References of "Luyten, F. P"
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See detailUncoupling of in-vitro identity of embryonic limb derived skeletal progenitors and their in-vivo bone forming potential
Verbeeck, L.; Geris, Liesbet ULiege; Tylzanowski, P. et al

in Scientific Reports (2019), 9(1),

The healing of large bone defects remains a major unmet medical need. Our developmental engineering approach consists of the in vitro manufacturing of a living cartilage tissue construct that upon ... [more ▼]

The healing of large bone defects remains a major unmet medical need. Our developmental engineering approach consists of the in vitro manufacturing of a living cartilage tissue construct that upon implantation forms bone by recapitulating an endochondral ossification process. Key to this strategy is the identification of the cells to produce such cartilage intermediates efficiently. We applied a cell selection strategy based on published skeletal stem cell markers using mouse embryonic limb cartilage as cell source and analysed their potential to form bone in an in vivo ectopic assay. FGF2 supplementation to the culture media for expansion blocked dedifferentiation of the embryonic cartilage cells in culture and enriched for stem cells and progenitors as quantified using the recently published CD marker set. However, when the stem cells and progenitors were fractionated from expanded embryonic cartilage cells and assessed in the ectopic assay, a major loss of bone forming potential was observed. We conclude that cell expansion appears to affect the association between cell identity based on CD markers and in vivo bone forming capacity. © 2019, The Author(s). [less ▲]

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See detailDeciphering the combined effect of bone morphogenetic protein 6 (BMP6) and calcium phosphate on bone formation capacity of periosteum derived cells-based tissue engineering constructs
Ji, W.; Kerckhofs, G.; Geeroms, C. et al

in Acta Biomaterialia (2018)

Cell based combination products with growth factors on optimal carriers represent a promising tissue engineering strategy to treat large bone defects. In this concept, bone morphogenetic protein (BMP) and ... [more ▼]

Cell based combination products with growth factors on optimal carriers represent a promising tissue engineering strategy to treat large bone defects. In this concept, bone morphogenetic protein (BMP) and calcium phosphate (CaP)-based scaffolds can act as potent components of the constructs to steer stem cell specification, differentiation and initiate subsequent in vivo bone formation. However, limited insight into BMP dosage and the cross-talk between BMP and CaP materials, hampers the optimization of in vivo bone formation and subsequent clinical translation. Herein, we combined human periosteum derived progenitor cells with different doses of BMP6 and with three types of clinical grade CaP-scaffolds (ChronOs® ReproBone™ & CopiOs®). Comprehensive cellular and molecular analysis was performed based on in vitro cell metabolic activity and signaling pathway activation, as well as in vivo ectopic bone forming capacity after 2 weeks and 5 weeks in nude mice. Our data showed that cells seeded on CaP scaffolds with an intermediate Ca2+ release rate combined with low or medium dosage of BMP6 demonstrated a robust new bone formation after 5 weeks, which was contributed by both donor and host cells. This phenomenon might be due to the delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of the canonical BMP signaling pathway that is required for in vivo bone formation. For high BMP6 dosage, we found that the BMP6 dosage overrides the effect of the Ca2+ release rate and this appeared to be a dominant factor for ectopic bone formation. Taken together, this study illustrates the importance of matching BMP dosage and CaP properties to allow an appropriate activation of canonical BMP signaling that is crucial for in vivo bone formation. It also provides insightful knowledge with regard to clinical translation of cell-based constructs for bone regeneration. Statement of Significance: The combination of bone morphogenetic proteins (BMP) and calcium phosphate (CaP)-based biomaterials with mesenchymal stromal cells represents a promising therapeutic strategy to treat large bone defects, an unmet medical need. However, there is limited insight into the optimization of these combination products, which hampers subsequent successful clinical translation. Our data reveal a delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of canonical BMP signaling required for in vivo bone formation. Our findings illustrate the importance of matching BMP dosage and CaP properties in the development of cell-based constructs for bone regeneration. © 2018 Acta Materialia Inc. [less ▲]

