Bio-Medical Materials and Engineering - Volume 19, issue 4-5

Authors: Stoltz, J.F. | Magdalou, J. | Netter, P. | de Isla, N.

Article Type: Other

DOI: 10.3233/BME-2009-0590

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 269-269, 2009

Authors: Schallmoser, Katharina | Rohde, Eva | Bartmann, Christina | Obenauf, Anna C. | Reinisch, Andreas | Strunk, Dirk

Article Type: Research Article

Abstract: Stem cell-based therapies are a promising prospect for regenerative medicine. Particularly, human multipotent mesenchymal stromal cells (MSCs) are currently in focus regarding their regenerative and immune modulating capacities. An increasing number of clinical trials investigating MSC efficiency and safety are ongoing. Ex vivo propagation of human MSCs is considered to be a prerequisite for MSC therapy. The to date standard use of fetal bovine serum in cell culture bears risks including xenoimmunization and transmission of pathogens. Alternatively, human platelet-derived growth factors have been efficiently implemented into routine MSC expansion protocols. In compliance with good manufacturing practice we established an effective …time- and resource-saving procedure for MSC propagation in an animal serum-free system. Bone marrow was seeded without manipulation directly in pooled human platelet lysate (pHPL) and L-glutamine supplemented minimum essential medium without antibiotics. Clinical scale expanded MSCs were harvested already after primary culture. MSC quality, identity, purity and function were assessed according to a defined panel of release criteria and comparative genomic hybridization was used to determine genomic stability. Because various potential risks of MSCs have recently been reported, further research is required to prove efficiency and long-term safety of human MSCs for cell therapy. Show more

Keywords: Multipotent mesenchymal stromal cells (MSCs), pooled human platelet lysate (pHPL), GMP-compliant propagation

DOI: 10.3233/BME-2009-0591

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 271-276, 2009

Authors: Galois, Laurent | Bensoussan, Danièle | Diligent, Jérôme | Pinzano, Astrid | Henrionnet, Cristel | Choufani, Elie | Stoltz, Jean-François | Mainard, Didier

Article Type: Research Article

Abstract: This article reports the technique of percutaneous autologous bone marrow injection as a minimally invasive method alternative to open grafting techniques in the treatment of delayed unions and non-unions. Despite continuous advances in the treatment of long bone fractures, disturbances of healing processes remain a difficult challenge for orthopaedic surgeons. Percutaneous administration of substances with osteoinductive and osteogenic properties offers the advantage of decreased morbidity associated with the classic open grafting techniques. This makes it worth exploring before embarking on more extensive open surgery. The authors present the main technical stages of the percutaneous bone marrow grafting (bone marrow aspiration, …concentration, intra-osseous re-injection and post-operative protocol) with a short literature review about this topic. Show more

Keywords: Bone marrow, non-union, percutaneous injection, grafting technique

DOI: 10.3233/BME-2009-0592

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 277-281, 2009

Authors: Lequeux, C. | Auxenfans, C. | Mojallal, A. | Sergent, M. | Damour, O.

Article Type: Research Article

Abstract: Our objective was to optimize a medium for preadipocyte differentiation into adipocytes. Methods: The differentiation medium contains fixed components as well as 7 variable ones. To perform this study, different experiments were designed and the study was carried out in 4 stages. The first two stages tested the influence of serum, dexamethasone, hydrocortisone and an cAMP activator. In the third stage, two new variables were added: rosiglitazone and insulin. In the final stage, the medium selected in stage 3 was validated. The differentiation selection criteria consisted of the number of mature adipocytes and adiponectin secretion. Results: We have …shown that each variable was indispensable and that positive interactions occurred between some variables. No negative interactions were found and it was possible to optimize the concentration of each variable. Conclusions: We selected the following medium, which provides optimal adipocyte size and adiponectin secretion: DMEM/HAMF12+10% Foetal Clone Serum (FCS)+2 nM triiodothyronine+10 nM hydrocortisone +0.5 mM IsoButyl Methyl Xanthine (IBMX)+500 nM dexamethasone+1 μM rosiglitazone+0.15 UI/ml insulin+antibiotics. Show more

Keywords: Adipocytes, adiponectine, preadipocytes, differentiation

DOI: 10.3233/BME-2009-0593

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 283-291, 2009

Authors: Decot, V. | Houzé, P. | Stoltz, J.-F. | Bensoussan, D.

