A three-dimensional traction/torsion bioreactor system for tissue engineering.

PURPOSE: The aim of this study was to design, develop and validate a simple, compact bioreactor system for tissue engineering. The resulting bioreactor was designed to achieve ease-of-use and low costs for automated cell-culturing procedures onto three-dimensional scaffolds under controlled torsion/traction regimes. METHODS: Highly porous poly-caprolactone-based scaffolds were used as substrates colonized by fibroblast cells (3T3 cell line). Constructs were placed within the cylindrical culture chamber, clumped at the ends and exposed to controlled sequences of torsional stimuli (forward/back-forward sequential cycles of 100 degrees from neutral position at a rate of 600 degrees/min) through a stepper-motor; working settings were defined via PC by an easy user-interface. Cell adhesion, morphology, cytoskeletal fiber orientation and gene expression of extracellular matrix proteins (collagen type I, tenascin C, collagen type III) were evaluated after three days of torsional stimulation in the bioreactor system. RESULTS AND CONCLUSIONS: The 3D bioreactor system was validated in terms of sterility, experimental reproducibility and flexibility. Cells adhered well onto the polymeric scaffolds. Collagen type I, tenascin C and collagen type III gene expression were significantly up-regulated when cells were cultured under torsion in the bioreactor for three days. In conclusion, we have developed a simple, efficient and versatile 3D cell-culture system to engineer ligament grafts. This system can be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system for possible clinical use in the field of regenerative medicine.

Patients with oral erosive and cutaneous lichen planus may have antibodies directed against the chronic ulcerative stomatitis protein antigen of 70-kDa.

OBJECTIVE: The aim of this study is to verify whether stratified epithelium-specific antinuclear antibodies are present in the sera of patients with erosive oral lichen planus and cutaneous lichen planus. METHODS: We studied the pre-immune and immune serum of a rabbit immunized with a peptide corresponding to the N-terminus of the 70-kDa antigen chronic ulcerative stomatitis protein; sera from two patients, one with oral erosive lichen planus and one with cutaneous lichen planus who presented stratified epithelium-specific antinuclear antibodies at high titer; and a third serum from a patient with cutaneous lichen planus without stratified epithelium-specific antinuclear antibodies. RESULTS: We demonstrated that the protein bands recognized by the serum of the rabbit immunized with an epitope of chronic ulcerative stomatitis protein co-migrated by SDS-PAGE with the protein bands recognized by the serum of a patient affected by oral erosive lichen planus and by the serum of a patient with cutaneous lichen planus, both containing antibodies directed against a 70-kDa antigen. CONCLUSIONS: Our results confirm that antibodies specifically directed against the chronic ulcerative stomatitis protein are not a distinctive marker of chronic ulcerative stomatitis, but may also be detected in oral erosive and cutaneous lichen planus.

Expression of serum amyloid A in chondrocytes and myoblasts differentiation and inflammation: possible role in cholesterol homeostasis.

Serum amyloid A (SAA) is synthesized by the liver during the acute phase. Local expression of SAA mRNA has been reported also in non-liver cells, a potential local source of SAA protein not related to the systemic acute phase response. SAA function has not been established yet. In the present study, we identified SAA as a protein expressed by chondrocytes and myoblasts in response to inflammatory stimula. In both cell systems, SAA mRNA and protein expression is strongly stimulated by bacterial lipopolysaccharide treatment. SAA mRNA expression is also enhanced during terminal differentiation of cells of the chondrogenic and myogenic lineage; mRNA is barely detectable in prechondrogenic cells and is highly expressed in differentiated hyperthrophic chondrocytes. An increased level of SAA mRNA was also observed in vivo when we compared mRNA extracted from tibiae of 10 day embryos, still fully cartilaginous, with tibiae from 18 day embryos, a stage when the endochondral ossification process has already started. p38 activation, a well-known event of the chondrogenesis signaling cascade, controls expression of SAA in cartilage following inflammatory stimuli. SAA secreted by stimulated chondrocytes is associated with cholesterol. Cholesterol is synthesized by the same chondrocytes and is also increased in inflammatory conditions. A role of SAA in cholesterol homeostasis in chondrocytes is proposed.

Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting.

Ex-FABP, an extracellular fatty acid binding lipocalin, is physiologically expressed by differentiating chicken chondrocytes and myoblasts. Its expression is enhanced after cell treatment with inflammatory stimuli and repressed by anti-inflammatory agents, behaving as an acute phase protein. Chicken liver fragments in culture show enhanced protein expression after bacterial endotoxin treatment. To investigate the biological role of Ex-FABP, we stably transfected proliferating chondrocytes with an expression vector carrying antisense oriented Ex-FABP cDNA. We observed a dramatic loss of cell viability and a strong inhibition of cell proliferation and differentiation. When chondrocytes were transfected with the antisense oriented Ex-FABP cDNA we observed that Ex-FABP down-modulation increased apoptotic cell number. Myoblasts transfected with the same expression vector showed extensive cell death and impaired myotube formation. We suggest that Ex-FABP acts as a constitutive survival protein and that its expression and activation are fundamental to protect chondrocytes from cell death.

Myotubularin-related 2 protein phosphatase and neurofilament light chain protein, both mutated in CMT neuropathies, interact in peripheral nerve.

Charcot-Marie-Tooth disease type 4B1, CMT4B1, is a severe, autosomal-recessive, demyelinating peripheral neuropathy, due to mutations in the Myotubularin-related 2 gene, MTMR2. MTMR2 is widely expressed and encodes a phosphatase whose substrates include phosphoinositides. However, this does not explain how MTMR2 mutants specifically produce demyelination in the peripheral nerve. Therefore, we analysed the cellular and subcellular distribution of Mtmr2 in nerve. Mtmr2 was detected in all cytoplasmic compartments of myelin-forming Schwann cells, as well as in the cytoplasm of non-myelin-forming Schwann cells and both sensory and motorneurons. In contrast, Mtmr2 was detected in the nucleus of Schwann cells and motorneurons, but not in the nucleus of sensory neurons. As Mtmr2 is diffusely present also within the nerve, a specific function could derive instead from nerve-specific interacting proteins. Therefore, we performed two yeast two-hybrid screenings, using either fetal brain or peripheral nerve cDNA libraries. The neurofilament light chain protein, NF-L, was identified repeatedly in both screenings, and found to interact with MTMR2 in both Schwann cells and neurons. Interestingly, NF-L, encoding NF-L, is mutated in CMT2E. These data may provide a basis for the nerve-specific pathogenesis of CMT4B1, and further support for the notion that hereditary demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

A chondrogenesis-related lipocalin cluster includes a third new gene, CALgamma.

We have previously reported the modulation, during chondrogenesis and/or inflammation, of two chicken genes laying in the same genomic locus and coding for two polypeptides of the lipocalin protein family, the extracellular fatty acid binding protein (ExFABP) and the chondrogenesis associated lipocalin beta (CALbeta). A third gene, located within the same cluster and coding for a new lipocalin, CALgamma, has been identified and is here characterized. Tissue distribution analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction in chicken embryos shows a ubiquitous expression with predominant levels of mRNA transcripts in the liver and the brain. In the developing tibia, a high expression of CALgamma mRNA was evidenced by in situ hybridization within the pre-hypertrophic and the hypertrophic zones of the bone-forming cartilage. In agreement, dedifferentiated chondrocytes in vitro express the transcripts to the highest level when they re-differentiate reaching hypertrophy. Such peculiar developmental pattern of expression that is analogous to those already described for Ex-FABP and CALbeta suggests that all three proteins may act synergistically in the process of endochondral bone formation. Moreover, like Ex-FABP and CALbeta, CALgamma is also highly induced in dedifferentiated chondrocytes upon stimulation with lypopolysaccharides, indicating that the whole cluster quite possibly is transcriptionally activated not only in physiological morphogenic differentiation but also in pathological acute phase response.