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See detailAdvancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.
Mendes, L. F.; Katagiri, H.; Tam, W. L. et al

in Stem Cell Research and Therapy (2018), 9(1), 42

BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue ... [more ▼]

BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-beta1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (microCT) and immunohistochemistry. RESULTS: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. CONCLUSION: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units. [less ▲]

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See detailAn Integrated Bioprocess for the Expansion and Chondrogenic Priming of Human Periosteum-Derived Progenitor Cells in Suspension Bioreactors
Gupta, P.; Geris, Liesbet ULiege; Luyten, F. P. et al

in Biotechnology Journal (2017)

The increasing use of microcarrier-based suspension bioreactors for scalable expansion of adult progenitor cells in recent years reveals the necessity of such approaches to address bio manufacturing ... [more ▼]

The increasing use of microcarrier-based suspension bioreactors for scalable expansion of adult progenitor cells in recent years reveals the necessity of such approaches to address bio manufacturing challenges of advanced therapeutic medicinal products. However, the differentiation of progenitor cells within suspension bioreactors for the production of tissue modules is of equal importance but not well investigated. This study reports on the development of a bioreactor-based integrated process for expansion and chondrogenic priming of human periosteum-derived stem cells (hPDCs) using Cultispher S microcarriers. Spinner flask-based expansion and priming of hPDCs were carried out over 12 days for expansion and 14 days for priming. Characterization of the cells were carried out every 3rd day. Our study showed that hPDCs were able to expand till confluency with fold increase of 3.2±0.64 and to be subsequently primed toward a chondrogenic state within spinner flasks. During expansion, the cells maintained their phenotypic markers, trilineage differentiation capabilities and viability. Upon switching to TGF-β containing media the cells were able to differentiate toward chondrogenic lineage by clustering into mm-sized macrotissues containing hundreds of microcarriers. Chondrogenic priming was further evidenced by the expression of relevant markers at the mRNA level while maintaining their viability. Ectopic implantation of macrotissues highlighted that they were able to sustain their chondrogenic properties for 8 weeks in vivo. The method indicated here, suggests that expansion and relevant priming of progenitor cells can be carried out in an integrated bioprocess using spinner flasks and as such could be potentially extrapolated to other stem and progenitor cell populations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. [less ▲]

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See detailWarning about the Use of Critical-Size Defects for the Translational Study of Bone Repair: Analysis of a Sheep Tibial Model
Lammens, J.; Maréchal, Marina; Geris, Liesbet ULiege et al

in Tissue Engineering. Part C, Methods (2017), 23(11), 694-699

The repair of large long bone defects requires complex surgical procedures as the bone loss cannot simply be replaced by autologous grafts due to an insufficient bone stock of the human body. Tissue ... [more ▼]

The repair of large long bone defects requires complex surgical procedures as the bone loss cannot simply be replaced by autologous grafts due to an insufficient bone stock of the human body. Tissue engineering strategies and the use of Advanced Therapy Medicinal Products (ATMPs) for these reconstructions remain a considerable challenge, in particular since robust outcomes in well-defined large animal models are lacking. To be suitable as a model for treatment of human sized bone defects, we developed a large animal model in both skeletally immature and mature sheep and made close observations on the spontaneous healing of defects. We warn for the spontaneous repair of large defects in immature animals, which can mask the (in)effectiveness of ATMP therapies, and propose the use of large 4.5 cm defects that are pretreated with a polymethylmethacrylate (PMMA) spacer in skeletally mature animals. © Copyright 2017, Mary Ann Liebert, Inc. 2017. [less ▲]

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See detailChanges in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography.
Kerckhofs, G.; Durand, M.; Vangoitsenhoven, R. et al

in Scientific Reports (2016), 6

High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 ... [more ▼]

High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 mum scale, no change was observed in the intracortical structure. At 1 mum scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging. [less ▲]

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See detailCombining microCT-based characterization with empirical modelling as a robust screening approach for the design of optimized CaP-containing scaffolds for progenitor cell-mediated bone formation.
Kerckhofs, G.; Chai, Y. C.; Luyten, F. P. et al

in Acta Biomaterialia (2016), 35

Biomaterials are a key ingredient to the success of bone tissue engineering (TE), which focuses on the healing of bone defects by combining scaffolds with cells and/or growth factors. Due to the widely ... [more ▼]

