Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Influence of External Beam Radiotherapy on the Properties of Polymethyl Methacrylate-Versus Silicone-Induced Membranes in a Bilateral Segmental Bone Defect in Rats.

INTRODUCTION: Standard care for malignant tumors arising next to a bone structure is surgical removal with safety margins, followed by external beam radiotherapy (EBRT). Complete tumor removal can result in large bone defects. A two-step bone reconstruction technique using the induced membrane (IM) technique has proven its efficacy to bridge gap nonunion. During the first step, a spacer is placed in the bone gap. The spacer then is removed and the IM around it is filled with autologous cancellous bone graft. However, the feasibility of this technique with the addition of adjuvant EBRT between the two reconstruction steps has not yet been studied. Polymethyl methacrylate (PMMA) used to be the standard spacer material for the first step. Silicone spacers could replace them owing to their good behavior when submitted to EBRT and their easier removal from the surgical site during the second step. The aim of this study was to evaluate the influence of EBRT on the histological and biochemical properties of IM induced using PMMA or silicone as spacer. MATERIALS AND METHODS: The analyses were performed on PMMA- or silicone-IM with and without EBRT in a 6-mm bilateral femoral defect in 32 rats. Thickness and vessel content were measured in both groups. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) content in lysates of the crushed membranes were measured by enzyme immunoassay. Finally, alkaline phosphatase activity was analyzed in human bone marrow stromal cell cultures in contact with the same lysates. RESULTS: EBRT did not change the histological structure of the cellular internal layer or the fibrous outer layer. The nature of the spacer only influenced IM thickness, PMMA-IM with external radiotherapy being significantly thicker. EBRT decreased the vascular density of IM but was less effective on VEGF/BMP2 production. In vitro, IM could have an osteoinductive potential on human bone marrow stem cells. CONCLUSION: EBRT did not modify the histological properties of IMs but decreased their vascular density. VEGF and BMP2 production within IMs was not affected by EBRT. Silicone spacers are able to induce membranes with similar histological characteristics to PMMA-IM.

Influence of the three-dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3.

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.

Characterization of printed PLA scaffolds for bone tissue engineering.

Autografts remain the gold standard for orthopedic transplantations. However, to overcome its limitations, bone tissue engineering proposes new strategies. This includes the development of new biomaterials such as synthetic polymers, to serve as scaffold for tissue production. The objective of this present study was to produce poly(lactic) acid (PLA) scaffolds of different pore size using fused deposition modeling (FDM) technique and to evaluate their physicochemical and biological properties. Structural, chemical, mechanical, and biological characterizations were performed. We successfully fabricated scaffolds of three different pore sizes. However, the pore dimensions were slightly smaller than expected. We found that the 3D printing process induced decreases in both, PLA molecular weight and degradation temperatures, but did not change the semicrystalline structure of the polymer. We did not observe any effect of pore size on the mechanical properties of produced scaffolds. After the sterilization by γ irradiation, scaffolds did not exhibit any cytotoxicity towards human bone marrow stromal cells (HBMSC). Finally, after three and seven days of culture, HBMSC showed high viability and homogenous distribution irrespective of pore size. Thus, these results suggest that FDM technology is a fast and reproducible technique that can be used to fabricate tridimensional custom-made scaffolds for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 887-894, 2018.

Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

The proangiogenic potential of a novel calcium releasing composite biomaterial: Orthotopic in vivo evaluation.

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca2+ release rate, with the faster Ca2+ release composite gel showing improved bone repair at 3weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. STATEMENT OF SIGNIFICANCE: In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration.

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

The proangiogenic potential of a novel calcium releasing biomaterial: Impact on cell recruitment.

In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications. STATEMENT OF SIGNIFICANCE: In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass - poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

Colorectal tissue engineering: A comparative study between porcine small intestinal submucosa (SIS) and chitosan hydrogel patches.

