Fish bone‐derived cell lines: an alternative in vitro cell system to study bone biology

Human bone diseases represent a major health problem worldwide and effective therapies have still to be developed. Despite numerous studies using mammalian systems, cellular and molecular processes governing bone and cartilage homeostasis in vertebrates are still not fully understood. Recently, fish have emerged as a suitable model and a promising alternative to the classical mammalian systems to study vertebrate development, in particular skeletogenesis. To complement in vivo developmental studies and identify signalling pathways involved in development processes, fish cell lines have been developed, in particular bone‐derived cells. This work intends to review what is presently known about fish bone‐derived cell lines, focusing on their relevance for bone biology studies.     

Proteomics of filamentous fungi

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.     

Osteoblast recruitment to sites of bone formation in skeletal development,homeostasis, and regeneration

During endochondral bone development, bone‐forming osteoblasts have to colonize the regions of cartilage that will be replaced by bone. In adulthood, bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, as a prerequisite for skeletal health. A failure of osteoblasts to reach the sites in need of bone formation may contribute to impaired fracture repair. Conversely, stimulation of osteogenic cell recruitment may be a promising osteo‐anabolic strategy to improve bone formation in low bone mass disorders such as osteoporosis and in bone regeneration applications. Yet, still relatively little is known about the cellular and molecular mechanisms controlling osteogenic cell recruitment to sites of bone formation. In vitro, several secreted growth factors have been shown to induce osteogenic cell migration. Recent studies have started to shed light on the role of such chemotactic signals in the regulation of osteoblast recruitment during bone remodeling. Moreover, trafficking of osteogenic cells during endochondral bone development and repair was visualized in vivo by lineage tracing, revealing that the capacity of osteoblast lineage cells to move into new bone centers is largely confined to undifferentiated osteoprogenitors, and coupled to angiogenic invasion of the bone‐modeling cartilage intermediate. It is well known that the presence of blood vessels is absolutely required for bone formation, and that a close spatial and temporal relationship exists between osteogenesis and angiogenesis. Studies using genetically modified mouse models have identified some of the molecular constituents of this osteogenic–angiogenic coupling. This article reviews the current knowledge on the process of osteoblast lineage cell recruitment to sites of active bone formation in skeletal development, remodeling, and repair, considering the role of chemo‐attractants for osteogenic cells and the interplay between osteogenesis and angiogenesis in the control of bone formation. Birth Defects Research (Part C) 99:170–191, 2013. © 2013 Wiley Periodicals, Inc.     

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis

Background

Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints.

Results

Two lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha).

Conclusion

This study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-947) contains supplementary material, which is available to authorized users.     

Nonuniform swelling-induced residual strains in articular cartilage

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swelling-induced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 M Pa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.     

Cartilage stress-relaxation proceeds slower at higher compressive strains

Articular cartilage is the connective tissue which covers bone surfaces and deforms during in vivo activity. Previous research has investigated flow-dependent cartilage viscoelasticity, but relatively few studies have investigated flow-independent mechanisms. This study investigated polymer dynamics as an explanation for the molecular basis of flow-independent cartilage viscoelasticity. Polymer dynamics predicts that stress-relaxation will proceed more slowly at higher volumetric concentrations of polymer. Stress-relaxation tests were performed on cartilage samples after precompression to different strain levels. Precompression increases the volumetric concentration of cartilage biopolymers, and polymer dynamics predicts an increase in relaxation time constant. Stress-relaxation was slower for greater precompression. There was a significant correlation between the stress-relaxation time constant and cartilage volumetric concentration. Estimates of the flow-dependent timescale suggest that flow-dependent relaxation occurs on a longer timescale than presently observed. These results are consistent with polymer dynamics as a mechanism of cartilage viscoelasticity.     

Distribution and Immunologic Cross Reactivity of a Phosphohydrolytic Activity of Calcifying Cartilage and Metaphyseal Bone

The increased phosphohydrolytic activity found in calcifying cartilage has breen implicated in the process of normal calcification. Part of this multipotential activity was found to be associated with an extracellular vesicle presumed to be the initial side of calcium salt deposition.
The phosphohydrolytic activity of water extracts from calcifying cartilage and metaphyseal bone has been resolved into three enzymatic entities by DEAE-cellulose column chromatography. The activity which was eluted first, phosphatase I (pyrophosphatase I), increases as cartilage differentiates and calcifies. This increase could serve as a marker for cartilage differentiation and/or calcification. Antibodies to this enzyme isolated from calcifying cartilage or metaphyseal bone cross-react suggesting that the enzymes might, at least in part, be similar.
Cartilage and bone also possess an inorganic pyrophosphatase, pyrophosphatase II, eluted second through the DEAE-cellulose column and another phosphatase, phosphatase II, which was eluted last. By enzymatic and immunologic criteria, it appears that bone and cartilage have the same phosphate-releasing activities indicative of tissues with common cellular origin.
The possible transformation of the differentiating chondrocyte into an osteoblast or osteocyte has been postulated as the cellular mechanism whereby calcified cartilage is replaced by bone. The similarity between the phosphatase I found in epiphyseal cartilage and metaphyseal bone suggests that such transformation is quite likely.     

Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos.

The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.     

Measurements of mineralization process in the femur growth plate and rib cartilage of the mouse using pixe in combination with a proton microprobe

The femoral bone from the 18-day pregnancy embryo and an rib cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and the results of quantitative measurement of P, S, Cl, K, Ca, Fe and Zn have been correlated with the histochemical localization of inorganic deposits. It has been found that in calcifying and degenerating cartilage of the growth plate there is substantial loss of S; this element being indicative for sulphate groups of glycosaminoglycans. This change seems to be an important factor conditioning the process of mineralization. Zn is found in higher concentration in mineralized tissues, both in embryonal and mature cartilage as well as in the bone, and this suggests that Zn is also involved in the mineralization process. The mineralization of rib cartilage exceeds that of embryonal bone, and the Ca/P ratio is higher in the former than in the hydroxyapatite of the latter. The method described is a useful analytical tool especially for such types of studies in which elements are not easily redistributed by freezing, cutting and drying; e.g. in investigations of mineral deposits.     

Developmental control of chondrogenesis and osteogenesis

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.     

Measurements of mineralization process in the femur growth plate and rib cartilage of the mouse using pixe in combination with a proton microprobe

Summary The femoral bone from the 18-day pregnancy embryo and an rib cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and the results of quantitative measurement of P, S, Cl, K, Ca, Fe and Zn have been correlated with the histochemical localization of inorganic deposits. It has been found that in calcifying and degenerating cartilage of the growth plate there is substantial loss of S; this element being indicative for sulphate groups of glycosaminoglycans. This change seems to be an important factor conditioning the process of mineralization. Zn is found in higher concentration in mineralized tissues, both in embryonal and mature cartilage as well as in the bone, and this suggests that Zn is also involved in the mineralization process. The mineralization of rib cartilage exceeds that of embryonal bone, and the Ca/P ratio is higher in the former than in the hydroxyapatite of the latter. The method described is a useful analytical tool especially for such types of studies in which elements are not easily redistributed by freezing, cutting and drying; e.g. in investigations of mineral deposits.     

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by “bottom-up” approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.     

Symbiosio of biotechnology and biomaterials: Applications in tissue engineering of bone and cartilage

The three ingredients for the successful tissue engineeping of bone and cartilage are ragulatory signals, cells and extracellular matrix. Recent advance in cellular and molecular biology of thde growth and differentiation factors have set the stage for a symbiosis of biotechnology and biomaterials. Recent advances permit one to enunciate the rules of architechure for tissue engineering of bone and cartilage. The purification and cloning of bone morphogenetic proteins (BMPs) and growth factors such as platelet derived growth factors (PDGF), tranforming growth factor-β (TGF-β), and insulin-like growth factors (IGF-I) Will allow the design of an optimal combinatiol of signals to initiate and promote development of skeletal stem cells into cartilage and bone. Successful and optimal bone and motion. BMPs function as inductive signals. Biomaterials (Both natural and synthetic) mimic the extracellular matrix and play a role in conduction of bone and cartiage. Examples of biomaterials include hydroxyapatite, polyanhydrides, polyphosphoesters, polylactic acid, and polyglycolic acid. The prospects for novel biomaterials are immense, and they likely will be a fertile erowth industpy. Cooperative ventures between academia and industry and teahnology transfer from the federal government augur well for an exciting future fop clinical applications.     

Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry

The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract.     

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31 mg/mL to 44 ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

细胞外基质硬度调控间充质干细胞分化

新近研究表叽细胞外基质（extracellularmatrix,ECM）的物理性质,特别是硬度或弹性,能对细胞的黏附、铺展、迁移、增殖、分化和凋亡等多种功能和行为产生重要影响。间充质干细胞（mesenchymalstemcells,MSCs）是组织工程和细胞治疗的理想种子细胞。ECM硬度可诱导MSCs向脂肪、软骨、神经、肌肉和骨等方向分化。该文综合论述了ECM硬度对干细胞分化的影响,涵盖了构建ECM硬度的测量、调控与表征等,不同培养条件下干细胞对硬度的响应和分化以及硬度和其他因素的联合作用;在此基础上,进一步论述了干细胞分化过程中细胞感应ECM硬度并转化为生物学信号的机制和信号通路。该文还总结了在ECM硬度调控干细胞分化行为领域最新的研究进展情况,较为系统地分析了材料学、细胞生物学、分子生物学水平的主要影响因素,并对本领域未来需要重点研究的问题进行了展望。     

The Rapid Manufacture of Uniform Composite Multicellular-Biomaterial Micropellets,Their Assembly into Macroscopic Organized Tissues,and Potential Applications in Cartilage Tissue Engineering

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100–500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix “cartilage dust (CD)”. Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.     

Adrenomedullin--a regulator of bone formation

Bone growth and maintenance are highly regulated processes. Throughout life, bone constantly undergoes remodelling, maintaining a balance between bone formation by osteoblasts and bone resorption by osteoclasts. This balance depends on the coordinated activities of many systemic hormones and locally acting factors in the bone microenvironment. Understanding the mechanisms of action of these factors provides a better appreciation of the cellular and molecular basis of bone remodelling.Adrenomedullin has recently been found to stimulate the proliferation of osteoblasts in vitro, and to increase indices of bone formation when administered either locally or systemically in vivo. Adrenomedullin receptors, as well as adrenomedullin itself, are expressed in primary osteoblasts and in osteoblast-like cell lines. In this paper we discuss the anabolic effect of adrenomedullin in bone, and present new evidence for a possible role of adrenomedullin in the regulation of cartilage cells. We show that adrenomedullin stimulates proliferation of primary chondrocytes in culture and that mRNA for adrenomedullin and for adrenomedullin receptors are expressed in these cells.Studies of structure-activity relationships have demonstrated that osteotropic effects of adrenomedullin can be retained in peptide fragments of the molecule which lack the parent molecule's vasodilatory properties. Thus, these small peptides, or their analogues, are attractive candidates as anabolic therapies for osteoporosis.