Biomaterials are a key ingredient to the success of bone tissue engineering (TE), which focuses on the healing of bone defects by combining scaffolds with cells and/or growth factors. Due to the widely variable material characteristics and patient-specificities, however, current bone TE strategies still suffer from low repeatability and lack of robustness, which hamper clinical translation. Hence, optimal TE construct (i.e. cells and scaffold) characteristics are still under debate. This study aimed to reduce the material-specific variability for cell-based construct design, avoiding trial-and-error, by combining microCT characterization and empirical modelling as an innovative and robust screening approach. Via microCT characterization we have built a quantitative construct library of morphological and compositional properties of six CE approved CaP-based scaffolds (CopiOs(R), BioOss, Integra Mozaik, chronOS Vivify, MBCP and ReproBone), and of their bone forming capacity and in vivo scaffold degradation when combined with human periosteal derived cells (hPDCs). The empirical model, based on the construct library, allowed identification of the construct characteristics driving optimized bone formation, i.e. (a) the percentage of beta-TCP and dibasic calcium phosphate, (b) the concavity of the CaP structure, (c) the average CaP structure thickness and (d) the seeded cell amount (taking into account the seeding efficiency). Additionally, the model allowed to quantitatively predict the bone forming response of different hPDC-CaP scaffold combinations, thus providing input for a more robust design of optimized constructs and avoiding trial-and error. This could improve and facilitate clinical translation. STATEMENT OF SIGNIFICANCE: Biomaterials that support regenerative processes are a key ingredient for successful bone tissue engineering (TE). However, the optimal scaffold structure is still under debate. In this study, we have provided a useful innovative approach for robust screening of potential biomaterials or constructs (i.e. scaffolds seeded with cells and/or growth factors) by combining microCT characterization with empirical modelling. This novel approach leads to a better insight in the scaffold parameters influencing progenitor cell-mediated bone formation. Additionally, it serves as input for more controlled and robust design of optimized CaP-containing bone TE scaffolds. Hence, this novel approach could improve and facilitate clinical translation. [less ▲]

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See detailThe combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells.
Bolander, J.; Ji, W.; Geris, Liesbet ULiege et al

in European Cells and Materials (2016), 31

When combining osteogenic progenitor cells such as human periosteum derived cells (hPDCs) with osteoconductive biomaterials like calcium phosphate (CaP)-scaffolds, in vivo bone formation can be achieved ... [more ▼]

When combining osteogenic progenitor cells such as human periosteum derived cells (hPDCs) with osteoconductive biomaterials like calcium phosphate (CaP)-scaffolds, in vivo bone formation can be achieved. This process is dependent on the early activation of Bone morphogenetic protein (BMP)-signalling. However, the bone forming process is slow and routinely only a limited amount of bone and bone marrow is formed. Therefore, we hypothesised that a robust clinically relevant outcome could be achieved by adding more physiological levels of potent BMP-ligands to these cell- and CaP-based constructs. For this, hPDCs were characterised for their responsiveness to BMP-ligands upon in vitro 2D stimulation. BMP-2, -4, -6 and -9 robustly induced osteochondrogenic differentiation. Subsequently, these ligands were coated onto clinically approved CaP-scaffolds, BioOss(R) and CopiOs(R), followed by hPDC-seeding. Protein lysates and conditioned media were investigated for activation of BMP signalling pathways. Upon in vivo implantation, the most abundant bone formation was found in BMP-2 and BMP-6-coated scaffolds. Implanted cells actively contributed to the newly formed bone. Remnants of cartilage could be observed in BMP-coated CopiOs(R)-constructs. Computational analysis displayed that the type of BMP-ligand as well as the CaP-scaffold affects skeletal tissue formation, observed in a qualitative as well as quantitative manner. Furthermore, the in vitro mechanism appears to predict the in vivo outcome. This study presents further evidence for the potential of BMP-technology in the development of clinically relevant cell-based constructs for bone regenerative strategies. [less ▲]

