Juliet Abadie Jean-Luc

Dupouey Catherine

Avon Xavier

Rochel Thierry

Tatoni Laurent Berge

J Abadie

J.-L Dupouey

L Berge

Forest recovery since 1860 in a Mediterranean region: drivers and implications for land use and land cover spatial distribution

Keywords: Forest transition, Historical maps, Land cover change, Land use change, Logistic regression, Long-term, Northern Mediterranean, Transition matrix

Context Land use and land cover (LULC) change is a major part of environmental change. Understanding its long-term causes is a major issue in landscape ecology.

Objectives Our aim was to characterise LULC transitions since 1860 and assess the respective and changing effects of biophysical and socioeconomic drivers on forest, arable land and pasture in 1860, 1958 and 2010, and of biophysical, socioeconomic and distance from pre-existing forest on forest recovery for the two time intervals.

Methods

We assessed LULC transitions by superimposing 1860, 1958 and 2010 LULCs using a regular grid of 1 9 1 km points, in a French Mediterranean landscape (195,413 ha). We tested the effects of drivers using logistic regressions, and quantified pure and joint effects by deviance partitioning. Results Over the whole period, the three main LULCs were spatially structured according to land accessibility and soil productivity. LULC was driven more by socioeconomic than biophysical drivers in 1860, but the pattern was reversed in 2010. A widespread forest recovery mainly occurred on steeper slopes, far from houses and close to pre-existing forest, due to traditional practice abandonment. Forest recovery was better explained by biophysical than by socioeconomic drivers and was more dependent

Introduction

Human activities are estimated to have transformed or degraded some 50% of the land surface up to now, mostly through agriculture [START_REF] Kareiva | Domesticated nature: shaping landscapes and ecosystems for human welfare[END_REF]. One major issue of land use and land cover (LULC) change worldwide is biodiversity loss, through habitat loss and fragmentation and landscape homogenisation [START_REF] Foley | Global consequences of land use[END_REF][START_REF] Lambin | Land use transitions: socioecological feedback versus socio-economic change[END_REF]. The study of LULC change is of crucial importance for understanding how the past shapes current landscapes and ecosystem functioning, and to better anticipate the consequences of current landscape modifications [START_REF] Antrop | Why landscapes of the past are important for the future[END_REF][START_REF] Kaim | Broad scale forest cover reconstruction from historical topographic maps[END_REF]. LULC change is predominantly induced by human activities, which are conditioned by external constraints or facilitations [START_REF] Lambin | The causes of land-use and landcover change: moving beyond the myths[END_REF]Bu ¨rgi et al. 2004;[START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF]. Bu ¨rgi et al. (2004) defined five types of LULC change: natural, socioeconomic, political, technological and cultural but two groups are mostly considered: natural (or biophysical) and socioeconomic drivers [START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF]. Biophysical drivers (climate, topography, soil) contribute to the development of adapted vegetation and inhibit or ease land use, while socioeconomic drivers (population size and composition, land prices, infrastructure network, etc.) reflect direct or indirect human pressure on land [START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF]. These two groups of drivers can be variably correlated or intrinsically intertwined [START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF], and so causal relationships between drivers and LULC change may be difficult to elucidate. Depending on the geographical context (lowlands or mountains), biophysical attributes can strongly guide human activities (density of settlement, transport infrastructures, proportion of agriculture) and thus be a major driver of land use compared with socioeconomic drivers [START_REF] Odgaard | Human-driven topographic effects on the distribution of forest in a flat, lowland agricultural region[END_REF].

After the last glaciation, forest cover became gradually predominant in European landscapes [START_REF] Kaplan | Large scale anthropogenic reduction of forest cover in last glacial maximum Europe[END_REF]. Since the Neolithic, forest cover has lost ground to agriculture, with short periods of limited recovery [START_REF] Kaplan | The prehistoric and preindustrial deforestation of Europe[END_REF][START_REF] Barbier | The forest transition: towards a more comprehensive theoretical framework[END_REF]. A sudden upturn in forest cover, known as the forest transition [START_REF] Mather | The forest transition[END_REF], occurred during the nineteenth century in Europe, due to land abandonment and land protection. In France, like in several other European countries, the drivers of forest transition were rural depopulation, improved crop yields, which reduced the need for agricultural expansion, the development of new sources of energy in addition to fuelwood and the development of transport infrastructures, which reduced the need for local subsistence [START_REF] Mather | The course and drivers of the forest transition: the case of France[END_REF][START_REF] Koerner | Evolution des surfaces boise ´es en France depuis le de ´but du XIXe sie `cle: identification et localisation des boisements des territoires agricoles abandonne ´s[END_REF].

Factors frequently related to forest recovery, or land abandonment leading to forest recovery, are slope, soil productivity or fertility, distance from roads, and distance from pre-existing forest. Steeper slopes and unproductive soils appear to maintain forest cover or enhance forest recovery [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF][START_REF] Lieskovsky | The abandonment of traditional agricultural landscape in Slovakia-analysis of extent and driving forces[END_REF], and forest recovery or land abandonment is more likely to occur far from roads [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF][START_REF] Schweizer | Factors driving land use change and forest distribution on the coastal plain of Mississippi, USA[END_REF]. Forest recovery has also mostly occurred closer to preexisting forest [START_REF] Crk | Forest recovery in a tropical landscape: what is the relative importance of biophysical, socioeconomic, and landscape variables?[END_REF][START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Puddu | Forest changes over a century in Sardinia: implications for conservation in a Mediterranean hotspot[END_REF]). Among studies that quantified the amount of variability of LULC or LULC change explained by different drivers, along with the pure and joint effects of each group of drivers [START_REF] Van Doorn | The destination of arable land in a marginal agricultural landscape in south Portugal: an exploration of land use change determinants[END_REF][START_REF] Wulf | Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period[END_REF], none have included the effect of distance from pre-existing forest. In this paper, we quantified the additive effects of three types of drivers (biophysical, socioeconomic and distance from pre-existing forest) to disentangle their pure and joint effects.

The Mediterranean region is characterised by strong biophysical constraints compared to temperate regions: water limitations due to hot and dry summers and a hard calcareous substrate with a high stone content result in a specific agriculture (vineyards, olive trees, lavender). A rugged topography led farmers to cultivate slopes, resulting in the traditional Mediterranean landscape characterised by terraced crops and extensive grazing. Studying Mediterranean landscapes is thus of particularly interest when seeking to quantify and disentangle the respective effects of drivers of LULC and LULC change. Also, drivers of LULC and forest recovery may differ in the Mediterranean region compared to temperate or lowland regions, where biophysical and socio-historical contexts are different.

