Immersed Boundary Models for Quantifying Flow-Induced Mechanical Stimuli on Stem Cells Seeded on 3D Scaffolds in Perfusion Bioreactors.
; ; et al
in PLoS Computational Biology (2016), 12(9), 1005108
Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold ... [more ▼]
Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/mum, at the lower end of the range reported in literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell's micro-environment, which can further aid in the achievement of robust bioprocess control for regenerative medicine applications. [less ▲]Detailed reference viewed: 17 (4 ULg)
A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy.
; ; et al
in PLoS ONE (2016), 11(8), 0162052
Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological ... [more ▼]
Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model's results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis. [less ▲]Detailed reference viewed: 15 (2 ULg)
Early BMP, Wnt and Ca(2+)/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates.
; ; Geris, Liesbet et al
in Biomaterials (2016), 86
The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the ... [more ▼]
The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the spatiotemporal activation of signalling pathways has hampered the optimisation of in vivo bone formation and subsequent clinical translation. To gain further knowledge regarding the early molecular events governing bone tissue formation, we combined human periosteum derived progenitor cells with three types of clinically used CaP-scaffolds, to obtain constructs with a distinct range of bone forming capacity in vivo. Protein phosphorylation together with gene expression for key ligands and target genes were investigated 24 hours after cell seeding in vitro, and 3 and 12 days post ectopic implantation in nude mice. A computational modelling approach was used to deduce critical factors for bone formation 8 weeks post implantation. The combined Ca(2+)-mediated activation of BMP-, Wnt- and PKC signalling pathways 3 days post implantation were able to discriminate the bone forming from the non-bone forming constructs. Subsequently, a mathematical model able to predict in vivo bone formation with 96% accuracy was developed. This study illustrates the importance of defining and understanding CaP-activated signalling pathways that are required and sufficient for in vivo bone formation. Furthermore, we demonstrate the reliability of mathematical modelling as a tool to analyse and deduce key factors within an empirical data set and highlight its relevance to the translation of regenerative medicine strategies. [less ▲]Detailed reference viewed: 26 (5 ULg)
Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.
; ; et al
in Tissue Engineering. Part C, Methods (2016), 22(5), 473-86
Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the ... [more ▼]
Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-beta1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (muMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of muMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-beta1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control muMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated muMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches. [less ▲]Detailed reference viewed: 32 (0 ULg)
Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.
; ; et al
in PloS one (2015), 10(6), 0130227
To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells ... [more ▼]
To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology. [less ▲]Detailed reference viewed: 47 (3 ULg)
In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.
; Geris, Liesbet ; et al
in BioResearch open access (2014), 3(6), 265-77
Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem ... [more ▼]
Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions. [less ▲]Detailed reference viewed: 63 (0 ULg)
Contrast-enhanced nanofocus computed tomography for virtual 3D histopathology and morphometric analysis of multiple skeletal tissues
Kerckhofs, Greet ; ; et al
Conference (2013)Detailed reference viewed: 17 (2 ULg)
Relating the Chondrocyte Gene Network to Growth Plate Morphology: From Genes to Phenotype
Kerkhofs, Johan ; ; et al
in PLoS ONE (2012)
During endochondral ossification, chondrocyte growth and differentiation is controlled by many local signalling pathways. Due to crosstalks and feedback mechanisms, these interwoven pathways display a ... [more ▼]
During endochondral ossification, chondrocyte growth and differentiation is controlled by many local signalling pathways. Due to crosstalks and feedback mechanisms, these interwoven pathways display a network like structure. In this study, a large-scale literature based logical model of the growth plate network was developed. The network is able to capture the different states (resting, proliferating and hypertrophic) that chondrocytes go through as they progress within the growth plate. In a first corroboration step, the effect of mutations in various signalling pathways of the growth plate network was investigated. [less ▲]Detailed reference viewed: 31 (12 ULg)
Ectopic bone formation by 3D porous calcium phosphate-Ti6Al4V hybrids produced by perfusion electrodeposition.
; Kerckhofs, Greet ; et al
in Biomaterials (2012), 33(16), 4044-58
Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised ... [more ▼]
Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised porous calcium phosphate-Ti6Al4V (CaP-Ti) hybrids were produced by perfusion electrodeposition, and the in vitro and in vivo biological performances were evaluated using human periosteum derived cells (hPDCs). By applying various current densities at the optimised deposition conditions, CaP coatings with sub-micrometer to nano-scale porous crystalline structures and different ion dissolution kinetics were deposited on the porous Ti6Al4V scaffolds. These distinctive physicochemical properties caused a significant impact on in vitro proliferation, osteogenic differentiation, and matrix mineralisation of hPDCs. This includes a potential role of hPDCs in mediating osteoclastogenesis for the resorption of CaP coatings, as indicated by a significant down-regulation of osteoprotegerin (OPG) gene expression and by the histological observation of abundant multi-nucleated giant cells near to the coatings. By subcutaneous implantation, the produced hybrids induced ectopic bone formation, which was highly dependent on the physicochemical properties of the CaP coating (including the Ca(2+) dissolution kinetics and coating surface topography), in a cell density-dependent manner. This study provided further insight on stem cell-CaP biomaterial interactions, and the feasibility to produced bone reparative units that are predictively osteoinductive in vivo by perfusion electrodeposition technology. [less ▲]Detailed reference viewed: 152 (0 ULg)
A Boolean network approach to developmental engineering
Kerkhofs, Johan ; ; et al
Conference (2011, June 13)Detailed reference viewed: 18 (0 ULg)
The combined bone forming capacity of human periosteal derived cells and calcium phosphates.
; Geris, Liesbet ; Kerckhofs, Greet et al
in Biomaterials (2011), 32(19), 4393-405
Current knowledge suggests that the periosteum, a fibrous tissue which covers the surface of all bones, contains a population of progenitor cells which mediate the repair of bone defects. In an effort to ... [more ▼]
Current knowledge suggests that the periosteum, a fibrous tissue which covers the surface of all bones, contains a population of progenitor cells which mediate the repair of bone defects. In an effort to optimise the utilisation of this source of cells for bone engineering, herein we describe the rational selection of calcium phosphate (CaP) containing materials, based on biomaterial properties, and evaluation of their combined bone forming capacity. Five different commercially available orthopaedic 3D matrices composed of CaP particles in an open collagen network (NuOss, CopiOs, Bio-Oss((R)), Collagraft and Vitoss((R))) were evaluated in vitro and in vivo with human periosteal derived cells (hPDCs). It was found that the cell-material combinations behaved quite differently in vivo, despite apparent in vitro similarities in gene expression profiles. Bone formation was highest within the NuOss/hPDC implant at 13.03%, which also contained the highest incidence of bone marrow formation. The bone formed in this implant was chimeric with approximately 65% originating from implanted cells. Upon analysis of human specific gene expression, although it was found that predominantly osteogenic differentiation was observed within NuOss/hPDC implants, a lesser induction of chondrogenic genes was also observed. The formation of a cartilage intermediate was confirmed by histology. Additionally the NuOss/hPDC implant integrated into the mouse environment with apparent active scaffold resorption. This study demonstrates the importance of matching a cell support/biological matrix with a cell type and subsequently has outlined parameters which can be used for the rational selection of biomaterials for bone engineering. [less ▲]Detailed reference viewed: 98 (0 ULg)
BMP signalling in growth plate chondrocytes: a Boolean modelling approach
Kerkhofs, Johan ; ; et al
Poster (2010, September 15)Detailed reference viewed: 11 (0 ULg)