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: 18 (0 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: 23 (0 ULg)
Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates.
; ; Kerckhofs, Greet et al
in Biomaterials (2013), 34(4), 922-9
Molecular oxygen is a main regulator of various cell functions. Imaging methods designed as screening tools for fast, in situ, 3D and non-interfering measurement of oxygen tension in the cellular ... [more ▼]
Molecular oxygen is a main regulator of various cell functions. Imaging methods designed as screening tools for fast, in situ, 3D and non-interfering measurement of oxygen tension in the cellular microenvironment would serve great purpose in identifying and monitoring this vital and pivotal signalling molecule. We describe the use of dual luminophore oxygen sensitive microbeads to measure absolute oxygen concentrations in cellular aggregates. Stable microbead integration, a prerequisite for their practical application, was ensured by a site-specific delivery method that is based on the interactions between streptavidin and biotin. The spatial stability introduced by this method allowed for long term measurements of oxygen tension without interfering with the cell aggregation process. By making multiple calibration experiments we further demonstrated the potential of these sensors to measure local oxygen tension in optically dense cellular environments. [less ▲]Detailed reference viewed: 28 (1 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: 130 (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: 92 (0 ULg)
Micro-CT: a powerful tool for the characterization and evaluation of the bone formatting capacity of calcium phosphate–stem cells tissue engineering constructs
Kerckhofs, Greet ; ; et al
in Abstract book SkyScan User Meeting 2011 (2011)Detailed reference viewed: 4 (0 ULg)
BMP signalling in growth plate chondrocytes: a Boolean modelling approach
Kerkhofs, Johan ; ; et al
Poster (2010, September 15)Detailed reference viewed: 10 (0 ULg)