Nathalie Groen

A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells

The response of osteoprogenitors to calcium (Ca(2+)) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca(2+) released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca(2+) concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca(2+) enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6h and pharmaceutical interference with a number of proteins involved in Ca(2+) sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca(2+) and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca(2+) and using microarray analysis, we have identified c-Fos as an early Ca(2+) response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca(2+), a simple and economic way of priming hMSCs for bone tissue engineering applications.

Endothelial Differentiation of Mesenchymal Stromal Cells

Human mesenchymal stromal cells (hMSCs) are increasingly used in regenerative medicine for restoring worn-out or damaged tissue. Newly engineered tissues need to be properly vascularized and current candidates for in vitro tissue pre-vascularization are endothelial cells and endothelial progenitor cells. However, their use in therapy is hampered by their limited expansion capacity and lack of autologous sources. Our approach to engineering large grafts is to use hMSCs both as a source of cells for regeneration of targeted tissue and at the same time as the source of endothelial cells. Here we investigate how different stimuli influence endothelial differentiation of hMSCs. Although growth supplements together with shear force were not sufficient to differentiate hMSCs with respect to expression of endothelial markers such as CD31 and KDR, these conditions did prime the cells to differentiate into cells with an endothelial gene expression profile and morphology when seeded on Matrigel. In addition, we show that endothelial-like hMSCs are able to create a capillary network in 3D culture both in vitro and in vivo conditions. The expansion phase in the presence of growth supplements was crucial for the stability of the capillaries formed in vitro. To conclude, we established a robust protocol for endothelial differentiation of hMSCs, including an immortalized MSC line (iMSCs) which allows for reproducible in vitro analysis in further studies.

A mesenchymal stromal cell gene signature for donor age

Human aging is associated with loss of function and regenerative capacity. Human bone marrow derived mesenchymal stromal cells (hMSCs) are involved in tissue regeneration, evidenced by their capacity to differentiate into several lineages and therefore are considered the golden standard for cell-based regeneration therapy. Tissue maintenance and regeneration is dependent on stem cells and declines with age and aging is thought to influence therapeutic efficacy, therefore, more insight in the process of aging of hMSCs is of high interest. We, therefore, hypothesized that hMSCs might reflect signs of aging. In order to find markers for donor age, early passage hMSCs were isolated from bone marrow of 61 donors, with ages varying from 17–84, and clinical parameters, in vitro characteristics and microarray analysis were assessed. Although clinical parameters and in vitro performance did not yield reliable markers for aging since large donor variations were present, genome-wide microarray analysis resulted in a considerable list of genes correlating with human age. By comparing the transcriptional profile of aging in human with the one from rat, we discovered follistatin as a common marker for aging in both species. The gene signature presented here could be a useful tool for drug testing to rejuvenate hMSCs or for the selection of more potent, hMSCs for cell-based therapy.

Predicting the therapeutic efficacy of MSC in bone tissue engineering using the molecular marker CADM1

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering.

UNLABELLED The research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. STATEMENT OF SIGNIFICANCE In this opinion paper, we postulate that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field.

High content imaging in the screening of biomaterial-induced MSC behavior

Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: The effect of silanol groups

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.

Linking the Transcriptional Landscape of Bone Induction to Biomaterial Design Parameters

New engineering possibilities allow biomaterials to serve as active orchestrators of the molecular and cellular events of tissue regeneration. Here, the molecular control of tissue regeneration for calcium phosphate (CaP)-based materials is established by defining the parameters critical for tissue induction and those are linked to the molecular circuitry controlling cell physiology. The material properties (microporosity, ion composition, protein adsorption) of a set of synthesized osteoinductive and noninductive CaP ceramics are parameterized and these properties are correlated to a transcriptomics profile of osteogenic cells grown on the materials in vitro. Using these data, a genetic network controlling biomaterial-induced bone formation is built. By isolating the complex material properties into single-parameter test conditions, it is verified that a subset of these genes is indeed controlled by surface topography and ions released from the ceramics, respectively. The gene network points to a decisive role for extracellular matrix deposition in osteoinduction by genes such as tenascin C and hyaluronic acid synthase 2, which are controlled by calcium and phosphate ions as well as surface topography. This work provides insight into the biomaterial composition and material engineering aspects of bone void filling and can be used as a strategy to explore the interface between biomaterials and tissue regeneration.

Bioinformatics-based selection of a model cell type for in vitro biomaterial testing.

Biomaterial properties can be tailored for specific applications via systematic and high-throughput screening of biomaterial-cell interactions. However, progress in material development is often hampered by the lack of adequate in vitro testing methods, frequently due to incomplete understanding of involved in vivo mechanisms. In line with drug discovery in pharmacology, a crucial step in assay development for biomaterial screening is the identification of a target to direct the screen against. Herein, the cell type to be used for screening is of essential importance and has to be carefully chosen. So far, few attention has been put on selecting a cell type specifically suitable for in vitro testing of materials for predefined applications. In this manuscript, we describe an approach to define a suitable cell type for screening assays, for which biomaterials for bone regeneration served as example. Using a bioinformatics methodology, different cell lines are compared on three well-characterized model materials. The transcriptional profiles of MG63, iMSC, SV-HFO, hPPCT, hBPCT and SW480 cells are assessed on 3 calcium phosphate-based materials to evaluate their potential application in a screening assay. We show that MG63 is the most suitable cell line to evaluate biomaterials for bone regeneration applications, evidenced by their robust gene expression differences between the 3 model materials. The gene expression differences between the cell lines were assessed based on the overall gene expression profiles and specific subsets of genes and pathways related to osteogenesis and bone homeostasis in response to the 3 materials tested. In the next phase, this cell line will be used to identify a target correlating with in vivo biomaterial performance and henceforth to develop an in vitro screening system.