Since current landscapes result from very long-term changes [START_REF] Foley | Global consequences of land use[END_REF], access to LULC data dating back more than only a few decades is of crucial importance [START_REF] Bender | Analysis of land-use change in a sector of Upper Franconia (Bavaria, Germany) since 1850 using land register records[END_REF][START_REF] Bieling | Patterns and causes of land change: empirical results and conceptual considerations derived from a case study in the Swabian Alb, Germany[END_REF][START_REF] Wulf | Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period[END_REF]. Besides, the effect of former land use on ecosystems can modify forest plant communities and soil properties for centuries [START_REF] Dupouey | Irreversible impact of past land use on forest soils and biodiversity[END_REF]. Throughout Europe, the recent availability of precise historical maps dating back to the beginning of the nineteenth century now allows the reconstruction of remote-past landscapes and helps gain a better understanding of long-term change [START_REF] Kaim | Broad scale forest cover reconstruction from historical topographic maps[END_REF]). However, drivers of forest cover change have rarely been assessed on both large spatial and temporal scales (more than 1000 km 2 and more than 100 years) (but see [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Wulf | Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period[END_REF]. Assessing LULC change with more than two dates allows proper quantification of LULC turn-over and identification of temporal discontinuities in LULC change [START_REF] Zhou | 90 Years of forest cover change in an urbanizing watershed: spatial and temporal dynamics[END_REF]). Yet studies of LULC change over a long time span, with a high temporal resolution and over a large spatial extent are very scant [START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Keersmaeker | The analysis of spatio-temporal forest changes (1775-2000) in Flanders (northern Belgium) indicates habitat-specific levels of fragmentation and area loss[END_REF].

Forest cover dynamics can only be fully understood by combining LULC transitions (where did forest cover come from?) and drivers of forest cover change (why did this transition occur here?). We set out to assess the effect of drivers on forest recovery, and to combine it with the changing effect of drivers on LULC over time. We assessed LULC spatial distribution and forest recovery since 1860 in the Regional Natural Park of Luberon (PNRL), a typical Mediterranean landscape. Firstly, we characterised LULC change and persistence since 1860 by focusing on the four main LULC available at each date: forest, arable land, pasture and built areas. Secondly, we quantified the effects of biophysical and socioeconomic drivers on forest, arable land and pasture in 1860, 1958 and 2010, and the effects of biophysical and socioeconomic drivers and distance from pre-existing forest on forest recovery for the two time intervals. We specifically addressed the following questions:

-At the expense of what LULC did forest recovery take place? -What biophysical and socioeconomic drivers explain the spatial distribution of LULC and how did their effects change between 1860 and 2010? -What biophysical, socioeconomic and landscape drivers explain the spatial distribution of forest recovery and how did their effects change between 1860 and 2010?

Methods

Study area

The PNRL is located in the eastern French Mediterranean region (43°39 0 N 44°02 0 N, 4°58 0 E 5°55 0 E) and covers 195,413 ha (Fig. 1). The climate is Mediterranean with mild humid winters and hot dry summers (annual mean precipitation 710 mm, mean temperature 13 °C) [START_REF] Varese | Pre ´-e ´tude en vue d'une typologie des stations forestie `res du Luberon[END_REF]. Elevation ranges from 51 to 1252 m. The land is mostly calcareous (95%) except for an acidic substrate strip in its northern part. This rural area is currently composed of 81 small towns (35-4775 inhabitants) and 4 mediumsize towns (12,000-25,000 inhabitants). More than half of the PNRL is now covered by forest (55%). Agricultural land supports annual crops (59%), vineyards (26%), tree crops (13%) and meadows (2%). Forest transition occurred in Provence during the nineteenth century. Until the mid-nineteenth century the economy of the region was based on an agrosylvo-pastoral system: sheep and goats, bred for wool and milk, roamed in forest and garrigue which served as pasture; their manure and some forest products (boxwood and dead leaves) were used to fertilise crops. Forests were mostly coppiced for firewood, tanning (oak bark) and grazing. The rural population peaked in the period 1830-1860, and forest cover shrank correspondingly (Appendix S1). The economic system was no longer compatible with population growth and economic pressure from the national and regional markets [START_REF] Chalvet | Paysages et conflits en Provence[END_REF]. Administrative laws were enacted in 1860 and 1864 to restore forest cover and curb soil erosion [START_REF] Fourchy | Les lois du 28 juillet 1860 et 8 juin 1864 sur le reboisement et le gazonnement des montagnes[END_REF][START_REF] Gilbert | Elevage, fore ˆt et socie ´te ´. Analyse socio-historique[END_REF].

Forest grazing regulation and the setting-up of a new economic market made pastoralism a less profitable activity, so local farmers and shepherds gradually changed their practices [START_REF] Chalvet | Paysages et conflits en Provence[END_REF]. In this context, also characterised by the industrial revolution, the region experienced great rural exodus. Land abandonment allowed forest to recover naturally (monospecific plantations were of minor importance) and since the early nineteenth century, forest cover gradually increased (Fig. 2, Appendix S2). The rural population continued to decline during the first half of the twentieth century, partly due to the Great War of 1914-1918 (Appendix S1). After 1960, rural population rose again following 'rurbanisation'. Since then, the tertiary sector has been growing rapidly at the expense of agriculture. In 1977, the PNRL was created with the main objective of reconciling economic development and the preservation of traditional rural activities.

Data

We created a systematic 1000 9 1000 m grid of points covering the whole study area (1916 points), and extracted LULC at each date and data for each spatially explicit factor (see [START_REF] Kaim | Broad scale forest cover reconstruction from historical topographic maps[END_REF].

LULC maps

The 1860 LULC map was digitised from the E ´tat-Major map (1858-1861 in the study area) [START_REF] Salvaudon | Notice de la carte des fore ˆts anciennes du Parc Naturel Re ´gional du Lube ´ron (1:40000) avec re ´fe ´rence aux autres usages du sol[END_REF]). The map scale was 1:40,000 and the median position error about 26 m after georeferencing. LULC in 1958 was photo-interpreted at point scale using 1953 and 1958 aerial orthophotographs. The 2010 LULC map resulted from a combination of contemporary maps [forest cover map (BD Fore ˆtÒ v2), built area map (BD TOPO Ò Ba ˆti) and regional land cover]. For all three dates, five land use categories were defined in accordance with the LULCs defined on the E ´tat-Major map: forest, arable land, pasture, built area, other. Arable land included all types of crops and hay meadows, while pasture corresponded to garrigue (Mediterranean sclerophyllous scrubland) and pastured grasslands. For further details, see Appendix S3.