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See detailCoupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold.
Guyot, Y.; Papantoniou, I.; Luyten, F. P. et al

in Biomechanics and Modeling in Mechanobiology (2016), 15(1), 169-80

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide ... [more ▼]

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth. [less ▲]

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See detailA Three-Dimensional Computational Fluid Dynamics Model Of Shear Stress Distribution During Neotissue Growth In A Perfusion Bioreactor.
Guyot, Yann ULiege; Luyten, F. P.; Schrooten, J. et al

in Biotechnology and Bioengineering (2015)

Bone tissue engineering strategies use flow through perfusion bioreactors to apply mechanical stimuli to cells seeded on porous scaffolds. Cells grow on the scaffold surface but also by bridging the ... [more ▼]

Bone tissue engineering strategies use flow through perfusion bioreactors to apply mechanical stimuli to cells seeded on porous scaffolds. Cells grow on the scaffold surface but also by bridging the scaffold pores leading a fully filled scaffold following the scaffold's geometric characteristics. Current computational fluid dynamic approaches for tissue engineering bioreactor systems have been mostly carried out for empty scaffolds. The effect of 3D cell growth and extracellular matrix formation (termed in this study as neotissue growth), on its surrounding fluid flow field is a challenge yet to be tackled. In this work a combined approach was followed linking curvature driven cell growth to fluid dynamics modeling. The level-set method (LSM) was employed to capture neotissue growth driven by curvature, while the Stokes and Darcy equations, combined in the Brinkman equation, provided information regarding the distribution of the shear stress profile at the neotissue/medium interface and within the neotissue itself during growth. The neotissue was assumed to be micro-porous allowing flow through its structure while at the same time allowing the simulation of complete scaffold filling without numerical convergence issues. The results show a significant difference in the amplitude of shear stress for cells located within the micro-porous neo-tissue or at the neotissue/medium interface, demonstrating the importance of taking along the neotissue in the calculation of the mechanical stimulation of cells during culture.The presented computational framework is used on different scaffold pore geometries demonstrating its potential to be used a design as tool for scaffold architecture taking into account the growing neotissue. This article is protected by copyright. All rights reserved. [less ▲]

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See detailCell based advanced therapeutic medicinal products for bone repair: Keep it simple?
Leijten, J.; Chai, Y. C.; Papantoniou, I. et al

in Advanced Drug Delivery Reviews (2015), 84

The development of cell based advanced therapeutic medicinal products (ATMPs) for bone repair has been expected to revolutionize the health care system for the clinical treatment of bone defects. Despite ... [more ▼]

The development of cell based advanced therapeutic medicinal products (ATMPs) for bone repair has been expected to revolutionize the health care system for the clinical treatment of bone defects. Despite this great promise, the clinical outcomes of the few cell based ATMPs that have been translated into clinical treatments have been far from impressive. In part, the clinical outcomes have been hampered because of the simplicity of the first wave of products. In response the field has set-out and amassed a plethora of complexities to alleviate the simplicity induced limitations. Many of these potential second wave products have remained "stuck" in the development pipeline. This is due to a number of reasons including the lack of a regulatory framework that has been evolving in the last years and the shortage of enabling technologies for industrial manufacturing to deal with these novel complexities. In this review, we reflect on the current ATMPs and give special attention to novel approaches that are able to provide complexity to ATMPs in a straightforward manner. Moreover, we discuss the potential tools able to produce or predict 'goldilocks' ATMPs, which are neither too simple nor too complex. [less ▲]

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See detailCurrent views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies.
Chai, Y. C.; Carlier, Aurélie ULiege; Bolander, J. et al

in Acta Biomaterialia (2012), 8(11), 3876-87

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP ... [more ▼]

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca(2)(+)) and phosphate (PO(4)(3)(-)) ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the material's osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell-material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed. [less ▲]

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See detailMechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers.
Chai, Y. C.; Roberts, S. J.; Desmet, E. et al

in Biomaterials (2012)

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for ... [more ▼]

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (muCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials. [less ▲]

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