Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells.

In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite(©)), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions.

Baseline simple and complex reaction times in female compared to male boxers.

AIM: The aim of the study was to compare baseline cognitive performance of female in respect to male amateur boxers. METHODS: Study population included 28 female amateur boxers. Fifty-six male boxers, matched for age, employment and competitive level to female athletes, formed the control group. All boxers had no history of head concussions (except boxing). Each boxer was requested to: 1) fulfill a questionnaire collecting demographic data, level of education, occupational status, boxing record and number of head concussions during boxing; 2) undergo a baseline computerized neuropsychological (NP) test (CogSport) measuring simple and complex reaction times (RT). RESULTS: Female were lighter than male boxers (56±7 vs. 73.1±9.8 kg, P<0.0001). No significant differences at CogSport scores were observed between groups. Male boxers showed a longer simple-RT at the end of the NP battery than at the beginning (0.247±0.007 vs. 0.243±0.007 s, P=0.02), however, with a significant lower rate of mistakes (0.7±1.6 vs. 2.0±3.1%, P=0.005), observed also in the female group (0.5±1.1 vs. 2.2±3.0%, P=0.005). No boxing activity parameter (record, number of knock-outs, etc.) correlated with NP scores. CONCLUSION: Female and male Olympic-style boxers have no (or minimal) differences in baseline cognitive performance. Further research with larger series of female boxers is required to confirm these findings.

Forced chondrocyte expression of sonic hedgehog impairs joint formation affecting proliferation and apoptosis.

Proliferation and apoptosis are two fundamental processes that occur during limb development, and in particular in joint formation. To study the role of hedgehog proteins in limbs, we have misexpressed Sonic Hedgehog specifically in chondrocytes. We found that the appendicular skeleton was severely misshapen while pelvic and shoulder girdles developed normally. In particular, we detected fusion of the elbow/knee joint, no definite carpal/tarsal, metacarpal/metatarsal bones and absence of distinct phalanges, fused in a continuous cartilaginous rod. Molecular markers of joints, such as Gdf5 and sFrp2 were absent at presumptive joint sites and Tenascin C, a molecule associated with joint formation and expressed in permanent cartilage, was expressed in a wider region in transgenic animals as compared to the wild type. The ratio of proliferating to non-proliferating chondrocytes was about two times higher in transgenic developing cartilage as compared to the wild type. Accordingly, the proapoptotic gene Bax was barely detectable in the growth plate of transgenic mice and Tunel assay showed the absence of apoptosis in presumptive joints at E15.5. Taken together, these results suggest that misexpression of Sonic Hedgehog causes apoptosis and proliferation defects leading to the lack of joint cavity and fusion of selected limb skeletal elements.

Regulated expression of fibronectin, laminin and related integrin receptors during the early chondrocyte differentiation.

We have investigated the expression and localization of fibronectin, laminin, and their receptors, and we used an in vitro chick chondrocyte differentiation model to define a time hierarchy for their appearance in early chondro-genesis and to determine their role in the cell condensation process. By serum fibronectin depletion/reconstitution, or GRGDSP peptide competition experiments, we show that fibronectin contributes to the initial cell-cell interactions that occur during condensation. In later stages, a down-regulation of both fibronectin and of its alpha5beta1 integrin receptor occur, as demonstrated by mRNA and protein kinetics. Immunolocalisation studies suggest that the reduction of fibronectin in discrete areas is involved in local activation of the cell differentiation program. Furthermore, we show that laminin is expressed during the in vitro cell condensation process in areas that are negative for fibronectin staining. The types of laminin as well as the timing of expression have been determined by northern blot and RT-PCR analyses. The highest levels of expression are coincident with maximal cell aggregation. The alpha3beta1 laminin receptor, highly expressed in dedifferentiated cells, follows later on the ligand trend. During in vitro chondrogenesis, a down-regulation in the B isoform, and an up-regulation of the A isoform, of the alpha subunit of the alpha6beta1 laminin receptor occurs. Immunolocalisation studies suggest that laminin is involved in the definition of differentiating areas as opposed to non differentiating areas of the condensed region, i.e. the periphery, which eventually gives rise to the perichondrium.

N-CAM and N-cadherin expression during in vitro chondrogenesis.