Biophysical factors

Elevation, slope and aspect were extracted from a digital elevation model (DEM) with a resolution of 25 m, retrieved from the French database BD ALTI Ò (Table 1). Aspect was transformed into northness (the cosine of aspect). Topographic position index (TPI) indicates the relative elevation of a point according to its surrounding environment: above (positive value) or below (negative value). It was derived from the DEM using the Topography Toolbox for ArcGIS 10.3 [START_REF] Jenness | Land facet corridor designer: extension for ArcGIS[END_REF], using a circular neighbourhood method. After testing differing radii (4, 20, 40 and 80 cells), the TPI calculated within a four-cell radius was retained, as this scale explained the higher deviance for all LULCs. We used a 1:50,000 geological vector map (ÓBRGM French Geological Survey 1998) to assess soil potential productivity for agriculture. The 52 geological formations were simplified into three groups according to their potential agricultural productivity: loose substrates (alluvial and colluvial deposits, molasse), hard substrates (hard limestone and conglomerates) and other substrates (acidic, marl and clay soils). We also used physical characteristics of topsoil (top 20 cm) from the European Soil Data Centre (ESDAC, http://esdac.jrc.ec.europa.eu/; Panagos et al. 2012) with a resolution of 500 m: available water capacity (AWC), sand, silt, clay and coarse fragment content (COARSE.F) [START_REF] Ballabio | Mapping topsoil physical properties at European scale using the LUCAS database[END_REF]. Finally, we retrieved the natural waterway network from the BD Carthage Ò 2013 (http://professionnels. ign.fr/bdcarthage), keeping only the main rivers, and then calculated the distance from the nearest river (D.RIVER). Biophysical factors were assumed to be stable throughout the study period.

Socioeconomic factors

We retrieved the 1861 communal population and 1851 farmer population figures from the departmental archives (http://archives.vaucluse.fr; http://www. archives04.fr) and 1954 and 2012 communal population and 1968 and 1999 farmer population figures from the National Institute of Statistics and Economic Studies (INSEE, https://www.insee.fr). Communal population density and farmer density (POP.DENS and FARMER.DENS, respectively) were obtained from the census data for each municipality divided by the municipal area (stable throughout the study period). To assess the effect of distance from transport infrastructure (D.ROAD) or houses (D.HOUSE), we used the main road network and the built area cover at each date (see sources in Table 1). We also examined distance from town centre (D.CENTRE), elevation difference with town centre (ELE.DIFF) and distance from the nearest canal (D.CANAL). For each point, the town centre corresponded to the old village part of its municipality and was extracted from the 1860 LULC map. The canal network was retrieved from the BD Carthage Ò 2013. Distance variables were the Euclidean distance between each point and the nearest edge of the focal object on the corresponding map. Socioeconomic factors corresponded to the data available closest to each date, except for town centre and canal network, assumed to be stable throughout the study period (Table 1). Finally, we calculated the distance from the nearest pre-existing forest edge from LULC maps, and only tested its effect on forest recovery. Analysis LULC transitions were obtained by crossing 1860, 1958 and 2010 LULC at point scale and synthesised in a transition matrix that quantified total gain and loss, net change and persistence, and the past and next transitions of each LULC [START_REF] Pontius | Detecting important categorical land changes while accounting for persistence[END_REF]). We converted the transition matrix into a transition diagram, following [START_REF] Cousins | Analysis of land-cover transitions based on 17th and 18th century cadastral maps and aerial photographs[END_REF].

The effect of all the drivers on LULC and forest recovery was assessed by logistic regressions (Van Doorn and Bakker 2007; Schweizer and Matlack 2014): we fitted models of LULC or forest recovery as a smooth function of the different drivers in a generalised additive model (GAM) with the R package mgcv [START_REF] Wood | Generalized additive models: an introduction with R. Chapman and Hall/CRC[END_REF]) using a logit link function. To take into account spatial autocorrelation in our models [START_REF] Beale | Regression analysis of spatial data[END_REF][START_REF] Saas | Comparison of regression methods for spatially-autocorrelated count data on regularly-and irregularly-spaced locations[END_REF], we incorporated spatial effects as covariates using a smooth function of geographical coordinates (UTM northing and easting), as proposed by [START_REF] Eitzel | Challenges and opportunities in synthesizing historical geospatial data using statistical models[END_REF]. A Moran test was applied to assess the global autocorrelation in the model residuals using the R package spdep [START_REF] Bivand | Driving forces of landscape change-current and new directions[END_REF]; we defined point neighbourhood by Euclidean distance using a binary neighbours list and a maximal threshold distance of 2000 m (Appendix S4).

First, we ran nine models to analyse the effect of biophysical and socioeconomic factors on forest, arable land and pasture in 1860, 1958 and 2010, as they were the most important land uses in terms of area covered and economic interest. Demographic data were log-transformed, and distance data were square root-transformed before analysis to respect normality. Second, we analysed the effect of biophysical and socioeconomic factors and distance from pre-existing forest on forest recovery between 1860 and 1958 using the 1400 points not located in forest in 1860, and between 1958 and 2010 using the 1115 points not located in forest in 1958.

For each LULC at each date, each predictor was tested independently with a univariate model, then a multiple regression was performed using all the significant predictors (full model); if two predictors were correlated (R [ 0.6), only the one explaining a higher deviance was included in the full model and the other was dropped (Appendix S5). Then, a parsimonious final model was obtained by sequentially dropping the single term with the highest non-significant p value from the full model and re-fitting the model until only significant predictors remained. Model fit was followed by deviance partitioning to determine pure and joint effects of each group of predictors (Van Doorn and Bakker 2007): biophysical, socioeconomic and spatial factors for the nine land use models, and biophysical, socioeconomic, spatial factors and distance from pre-existing forest for the two forest recovery models (Appendix S6). As elevation difference with town centre could be considered as a biophysical as well as a socioeconomic factor, we averaged the two decomposition models with the variable considered first as a biophysical and then as a socioeconomic factor.

All the mapping data were processed using ArcGIS 10.3; statistical analysis was carried out using R software v. 3.2.5 (R Core Team 2016).

Results

LULC transitions

In 1860, forest, arable land and pasture were the three main LULCs and arable land was predominant, while in 2010, forest became predominant and pasture was no longer a main LULC (Fig. 3). Forest and built-up area increased continuously from 1860 to 2010 (?103 and ?664%, respectively), while arable land and pasture showed a continuous decrease (-31 and -72%, respectively). Over the whole period, 16.3% of 1860 forest was lost and 46% of the total area experienced at least one LULC change involving one of the five categories, with 30 and 23% of global LULC change between 1860and 1958, and between 1958and 2010, respectively (Table S7-4), respectively (Table S7-4). Forest, arable land and pasture persistence since 1860 constituted 84, 59 and 3% of their original cover, respectively (Fig. 3; Table S7-3). In 1958, 56% of the forest cover originated from forest present in 1860, 27% from pasture and 16% from arable land. In 2010, 74% of the forest cover came from forest present in 1958, 12% from arable land and 13% from pasture. About 79% of 1860 pasture was reforested in 2010, and 28% of 1860 arable land was reforested in 2010. To a lesser extent, arable land was turned into pasture (5%, mostly between 1958 and 2010) or built area (7%, mostly between 1958 and 2010, Table S7-4). Annual forest recovery rate was 0.45% in the first period (1860-1958) and 0.51% in the second one .

