Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Advances in vascular tissue engineering

Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the Supply Of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials. Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries. Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture. Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties. Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells. Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery. Tissue-engineered blood vessels may also be formed from layers of human vascular cells grown in culture. These engineered vessels have many of the characteristics of arteries formed in vivo. Artificial arteries may be also be derived from peritoneal granulation tissue in body bioreactors by adapting the body's natural wound healing response to produce a hollow tube. (C) 2003 Elsevier Inc. All rights reserved.

Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation

This study demonstrates a novel approach to characterizing hydrated bone's viscoelastic behavior at lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique for analyzing local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying subsurface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods. © 2010 Elsevier Ltd.

Tissue engineering of co-cultured skin substitutes using biocomposite membranes based on collagen and polycaprolactone

The preparation and characterisation of collagen: PCL, gelatin: PCL and gelatin/collagen:PCL biocomposites for manufacture of tissue engineered skin substitutes are reported. Films of collagen: PLC, gelatin: PCL (1:4, 1:8 and 1:20 w/w) and gelatin/collagen:PCL (1:8 and 1:20 w/w) biocomposites were prepared by impregnation of lyophilised collagen and/or gelatin mats by PCL solutions followed by solvent evaporation. In vitro assays of total protein release of collagen:PCL and gelatin: PCL biocomposite films revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the biocomposite samples that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. Good compatibility of all biocomposite groups was proven by interaction with 3T3 fibroblasts, normal human epidermal keratinocytes (NHEK), and primary human epidermal keratinocytes (PHEK) and dermal fibroblasts (PHDF) in vitro respectively. The 1:20 collagen: PCL materials exhibiting good cell growth curves and mechanical characteristics were selected for engineering of skin substitutes in this work. The tissue-engineered skin model based on single-donor PHEK and PHDF with differentiated confluent epidermal layer and fibrous porous dermal layer was then developed successfully in vitro proven by SEM and immunohistochemistry assay. The following in vivo animal study on athymic mice revealed early complete wound healing in 10 days and good integration of co-cultured skin substitutes with adjacent mice skin structures. Thus the co-cultured skin substitutes based on 1:20 collagen: PCL biocomposite membranes was proven in principle. The approach to skin modelling reported here may find application in wound treatment, gene therapy and screening of new pharmaceuticals.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair. © 2003 Elsevier Ltd. All rights reserved.

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Validation of single photon absorptiometry for on-farm measurement of density and mineral content of tail bone in cattle

A validation study examined the accuracy of a purpose-built single photon absorptiometry (SPA) instrument for making on-farm in vivo measurements of bone mineral density (BMD) in tail bones of cattle. In vivo measurements were made at the proximal end of the ninth coccygeal vertebra (Cy9) in steers of two age groups (each n = 10) in adequate or low phosphorus status. The tails of the steers were then resected and the BMD of the Cy9 bone was measured in the laboratory with SPA on the resected tails and then with established laboratory procedures on defleshed bone. Specific gravity and ash density were measured on the isolated Cy9 vertebrae and on 5-mm2 dorso-ventral cores of bone cut from each defleshed Cy9. Calculated BMD determined by SPA required a measure of tail bone thickness and this was estimated as a fraction of total tail thickness. Actual tail bone thickness was also measured on the isolated Cy9 vertebrae. The accuracy of measurement of BMD by SPA was evaluated by comparison with the ash density of the bone cores measured in the laboratory. In vivo SPA measurements of BMD were closely correlated with laboratory measurements of core ash density (r = 0.92). Ash density and specific gravity of cores, and all SPA measures of BMD, were affected by phosphorus status of the steers, but the effect of steer age was only significant (P < 0.05) for steers in adequate phosphorus status. The accuracy of SPA to determine BMD of tail bone may be improved by reducing error associated with in vivo estimation of tail bone thickness, and also by adjusting for displacement of soft tissue by bone mineral. In conclusion a purpose-built SPA instrument could be used to make on-farm sequential non-invasive in vivo measurements of the BMD of tailbone in cattle with accuracy acceptable for many animal studies.