Article Type: Research Article

Abstract: Dimethylsulfoxide (DMSO) is a cryoprotective substance often used to allow long term storage of stem cells or tissue grafts. However, a high frequency of adverse events is associated with the infusion of thawed cells. These events are in part due to DMSO, leading many cell therapy facilities to introduce a washing step before the delivery of the grafts. The lack of method for evaluating the residual quantities of this substance in the reinfused cells led us to develop a technique, based on capillary zone electrophoresis for assaying DMSO. The cryoprotectant was measured in 55 hematopoietic stem cell grafts, 6 parathyroids …and 5 blood vessels immediately after thawing and after washing or bathing in a saline solution. The results showed that DMSO reduction in stem cell grafts reached more than 90% after the washing procedure. Furthermore, this study has shown that 2 washing steps significantly improved DMSO elimination as compared to 1 washing step. For parathyroids and blood vessels, bathing the tissues after thawing in a saline solution allowed more than 95% DMSO reduction. This study demonstrated that the technique of DMSO measurement used here, is simple and feasible on complex matrices such as protein samples after dilution. It is an appropriate method for residual quantification of the cryoprotectant before graft. Show more

Keywords: DMSO, stem cells cryopreservation, tissue grafts, capillary zone electrophoresis

DOI: 10.3233/BME-2009-0594

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 293-300, 2009

Authors: Chen, H.H. | Decot, V. | Ouyang, J.P. | Stoltz, J.F. | Bensoussan, D. | de Isla, N.G.

Article Type: Research Article

Abstract: In the last years, there were many studies based on the use of human bone marrow mesenchymal stem cells (hMSCs) in cell therapy and tissue engineering. Although hMSCs can be easily obtained and expanded in culture, a large number of cells are often needed. The expansion of hMSCs depends on the culture conditions, such as media, cell density or culture flasks. Moreover, growth factors are often added to improve cell proliferation. In this study, we compared the effect of two culture media (DMEM and α-MEM), two culture flasks (75 or 25 cm2 ) and two different mononuclear cell seeding densities …(1×104 or 5×104 MNC/cm2 ) on the isolation of hMSCs from bone marrow samples and analyzed if the isolation conditions affected the expansion of these cells in the first two passages. Experiments were performed without the addition of exogenous growth factors. Our results showed that α-MEM is the optimal culture medium for both, isolation and expansion of mesenchymal stem cells. Moreover, the cell seeding density of 50,000 MNC/cm2 in 25 cm2 culture flasks seems to be the best condition for the isolation step. Show more

Keywords: Mesenchymal stem cells, isolation, expansion, culture conditions

DOI: 10.3233/BME-2009-0595

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 301-309, 2009

Authors: Henrotin, Y. | Pesesse, L. | Sanchez, C.

Article Type: Research Article

Abstract: Osteoarthritis (OA) is the most common form of arthritic disease, and it is a major cause of disability and impaired quality of life in the elderly. A hallmark of the disease is progressive degeneration of articular cartilage and subsequent joint space narrowing. However, OA is a complex disease not limited to cartilage degeneration, but involving also synovial membrane and subchondral bone, thereby presenting alternatives approaches for treatment. In this paper, we propose a short review of the recent advances in the understanding of the role played by subchondral bone in OA.

Keywords: Bone, osteoarthritis

DOI: 10.3233/BME-2009-0596

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 311-316, 2009

Authors: de Mel, Achala | Punshon, Geoffrey | Ramesh, Bala | Sarkar, Sandip | Darbyshire, Arnold | Hamilton, George | Seifalian, Alexander M.

