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Tissue engineered constructs have the potential to respond to the unmet medical need of treating deep osteochondral defects. However, current tissue engineering strategies struggle in the attempt to create patterned constructs with biologically distinct functionality. In this work, a developmentally-inspired modular approach is proposed, whereby distinct cartilaginous organoids are used as living building blocks. First, a hierarchical construct was created, composed of three layers of cartilaginous tissue intermediates derived from human periosteum-derived cells: (i) early (SOX9), (ii) mature (COL2) and (iii) (pre)hypertrophic (IHH, COLX) phenotype. Subcutaneous implantation in nude mice generated a hybrid tissue containing one mineralized and one non-mineralized part. However, the non-mineralized part was represented by a collagen type I positive fibrocartilage-like tissue. To engineer a more stable articular cartilage part, iPSC-derived cartilage microtissues (SOX9, COL2; IHH neg) were generated. Subcutaneous implantation of assembled iPSC-derived cartilage microtissues resulted in a homogenous cartilaginous tissue positive for collagen type II but negative for osteocalcin. Finally, iPSC-derived cartilage microtissues in combination with the pre-hypertrophic cartilage organoids (IHH, COLX) could form dual tissues consisting of i) a cartilaginous safranin O positive and ii) a bony osteocalcin positive region upon subcutaneous implantation, corresponding to the pre-engineered zonal pattern. The assembly of functional building blocks, as presented in this work, opens possibilities for the production of complex tissue engineered implants by embedding zone-specific functionality through the use of pre-programmed living building blocks., (Copyright © 2021. Published by Elsevier Ltd.)

Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Regenerative cell-based implants using periosteum-derived stem cells were developed for the treatment of large 3 cm fresh and 4.5 centimeter biological compromised bone gaps in a tibial sheep model and compared with an acellular ceramic-collagen void filler. It was hypothesized that the latter is insufficient to heal large skeletal defects due to reduced endogenous biological potency. To this purpose a comparison was made between the ceramic dicalciumphosphate scaffold (CopiOs®) as such, the same ceramic coated with clinical grade Bone Morphogenetic Protein 2 and 6 (BMP) only or a BMP coated cell-seeded combination product. These implants were evaluated in 2 sheep models, a fresh 3 cm critical size tibial defect and a 4.5 cm biologically exhausted tibial defect. For the groups in which growth factors were applied, BMP-6 was chosen at a dose of 344 μg for 3 cm and 1.500 μg or 3.800 μg for 4.5 cm defects. An additional group in the 4.5 cm defect was tested using BMP-2 in a dose of 1.500 μg. For all the cell based implants autologous periosteum-derived cells were used which were cultured in monolayer during 6 weeks. For the fresh defect 408 million cells and for the biologically exhausted tibial defect 612 million cells were drop-seeded on the BMP coated scaffolds. Bone healing was studied during 16 weeks postimplantation, using standard radiographs. While fresh defects responded to all treatments, regardless the use of cells, the biologically hampered defects responded in half of the cases and only if the BMP-cell combination product was used, supporting the concept that cell-based therapies may become attractive in treating defects with a compromised biological status., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Biological tissues have a complex and heterogeneous 3D structure, which is only partially revealed by standard histomorphometry in 2D. We here present a novel chemical compound for contrast-enhanced microfocus computed tomography (CE-CT), a Hafnium-based Wells-Dawson polyoxometalate (Hf-POM), which allows simultaneous 3D visualization of mineralized and non-mineralized skeletal tissues, such as mineralized bone and bone marrow vasculature and adipocytes. We validated the novel contrast agent, which has a neutral pH in solution, by detailed comparison with (immuno)histology on murine long bones as blueprint, and showed that Hf-POM-based CE-CT can be used for virtual 3D histology. Furthermore, we quantified the 3D structure of the different skeletal tissues, as well as their spatial relation to each other, during aging and diet-induced obesity. We discovered, based on a single CE-CT dataset per sample, clear differences between the groups in bone structure, vascular network organization, characteristics of the adipose tissue and proximity of the different tissues to each other. These findings highlight the complementarity and added value of Hf-POM-based CE-CT compared to standard histomorphometry. As this novel technology provides a detailed 3D simultaneous representation of the structural organization of mineralized bone and bone marrow vasculature and adipose tissue, it will enable to improve insight in the interactions between these three tissues in several bone pathologies and to evaluate the in vivo performance of biomaterials for skeletal regeneration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Progress in medicine and higher expectation of quality of life has led to a higher demand for several dental and medical treatments. This increases the occurrence of situations in which orthodontic treatment is complicated by pathological conditions, medical therapies and drugs. Together with experiments, computer models might lead to a better understanding of the effect of pathologies and medical treatment on tooth movement. This study uses a previously presented mechanobiological model of orthodontic tooth displacement to investigate the effect of pathologies and (medical) therapies on the result of orthodontic treatment by means of three clinically relevant case studies looking at the effect of estrogen deficiency, the