Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering.

For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

[Influences of induced membranes on heterotopic bone formation within an osteo-inductive complex. Experimental study in rabbits]. / Influence des membranes induites sur l'ostéogenèse hétérotopique au sein d'un complexe ostéo-inducteur. Etude expérimentale chez le lapin.

UNLABELLED: Based on a new concept, a procedure combining induced membranes and cancellous autografts allows the reconstruction of wide diaphyseal defects. To date, this procedure is limited by the amount of cancellous bone available from the patient and by the related morbidity at the donor site. The aim of this study was to evaluate the biological effect of induced membranes on a cylindrical-shaped ceramic implants loaded with OP-1 in heterotopic site. MATERIALS AND METHODS: Sixty hydroxyapatite tricalcium phosphate (HA-TCP) implants, 20 of which being loaded with a bone growth factor (rhOP-1) were inserted either in a subcutaneous tunnel or within a previously induced membrane on the back of rabbits. There were two time-points at four and 16 weeks. Implants were investigated at three different levels (extremities and middle). RESULTS: None of the untreated implants showed any evidence of bone formation. Implants inserted in an induced membrane presented with less resorption. Bone ingrowth within the pores of the materials was significantly higher when the implants were inserted into the induced membrane whatever the time-point considered. CONCLUSION: The membrane seems to play the role it was assigned, i.e. to protect and revascularize the implant, thus favouring osteogenesis that occurs in 80% of the implants after four months.

Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function.

Osteogenesis occurs in striking interaction with angiogenesis. There is growing evidence that endothelial cells are involved in the modulation of osteoblast differentiation. We hypothesized that primary human umbilical vein endothelial cells (HUVEC) should be able to modulate primary human osteoprogenitors (HOP) function in an in vitro co-culture model. In a previous study we demonstrated that a 3 day to 3 week co-culture stimulates HOP differentiation markers such as Alkaline Phosphatase (ALP) activity and mineralization. In the present study we addressed the effects induced by the co-culture on HOP within the first 48 hours. As a prerequisite, we validated a method based on immuno-magnetic beads to separate HOP from HUVEC after co-culture. Reverse transcription-real time quantitative PCR studies demonstrated up-regulation of the ALP expression in the co-cultured HOP, confirming previous results. Surprisingly, down-regulation of runx2 and osteocalcin was also shown. Western blot analysis revealed co-culture induced down-regulation of Connexin43 expression in both cell types. Connexin43 function may be altered in co-cultured HOP as well. Stimulation of the cAMP pathway was able to counterbalance the effect of the co-culture on the ALP activity, but was not able to rescue runx2 mRNA level. Co-culture effect on HOP transcriptome was analyzed with GEArray cDNA microarray showing endothelial cells may also modulate HOP extracellular matrix production. In accordance with previous work, we propose endothelial cells may support initial osteoblastic proliferation but do not alter the ability of the osteoblasts to produce extracellular mineralizing matrix.

Functionalization of biomaterials and cell to cell communication.

In the field of osseous substitution, the possibilities being offered to the surgeons prove sometimes difficult to apply in particular in the case of great losses of osseous substance. For these reasons, it is necessary to develop innovative techniques to satisfy the request increasing for substitutes and to see appearing on the market solutions combining availability, perenniality and biosecurity of the implants. The implantation of stem cells in a biomaterial opens a way of development of therapeutic substitute. Moreover, in order to optimize the rehabitation of the biomaterials by the cells and the host tissues, the second approach consists in modifying the surface of materials by the coating or the grafting of adhesive factors in order to stimulate their colonization. At least, one cannot consider a tissue mechanism of repair without a better knowledge of the respective role of the various cell populations implied in the rebuilding of this tissue and their cell to cell communication processes.

A novel hybrid nanofibrous strategy to target progenitor cells for cost-effective in situ angiogenesis.

Although the impact of composites based on Ti-doped calcium phosphate glasses is low compared with that of bioglass, they have been already shown to possess great potential for bone tissue engineering. Composites made of polylactic acid (PLA) and a microparticle glass of 5TiO2-44.5CaO-44.5P2O5-6Na2O (G5) molar ratio have already demonstrated in situ osteo- and angiogenesis-triggering abilities. As many of the hybrid materials currently developed usually promote osteogenesis but still lack the ability to induce vascularization, a G5/PLA combination is a cost-effective option for obtaining new instructive scaffolds. In this study, nanostructured PLA-ORMOGLASS (organically modified glass) fibers were produced by electrospinning, in order to fabricate extra-cellular matrix (ECM)-like substrates that simultaneously promote bone formation and vascularization. Physical-chemical and surface characterization and tensile tests demonstrated that the obtained scaffolds exhibited homogeneous morphology, higher hydrophilicity and enhanced mechanical properties than pure PLA. In vitro assays with rat mesenchymal stem cells (rMSCs) and rat endothelial progenitor cells (rEPCs) also showed that rMSCs attached and proliferated on the materials influenced by the calcium content in the environment. In vivo assays showed that hybrid composite PLA-ORMOGLASS fibers were able to promote the formation of blood vessels. Thus, these novel fibers are a valid option for the design of functional materials for tissue engineering applications.

RGD-functionalized spherulites as targeted vectors captured by adherent cultured cells.

Spherulites are multilamellar vesicles consisting of concentric shells that can encapsulate small organic molecules or macromolecules. We investigate the possibility of targeting neutral spherulites to adherent culture cells by functionalizing their surface with RGD-containing ligands. The strength and specificity of association of RGD spherulites with several cell lines (EAhy 926 endothelial cell line, human umbilical vein endothelial cell (HUVEC) and human osteoprogenitor (HOP) primary cells) was studied, and the molecular interaction of RGD spherulites with the EAhy 926 cell surface was investigated. We show that, after binding to cells, spherulites are internalized.

Effect of several sterilisation techniques on homogeneous self assembled monolayers.

Understanding how cells sense their environment and are able to regulate their metabolism is of great importance for the success of biomaterials implantation. Self assembled monolayers (SAMs) are in use nowadays to model the surface of such materials. They permit the control of different surface parameters (like chemistry, surface energy and topography) enabling to get a greater insight in cells behaviour when interacting with surfaces and thus, in the future, to enhance surface properties of biomaterials. As sterilisation is the compulsory step for in vitro and in vivo assays with living biological materials, it is important to know how SAMs react under sterilisation techniques in use on biomaterials. In this work, the effect of three types of sterilisation techniques: gamma-irradiation, mostly used on biomaterials, dry heat and steam autoclaving, have been investigated on NH2 and CH3 terminated SAMs. Gamma-irradiation destructs drastically the NH2 and partially the CH3 monolayers by producing oxidative compounds (COOH, C=O, C-OH). The main product induced by gamma-irradiation on NH2 monolayers is carboxylic acid, whereas CH3 shows an important increase in the amount of alcoholic groups. This difference in deterioration is assumed to be due to the higher stability of the CH3 monolayer. Steam autoclaving to a lesser extent gives the same results on NH2 monolayers. Dry heat seems to be the most reliable technique, which can be used on such surfaces as it removes physically adsorbed organic contaminants without affecting the integrity of the surface.

Colonization of a calcium phosphate/elastin-solubilized peptide-collagen composite material by human osteoblasts.

The evaluation of the suitability of a new biomaterial as a possible substitute for bone tissue is described here. This biomaterial is based on calcium phosphate particles linked to an artificial connective matrix, the elastin-solubilized peptides (ESP) associated with type I and III collagens. This work demonstrates the feasibility of shaping this composite material into discs, describes its microstructural characteristics, and evaluates its capacity as a substrate for the proliferation of human osteoblasts without loss of their phenotypic expression.

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics.

The main problem for macroporous structures used as bone substitutes is their lower resistances when compared to that of cancellous bone. The present investigation aimed to improve the strength of ceramics with 65% porosities based on beta-TCP. The initial mixtures were rendered plastic by addition of non-ionic carbohydrate binders. Macropores were created using substances which were eliminated by heat. Mechanical tests indicated that the resistance of the ceramics depended more on the quantity than the nature of the binders. Porosity measurements were done with a mercury porosimeter, and cellular biocompatibility was evaluated by performing cellular attachment tests and observing the proliferation of differentiated cells.

Various evaluation techniques of newly formed bone in porous hydroxyapatite loaded with human bone marrow cells implanted in an extra-osseous site.

The purpose of this work was to develop qualitative methods for in situ analysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBON), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed in order to observe inflammatory cells, vessels, newly formed bone, woven and lamellar bone. Enzymohistochemistry was carried out to detect positive tartrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry using antibodies against type I collagen and osteocalcin permitted us to characterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human cells in osteogenesis. Results demonstrated an early formation of lamellar bone only in the pores of the studied HAP loaded with HBMSC. This bone contained a matrix showing positive reaction for type I collagen and osteocalcin. In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed positive cells for the human DNA probe. All these results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.