Article Type: Research Article

Abstract: Endothelial dysfunction or the lack of an endothelium associated with cardiovascular grafts is a major cause of graft failure which is linked to thrombosis and related complications. This study was aimed to (1) biofunctionalise a nanocomposite biomaterial, Polyhedral Oligomeric silsesquioxane modified polycarbonate urea-urethane (POSS–PCU), based small diameter vascular graft and to (2) induce endothelialisation with EPC containing monocytes, which were extracted from peripheral blood. (1) Biofunctionalisation of the nanocomposite polymer: bioactive RGD peptide, which is a functional domain of an extracellular matrix component, fibronectin, was synthesised using fmoc chemistry. A lauric acid hydrophobic “tail” was attached to optimise the RGD …orientation on the biomaterial. The peptide was cross linked to POSS–PCU. The presence of RGD on the nanocomposite was tested with water contact angle measurements and specificity tests were carried out with the peptide RAD (2) Progenitor cells were extracted from peripheral blood of adult healthy volunteers and cultured on porous biofunctionalised nanocomposite polymer under static conditions. Cells were also introduced to a circuit to which the grafts are connected and non static pulsatile flow conditions were introduced after 72 h following cell introduction. The degree of cell growth was tested with Alamar Blue assay. Endothelialisation was confirmed with SEM and by immunostaining for endothelial cell markers, CD34, CD31 and eNOS. Water contact angle measurement indicated that biofunctionalisation had increased hydrophilicity of the nanocomposite polymer. Alamar blue indicated a greater presence of cells on biofunctionalised nanocomposite and this relative increase in cell viability was specific to RGD as confirmed with RAD peptides. SEM provided evidence for endothelial cell morphology and this was confirmed with endothelial cell markers with immunostaining. Biofunctionalised nanocomposite polymer-based small diameter bypass graft demonstrated the potential for relatively rapid endothelialisation from cells extracted from peripheral blood. Show more

Keywords: Endothelialisation, RGD peptides, progenitor stem cells, polyhedral oligomeric silsesquioxane, vascular grafts, nanocomposite

DOI: 10.3233/BME-2009-0597

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 317-331, 2009

Authors: Piterina, A.V. | Callanan, A. | Davis, L. | Meaney, C. | Walsh, M. | McGloughlin, T.M.

Article Type: Research Article

Abstract: An alternative non-vascular extracellular material was obtained by decellularisation of porcine urinary bladder and examined for its potential as scaffold for vascular tissue engineering. Analysis using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Laser Scanning Microscopy (LSCM) showed a porous interconnective microarchitecture, an abundance of well preserved fibers on the abluminal side and a micropatterned flat luminal surface. Uniaxial tensile testing revealed a strength of 1.9±0.3 MPa for the rehydrated material in a phosphate buffered saline medium for the ECM-UBM sheet and these results comparable to those of native artery of a middle aged human. Multilamination of the …UBM increases the tensile properties in general (9±0.45 MPa for 2 layer, 30±0.6 MPa for 4 layers construct), with no effect on elongation capacities (38–40%) of the material. Contact-angle measurements indicated that the ECM-UBM surface exhibited a hydrophylic characteristic and better wettability than any vascular synthetic materials. Comparison of the initial adhesion in the multiplayer ECM constructs was evaluated and was found not to be altered by the preparation. The HAECs and HSMC shown an excellent adherence, spread and proliferation on the ECM-UBM material with the preservation of the cell phenotype. The level of MMP-1 and MMP-9 produced by endothelial cells was evaluated in this study and provides some data on the remodelling capacity of the scaffold. Vascular cell seeded-UBM constructs may also provide a suitable and affordable in vitro model for cell-physiology and drug development studies, which may elucidate to the mechanisms of vascular disease formation, and its prevention and treatment. Show more

Keywords: ECM, UBM, vascular tissue-engineering, smooth muscle cells, endothelial cells, tissue model

DOI: 10.3233/BME-2009-0598

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 333-348, 2009

Authors: Salmon, N. | Paternotte, E. | Decot, V. | Stoltz, J.-F. | Menu, P. | Labrude, P.

Article Type: Research Article

Abstract: Background: recent studies in bio-engineering have showed the influence of Polyelectrolyte Multilayer (PEM) films on endothelial cells (ECs), especially poly(sodium-4-styrene-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). They were tested either with human mature ECs or rabbit immature endothelial progenitor cells (EPCs), but never on human EPCs. In view to obtain an EC covered surface, human cord blood (HCB) EPCs were cultivated on PSS/PAH films. Material and methods: PEMs were obtained by 7 alternate depositions of cationic PAH and anionic PSS layers. HCB mononuclear cells were isolated by centrifugation through density gradient. 7 days after seeding on PEM, unattached cells were …removed and adherent EPCs were cultivated in endothelial specific medium until P6. Appearance of CD31 and vWF was evaluated by confocal microscopy. Results: EPCs not only successfully adhered on PEM, but also spread and proliferated. Moreover, cells differentiated into a typical endothelial cobblestone monolayer within 2 weeks. Immunostaining of CD31 and vWF confirmed the formation of an EC-like confluent monolayer. Furthermore, these cells showed after 6 passages a good phenotypic stability while reseeded on the PEM film. Conclusion: these results show an easy way to obtain mature ECs from human stem cells, which may open new applications for a scaffold cellularization in tissue bio-engineering. Show more

