Functional traits and climate drive interspecific differences in disturbance-induced tree mortality.

With climate change, natural disturbances such as storm or fire are reshuffled, inducing pervasive shifts in forest dynamics. To predict how it will impact forest structure and composition, it is crucial to understand how tree species differ in their sensitivity to disturbances. In this study, we investigated how functional traits and species mean climate affect their sensitivity to disturbances while controlling for tree size and stand structure. With data on 130,594 trees located on 7617 plots that were disturbed by storm, fire, snow, biotic or other disturbances from the French, Spanish, and Finnish National Forest Inventory, we modeled annual mortality probability for 40 European tree species as a function of tree size, dominance status, disturbance type, and intensity. We tested the correlation of our estimated species probability of disturbance mortality with their traits and their mean climate niches. We found that different trait combinations controlled species sensitivity to disturbances. Storm-sensitive species had a high height-dbh ratio, low wood density and high maximum growth, while fire-sensitive species had low bark thickness and high P50. Species from warmer and drier climates, where fires are more frequent, were more resistant to fire. The ranking in disturbance sensitivity between species was overall consistent across disturbance types. Productive conifer species were the most disturbance sensitive, while Mediterranean oaks were the least disturbance sensitive. Our study identified key relations between species functional traits and disturbance sensitivity, that allows more reliable predictions of how changing climate and disturbance regimes will impact future forest structure and species composition at large spatial scales.

Significant increase in natural disturbance impacts on European forests since 1950.

Over the last decades, the natural disturbance is increasingly putting pressure on European forests. Shifts in disturbance regimes may compromise forest functioning and the continuous provisioning of ecosystem services to society, including their climate change mitigation potential. Although forests are central to many European policies, we lack the long-term empirical data needed for thoroughly understanding disturbance dynamics, modeling them, and developing adaptive management strategies. Here, we present a unique database of >170,000 records of ground-based natural disturbance observations in European forests from 1950 to 2019. Reported data confirm a significant increase in forest disturbance in 34 European countries, causing on an average of 43.8 million m3 of disturbed timber volume per year over the 70-year study period. This value is likely a conservative estimate due to under-reporting, especially of small-scale disturbances. We used machine learning techniques for assessing the magnitude of unreported disturbances, which are estimated to be between 8.6 and 18.3 million m3 /year. In the last 20 years, disturbances on average accounted for 16% of the mean annual harvest in Europe. Wind was the most important disturbance agent over the study period (46% of total damage), followed by fire (24%) and bark beetles (17%). Bark beetle disturbance doubled its share of the total damage in the last 20 years. Forest disturbances can profoundly impact ecosystem services (e.g., climate change mitigation), affect regional forest resource provisioning and consequently disrupt long-term management planning objectives and timber markets. We conclude that adaptation to changing disturbance regimes must be placed at the core of the European forest management and policy debate. Furthermore, a coherent and homogeneous monitoring system of natural disturbances is urgently needed in Europe, to better observe and respond to the ongoing changes in forest disturbance regimes.

Asymmetric competition, ontogenetic growth and size inequality drive the difference in productivity between two-strata and one-stratum forest stands.

Size inequality has been considered a key feature of plant population structure with impacts on ecosystem functions. In forest ecosystems, studies examining the relationship between tree size inequality and stand productivity have produced mixed outcomes. These studies found positive, neutral or negative relationships and discussed how this could be influenced by competition for light between trees (e.g. light interception efficiency), but far less attention has been paid to the role played by tree ontogenetic growth. In this article, we present a simple mathematical model that predicts the basal area growth of a two-strata stand as a function of tree basal areas and asymmetric competition. Comparing the growth of this stand to the growth of a spatially homogeneous one-stratum stand and a spatially heterogeneous one-stratum stand, we show that higher growth of the two-strata stand is achieved for concave shape, increasing functions of ontogenetic growth and for low intensities of absolute size-asymmetric competition. We also demonstrate that the difference in growth between the two-strata stand and the one-stratum stands depends on tree size inequality, mean tree basal area and total basal area in the two-strata stand. We finally found that the relationships between tree size inequality and productivity can vary from positive to negative and even non-monotonous. However, we highlight that negative relationships may be more frequent. As a conclusion, our results indicate that ontogenetic growth can have a major impact on the form and the magnitude of the size inequality-productivity relationship.

Ecological research and environmental management: We need different interfaces based on different knowledge types.