Drivers of LULC and forest recovery

Spatial autocorrelation in the model residuals was never significant for the threshold distance retained (Appendix S4) and the 11 models tested. The spatial term was always significant (Tables 2,3).

Forest, arable land and pasture in 1860, 1958 and 2010 Slope, TPI, substrate type and D.HOUSE were the main drivers of LULC at each date (Table 2). Elevation had no effect on the presence of arable land, but influenced pasture in 1860 and 1958, and forest in 2010: both pasture and forest frequencies increased on higher land (Fig. S8-1). Forest frequency was higher on steeper slopes in 1860, and reached an optimum for slopes between 30 and 45% from 1958 onwards; pasture frequency was higher on intermediate and steep slopes (from 20%) and arable land frequency decreased with increasing slope (Fig. S8-2). According to v 2 values, the effect of slope on forest and arable land frequency increased gradually from 1860 to 2010, but decreased for pasture. Arable land was always more frequent on flat topography (TPI = 0), pasture on convex topography (TPI [ 0), and forest on Fig. 3

123

concave topography (TPI \ 0; Table 2; Fig. S8-3). Globally, forest was more frequent on hard substrates and less frequent on loose substrates at each date, whereas arable land showed the opposite pattern, and pasture was more often present on other substrates in 1860 (Fig. 4). The effect of substrate type on forest and arable land decreased gradually from 1860 to 2010, and was non-significant for pasture in 1958 and S8-4). Forest occurrence increased farther from houses up to 600 to 1000 m; arable land occurrence was maximal at 50 m from houses and then decreased with distance from houses (Fig. S8-5), and this pattern became more pronounced in 1958 and 2010. In 1860, pasture occurrence peaked at 100-600 m from houses, but was not influenced by distance from houses after this date. Globally, forest had higher elevation than town centre, whereas arable land decreased with ELE.DIFF (Fig. S8-6).

The forest and arable land models in 1860, 1958 and 2010 globally explained between 44 and 55% of the total deviance. The pasture models explained 28% of the total deviance in 1860 but only 11% in 2010 (Table 2). From 1860 to 2010, the ratio of the pure effects of biophysical drivers to the pure effect of socioeconomic drivers strongly increased for forest (from 0.9 to 4.1), slightly rose for arable land (from 1 to 1.5), and soared for pasture (from 1.7 to no significant effect of socioeconomic factors). The joint effect of biophysical, socioeconomic and spatial factors (for the three land uses and for each date) was always higher than the pure effect of any group of factors, except for pasture. The pure effect of the spatial factor on each LULC decreased from 1860 to 2010.

Forest recovery

Forest recovery from 1860 to 1958 depended on slope, substrate type, soil COARSE.F, and distance from houses and from pre-existing forest (Table 3): it occurred more frequently on slopes between 35 and 40%, on soils with higher COARSE.F, mostly marls and clays, farther from houses, and closer to the 1860 forest edge (Fig. 5). Forest recovery from 1958 to 2010 depended on slope, distance from houses and from preexisting forest, but was not influenced anymore by COARSE.F and substrate type: it increased with increasing slope, with a local peak around 40%, peaked around 500 m from houses and increased with proximity to 1958 forest edge (Fig. 5). Slope and distance from houses had a stronger effect on forest recovery during the first period than during the second one , while proximity to preexisting forest displayed the reverse trend.

The models of forest recovery explained a similar proportion of deviance for the two periods (38 and 33%). During the first period, the pure effect of biophysical factors was predominant (at least twice as Landscape Ecol much as the other pure or joint effects). During the second period, the joint effect of biophysical, distance from pre-existing forest and spatial factors became predominant; and the pure effect of distance from preexisting forest was higher than biophysical and socioeconomic pure effects. We note that all the terms including biophysical and socioeconomic factors (pure and joint effects) decreased and all the terms including distance from pre-existing forest (pure and joint effects) increased between the first and the second period.

Discussion

The doubling of forest cover and the very low rate of deforestation over the whole period confirmed the forest recovery context in the northern Mediterranean region [START_REF] Nogues-Bravo | Assessing the effect of environmental and anthropogenic factors on land-cover diversity in a Mediterranean mountain environment[END_REF][START_REF] Falcucci | Changes in landuse/land-cover patterns in Italy and their implications for biodiversity conservation[END_REF][START_REF] Puddu | Forest changes over a century in Sardinia: implications for conservation in a Mediterranean hotspot[END_REF]. The overall LULC transformation (46% over 150 years) echoes the huge proportion of land degradation or transformation reported at the world scale [START_REF] Antrop | Why landscapes of the past are important for the future[END_REF][START_REF] Kareiva | Domesticated nature: shaping landscapes and ecosystems for human welfare[END_REF]). An even lower rate of LULC persistence could be found with a higher time resolution, as demonstrated by De [START_REF] Keersmaeker | The analysis of spatio-temporal forest changes (1775-2000) in Flanders (northern Belgium) indicates habitat-specific levels of fragmentation and area loss[END_REF] for ancient forest rate. Yet, the reconstruction of the forest area since 1776 in our study area (Fig. 2) indicated that no period of net forest loss occurred since 1860, which means that a reversal of trend was very unlikely to happen between 1860 and 2010. Also, the accuracy of the historical and current map may differ due to position errors [START_REF] Vuorela | Systematic assessment of maps as source information in landscape-change research[END_REF][START_REF] Leyk | Saliency and semantic processing: extracting forest cover from historical topographic maps[END_REF]) but these problems were reduced as far as possible by applying a precise georeferencing correction protocol (Appendix S3). However, we must keep in mind that the E ´tat-Major map was based on land uses, while 1958 and 2010 maps were based on land covers, thus pastures or forests in 1860 might not look the same as in 1958 and 2010. This difference in data transcription between ancient and recent sources, the potential limited precision in historical georeferencing and the unknown minimum mapping unit in these historical maps strongly suggest that ancient land use maps must be analysed with caution.