Keywords: Polyelectrolyte multilayer, human cord blood cells, endothelial progenitor cells, cells differentiation

DOI: 10.3233/BME-2009-0599

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 349-354, 2009

Authors: Petitpain, Nadine | Gambier, Nicolas | Wahl, Denis | Chary-Valckenaere, Isabelle | Loeuille, Damien | Gillet, Pierre

Article Type: Research Article

Abstract: Background: Systemic inflammation such as rheumatoid arthritis (RA) and Crohn's disease (CD) may be responsible for vascular comorbidity. TNF-α blockade was expected to lower these comorbidities but several cases of arterial and venous thromboembolic events (TE) have been reported. Objectives: The aim of this work was to study retrospectively the main characteristics of spontaneously notified TNF-α blockers related TE over a 7-year period. Methods: TE related to infliximab, etanercept and adalimumab spontaneously notified to the French adverse drug reporting system database between January 2000 and December 2006 were analyzed. Separate analysis of arterial TE and venous TE was …performed. Risk factors for each category of TE were assessed with consensual criteria. Results: 85 TE were analyzed, representing 4.5% of all the spontaneously notified adverse reactions of the 3 TNF-α blockers in the database. 42 were arterial events and 43 were venous events. The incidence was not significantly different between the 3 TNF-α blockers. Mean delay of TE onset after treatment initiation was 10.6 months. It was significantly shorter for etanercept (6.1 months, p=0.001) especially for venous TE (2.4 months). 16 among the 42 patients with arterial TE had 2 or more risk factors whereas 39 among the 43 patients with venous TE had no RF or only one. Most of patients (79/85) received concomitant systemic corticosteroids and/or methotrexate and/or COX-2 selective inhibitors. 23 patients had been investigated for autoimmunity, 13 had antinuclear and/or antiphospholipid antibodies. Main limitations of this study were underreporting and heterogeneous report contents. Conclusion: Despite its limitations, this study suggests that venous TE could be favoured by TNF-α blockers therapy since they occurred in patients with no or few risk factors for venous thrombosis. However, this needs to be more evaluated by controlled studies. Show more

Keywords: Arthritis, rheumatoid, complications, drug therapy, physiopathology, cardiovascular diseases, etiology, mortality, tumor necrosis factor-alpha, antagonists and inhibitors

DOI: 10.3233/BME-2009-0600

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 355-364, 2009

Authors: Auxenfans, Céline | Thépot, Amélie | Justin, Virginie | Hautefeuille, Agnès | Shahabeddin, Lili | Damour, Odile | Hainaut, Pierre

Article Type: Research Article

Abstract: Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, …murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of ΔN to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. Show more

Keywords: Feeder layer, 3T3 fibroblasts, human fibroblasts, keratinocytes culture, ΔN/TAp63

DOI: 10.3233/BME-2009-0601

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 365-372, 2009

Authors: Caunday, Olivia | Bensoussan, Danièle | Decot, Véronique | Bordigoni, Pierre | Stoltz, Jean François

Article Type: Review Article

Abstract: In 1997, the Joint Accreditation Committee ISCT & EBMT (JACIE) was created. The following year, it approved the first edition of standards for haemopoietic progenitor cell collection, processing and transplantation. The purpose of the standards is to ensure a minimal level of quality, alertness and homogeneity in the implementation of autologous and allogeneic haemopoietic stem cell transplantation (HSCT) programme in onco-hematology. The acquisition of accreditation is based upon the system of examination by trained medical professionals according to countries endorsements with the national regulation obligations applicable to HSCT. In 2008, the fourth edition has been launched. The range …of application of the standards comprises both donors and recipients, and all phases of collection, processing, storage and administration of haemopoietic progenitor cells. Among the accredited processing facilities, a few have been integrated JACIE standards into their existing management quality system which is inspected by national health authority. In France, the comparison between JACIE standards and the good manufacturing practices of cellular therapy product reveals some common points and some differences to apply. Show more

DOI: 10.3233/BME-2009-0602

Citation: Bio-Medical Materials and Engineering, vol. 19, no. 4-5, pp. 373-379, 2009