The role of ecological science in environmental management has been discussed by many authors who recognize that there is a persistent gap between ecological science and environmental management. Here we develop theory through different perspectives based on knowledge types, research categories and research-management interface types, which we combine into a common framework. To draw out insights for bridging this gap, we build our case by:We point out the complementarities as well as the specificities and limitations of the different types of ecological research, ecological knowledge and research-management interfaces, which is of major importance for environmental management and research policies.

Long-term tree inventory data from mountain forest plots in France.

We present repeated tree measurement data from 63 permanent plots in mountain forests in France. Plot elevations range from 800 (lower limit of the montane belt) to 1942 m above sea level (subalpine belt). Forests mainly consist of pure or mixed stands dominated by European beech (Fagus sylvatica), Silver fir (Abies alba), and Norway spruce (Picea abies), in association with various broadleaved species at low elevation and with Arolla pine (Pinus cembra) at high elevation. The plot network includes 23 plots in stands that have not been managed for the last 40 years (at least) and 40 plots in plots managed according to an uneven-aged system with single-tree or small-group selection cutting. Plot sizes range from 0.2 to 1.9 ha. Plots were installed from 1994 to 2004 and remeasured two to five times during the 1994-2015 period. During the first census (installation), living trees more than 7.5 cm in dbh were identified, their diameter at breast height (dbh) was measured and their social status (strata) noted. Trees were spatially located, either with x, y, and z coordinates (40 plots) or within 0.25-ha square subplots (23 plots). In addition, in a subset of plots (58 plots), tree heights and tree crown dimensions were measured on a subset of trees and dead standing trees and stumps were included in the census. Remeasurements after installation include live tree diameters (including recruited trees), tree status (living, damaged, dead, stump), and for a subset of trees, height. At the time of establishment of the plots, plot densities range from 181 to 1328 stems/ha and plot basal areas range from 13.6 to 81.3 m2 /ha.

Hsp27 regulates EGF/ß-catenin mediated epithelial to mesenchymal transition in prostate cancer.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3ß phosphorylation and ß-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of ß-catenin on tyrosine 142 and 654, enhances ß-catenin ubiquitination and degradation, prevents ß-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the ß-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

Modelling and predicting the spatial distribution of tree root density in heterogeneous forest ecosystems.

BACKGROUND AND AIMS: In mountain ecosystems, predicting root density in three dimensions (3-D) is highly challenging due to the spatial heterogeneity of forest communities. This study presents a simple and semi-mechanistic model, named ChaMRoots, that predicts root interception density (RID, number of roots m(-2)). ChaMRoots hypothesizes that RID at a given point is affected by the presence of roots from surrounding trees forming a polygon shape. METHODS: The model comprises three sub-models for predicting: (1) the spatial heterogeneity - RID of the finest roots in the top soil layer as a function of tree basal area at breast height, and the distance between the tree and a given point; (2) the diameter spectrum - the distribution of RID as a function of root diameter up to 50 mm thick; and (3) the vertical profile - the distribution of RID as a function of soil depth. The RID data used for fitting in the model were measured in two uneven-aged mountain forest ecosystems in the French Alps. These sites differ in tree density and species composition. KEY RESULTS: In general, the validation of each sub-model indicated that all sub-models of ChaMRoots had good fits. The model achieved a highly satisfactory compromise between the number of aerial input parameters and the fit to the observed data. CONCLUSIONS: The semi-mechanistic ChaMRoots model focuses on the spatial distribution of root density at the tree cluster scale, in contrast to the majority of published root models, which function at the level of the individual. Based on easy-to-measure characteristics, simple forest inventory protocols and three sub-models, it achieves a good compromise between the complexity of the case study area and that of the global model structure. ChaMRoots can be easily coupled with spatially explicit individual-based forest dynamics models and thus provides a highly transferable approach for modelling 3-D root spatial distribution in complex forest ecosystems.

Reconciling Biodiversity Conservation and Timber Production in Mixed Uneven-Aged Mountain Forests: Identification of Ecological Intensification Pathways.

Mixed uneven-aged forests are considered favorable to the provision of multiple ecosystem services and to the conciliation of timber production and biodiversity conservation. However, some forest managers now plan to increase the intensity of thinning and harvesting operations in these forests. Retention measures or gap creation are considered to compensate potential negative impacts on biodiversity. Our objectives were to assess the effect of these management practices on timber production and biodiversity conservation and identify potential compensating effects between these practices, using the concept of ecological intensification as a framework. We performed a simulation study coupling Samsara2, a simulation model designed for spruce-fir uneven-aged mountain forests, an uneven-aged silviculture algorithm, and biodiversity models. We analyzed the effect of parameters related to uneven-aged management practices on timber production, biodiversity, and sustainability indicators. Our study confirmed that the indicators responded differently to management practices, leading to trade-offs situations. Increasing management intensity had negative impacts on several biodiversity indicators, which could be partly compensated by the positive effect of retention measures targeting large trees, non-dominant species, and deadwood. The impact of gap creation was more mitigated, with a positive effect on the diversity of tree sizes and deadwood but a negative impact on the spruce-fir mixing balance and on the diversity of the understory layer. Through the analysis of compensating effects, we finally revealed the existence of possible ecological intensification pathways, i.e., the possibility to increase management intensity while maintaining biodiversity through the promotion of nature-based management principles (gap creation and retention measures).