LULC distribution structured by land accessibility and productivity

Between 1860 and 2010, forest, arable land and pasture were mostly structured by slope, topography position, substrate type and distance from the nearest house, reflecting a spatial distribution that depended on land accessibility and soil productivity. Forests were confined to less productive land (steeper slopes, hard substrates, and soils with large amounts of coarse fragments), and less accessible land (far from houses), while arable land subsisted on the sites that were the most productive (flat terrain and loose substrates) and accessible (near houses). Pastures were preferentially located on unproductive land and with either intermediate or limited accessibility. Our study confirms the major role of slope and soil productivity on LULC and especially on forest cover [START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF][START_REF] Sandel | Human impacts drive a global topographic signature in tree cover[END_REF][START_REF] Lieskovsky | The abandonment of traditional agricultural landscape in Slovakia-analysis of extent and driving forces[END_REF].

Steep slopes require more effort to work on the land (human force, agricultural equipment). Also, the productivity of soils with high nutrient availability (most PNRL soils) depends mainly on their physical properties: loose substrates such as alluvial or colluvial deposits are much easier to till and are associated with deep soils, unlike hard substrates such as conglomerates and hard limestone (''karst'' systems provide very shallow soils). Land accessibility was reflected by distance from houses, but not by distance from roads like elsewhere [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF]. In our study, distance from roads and distance from houses were correlated, and only the effect of the second was retained in the final model. Hence, forests were confined to remote and unproductive areas, where agricultural pressure is likely to have remained nil and which may never have been deforested in the past.

Our results emphasise that socioeconomic and biophysical drivers had an additive effect on LULC spatial distribution. However, their joint effect represented a substantial share of the deviance explained by the statistical models at all three dates, confirming the intertwining of these two groups of drivers [START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF]. A large part of the variability was not explained by the LULC drivers selected. Technological, other economic and political drivers (development of industry, global trading, etc.) together with the human cultural environment and prerequisites (beliefs, knowledge, attitudes, age, health Bu ¨rgi et al. 2004) may be responsible for the unexplained part of LULC, but they were not included in our study because difficult to obtain or spatialize. Moreover, some drivers act at a national or international scale, and assessing them would require a study with a much broader scope, as demonstrated at the world scale by [START_REF] Sandel | Human impacts drive a global topographic signature in tree cover[END_REF].

Abandonment of traditional agriculture and pastoralism led to forest recovery Forest recovered mostly at the expense of arable land and pasture, as a consequence of their abandonment, exemplifying the regional [START_REF] Taillefumier | Contemporary land use changes in prealpine Mediterranean mountains: a multivariate GIS-based approach applied to two municipalities in the Southern French Prealps[END_REF][START_REF] Fabre | E ´volution de l'occupation du sol dans les Alpes-de-Haute-Provence (de ´but du XIXe sie `cle -fin du XXe sie `cle)[END_REF] and Mediterranean trend [START_REF] Nogues-Bravo | Assessing the effect of environmental and anthropogenic factors on land-cover diversity in a Mediterranean mountain environment[END_REF][START_REF] Falcucci | Changes in landuse/land-cover patterns in Italy and their implications for biodiversity conservation[END_REF][START_REF] Sereda | Assessment of changes in landuse development in the Magura and the Eastern Tatras in the years 1772-2003[END_REF]. Our study area suffered from a dramatic abandonment of pasture after 1860, mostly occurring in the first period , while arable land gradually decreased during the two periods. Forest recovery from arable land and pasture resulted from (i) socioecological feedback, and (ii) socioeconomic exogenous drivers (Lambin and Meyfroidt 2010):

(i) The drastic reduction in forest cover in the eighteenth century led to land erosion, and resulted in the implementation of forest protection policy and forest grazing regulation (1860, 1864 and 1882 reforestation laws) and financial incentives to restore forests [START_REF] Fourchy | Les lois du 28 juillet 1860 et 8 juin 1864 sur le reboisement et le gazonnement des montagnes[END_REF][START_REF] Chalvet | Paysages et conflits en Provence[END_REF]. At the same time, the agricultural crisis caused the end of sericulture and madder growing, together with a temporary agricultural shrinkage due to the Phylloxera crisis. All this led to outward migration, as indicated by the decline in rural population registered during the first period (Appendix S1). (ii) The political and economic context reinforced forest management and agricultural practices focused on productivity. The improvement of agricultural yields reduced the need for arable land, and enabled farmers to feed their herds with fodder from meadows and crops, and thus abandon pastoral use of forest, garrigue and grassland [START_REF] Gilbert | Elevage, fore ˆt et socie ´te ´. Analyse socio-historique[END_REF]), as also observed in Germany [START_REF] Bieling | Patterns and causes of land change: empirical results and conceptual considerations derived from a case study in the Swabian Alb, Germany[END_REF].

Such LULC change drivers are common in the northern Mediterranean area [START_REF] Nogues-Bravo | Assessing the effect of environmental and anthropogenic factors on land-cover diversity in a Mediterranean mountain environment[END_REF][START_REF] Falcucci | Changes in landuse/land-cover patterns in Italy and their implications for biodiversity conservation[END_REF][START_REF] Sereda | Assessment of changes in landuse development in the Magura and the Eastern Tatras in the years 1772-2003[END_REF], but in the southern Mediterranean where droughts can be very frequent, climate may be a major driver of LULC change [START_REF] Nash | Regional assessment of landscape and land use change in the Mediterranean region-Morocco case study[END_REF].

Forest cover increased at the expense of the pasture and arable land located on the steeper slopes, consistent with the global trend [START_REF] Van Doorn | The destination of arable land in a marginal agricultural landscape in south Portugal: an exploration of land use change determinants[END_REF][START_REF] Sereda | Assessment of changes in landuse development in the Magura and the Eastern Tatras in the years 1772-2003[END_REF][START_REF] Schweizer | Factors driving land use change and forest distribution on the coastal plain of Mississippi, USA[END_REF][START_REF] Lieskovsky | The abandonment of traditional agricultural landscape in Slovakia-analysis of extent and driving forces[END_REF]. Globally, forest is reported to recover preferentially on less productive, formerly cultivated soils [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Schweizer | Factors driving land use change and forest distribution on the coastal plain of Mississippi, USA[END_REF]. Here, forest significantly recovered on unproductive soils in the first period, but as it recovered on arable land and close to rivers in the second period, it also became frequent on productive soils, and substrate type no longer had any effect on forest recovery between 1958 and 2010. Arable land farther from houses was reforested, while individual housing and cities sprawled mostly at the expense of the surrounding arable land. Less productive and accessible arable land was abandoned and reforested because poorly suited to the new agricultural system directed towards yield increase (large fields, motorised equipment). Traditional terraces were mostly set aside, a major trend throughout Mediterranean Europe [START_REF] Sanz | Long-term forest dynamics and land-use abandonment in the Mediterranean mountains[END_REF][START_REF] Lasanta | Space-time process and drivers of land abandonment in Europe[END_REF]. Meanwhile, urban sprawl replaced former arable land around urban areas (built area was multiplied by 7 between 1860 and 2010) and included the most productive soils (Fig. 4). Traditional agriculture abandonment combined with rapid urban expansion represents a major issue for arable land conservation throughout Europe, both temperate and Mediterranean (Vos and Meekes 1999; [START_REF] Price | Future landscapes of Switzerland: risk areas for urbanisation and land abandonment[END_REF] and around the Mediterranean Basin, north and south [START_REF] Marraccini | Common features and different trajectories of land cover changes in six Western Mediterranean urban regions[END_REF].