Mesenchymal cell condensation in chick limb bud occurs at embryonic stage 22 and is the starting event of chondrogenesis. Several mechanisms have been proposed to have an active role in the induction of this process. Among them the establishment of cell-cell contacts represents a key event. Here we have investigated the modulation of N-CAM and N-cadherin gene expression in an in vitro culture system which allows chondrocyte differentiation to proceed from condensation of prechondrogenic cells to hypertrophic chondrocytes and eventually to osteoblast-like cells. Both Northern and Western blots demonstrated that they were developmentally regulated in differentiating chondrocytes. Both cell adhesion proteins were detectable in prechondrogenic cells, increased during cell aggregation, became undetectable in hypertrophic chondrocytes, and resulted in reexpression during their maturation to osteoblast-like cells. The timing of appearance of N-cadherin and N-CAM suggests that N-cadherin initiates the in vitro cell condensation thereafter stabilized by N-CAM. In agreement with the above findings, the immunolocalization of these molecules in the cell aggregates revealed that N-CAM and N-cadherin appear, after 12 h of suspension culture, on the surface of all cells at the membrane regions participating in cell-cell contacts. At 72 h N-CAM became restricted to cells at the aggregate periphery, while N-cadherin was detected both in type II collagen-negative and -positive regions. At this time of culture, electron microscopy shows a number of cell-cell contacts at the perifery of the cell aggregates, while only a few of them were observed in the aggregate interior. The expression of N-CAM and type II collagen by chondrocytes was mutually exclusive and a sorting out between differentiating and nondifferentiating cells occurred.

Modulation of tensin and vimentin expression in chick embryo developing cartilage and cultured differentiating chondrocytes.

It has been proposed that tensin, in association with several other proteins, mediates the micro-filament-integrin link. Here we describe the isolation of clones spanning about 5 kb from the 3' end of tensin mRNA from cultured chick embryo chondrocyte and embryonic heart cDNA libraries. Tensin expression was investigated in cultured chick embryo cells. It was observed that tensin expression is dependent upon substrate adhesion and it is turned off after 7 days of suspension culture. This process is reversible. Tensin expression is also regulated during cartilage cell differentiation in vivo; at Hamburger and Hamilton stage 39-40, non-hypertrophic tibial chondrocytes express both RNA and protein while hypertrophic chondrocytes do not. In the culture system the expression of vimentin, a major component of intermediate filaments, showed an opposite behaviour since the suspension culture enhances the accumulation of both vimentin and its mRNAs. Therefore in chick embryo cultured chondrocytes and in vivo, during cartilage development, cell shape changes and/or integrin-extracellular matrix protein interactions may be involved in the regulation of these two genes coding for cytoskeletal proteins.

Tissue-specific expression of type XIV collagen--a member of the FACIT class of collagens.

The collagens represent a highly diverse superfamily of extracellular matrix proteins that can be divided into several distinct families. One of the families, called FACIT (fibril-associated collagens with interrupted triple-helices) family, contains molecules that appear to be associated with cross-striated fibrils composed of members of the fibrillar collagen family. We have determined a portion of the primary structure of a recently discovered member of the FACIT family, chicken alpha 1(XIV) collagen, based on cloning and sequencing cDNAs. A synthetic oligopeptide from within the carboxy-terminal non-triple-helical domain of the alpha 1(XIV) chain has been used for generating specific polyclonal antibodies. The antiserum, PS1, recognizes a 220 kDa polypeptide in immunoblots of extracts of chicken skin, tendons, and cartilage. Sequencing of a tryptic peptide generated from purified, immunoreactive material, gives a sequence identical to that derived from cDNA sequencing, providing strong support for the type XIV-specificity of PS1. We have examined the expression of type XIV collagen in developing chick embryos by immunostaining of sections from 12-day-old embryos with PS1 and by Northern blot analysis of RNA from several tissues from both 12- and 17-day-old embryos. The results show that type XIV collagen is prevalent within relatively dense connective tissues such as dermis, tendons, perichondrium, perimysium, the stroma of lungs and liver, and blood vessels.

Cell condensation in chondrogenic differentiation.

Reduction of intercellular spaces in the areas of prospective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may represent an important step in the process of cartilage differentiation during limb skeletogenesis. We have attempted to clarify the role of the microenvironment established during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., increased cell density due to cell aggregation) and chondrogenic differentiation (i.e., synthesis of cartilage-specific extracellular matrix proteins, such as type II collagen and acquisition of a chondrocyte morphology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in carboxymethylcellulose and changes in the differentiation pathway were evaluated. In another series of experiments, we have separated single cells from the aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II collagen expression, at both the protein and the mRNA level, show that a reduced rate of cell clustering and cell to cell contact parallels a reduction of cell recruitment into the differentiation program. On the basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte differentiation: (a) the acquisition of the ability to establish cell to cell contacts, (b) the formation of a permissive environment capable of activating the differentiation program, and (c) the expression of differentiation markers.