OBJECTIVE: Tissue engineering may provide new operative tools for colorectal surgery in elective indications. The aim of this study was to define a suitable bioscaffold for colorectal tissue engineering. METHODS: We compared 2 bioscaffolds with in vitro and in vivo experiments: porcine small intestinal submucosa (SIS) versus chitosan hydrogel matrix. We assessed nontoxicity of the scaffold in vitro by using human adipose-derived stem cells (hADSC). In vivo, a 1 × 2-cm colonic wall defect was created in 16 rabbits. Animals were divided randomly into 2 groups according to the graft used, SIS or chitosan hydrogel. Graft area was explanted at 4 and 8 weeks. The end points of in vivo experiments were technical feasibility, behavior of the scaffold, in situ putative inflammatory effect, and the quality of tissue regeneration, in particular smooth muscle layer regeneration. RESULTS: In vitro, hADSC attachment and proliferation occurred on both scaffolds without a substantial difference. After proliferation, hADSCs kept their mesenchymal stem cell characteristics. In vivo, one animal died in each group. Eight weeks after implantation, the chitosan scaffold allowed better wound healing compared with the SIS scaffold, with more effective control of inflammatory activity and an integral regeneration of the colonic wall including the smooth muscle cell layer. CONCLUSION: The outcomes of in vitro experiments did not differ greatly between the 2 groups. Macroscopic and histologic findings, however, revealed better wound healing of the colonic wall in the chitosan group suggesting that the chitosan hydrogel could serve as a better scaffold for colorectal tissue engineering.

Comparative study of membranes induced by PMMA or silicone in rats, and influence of external radiotherapy.

The induced membrane technique has been used for long bone defect reconstruction after traumatism. One of the major drawbacks of this method is the difficult removal of the polymethyl methacrylate spacer after membrane formation. We therefore replaced the stiff PMMA spacer with a semi-flexible medical grade silicone spacer. This study aimed to compare subcutaneously formed membranes, induced by PMMA and silicone, in the irradiated or not irradiated areas within 28 rats that received the spacers. Histological analysis was performed to evaluate the composition of the membrane and to quantify the amount of vessels. Histomorphometric measurements were used to evaluate membranes' thickness, while fibrosis and inflammation were scored. The expression of VEGF and BMP-2 in lysates of the crushed membranes was determined by Western blotting. ALP expression was analyzed in HBMSC cultures in contact with the same lysates. Non-irradiated membranes induced by the two spacer types were non-inflammatory, fibrous and organized in layers. Irradiation did not change the macroscopic properties of membranes that were induced by silicone, while PMMA induced membranes were sensitive to the radiotherapy, resulting in thicker, strongly inflammatory membranes. Irradiated membranes showed an overall reduced osteogenic potential. Medical grade silicone is safe for the use in radiotherapy and might therefore be of great advantage for patients in need of cancer treatment.

The use of total human bone marrow fraction in a direct three-dimensional expansion approach for bone tissue engineering applications: focus on angiogenesis and osteogenesis.

Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion.

The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress.

Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Tissue engineering strives to produce bio-artificial conduits to provide resistance to thrombosis. The objectives of our study were to culture endothelial cells (EC) on composite assemblies of extracellular matrix proteins, and to evaluate the cellular phenotype under flow. Cell-adhesive assemblies were fabricated on glass slides as combinations of collagen (Co), laminin (LM), and fibronectin (FN), resulting in three samples: Co, Co/LM, and Co/FN. Surface topography, roughness, and wettability were determined. Human saphenous vein EC were harvested from cardiac patients, cultured on the assemblies and submitted to laminar shear stress (SS) of 12 dyn/cm(2) for 40, 80, and 120 min. Cell retention was assessed and qRT-PCR of adhesion genes (VE-cadherin, vinculin, KDR, CD-31 or PECAM-1, ß1-integrins) and metabolic genes (t-PA, NF-κB, eNOS and MMP-1) was performed. Quantitative immunofluorescence of VE cadherin, vinculin, KDR, and vonWillebrand factor was performed after 2 and 6 h of flow. Static samples were excluded from shearing. The cells reached confluence with similar growth curves. The cells on Co/LM and Co/FN were resistant to flow up to 120 min but minor desquamation occurred on Co corresponding with temporary downregulation of VE cadherin and vinculin-mRNA and decreased fluorescence of vinculin. The cells seeded on Co/LM initially more upregulated vinculin-mRNA and also the inflammatory factor NF-κB, and the cells plated on Co/FN changed the expression profile minimally in comparison with the static control. Fluorescence of VE cadherin and vonWillebrand factor was enhanced on Co/FN. The cells cultured on Co/LM and Co/FN increased the vinculin fluorescence and expressed more VE cadherin and KDR-mRNA than the cells on Co. The cells plated on Co/FN upregulated the mRNA of VE cadherin, CD-31, and MMP 1 to a greater extent than the cells on Co/LM and they enhanced the fluorescence of VE cadherin, KDR, and vonWillebrand factor. Some of these changes sustained up to 6 h of flow, as confirmed by immunofluorescence. Combined matrices Co/LM and Co/FN seem to be more suitable for EC seeding and retention under flow. Moreover, Co/FN matrix promoted slightly more favorable cellular phenotype than Co/LM under SS of 2-6 h.