From an agro-sylvo-pastoral to a simplified landscape In 1860, the LULC spatial distribution corresponded to the agro-sylvo-pastoral system, specific of the Mediterranean area, based on the complementary use of forest, pasture and arable land [START_REF] Chalvet | Paysages et conflits en Provence[END_REF][START_REF] Nogues-Bravo | Assessing the effect of environmental and anthropogenic factors on land-cover diversity in a Mediterranean mountain environment[END_REF][START_REF] Puddu | Forest changes over a century in Sardinia: implications for conservation in a Mediterranean hotspot[END_REF]: arable land, which required frequent maintenance, was located close to built areas, while forest was confined to remote areas. Pasture was either in intermediate locations between arable land and forest, or even farther out than forest (Fig. 6). Sheep or goat herds were kept near crops to enrich the soil with their manure, and grazed on cropland during the late season. As they also needed the proximity of forests to supplement their diet, pasture was in intermediate locations. Located farther from houses were upland pastures grazed during summer, where shepherds could more easily watch over their flocks, and the topography was smoother. This may explain why pasture location was less explained by biophysical and socioeconomic drivers compared to forest and arable land.

The socioeconomic system developed in the late nineteenth century transformed the landscape through the specialisation of production systems. The nineteenth century landscape gradually turned into a dichotomy of arable land and forest during the twentieth century, as described by [START_REF] Barbier | The forest transition: towards a more comprehensive theoretical framework[END_REF], and showed the early stages of a landscape dominated by forest, arable land and artificialized areas for the twenty first century. As slope and distance from houses were drivers of forest recovery, their effects were reinforced between 1860 and 2010, leading to a sharper spatial partitioning of forest and arable land. This spatial discontinuity has been emphasised over the last 150 years throughout the northern Mediterranean area [START_REF] Taillefumier | Contemporary land use changes in prealpine Mediterranean mountains: a multivariate GIS-based approach applied to two municipalities in the Southern French Prealps[END_REF][START_REF] Nogues-Bravo | Assessing the effect of environmental and anthropogenic factors on land-cover diversity in a Mediterranean mountain environment[END_REF]. The traditional Mediterranean landscape has thus progressively turned into a landscape comparable to temperate lowland regions, losing its specificity due to the abandonment of traditional practices.

Our results underlined the importance of geographical context regarding the relative effects of biophysical and socioeconomic drivers. We showed that biophysical drivers explain forest recovery better than socioeconomic drivers, as in [START_REF] Wulf | Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period[END_REF], but above all that socioeconomic drivers had a stronger influence on LULC spatial distribution in 1860 than subsequently. This trend might be specific to Mediterranean or mountainous landscapes, as it is in accordance with Van Doorn and Bakker (2007), while [START_REF] Wulf | Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period[END_REF] and [START_REF] Schweizer | Factors driving land use change and forest distribution on the coastal plain of Mississippi, USA[END_REF] found that the effect of socioeconomic drivers on LULC increased with time. In our study area, the LULC distribution observed in 1860 resulted from a very long agriculture and pastoralism development in the previous centuries: during this ancient period, the importance of agriculture and pastoralism had partly overridden the biophysical constraints, and thus had weakened the statistical relationship between biophysical drivers and LULC. With the relaxation of agriculture and pastoralism pressure after 1860, the sites that were previously cultivated or grazed despite biophysical constraints (particularly steep slope) were preferentially set aside owing to their lower economic profitability [START_REF] Taillefumier | Contemporary land use changes in prealpine Mediterranean mountains: a multivariate GIS-based approach applied to two municipalities in the Southern French Prealps[END_REF]; this resulted in strengthening the statistical link between biophysical drivers and LULCs, as detected in 1958 and 2010.

Forest recovery has become independent of human decisions

As expected, proximity to pre-existing forest was a significant driver of forest recovery [START_REF] Serra | Land-cover and land-use change in a Mediterranean landscape: a spatial analysis of driving forces integrating biophysical and human factors[END_REF][START_REF] Crk | Forest recovery in a tropical landscape: what is the relative importance of biophysical, socioeconomic, and landscape variables?[END_REF][START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Puddu | Forest changes over a century in Sardinia: implications for conservation in a Mediterranean hotspot[END_REF]. Interestingly, we showed that the importance of this driver increased between the first and second period. From 1860 to 1958, forest recovery was mainly shaped by biophysical and socioeconomic drivers (slope and distance from the nearest house): farmers abandoned first the least productive and least accessible land. From 1958 to 2010 however, forest recovery was much more strongly determined by proximity to tree seed sources. Our results also indicated a slight acceleration of forest recovery during the last decades. All these findings suggest that forest recovery has become an increasingly self-sustained process, implying a generalisation of land abandonment in the study area: human activities claim space closer to built areas, encroaching agricultural land, but no longer in remote areas, letting forest expand from already existing forest patches. A finer time resolution would enable us to detect whether this trend accelerated or decelerated during the last decades.

The literature records that forest recovery and deforestation are influenced by the same main drivers, such as slope, soil productivity and distance from roads [START_REF] Flinn | Environmental causes and consequences of forest clearance and agricultural abandonment in central New York, USA[END_REF][START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF], and occur close to pre-existing forest [START_REF] Wulf | Forest cover changes in the Prignitz region (NE Germany) between 1790 and 1960 in relation to soils and other driving forces[END_REF][START_REF] Mitsuda | A review of spatial-explicit factors determining spatial distribution of land use/land-use change[END_REF][START_REF] Schweizer | Factors driving land use change and forest distribution on the coastal plain of Mississippi, USA[END_REF]. We can therefore assume that the areas reforested since 1860 may have been those that were preferentially deforested before 1860, and that forest expansion and regression phases in previous centuries may have mainly occurred in the same areas [START_REF] Paulet | Fore ˆts et pression de ´mographique en Basse Provence[END_REF]. Thus we can expect future LULC changes to operate preferentially in these fluctuating zones.