Using the Viability Theory to Assess the Flexibility of Forest Managers Under Ecological Intensification.

Greater demand for wood material has converged with greater demand for biodiversity conservation to make balancing forest ecosystem services a key societal issue. Forest managers, owners, or policymakers need new approaches and methods to evaluate their ability to adapt to this dual objective. We analyze the ability of forest owners to define sustainable forest management options based on viability theory and a new flexibility index. This new indicator gauges the adaptive capacity of forest owners based on the number of sustainable actions available to them at a given time. Here we study a public forest owner who regulates harvest intensity and frequency in order to meet demand for timber wood at forest scale and to meet a biodiversity recommendation via a minimum permanently maintained volume of deadwood per hectare at stand scale. Dynamical systems theory was used to model uneven-aged forest dynamics-including deadwood dynamics-and the dynamics of timber wood demand and tree removals. Uneven-aged silver fir forest management in the "Quatre Montagnes region" (Vercors, France) is used as an illustrative example. The results explain situations where a joint increase in wood production and deadwood retention does not reduce the flexibility index more than increasing either one dimension alone, thus opening up ecological intensification options. To conclude, we discuss the value of the new flexibility index for addressing environmental management and ecological intensification issues.

Diameter, height and species of 42 million trees in three European landscapes generated from field data and airborne laser scanning data.

Ecology and forestry sciences are using an increasing amount of data to address a wide variety of technical and research questions at the local, continental and global scales. However, one type of data remains rare: fine-grain descriptions of large landscapes. Yet, this type of data could help address the scaling issues in ecology and could prove useful for testing forest management strategies and accurately predicting the dynamics of ecosystem services. Here we present three datasets describing three large European landscapes in France, Poland and Slovenia down to the tree level. Tree diameter, height and species data were generated combining field data, vegetation maps and airborne laser scanning (ALS) data following an area-based approach. Together, these landscapes cover more than 100 000 ha and consist of more than 42 million trees of 51 different species. Alongside the data, we provide here a simple method to produce high-resolution descriptions of large landscapes using increasingly available data: inventory and ALS data. We carried out an in-depth evaluation of our workflow including, among other analyses, a leave-one-out cross validation. Overall, the landscapes we generated are in good agreement with the landscapes they aim to reproduce. In the most favourable conditions, the root mean square error (RMSE) of stand basal area (BA) and mean quadratic diameter (Dg) predictions were respectively 5.4 m 2.ha -1 and 3.9 cm, and the generated main species corresponded to the observed main species in 76.2% of cases.

Protein-polysaccharide complexes for enhanced protein delivery in hyaluronic acid templated calcium carbonate microparticles.

The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO3. In this study, we explored the use of polysaccharide-protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a ß-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO3 microparticles using supercritical CO2 (ScCO2) technology aided by the templating effect of HA on CaCO3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the microparticles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage.

Silica nanofibers as a new drug delivery system: a study of the protein-silica interactions.

Drug delivery systems are proposed for the in situ controlled delivery of therapeutic molecules in the scope of tissue engineering. We propose herein silica nanofibers as carriers for the loading and release of bioactive proteins. The influence of pH, time and concentration on the amount of adsorbed proteins was studied. The interactions allowing loading were then studied by means of electron microscopy, zeta potential measurements, electron energy loss spectroscopy and attenuated total reflectance Fourier transform infrared analysis. Release profiles were determined and biological activities were enzymatically assessed. The first part of the work was carried out with lysozyme as a model protein, and then bioactive growth factors TGF-ß1 and GDF-5 were used because their significance in human adipose stromal cell differentiation towards intervertebral disc nucleopulpocytes was previously assessed. It is demonstrated that protein-silica nanofiber interactions are mainly driven by hydrogen bonds and local electrostatic interactions. The present data thus provide a better understanding of the adsorption phenomenon involved, as well as a method to control protein adsorption and release. It is worth pointing out that the kinetic release of growth factors, up to 28 days, and their biological activity maintenance seem to be compatible with intervertebral disc regenerative medicine.