The Effect of Architecture and Shear Stress on Endothelialization of 3D Printed Vascular Networks

Despite significant progress in the field of tissue engineering within the last decade, a number of unsolved problems still remain. One of the most relevant issues is the lack of proper vascularization that limits the size of engineered tissues to smaller than clinically relevant dimensions. In particular, the growth of engineered tissue in vitro within bioreactors is plagued with this challenge. Specifically, the tubular perfusion system bioreactor has been used for large scale bone constructs; however these engineered constructs lack inherent vasculature and quickly develop a hypoxic core, where no nutrient exchange can occur, thus leading to cell death. Through the use of 3D printed vascular templates in conjunction with a tubular perfusion system bioreactor, we attempt to create an endothelial cell monolayer on 3D scaffolds that could potentially serve as the foundation of inherent vasculature within these engineered bone grafts.

Molecular phylogenetics of Chaetodon and the Chaetodontidae (Teleostei : Perciformes) with reference to morphology

Butterflyfish are colourful, pan-tropical coastal fish that are important and distinctive members of coral reef communities. A successful systematic scheme and a robust phylogeny is considered essential in understanding further their biogeography and ecology, although recent cladistic treatments of butterflyfish phylogeny, based on soft tissue and bone morphology and coded at the generic and subgeneric levels, differ in character coding and subsequently tree topology. This study provides an independent test of the morphologically based hypotheses, using molecular systematic data from two partial mitochondrial gene fragments, cytochrome b (cytb) and small subunit rRNA (rrnS), for 52 ingroup chaetodontids and seven pomacanthids used to root the molecular trees. Individual gene trees were largely compatible and a combined molecular phylogeny, inferred from Bayesian analysis, was used to test alternative hypotheses suggested by morphological analyses. The tree was also used to map the latest morphological matrix in order to evaluate potential synapomorphies for various nodes defining butterflyfish interrelationships. A clade comprised of Chelmon and Coradion was sister group to other chaetodontids. Heniochus and Hemitaurichthys were each resolved as monophyletic groups, and as sister taxa Of the taxa sampled, Prognothodes was resolved as the sister genus to Chaeotodon. Of the ten Chaetodon subgenera sampled, all were monophyletic but their interrelationships differed significantly from that inferred from morphological characters. Lepidochaetodon was the most basal subgenus followed by Exornator and the remaining subgenera. Molecular data support the sister group relationship between Corallochaetodon and Citharoedus suggested by morphology, but major differences occur among the remaining more derived taxa. Chaetodon trifascialis and C. oligacanthus were resolved as sister taxa adding weight to the inclusion of the latter in C. Megaprotodon. Of those pairs of taxa known to hybridize and sampled with molecular data, all were closely related phylogenetically, except those hybrids known to occur in the Rabdophorus subgenus. Two base changes separated C. pelewensis from C. paucifasciatus which have been regarded previously as a single species. Cytb provided greater resolution than rrnS and will likely provide additional resolution with greater taxon sampling.

Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells

The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The us e of hDPSC in NIS rats did not cause any graft. rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.

Rare case of unifocal Langerhans cell histiocytosis in four-month-old child

Langerhans cell histiocytosis (LCH) comprises a group of disorders, the common feature of which is Langerhans cell proliferation. The clinical presentation is highly varied. The severity and prognosis of the disease are dependent on the type and extent of organ involvement. This paper reports a rare case of a four-month-old white male with unifocal LCH limited exclusively to the mandible, discussing the diagnosis, radiographic and immunohistochemical aspects, treatment and monitoring multidisciplinary of the case. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

The mm of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR) Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20.000 cells well(-1) and Cultured for up to 21 days Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF Using a higher cell density. real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1. 10 and 14 Expression of the apoptotic genes bax, bcl-2 and Survivin was evaluated for both cultures hPDLF adhered and spread more oil P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater oil P(VDF-TrFE)/BT at 2 and 4 h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7. no signs of keratinocyte proliferation could be noticed for both membranes Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed oil P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion. spreading. proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR (C) 2009 Acta Materialia Inc Published by Elsevier Ltd All rights reserved