Conclusion

Our study confirmed a major trend throughout European and particularly Mediterranean landscapes in the last 150 years: widespread forest recovery as a result of the abandonment of traditional agriculture and pastoralism, located on the most remote and unproductive land. We confirmed that the three main drivers of forest recovery were slope, distance from house and distance from pre-existing forest. We specifically showed that the main drivers of LULC were similar at the three dates, which suggests that LULC spatial distribution obeyed the same rules over a much longer time frame. We also found that extreme biophysical conditions (arable land on loose substrate and flat topography vs. forest on hard, shallow soils and steep slopes) have undergone very little change over the past 150 years. Interestingly, we showed that the part of LULC distribution explained by biophysical and socioeconomic drivers changed in time differently between temperate and Mediterranean regions, due to different long-term use of the landscape.

Fig. 1

 1 Fig. 1 Study area and forest cover change since 1860. Forest cover change is represented according to 1860, 1958 and 2010 forest cover. Ancient forest: persistent since 1860, recent forest:

Fig. 2

 2 Fig. 2 Forest cover change (ha) in Vaucluse and Alpesde-Haute-Provence since 1776. See Appendix S2 for further details on the historical sources used

Fig. 4 Fig. 5

 45 Fig. 4 Proportion of the five LULCs on different substrates (loose, hard and other substrates) in 1860, 1958 and 2010

Fig. 6

 6 Fig. 6 Spatial distribution of LULC in 1860 and 2010, adapted from Verdier (2013). This diagram includes a productivity and accessibility gradient from centre to periphery. Red: built area, yellow: arable land, light green: pasture, dark green: forest. (Color figure online)

Table 1

 1 Biophysical, socioeconomic factors and distance from pre-existing forest: range, unit, source and date available in1860, 1958 and 2010

	Factors	Ranges and units	Sources	Date
				available
	Biophysical factors
	Elevation	55 to 1210 m	DEM 25 m (BD ALTI Ò )
	Slope	0 to 98%	Idem
	TPI	-26.2 to 25.9 m	Idem
	Northness	Exposure index -1 (south) to 1 (north)	Idem
	Substrate type	Three categories: 44% loose, 38% hard and 18% other	ÓBRGM lithology map
	AWC	0.08 to 0.13	ESDAC
	SAND	14.2 to 57.3%	Idem
	SILT	28.4 to 58.4%	Idem
	CLAY	12.3 to 37.2%	Idem
	COARSE.F	6.7 to 36.9%	Idem
	D.RIVER	0.8 to 7294 m	2013 BD Carthage Ò map
	Socioeconomic factors
	POP.DENS	1861: 5 to 163.1 inhab km -2 , 1954: 1.7 to 323.4 inhab km -2 ,	Archives and INSEE	1861,
		2012: 1.6 to 551.4 inhab km -2		1954,
				2012
			Idem	1851,
				1968,
				1999
	D.HOUSE	1860: 0 to 2414 m, 1958: 0 to 2598 m, 2010: 0 to 2697 m	1860, 1958 and 2010 land use	1860,
			map (built area)	1958,
				2010
	D.ROAD	1860: 1 to 7225 m, 1958: 0 to 2860 m, 2010: 0 to 2642 m	1860 and 1958 road maps,	1860,
			2009 BD Route Ò	1958,
				2009
	D.CENTRE	41.2 to 10,300 m	Town centre
	ELE.DIFF	-294 to 789 m	DEM 25 m
	D.CANAL	0.1 to 19,060 m	2013 BD Carthage

FARMER.DENS 1851: 4.2 to 58 inhab km -2 , 1968: 0 to 23.7 inhab km -2 , 1999: 0 to 11.6 inhab km -2 Ò map D.PREEX.FOREST 1958: 0 to 3174 m, 2010: 0 to 3209 m 1860 and 1958 land use maps 1860, 1958

Table 2

 2 Final GAM models for major LULCs (forest, arable and pasture) at each date, and deviance partitioning (%)

	Landscape Ecol
		1860			1958			2010
		Forest	Arable	Pasture	Forest	Arable	Pasture Forest	Arable	Pasture
	LULC frequency	0.27	0.51	0.19	0.42	0.47	0.09	0.55	0.35	0.05
	Significance and direction of effects
	Biophysical factors
	Elevation	-	-	6* (?)	-	-	16* (?) 20*** (?) -	-
	Slope	29***	61***	36***	81***	102***	16**	138***	136***	-
		(?)	(-)	(?)	(opt)	(-)	(?)	(opt)	(-)
	TPI	-	25***	7** (?)	35***	48***	12**	18** (-)	27***	-
			(opt)		(-)	(opt)	(?)		(opt)
	Substrate type	26***	38***	27***	20***	28***	-	17***	14***	-
	COARSE.F	17***	-	-	19***	9** (-)	-	18*** (?) -	-
		(?)			(?)
	SAND	13*	-	-	-	-	-	-	-	-
		(opt)
	D.RIVER	-	7** (?)	-	-	31***	-	32*** (-) 35***	29***
						(?)			(?)	(-)
	Socioeconomic factors
	D.ROAD	9** (?) 15***	-	-	-	-	-	-	-
			(-)
	D.HOUSE	60***	137***	37***	101***	191***	-	99*** (?) 176***	-
		(?)	(-)	(opt)	(?)	(-)			(-)
	D.CENTRE	23***	20***	-	7** (?)	-	-	-	-	-
		(?)	(-)
	ELE.DIFF	9* (?)	10* (-)	-	9* (?)	8** (-)	-	7* (?)	17***	-
									(-)
	Spatial term	180***	99***	193***	136***	67***	146*** 17*	49***	40***
	Deviance partitioning
	PE(BIO)	4.65	5.45	4.10	8.95	14.20	5.20	10.15	13.95	2.55
	PE(SOC)	5.05	5.25	2.40	4.20	6.20	0.00	2.50	9.60	0.00
	PE(SPA)	11.90	4.90	14.00	7.90	3.10	17.70	1.20	2.90	7.00
	JE(BIO, SOC)	2.90	3.20	-0.10	3.75	3.90	0.00	3.85	-0.25	0.00
	JE(BIO, SPA)	5.40	6.75	6.36	4.15	3.20	6.50	9.00	5.25	1.55
	JE(SOC, SPA)	3.85	5.60	0.70	2.80	4.35	0.00	1.95	1.95	0.00
	JE(BIO, SOC, SPA) 15.85	13.15	0.74	16.35	20.05	0.00	18.05	15.10	0.00
	Total explained	49.60	44.30	28.20	48.10	55.00	29.40	46.70	48.50	11.10
	deviance
	The table gives v 2 values of each predictor in the final model and total deviance explained (%) for each model used for the deviance
	partitioning. All models were applied on 1916 points. Significance of the relation are provided by stars reflecting p values: 0\***\
	0.001\**\0.01\* \0.05. Northness, AWC, POP.DENS, FARMER.DENS and D.CANAL were tested, but were not significant.
	The direction of the relation between each LULC and each predictor is given by ? (positive relation), -(negative relation) and 'opt'
	(optimum at intermediate values). See Appendix S8 for detailed relations. All other lines correspond to the joint effect (JE) of the
	different combinations of these groups of factors
	PE(BIO) pure effect of biophysical factors, PE(SOC) pure effect of socioeconomic factors, PE(SPA) pure effect of spatial factor
										123

2010. COARSE.F had an effect at each date on forest location only: forest mostly occurred on soils with higher COARSE.F (Table

2

; Fig.