Composition and properties of silver-containing calcium carbonate-calcium phosphate bone cement.

The introduction of silver, either in the liquid phase (as silver nitrate solution: Ag(L)) or in the solid phase (as silver phosphate salt: Ag(S)) of calcium carbonate-calcium phosphate (CaCO3-CaP) bone cement, its influence on the composition of the set cement (C-Ag(L) and C-Ag(S) cements with a Ca/Ag atomic ratio equal to 10.3) and its biological properties were investigated. The fine characterisation of the chemical setting of silver-doped and reference cements was performed using FTIR spectroscopy. We showed that the formation of apatite was enhanced from the first hours of maturation of C-Ag(L) cement in comparison with the reference cement, whereas a longer period of maturation (about 10 h) was required to observe this increase for C-Ag(S) cement, although in both cases, silver was present in the set cements mainly as silver phosphate. The role of silver nitrate on the setting chemical reaction is discussed and a chemical scheme is proposed. Antibacterial activity tests (S. aureus and S. epidermidis) and in vitro cytotoxicity tests (human bone marrow stromal cells (HBMSC)) showed that silver-loaded CaCO3-CaP cements had antibacterial properties (anti-adhesion and anti-biofilm formation) without a toxic effect on HBMSC cells, making C-Ag(S) cement a promising candidate for the prevention of bone implant-associated infections.

Colorectal tissue engineering: prerequisites, current status and perspectives.

Gastrointestinal tissue engineering has emerged over the past 20 years and was often focused on esophagus, stomach or small intestine, whereas bioengineering researches of colorectal tissue are scarce. However, some promising results have been obtained in animal models. Refinements should be performed in scaffold and cell source selection to allow smooth muscle layer regeneration. Indeed, synthetic and natural polymers such as small intestinal submucosa and collagen sponge seeded with organoid units or smooth muscle cells did not allow smooth muscle regeneration. Mesenchymal stem cells derived from adipose tissue seeded on composite scaffold could represent an interesting way to achieve this goal. This article reviews potential indications, current status and perspectives of tissue engineering in the area of colorectal surgery.

Polyethylene terephthalate membrane grafted with peptidomimetics: endothelial cell compatibility and retention under shear stress.

The present work aimed to treat a polyethylene terephthalate (PET) surface to make the biomaterial more 'attractive' in terms of attachment and shear stress response to endothelial cells with a view to possible applications in vascular grafting. A surface wet-chemistry protocol was applied to graft track-etched PET membranes with RGD peptidomimetics based on the tyrosine template and active at the nano-level vs. isolated human αvß3 receptor, which was monitored by X-ray photoelectron spectroscopy, contact angle measurement and atomic force microscopy for characterization. A primary culture of human saphenous vein endothelial cells was used before and after sterilization of the membranes (heat treatment or γ-ray irradiation) to test the benefit of grafting. The optimal surface concentrations of grafted molecules were around 50 pmol/cm². Compared to GRGDS, the peptidomimetics promoted cell attachment with similar or slightly better performances. Endothelialized grafted supports were further exposed to 2 h of shear stress mimicking arterial conditions. Cells were lost on non-grafted PET whereas cells on grafted polymers sterilized by γ-ray irradiation withstood forces with no significant difference in focal contacts. At the mRNA level, cells on functionalized PET were able to respond to shear stress with NFkB upregulation. Thus, grafting of peptidomimetics as ligands of the αvß3 integrin could be a relevant strategy to improve the adhesion of human endothelial cells and to obtain an efficient endothelialized PET for the surgery of small-diameter vascular prostheses.

