Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

<p>Abstract</p> <p>Background</p> <p>Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.</p> <p>Methods</p> <p>A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured <it>in vitro </it>and <it>in vivo </it>in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific <it>in situ </it>hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.</p> <p>Results</p> <p>The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. <it>In vitro </it>and <it>in vivo </it>(subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels <it>in vitro </it>and <it>in vivo</it>, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the <it>in vitro </it>cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific <it>in situ </it>hybridization.</p> <p>Conclusions</p> <p>The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.</p

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion

Omics-based molecular techniques in oral pathology centred cancer: Prospect and challenges in Africa

: The completion of the human genome project and the accomplished milestones in the human proteome project; as well as the progress made so far in computational bioinformatics and “big data” processing have contributed immensely to individualized/personalized medicine in the developed world.At the dawn of precision medicine, various omics-based therapies and bioengineering can now be applied accurately for the diagnosis, prognosis, treatment, and risk stratifcation of cancer in a manner that was hitherto not thought possible. The widespread introduction of genomics and other omics-based approaches into the postgraduate training curriculum of diverse medical and dental specialties, including pathology has improved the profciency of practitioners in the use of novel molecular signatures in patient management. In addition, intricate details about disease disparity among diferent human populations are beginning to emerge. This would facilitate the use of tailor-made novel theranostic methods based on emerging molecular evidences

Conditional Deletion of Cytochrome P450 Reductase in Osteoprogenitor Cells Affects Long Bone and Skull Development in Mice Recapitulating Antley-Bixler Syndrome: Role of a Redox Enzyme in Development

<div><p>NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding <i>Por</i>^{<i>lox/lox</i>} and <i>Dermo1 Cre</i> mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the <i>Por</i>-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon <i>Por</i> deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.</p> </div

Skull phenotype.

<p>(A) Dome-shaped skull present in the case of 16-day-old CKO mouse; (B) dome-shaped skull and tooth malocclusion in 16-wk-old differentially stained skull (C) differentially stained skull base of 4-day old mice showing premature fusion of sphenooccipital synchondroses, SOS, black asterisk, and basioccipital-exoccipital synchondroses, BES, [black arrow], in case of CKO (sph: spheno-occipital bone, occ: occipital bone, exo: exo-occipital bone). These observations were made with n=4 animals in each case.</p

Body size/weight differences in littermates.

<p>(<b>A</b>) Photograph of 5-day-old mice; (<b>B</b>) differential stain of 4- and 16-day-old mice (images not taken to scale, precluding comparisons between age groups); and (<b>C</b>) compiled body weights of 4-, 16- and 16-week-old wild type (open bars) and CKO (solid bars) mice (*, p < 0.005 and **, p < 0.001).</p

Images of differentially stained femurs, tibias and rib cage of 4-day-old mice.

<p>Whole animal littermate pups are differentially stained using Alizarin red and Alcian blue and the images of (A) femur and tibia were taken after disarticulating them from the whole stained animal. This represent observations from n=3 for 4 days, n=4 for 16 weeks and n=7 for 16 weeks old animals. (B) The rib cage image of 4-day old pup was taken using a stereoscope. The rib cage images are not taken to scale. (C) Mid-substance grey-scale and three-dimensional views of proximal tibia trabecular bone in 16-week-old mice show reduced bone volume fraction (D). At 16 weeks, cortical bone thickness in the midshaft is also lower in the CKO mice (E), but little difference is seen in cortical bone at earlier time points. (F) Bone mineralization using primary calvarial osteoblasts: von Kossa staining was performed with primary osteoblasts isolated from calvaria of wild type and CKO littermate mice pups (3-day-old) grown in differentiation media. (G) The surface area of mineralized matrix was quantified using ImageJ from three independent mineralized experiments (n=2).</p

Immunocytochemistry of stably POR knockdown 2T3 cells.

<p>Stable clones of shRNA mediated POR knockdown 2T3 cells along with scrambled shRNA clone were stained for (A) POR, (B) pFGFR, and (C) pERK, using antibodies against these proteins and developed using fluorescent-labeled secondary antibodies. DAPI (blue fluorescence) was used for nuclear staining.</p