Ex-FABP, extracellular fatty acid binding protein, is a stress lipocalin expressed during chicken embryo development.

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.

Chondrocyte protein with a poly-proline region is a novel protein expressed by chondrocytes in vitro and in vivo.

Chondrogenic differentiation is a multistep process entailing the sequential activation and inhibition of the expression of a number of genes. To identify genes preferentially expressed at the hypertrophic stage rather than early differentiation stages of chicken chondrocyte differentiation, a subtracted cDNA library was generated. Here we describe the characterization of a cDNA isolated from this library and that of the encoded protein referred to as Chondrocyte Protein with a Poly-proline Region (CHPPR). The cDNA coding for CHPPR hybridizes with a 3.0-kb mRNA expressed at extremely low levels in dedifferentiated chondrocytes, cultured in adherent conditions, at low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes in suspension culture. The Parathyroid Hormone peptide [PTH (1-34)] enhances accumulation of CHPPR mRNA in cultured chondrocytes. This 3.0-kb mRNA is also detectable in several chick embryo tissues but at a lower extent when compared to that present in cartilage and in hypertrophic chondrocytes. The CHPPR cDNA has a complete open reading frame coding for a polypeptide with a calculated mass of 35.6 kDa containing a proline-rich region with a PPLP motif (single-letter amino acid code). We demonstrate by Western blot analysis that two CHPPR isoforms are detected in the cell lysates from cultured chondrocytes when they are not in the culture medium; furthermore, we find that the CHPPR gene is expressed in vivo by chick embryo chondrocytes at higher levels in the prehypertrophic and hypertrophic zones.

The Dlx5 homeobox gene is essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway.

In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear. In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed, characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however, sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of Dlx5(-/-) mice. The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the Dlx5(-/-) embryos, no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) was drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. We propose that development of the semicircular canals and the vestibular inner ear requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BPM4 expression and the regional organization of cell differentiation, proliferation, and apoptosis.

CALbeta, a novel lipocalin associated with chondrogenesis and inflammation.

We have previously demonstrated the association of the chicken lipocalin Ex-FABP with cartilage formation and inflammatory responses as a marker of these processes (Descalzi Cancedda et al., Biochim. Biophys. Acta 1482, 127-135, 2000). Here we report the isolation and characterisation of a new lipocalin gene laying upstream the Ex-FABP, thus representing the second member of a possible genomic cluster. This gene contains an open reading frame coding for a polypeptide of about 19 kDa. The amino-acid sequence revealed a conserved lipocalin secondary structure. Tissue distribution of the protein in developing embryos showed a preferential expression in the heart although mRNA transcripts could be detected also in muscle, lung and liver. The lowest expression was observed in the stomach, brain and skin. During endochondral formation of long bones, the protein is differentially distributed, as the transcripts, evidenced in the tibia by in situ hybridisation, are present in the hypertrophic cone of the cartilage and mostly absent in the area of the proliferating chondrocytes. Such developmental regulation was observed also in vitro in cultured chondrocytes where the transcripts were barely detectable in dedifferentiated cells but highly expressed in hypertrophic chondrocytes. The protein was also significantly induced by lipopolysaccharide stimulation of chondrocytes, indicating a possible involvement in acute phase response. Raising specific antibodies in a rabbit allowed validating, at the protein level, all the transcriptional data. Moreover, we gained evidence that the protein is actively secreted in the extracellular matrix surrounding the chondrocytes. Because of its peculiar expression in cartilage, this new protein was named chondrogenesis-associated lipocalin beta (thereafter referred to as CAL beta). The close similarity between Ex-FABP and CAL beta expression patterns supports the hypothesis of a genomic organisation in a cluster where both genes could be co-ordinately regulated.