Tree Size Inequality Reduces Forest Productivity: An Analysis Combining Inventory Data for Ten European Species and a Light Competition Model.

Plant structural diversity is usually considered as beneficial for ecosystem functioning. For instance, numerous studies have reported positive species diversity-productivity relationships in plant communities. However, other aspects of structural diversity such as individual size inequality have been far less investigated. In forests, tree size inequality impacts directly tree growth and asymmetric competition, but consequences on forest productivity are still indeterminate. In addition, the effect of tree size inequality on productivity is likely to vary with species shade-tolerance, a key ecological characteristic controlling asymmetric competition and light resource acquisition. Using plot data from the French National Geographic Agency, we studied the response of stand productivity to size inequality for ten forest species differing in shade tolerance. We fitted a basal area stand production model that included abiotic factors, stand density, stand development stage and a tree size inequality index. Then, using a forest dynamics model we explored whether mechanisms of light interception and light use efficiency could explain the tree size inequality effect observed for three of the ten species studied. Size inequality negatively affected basal area increment for seven out of the ten species investigated. However, this effect was not related to the shade tolerance of these species. According to the model simulations, the negative tree size inequality effect could result both from reduced total stand light interception and reduced light use efficiency. Our results demonstrate that negative relationships between size inequality and productivity may be the rule in tree populations. The lack of effect of shade tolerance indicates compensatory mechanisms between effect on light availability and response to light availability. Such a pattern deserves further investigations for mixed forests where complementarity effects between species are involved. When studying the effect of structural diversity on ecosystem productivity, tree size inequality is a major facet that should be taken into account.

Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles.

Calcium carbonate particles are promising candidates as proteins carriers for their controlled delivery in the body. The present paper aims at investigating the protein encapsulation by in situ precipitation of calcium carbonate particles prepared by a process based on supercritical CO2 and using a new type of degradable well-defined double hydrophilic block copolymers composed of poly(ethylene oxide) and polyphosphoester blocks acting as templating agent for the calcium carbonate. For this study, lysozyme was chosen as a model for therapeutic protein for its availability and ease of detection. It was found that by this green process, loading into the CaCO3 microparticles with a diameter about 2µm can be obtained as determined by scanning electron microscopy. A protein loading up to 6.5% active lysozyme was measured by a specific bioassay (Micrococcus lysodeikticus). By encapsulating fluorescent-labelled lysozyme (lysozyme-FITC), the confocal microscopy images confirmed its encapsulation and suggested a core-shell distribution of lysozyme into CaCO3, leading to a release profile reaching a steady state at 59% of release after 90min.

Sustained release of TGF-ß1 from biodegradable microparticles prepared by a new green process in CO2 medium.

The aim of this work was to encapsulate transforming growth factor ß1 (TGF-ß1) into PLGA microparticles for regenerative medicine applications. TGF-ß1 was firstly precipitated to ensure its stability during subsequent encapsulation within microparticles. A novel emulsification/extraction process in CO2 medium under mild conditions of pressure and temperature was used to encapsulate the protein. Interestingly, non-volatile injectable solvents, isosorbide dimethyl ether (DMI) and glycofurol (GF), were employed to precipitate the protein and to dissolve the polymer. Good encapsulation efficiency was obtained with preserved bioactivity of the protein. The microparticles were characterized in terms of size and zeta potential. In addition, the morphology and surface properties were determined using scanning electron microscopy (SEM) and atomic force microscopy (AFM) respectively. In vitro release study of the protein from microparticles was presented to assess the capacity of these systems to control the protein release. Moreover, cytotoxicity study was performed and showed an excellent cytocompatibility of the obtained microparticles. Thus, we described an effective and original process for TGF-ß1 encapsulation into PLGA microparticles. The obtained polymeric carriers could be used in many biomedical applications and were more specifically developed for cartilage regeneration.

Preparation of polymeric particles in CO2 medium using non-toxic solvents: discussions on the mechanism of particle formation.