Human saphenous vein endothelial cell adhesion and expansion on micropatterned polytetrafluoroethylene.

Intimal hyperplasia and thrombosis are responsible for the poor patency rates of small-diameter vascular grafts. These complications could be avoided by a rapid and strong adhesion of endothelial cells to the prosthetic surfaces, which typically consist of expanded polytetrafluoroethylene (PTFE) for small-diameter vessels. We have previously described two peptide micropatterning strategies that increase the endothelialization rates of PTFE. The micropatterns were generated either by inkjet printing 300 µm squares or by spraying 10.1 ± 0.1 µm diameter droplets of the CGRGDS cell adhesion peptide, while the remaining surface was functionalized using the CWQPPRARI cell migration peptide. We now directly compare these two micropatterning strategies and examine the effect of hydrodynamic stress on human saphenous vein endothelial cells grown on the patterned surfaces. No significant differences in cell adhesion were observed between the two micropatterning methods. When compared to unpatterned surfaces treated with a uniform mixture of the two peptides, the cell expansion was significantly higher on sprayed or printed surfaces after 9 days of static cell culture. In addition, after 6 h of exposure to hydrodynamic stress, the cell retention and cell cytoskeleton reorganization on the patterned surfaces was improved when compared to untreated or random treated surfaces. These results indicate that micropatterned surfaces lead to improved rates of PTFE endothelialization with higher resistance to hydrodynamic stress.

uPA and MMP-2 were involved in self-assembled network formation in a two dimensional co-culture model of bone marrow stromal cells and endothelial cells.

Two dimensional (2D) co-cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self-assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2) as well tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) were investigated in this 2D co-culture model. We found that the expression of uPA, MMP-2 in the co-cultured cells was significantly higher than those in mono-cultured cells. In opposite, PAI-1, expressed only by HUVECs is not regulated in the co-culture. Inhibition assays confirm that uPA played a critical role in the formation of self-assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP-2 prevented the formation of self-assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP-2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP-2 activity, and may contribute to endothelial cell migration and the formation of this self-assembled network observed in these 2D co-cultured cells.

Altered nanofeature size dictates stem cell differentiation.

The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

Strontium-loaded mineral bone cements as sustained release systems: Compositions, release properties, and effects on human osteoprogenitor cells.

This study aims to evaluate in vitro the release properties and biological behavior of original compositions of strontium (Sr)-loaded bone mineral cements. Strontium was introduced into vaterite CaCO3 -dicalcium phosphate dihydrate cement via two routes: as SrCO3 in the solid phase (SrS cements), and as SrCl2 dissolved in the liquid phase (SrL cements), leading to different cement compositions after setting. Complementary analytical techniques implemented to thoroughly investigate the release/dissolution mechanism of Sr-loaded cements at pH 7.4 and 37°C during 3 weeks revealed a sustained release of Sr and a centripetal dissolution of the more soluble phase (vaterite) limited by a diffusion process. In all cases, the initial burst of the Ca and Sr release (highest for the SrL cements) that occurred over 48 h did not have a significant effect on the expression of bone markers (alkaline phosphatase, osteocalcin), the levels of which remained overexpressed after 15 days of culture with human osteoprogenitor (HOP) cells. At the same time, proliferation of HOP cells was significantly higher on SrS cements. Interestingly, this study shows that we can optimize the sustained release of Sr(2+) , the cement biodegradation and biological activity by controlling the route of introduction of strontium in the cement paste.

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells.

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-ß3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-ß3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.