Robin Siadous

Whatever their differentiation status, human progenitor derived – or mature – endothelial cells induce osteoblastic differentiation of bone marrow stromal cells

Association of the bone‐forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co‐culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular‐like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co‐cultured with human endothelial progenitor cells in order to assess whether progenitor‐derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co‐culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non‐orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co‐cultures were undertaken: HBMSCs–PDECs, HBMSCs–HUVECs, HOPs–PDECs, HOPs–HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co‐culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real‐time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono‐ and co‐cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering. Copyright

Physicochemical modulation of chitosan‐based hydrogels induces different biological responses: Interest for tissue engineering

Polysaccharide-based hydrogels are remarkable materials for the development of tissue engineering strategies as they meet several critical requirements for such applications and they may partly mimic the extracellular matrix. Chitosan is widely envisioned as hydrogel in biomedical fields for its bioresorbability, biocompatibility, and fungistatic and bacteriostatic properties. In this study, we report that the modulation of the polymer concentration, the degree of acetylation, the gelation processes [or neutralization routes (NR)] in the preparation of different chitosan-based hydrogels lead to substantially and significantly different biological responses. We show that it is possible to tune the physicochemical characteristics, mechanical properties, and biological responses of such matrices. Physical hydrogels prepared from highly acetylated chitosan were softer, degraded quickly in vivo, and were not suitable for in vitro culture of human mesenchymal stem and progenitor derived endothelial cells. In contrast, for a same chitosan concentration and obtained by the same processing route, a low degree of acetylation chitosan hydrogel provided a more elastic material, better cell adhesion on its surface and tissue regeneration, and restored tissue neo-vascularization as well. This work offers promising and innovative perspectives for the design of hydrogel materials with tunable properties for tissue engineering and regenerative medicine.

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.

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.Graphical Abstract

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.

A new composite hydrogel combining the biological properties of collagen with the mechanical properties of a supramolecular scaffold for bone tissue engineering.

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long‐term cell survival in vivo. Moreover, it promoted the differentiation of human adipose‐derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.

Labeling and qualification of endothelial progenitor cells for tracking in tissue engineering: An in vitro study.

Purpose In order to track location and distribution of endothelial cells (ECs) within scaffolds in vitro, we chose lentiPGK-TdTomato transduction of human endothelial progenitor cells (EPCs) isolated and differentiated from cord blood. Because transduction could have a functional impact on cell behavior, we checked different parameters for qualification of labeled- EPCs as well as their use for potential applications in the context of vascular and bone tissue engineering. Methods After isolation and expansion, EPCs were classically characterized then transduced with the lentiviral vector containing the TdTomato protein gene under the control of the phosphoglycerate kinase (PGK) promoter. Conventional karyotyping, differentiation capacity, viability, proliferation assays were performed with labeled and unlabeled EPCs. Scaffolds and co-cultures were explored with labeled EPCs, in static or shear stress conditions. Results Our results show that cell labeling did not affect cell adhesion nor induce cell death. Cell labeling did not induce more chromosomal aberrations. Phenotypical characterization was not affected. In the context of tissue engineering applications, labeled EPCs maintained their ability to line 2D or 3D scaffolds, withstand physiological arterial shear stress, and form tubular networks in co-cultures with human osteoblast progenitor cells. Conclusions It is possible to label human EPCs with TdTomato without affecting their behavior by the transduction procedure. This creates an important tool for numerous applications. Our results provide a qualification of labeled EPCs in comparison with unlabeled ones for vascular and bone tissue engineering.

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.

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.

An easy-to-use and versatile method for building cell-laden microfibres

Fibre-shaped materials are useful for creating different functional three-dimensional (3D) structures that could mimic complex tissues. Several methods (e.g. extrusion, laminar flow or electrospinning) have been proposed for building hydrogel microfibres, with distinctive cell types and with different degrees of complexity. However, these methods require numerous protocol adaptations in order to achieve fibre fabricating and lack the ability to control microfibre alignment. Here, we present a simple method for the production of microfibers, based on a core shell approach, composed of calcium alginate and type I collagen. The process presented here allows the removal of the calcium alginate shell, after only 24 hours of culture, leading to stable and reproducible fibre shaped cellular constructs. With time of culture cells show to distribute preferentially to the surface of the fibre and display a uniform cellular orientation. Moreover, when cultured inside the fibres, murine bone marrow mesenchymal stem cells show the capacity to differentiate towards the osteoblastic lineage, under non-osteoinductive culture conditions. This work establishes a novel method for cellular fibre fabrication that due to its inherent simplicity can be easily upscaled and applied to other cell types.