effect of OPG injections and the influence of fluoride intake. When less estrogen was available, the model predicted bone loss and a rise in the number of osteoclasts present at the compression side, and a faster bone resorption. These effects were also observed experimentally. Experiments disagreed on the effect of estrogen deficiency on bone formation, while the mechanobiological model predicted very little difference between the pathological and the non-pathological case at formation sites. The model predicted a decrease in tooth movement after OPG injections or fluoride intake, which was also observed in experiments. Although more experiments and model analysis is needed to quantitatively validate the mechanobiological model used in this study, its ability to conceptually describe several pathological conditions is an important measure for its validity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 (μCT) 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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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(®), Collagraft™ and Vitoss(®)) 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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally-informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cell-seeded hydrogels., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In this study, a cell based lattice free modelling framework is proposed to study cell aggregate behaviour in bone tissue engineering applications. The model encompasses cell-to-cell and cell-environment interactions such as adhesion, repulsion and drag forces. Oxygen, nutrients, waste products, growth factors and inhibitors are explicitly represented in the model influencing cellular behaviour. Furthermore, a model for cell metabolism is incorporated representing the basic enzymic reactions of glycolysis and the Krebs cycle. Various types of cell death such as necrosis, apoptosis and anoikis are implemented. Finally, an explicit model of the cell cycle controls the proliferation process, taking into account the presence or absence of various metabolites, sufficient space and mechanical stress. Several examples are presented demonstrating the potential of the modelling framework. The behaviour of a synchronised cell aggregate under ideal circumstances is simulated, clearly showing the different stages of the cell cycle and the resulting growth of the aggregate. Also the difference in aggregate development under ideal (normoxic) and hypoxic conditions is simulated, showing hypoxia induced necrosis mainly in the centre of the aggregate grown under hypoxic conditions. The next step in this research will be the application of this modelling framework to specific experimental set-ups for bone tissue engineering applications., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Several mechanoregulatory tissue differentiation models have been proposed over the last decade. Corroboration of these models by comparison with experimental data is necessary to determine their predictive power. So far, models have been applied with various success rates to different experimental set-ups investigating mainly secondary fracture healing. In this study, the mechanoregulatory models are applied to simulate the implant osseointegration process in a repeated sampling in vivo bone chamber, placed in a rabbit tibia. This bone chamber provides a mechanically isolated environment to study tissue differentiation around titanium implants loaded in a controlled manner. For the purpose of this study, bone formation around loaded cylindrical and screw-shaped implants was investigated. Histologically, no differences were found between the two implant geometries for the global amount of bone formation in the entire chamber. However, a significantly larger amount of bone-to-implant contact was observed for the screw-shaped implant compared to the cylindrical implant. In the simulations, a larger amount of bone was also predicted to be in contact with the screw-shaped implant. However, other experimental observations could not be predicted. The simulation results showed a distribution of cartilage, fibrous tissue and (im)mature bone, depending on the mechanoregulatory model that was applied. In reality, no cartilage was observed. Adaptations to the differentiation models did not lead to a better correlation between experimentally observed and numerically predicted tissue distribution patterns. The hypothesis that the existing mechanoregulatory models were able to predict the patterns of tissue formation in the in vivo bone chamber could not be fully sustained.

The application of a bone chamber provides a controlled environment for the study of tissue differentiation and bone adaptation. The influence of different mechanical and biological factors on the processes can be measured experimentally. The goal of the present work is to numerically model the process of peri-implant tissue differentiation inside a bone chamber, placed in a rabbit tibia. 2D and 3D models were created of the tissue inside the chamber. A number of loading conditions, corresponding to those applied in the rabbit experiments, were simulated. Fluid velocity and maximal distortional strain were considered as the stimuli that guide the differentiation process of mesenchymal cells into fibroblasts, chondrocytes and osteoblasts. Mesenchymal cells migrate through the chamber from the perforations in the chamber wall. This process is modelled by the diffusion equation. The predicted tissue phenotypes as well as the process of tissue ingrowth into the chamber show a qualitative agreement with the results of the rabbit experiments. Due to the limited number of animal experiments (four) and the observed inter-animal differences, no quantitative comparison could be made. These results however are a strong indication of the feasibility of the implemented theory to predict the mechano-regulation of the differentiation process inside the bone chamber.