The aim of this work was to develop a novel formulation method, termed modified-PGSS (modified-Particle from Gas Saturated Solution), for the encapsulation of protein into polymeric microparticles in CO2 medium. In this study, isosorbide dimethyl ether (DMI), a non-toxic water-miscible solvent, was used for the formulation and lysozyme was chosen as a model protein for encapsulation into PLGA microparticles. First, the mechanism of particle formation has been extensively studied and was discussed in detail. Phase behavior was investigated by measuring the solubility of CO2 in DMI and volumetric expansion of DMI saturated in CO2. Here, we demonstrate the consistency of the experimental values with the data obtained from the mathematical (such as the neural network) and thermodynamic (such as the Peng-Robinson equation of state) models. These models were built to develop predictive tools in the chosen experimental space for microparticle formulation. Furthermore, these microparticles were characterized in terms of size and zeta potential. The morphology and protein distribution within PLGA microparticles were determined using scanning electron microscopy and confocal microscopy, respectively. High encapsulation efficiency (65%) was obtained as confirmed by lysozyme quantification using a specific bioassay (M. lysodeikticus). Moreover, the in vitro protein release profile from loaded microparticles was presented. In this study, we report an innovative and green process for lysozyme encapsulation into PLGA microparticles. Thus, this process could be applied to the encapsulation of therapeutic proteins requiring protection and controlled release such as growth factors for regenerative medicine.

Inhibition of the HER2-YB1-AR axis with Lapatinib synergistically enhances Enzalutamide anti-tumor efficacy in castration resistant prostate cancer.

Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

Combination AZD5363 with Enzalutamide Significantly Delays Enzalutamide-resistant Prostate Cancer in Preclinical Models.

UNLABELLED: The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt (PI3K/Akt) pathway is a key pathway activated in castrate-resistant prostate cancer (CRPC). This preclinical study evaluates targeting of Akt with AZD5363 alone and in combination with enzalutamide (ENZ) to prevent and delay resistance. Our results demonstrate AZD5363 has significant proapoptotic, antiproliferative activity as monotherapy in ENZ-resistant cell lines in vitro and significantly decreased tumour growth in ENZ-resistant xenograft. The combination of AZD5363 and ENZ showed synergistic decreases in cell proliferation and induced cell-cycle arrest and apoptosis in prostate cancer cell lines LNCaP and C4-2. Notably, the combination of AZD5363 and ENZ resulted in an impressive regression of castrate-resistant LNCaP xenograft tumours without any recurrence demonstrated, whereas progression occurred with both monotherapies. Serum prostate-specific antigen (PSA) levels were also continuously suppressed, and nadir PSA levels were lower in the combination arm compared to ENZ alone. Combination AZD5363 and ENZ at time of castration similarly resulted in significant regression of tumours, with greater relative suppression of PSA compared to when administered to castrate-resistant xenografts. In summary, combination AZD5363 and ENZ significantly delays the development of ENZ resistance in preclinical models through synergistic increases in apoptosis and cell cycle arrest. Our results also suggest greater efficacy may be seen with earlier combination treatment. This study provides preclinical data to support evaluation of combination targeting of the PI3K/Akt pathway and the androgen-receptor axis in the clinic using AZD5363 and ENZ, respectively. PATIENT SUMMARY: Targeting of the Akt and androgen receptor pathways with AZD5363 and enzalutamide, respectively, significantly delayed the development of enzalutamide-resistant prostate cancer through increased apoptosis and cell cycle arrest. This preclinical synergy provides a strong rationale for clinical evaluation of this combination.

Osteoblastic differentiation and potent osteogenicity of three-dimensional hBMSC-BCP particle constructs.

Bone tissue engineering usually consists of associating osteoprogenitor cells and macroporous scaffolds. This study investigated the in vitro osteoblastic differentiation and resulting in vivo bone formation induced by a different approach that uses particles as substrate for human bone marrow stromal cells (hBMSCs), in order to provide cells with a higher degree of freedom and allow them to synthesize a three-dimensional (3D) environment. Biphasic calcium phosphate (BCP) particles (35 mg, ~175 µm in diameter) were therefore associated with 4 × 10(5) hBMSCs. To discriminate the roles of BCP properties and cell-synthesized 3D environments, inert glass beads (GBs) of similar size were used under the same conditions. In both cases, high cell proliferation and extensive extracellular matrix (ECM) production resulted in the rapid formation of thick cell-synthesized 3D constructs. In vitro, spontaneous osteoblastic differentiation was observed in the 3D constructs at the mRNA and protein levels by monitoring the expression of Runx2, BMP2, ColI, BSP and OCN. The hBMSC-BCP particle constructs implanted in the subcutis of nude mice induced abundant ectopic bone formation after 8 weeks (~35%, n = 5/5). In comparison, only fibrous tissue without bone was observed in the implanted hBMSC-GB constructs (n = 0/5). Furthermore, little bone formation (~3%, n = 5/5) was found in hBMSC-macroporous BCP discs (diameter 8 × 3 mm). This study underlines the lack of correspondence between bone formation and in vitro differentiation assays. Furthermore, these results highlight the importance of using BCP as well as a 3D environment for achieving high bone yield of interest for bone engineering.