Evaluation of cell colonization on biomaterials: preventing cell attachment to plastic containers.

In biocompatibility evaluation involving cell culture models, we use samples of biomaterials of different forms and sizes. During cell seeding onto biomaterials of an inadequate size to cover the bottom of the culture wells completely, cells have the opportunity to attach to the plastic. As described in this report with two culture models and two biomaterials, we use an agarose gel sublayer to prevent this phenomenon.

New artificial connective matrix-like structure made of elastin solubilized peptides and collagens: elaboration, biochemical and structural properties.

Several improvements of the basic reaction between elastin peptides and type III collagen, specially the addition of heparan sulphate proteoglycans, are presented. The consequent elaboration of either cell culture support or membranes are described and illustrated by scanning electron microscopy. The material produced possesses composition, very well organized lamellar structure and some properties quite close to natural membranes such as subendothelial tissue.

Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption.

The effect of surface roughness of the titanium alloy Ti-6Al-4V (Ti alloy) on the short- and long-term response of human bone marrow cells in vitro and on protein adsorption was investigated. Three different values in a narrow range of surface roughness were used for the substrata (R(alpha): 0.320, 0.490 and 0.874 microm). Cell attachment, cell proliferation and differentiation (alkaline phosphatase specific activity) were determined past various incubation periods. The protein adsorption of bovine serum albumin and fibronectin, from single protein solutions, on rough and smooth Ti alloy surfaces was examined with two methods, X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cell attachment and proliferation were surface roughness sensitive and increased as the roughness of Ti alloy increased. No statistically significant difference was observed in the expression of ALP activity on all three Ti alloy surfaces and culture plastic. Both methods, XPS and protein radiolabeling, showed that human serum albumin was adsorbed preferentially onto the smooth substratum. XPS technique showed that the rough substratum bound a higher amount of total protein (from culture medium supplied with 10% serum) and fibronectin (10-fold) than did the smooth one. The cell attachment may be explained by the differential adsorption of the two proteins onto smooth and rough Ti alloy surfaces.

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion.

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration.

Based on a new concept, a procedure combining induced membranes and cancellous autografts allows the reconstruction of wide diaphyseal defects. In the first stage of this procedure, a cement spacer is inserted into the defect; the spacer is responsible for the formation of a pseudo-synovial membrane. In the second stage, the defect is reconstructed two months later by an autologous cancellous bone graft. The aim of this study was to evaluate the histological and biochemical characteristics of these membranes induced in rabbits. Histological studies carried out two, four, six, and eight weeks following implantation revealed a rich vascularization. Qualitative and quantitative immunochemistry showed production of growth factors (VEGF, TGFbeta1) and osteoinductive factors (BMP-2). Maximum BMP-2 production was obtained four weeks after the implantation, and, at this time, induced membranes favored human bone marrow stromal cell differentiation to the osteoblastic lineage. Should these results be confirmed in humans, bone reconstruction could be carried out earlier than previously thought and in better conditions than expected, the membrane playing the role of an in situ delivery system for growth and osteoinductive factors.

Radioimmunodetection of rat and rabbit cartilage using a monoclonal antibody specific to link proteins.

Biodistribution analysis using [125I]Fab-6F3 specific to link proteins from human articular cartilage performed in rats by autoradiography showed a high concentration of radioactivity in all cartilaginous tissues. Preliminary immunoscintigraphic assays were performed in rabbits. Front and side view images of whole animals exhibited high uptake in cartilage tissue of the knee articulation, in the invertebral disk and the humeral head. This fixation was still detected 24 h post-injection, although high washout of radioactivity was observed.

In vitro and in vivo stability of new multilamellar vesicles.

Factors affecting multilamellar vesicles transport to the blood compartment after oral administration to rats were evaluated first in vitro. A high entrapment of protein A was obtained when the vesicles were prepared by shearing a lyotropic lamellar phase composed of soybean phosphatidylcholine, cholesterol and polyoxyethylene alcohol (C12H25(OCH2CH2)4OH) as neutral detergent. In vitro tests showed that these vesicles (spherulites) were stabled in 50% of fetal calf serum, in acidic (pH 3) or basic (pH 10) buffers, in pancreatin but are partially lysed in 20mM bile salts. Oral administration of spherulites entrapping 111In-NTA in fasting rats showed a increase of radioacticivity in blood. This could be explained by passage of some spherulites in the enterocytes.

Human bone marrow stromal cells express an osteoblastic phenotype in culture.

This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two "clonings" and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of beta-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by "budding" structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with 45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.