Table 3

 3 Final GAM models for forest recovery from 1860 to 1958 (1400 points) and from 1958 to 2010 (1115 points), and deviance partitioning (%)

		Forest recovery
		1860-1958	1958-2010
	Forest recovery frequency	0.25	0.24
	Significance and direction of effects
	Biophysical factors
	Slope	97***	56***
	Substrate type	25***	-
	COARSE.F	6*	-
	Socioeconomic factors
	D.HOUSE	61***	24***
	D.PREEX.FORESTS	32***	60***
	Spatial term	74***	14***
	Deviance partitioning
	PE(BIO)	10.50	3.40
	PE(SOC)	3.80	1.80
	PE(D.FOR)	2.30	4.70
	PE(SPA)	7.00	1.50
	JE(BIO, SOC)	-0.10	-0.30
	JE(BIO, D.FOR)	1.30	3.90
	JE(SOC, D.FOR)	0.50	1.20
	JE(BIO, SPA)	4.80	2.70
	JE(SOC, SPA)	0.70	0.70
	JE(D.FOR, SPA)	0.20	0.50
	JE(BIO, SOC, D.FOR)	0.20	-0.10
	JE(BIO, SOC, SPA)	2.25	0.00
	JE(BIO, D.FOR, SPA)	5.05	11.00
	JE(SOC, D.FOR, SPA)	-0.10	1.50
	JE(BIO, SOC, D.FOR, SPA)	-0.60	0.60
	Total explained deviance	37.80	33.10
	The table gives v 2 values of each predictor in the final model
	and total deviance explained (%) for each model used for the
	deviance partitioning. Significance of the relation are provided
	by stars reflecting p values: 0\***\0.001\**\0.01\*\
	0.05. Elevation, northness, TPI, AWC, SAND, D.RIVER,
	D.ROAD, D.CENTRE, POP.DENS, FARMER.DENS,
	ELE.DIFF and D.CANAL were tested, but were not
	significant. All other lines correspond to the joint effect (JE)
	of the different combinations of these groups of factors
	PE(BIO) pure effect of biophysical factors, PE(SOC) pure
	effect of socioeconomic factors, PE(D.FOR) pure effect of
	distance from pre-existing forest, PE(SPA) pure effect of
	spatial factor

Acknowledgements The 1860 LULC and lithology maps were provided by the PNRL, and 1958 orthophotographs were provided by the National Institute of Geographic and Forestry Information. This work is part of a PhD Thesis supported by the Re ´gion Provence-Alpes-Co ˆte-d'Azur and IRSTEA. The UMR 1137 Forest Ecology and Ecophysiology is supported by a Grant overseen by the French National Research Agency (ANR) as part of the ''Investissements d'Avenir'' Program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). The authors thank J. Wu and two anonymous reviewers for their constructive and helpful comments. We also thank P. K. Roche and K. Verheyen for their advice.


MSc,* † Thomas Giraud

PhD Charlotte Jeanneau

MSc Madison Bergmann

PhD,* † Patrick Laurent

PhD Imad About
email: imad.about@univ-amu.fr

Tricalcium Silicate Capping Materials Modulate Pulp Healing and Inflammatory Activity In Vitro

Keywords: Pulp capping materials, pulp healing, pulp inflammation, tricalcium silicates

Introduction: On stimulation by lipoteichoic acid or by a physical injury, fibroblasts have been shown to play a major role in the initiation of the pulp inflammatory reaction and healing through secretion of complement proteins and growth factors. The application of direct pulp-capping materials on these cells may interfere with the inflammatory and the healing processes within the pulp's inextensible environment. This work was designed to study in vitro the effects of silicate-based materials on pulp fibroblast modulation of the initial steps of pulp inflammation and healing. Methods: The effects of Biodentine, TheraCal, and Xeno III eluates were studied on lipoteichoic acid-stimulated and physically injured fibroblasts. Cytokine secretion (interleukin 6, vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-b1) was quantified by enzyme-linked immunosorbent assay. Inflammatory THP-1 adhesion to endothelial cells and their migration and activation were studied in vitro. Human pulp fibroblast proliferation was investigated with the MTT test, and their migration to the injury site was studied with the scratch healing assay. Results: Interleukin 6 and vascular endothelial growth factor secretion increased with all materials but to a lesser extent with Biodentine. Fibroblast growth factor-2 and transforming growth factor-b1 secretion was significantly higher with Biodentine than with all other materials. THP-1 cell adhesion to endothelial cells and their activation were reduced by Biodentine and TheraCal. However, their migration decreased only with Biodentine. Fibroblast proliferation significantly increased with Biodentine but significantly decreased with Xeno III after day 6. Finally, only Biodentine induced fibroblast migration to the injury site in the scratch assay. Conclusions: These results confirm that pulp-capping materials affect the early steps of pulp inflammation and healing. They show that Biodentine had the highest pulp healing and anti-inflammatory potential when compared with the resin-containing materials. This highlights the interest of the material choice for direct pulp-capping. (

D eep carious or physical lesions may lead to dental pulp injuries. Subsequently, both pulpinfiltrating bacteria and injured tissues initiate an inflammatory reaction [START_REF] Massler | Pulpal reactions to dental caries[END_REF].

The inflammatory reaction implies secretion of cytokines that contribute to immune cell recruitment. These cells adhere on the vascular endothelium and transmigrate and reach the injured tissue guided by a chemotaxis gradient to be finally activated as macrophage-like cells [START_REF] Ley | Getting to the site of inflammation: the leukocyte adhesion cascade updated[END_REF]. Many cytokines such as interleukin (IL) 6, IL-8, and IL-1b are involved in the inflammatory events. Others, such as vascular endothelial growth factor (VEGF), play a role both in the vasodilation of the vessels after the initiation of the inflammation and in pulp tissue vascularization during the regenerative processes after pulp hypoxia [START_REF] Rombouts | