEs) are the products of non-enzymatic reactions between free amino groups of macromolecules and reducing sugars. AGEs accumulation in bone tissues changes the activity and function of bone cells by binding to their surface receptors, causing abnormalities in the process of bone remodeling. AGEs accumulation can also change the original structure and mineral deposition of bone collagen, affect the micro-mechanical properties of bone tissues, and further reduce bone strength and toughness, increase the bone fracture risk, which will lead to bone diseases and do great harm to human health. This article summarized the causes of AGEs and their detection methods, and reviewed previous studies about the effects of AGEs accumulation on bone biomechanics at micro and macro levels, so as to provide references for the early diagnosis and treatment of bone diseases in clinic.

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells.

BACKGROUND: Autologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient's dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells. PATIENTS AND METHODS: We compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation. RESULTS: The majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME's was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous melanoma cell lines with high expression of CTA. CONCLUSION: Our results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

The feasibility of locating the affected bone of BRONJ with indocyanine green.

The morbidity of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is also increasing with the use of bisphosphonates (BPs). Removing affected bone accurately is the most effective treatment. This study aimed to explore the feasibility of Indocyanine green (ICG)-based Near-Infrared fluorescence (NIF) imaging to remove BRONJ affected bone. Firstly, the rat model of BRONJ was constructed. And 5 mg/kg ICG were injected via tail vein, after 12 hr, the affected and healthy bone were dissected for ICG-based NIF imaging and quantification detection of fluorescence intensity. Finally, all the bone samples were sent for further pathological examination. All the affected bone tissues in rat BRONJ model were fluorescence developed with ICG. And the fluorescence developed regions were further confirmed as affected bone tissues with pathological examination. The fluorescence intensity in affected bone tissues, adjacent, and opposite bone tissues was 1.93*107 ± 2.08*106, 1.19*106 ± 2.33*105, and 1.24*106 ± 1.57*105, respectively (p < .05). Conclusions It was feasible that the extent of affected bone in rat model with BRONJ could be estimated intraoperative via ICG-based NIF imaging. This novel approach would become an auxiliary method in the treatment of patients with BRONJ in the future.

Quantitative imaging of platinum-based antitumor complexes in bone tissue samples using LA-ICP-MS.

There is a need for effective medication against bone metastases because todays drugs are not able to penetrate the bone and reach the affected areas. To analyze if current or future platinum-containing drugs are able to achieve this, a quantitative imaging method is urgently needed. In this study, the platinum distribution in thin sections of mice tibia was determined using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) in a spatially resolved manner. The hard bone tissue visible in microscopic images and signals found for calcium and phosphorous recorded via LA-ICP-MS and micro X-ray fluorescence spectroscopy (µXRF) correlate well. Furthermore, the platinum concentration was quantified using polymer-based matrix-matched standards. A limit of detection of 6 µg/g and a linearity of almost three decades could be achieved. Concentrations surpassing 300 µg/g could be found in the tibia samples. The method presented herein is a powerful approach for the visualization and quantification of platinum. As such, this method is a valuable tool to unravel the mechanism of delivery and optimize the therapeutic potency of platinum-containing drugs targeting bone diseases like bone metastases.

Hierarchical Biomechanical Properties and Constitutive Relationships of Bone Tissues / 医用生物力学

As the main organ of the body, the load-bearing ability of bone is closely connected to its biomechanical properties. Bone is a complex hierarchical biomaterial, whose biomechanical properties are determined by its own structure and biological characteristics. Because of its mechanical adaptability, bone tissues represent different biomechanical properties under different mechanical loading. To quantify the complicated properties of bone and provide an accurate theoretical basis for clinical research, it is necessary to give insight into the biomechanical properties of bone at different levels and the constitutive relationships of bone tissues. In this review, relative researches on constitutive relationships in recent years were summarized based on its hierarchical biomechanical properties.

Hierarchical Biomechanical Properties and Constitutive Relationships of Bone Tissues / 医用生物力学

As the main organ of the body, the load-bearing ability of bone is closely connected to its biomechanical properties. Bone is a complex hierarchical biomaterial, whose biomechanical properties are determined by its own structure and biological characteristics. Because of its mechanical adaptability, bone tissues represent different biomechanical properties under different mechanical loading. To quantify the complicated properties of bone and provide an accurate theoretical basis for clinical research, it is necessary to give insight into the biomechanical properties of bone at different levels and the constitutive relationships of bone tissues. In this review, relative researches on constitutive relationships in recent years were summarized based on its hierarchical biomechanical properties.

Fabrication of novel bioactive hydroxyapatite-chitosan-silica hybrid scaffolds: Combined the sol-gel method with 3D plotting technique.

Sol-gel derived organic/inorganic hybrids, in which organic and inorganic components form co-networks at the molecular level, have demonstrated great potential for providing improved mechanical properties and biological functions in tissue engineering applications. Here, a novel bioactive hydroxyapatite-chitosan-silica hybrid (HA-CSH) scaffold was successfully fabricated by combining the sol-gel method and 3D plotting technique. Physiochemical characterization confirmed that chitosan was hybridized homogeneously with the inorganic phase on nanoscale. The obtained scaffolds possessed precisely controllable and interconnected porous structures. The nano-sized HA formed in situ and dispersed uniformly in the hybrid network, which reduced the water absorption and increased the mechanical strength of the hybrid scaffold under humidity condition as compared to chitosan-silica hybrid (CSH) scaffold. Compression tests showed that the 3D plotted hybrid scaffolds under wet conditions had compressive strengths of 10-13 MPa and elastic moduli of 21-27 MPa and thus met the mechanical requirements of human trabecular bone. Studies on the mineralization process under simulated body fluid (SBF) conditions confirmed that the introduction of HA obviously increased the biological activity of hybrid scaffolds. In vitro cell results indicated that the HA-CSH scaffold not only supported adhesion and proliferation of mouse bone mesenchymal stem cells (mBMSCs), but also improved the osteoinductivity. The alkaline phosphatase activity and mineral deposition on the HA-CSH scaffold were higher than those on the CSH scaffold. These results suggested that the 3D plotted HA-CSH scaffold may be a promising bioactive material for bone tissues regeneration.

Influences of geometrical and mechanical properties of bone tissues in mandible behaviour - experimental and numerical predictions.

The properties and geometry of bone in the mandible play a key role in mandible behaviour during a person's lifetime, and attention needs to be paid to the influence of bone properties. We analysed the effect of bone geometry, size and bone properties in mandible behaviour, experimenting on cadaveric mandibles and FE models. The study was developed using the geometry of a cadaveric mandible without teeth. Three models of cadaveric condyles were experimentally tested with instrumented with four rosettes, and a condyle reaction of 300 N. Four finite element models were considered to validate the experiments and analyse mandible behaviour. One numeric model was simulated with 10 muscles in a quasi-static condition. The experimental results present different condyle stiffness's, of 448, 215 and 254 N/mm. The values presented in the rosettes are influenced by bone geometry and bone thickness; maximum value was -600 µÎµ in rosette #4, and the maximum strain difference between mandibles was 111%. The numerical results show that bone density decreases and strain distribution increases in the thinner mandible regions. Nevertheless, the global behaviour of the structure remains similar, but presents different strain magnitudes. The study shows the need to take into account bone characteristics and their evolutions in order to improve implant design and fixation throughout the patient life. The change in bone stiffness promotes a change in maximum strain distribution with same global behaviour.

Fósforo-32 e itrio-90 como opciones en el tratamiento del dolor óseo metastásico / Phosphorous-32 and Yttrium-90 as options in the treatment of metastatic bone pain

En el trabajo se estima la prevalencia del dolor óseo metastásico en nuestro país en no menos de 3 750 pacientes. Se examina, asimismo, el papel del Fosfato de sodio-[], de eficacia comparable y menor costo que el de otros radiofármacos, lo que hace posible el renacer de su uso. Aunque más radiotóxico, ello no lo invalida como opción, lo que se evidencia también en la experiencia cubana. El Centro de Isótopos (Centis) puso en marcha una línea de producción con posibilidad de obtener hasta 37 GBq (1 Ci) de de alta calidad cada semana. El hecho de que se produzca por separación electroquímica mediante un generador de radionúclidos, a partir del 90Sr, hace que no se requiera importación de materia prima radiactiva, prácticamente, por tiempo indefinido, es decir, que el radionúclido estaría disponible de manera sostenible. Al tener el una energía de emisión β- mayor, pudiera pensarse que debiera ser más radiotóxico que el . Sin embargo, se dan evidencias de que el en forma de fosfato es más radiotóxico que el 90Y en forma de citrato. En Europa un radiofármaco basado en un liofilizado de EDTMP y Cloruro de itrio-[] ha tenido buen efecto para el alivio del dolor en 191 pacientes. (Centis) tiene registrado el Cloruro de itrio-[] para marcaje y se trata de introducir en el mercado un radiofármaco con este precursor radioactivo y el liofilizado importado, a los efectos de dar adecuada atención al importante número de pacientes que se beneficiarían

In this paper the prevalence of metastatic bone pain is estimated in our country in no less than 3 750 patients. Moreover, the role of -phosphate, which effectiveness is comparable to other radiopharmaceuticals, and the possible return to its previous general use, as it is cheaper, is examined. The fact that is considered more radiotoxic doesn’t invalidate it as a treatment option, which is also evidenced by the Cuban experience. The Center of Isotopes (Centis) has established a production line of up to 37 GBq (1 Ci) per week of high quality . The fact of its being obtained through a radionuclide generator by electrochemical separation from makes the import of radioactive raw material practically not required for indefinite time. This implies that the radionuclide could be available in a sustainable way. By having the greater β- emission energy, it might be more radiotoxic than . Nevertheless, some evidences show that in the form of orthophosphate is more radiotoxic than 90Y citrate. In Europe, a radiopharmaceutical consisting of a lyophilized kit of EDTMP and Chloride solution was able to relief pain in 191 patients. Centis has registered the chloride solution for labeling. Then the task is to introduce in the market a radiopharmaceutical with this radioactive precursor and the imported lyophilized kit in order to increase the treatment to an important number of patients that, as a result, could be benefitted

Microwave bone imaging: a preliminary scanning system for proof-of-concept.

This Letter introduces a feasibility study of a scanning system for applications in biomedical bone imaging operating in the microwave range 0.5-4 GHz. Mechanical uncertainties and data acquisition time are minimised by using a fully automated scanner that controls two antipodal Vivaldi antennas. Accurate antenna positioning and synchronisation with data acquisition enables a rigorous proof-of-concept for the microwave imaging procedure of a multi-layer phantom including skin, fat, muscle and bone tissues. The presence of a suitable coupling medium enables antenna miniaturisation and mitigates the impedance mismatch between antennas and phantom. The three-dimensional image of tibia and fibula is successfully reconstructed by scanning the multi-layer phantom due to the distinctive dielectric contrast between target and surrounding tissues. These results show the viability of a microwave bone imaging technology which is low cost, portable, non-ionising, and does not require specially trained personnel. In fact, as no a-priori characterisation of the antenna is required, the image formation procedure is very conveniently simplified.

Free fibula and flap graft for reconstruction all the first metatarsal bone / 中华显微外科杂志

Objective To evaluate the clinical effects of free fibula and flap grafts on the repair of all the first metatarsal bone at one stage.Methods There were 9 cases with the first metatarsal bone defect from Janurary 2003 to December 2009 that treated with free vascularized fibular bone and free vascularized flap at one stage.In which 6 cases reconstructed at the primary stage and 3 cases reconstructed at the second stage.Seven cases reconstructed by free vascularized fibular combined with ALTPF,two cases reconstructed by free vascularized fibular combined with TAPF.The free vascularized fibular and flap restored the first metatarsal bone and the soft tissue defects respectively.Vascular anastomosis was the artery of flap anastomosis with anterior tibial artery and the vein of the flap anastomosis with great saphenous vein,the peroneal artery and accompany vein anastomosis with artery and vein of the flap.Results The grafted tissues survived smoothly in 8 cases,vein crisis happened in I case and the ALTPF necrosis after blood vessels expedition.So the TAPF was changed to cover the soft defect and survived smoothly.Followiy-up were done from 6 to 36 months in 9 cases.There were no ulcer on flaps and no fracture again,the fibulas had been bone healing.Evaluated by Maryland standards,six cases were excellent,two cases were fine,one case was good.Conclusion The fibula combined flap grafts provide a relatively better alternative to repair the first metatarsal bone compound tissue defects at one stage.In addition,the procedure decreased frequency of operations and short the course of treatment.Sensory function reconstruction of fibula flaps should be given full attention.As fine function of the reconstructed foot,it is a effective method for reconstruction the burdened area of the foot.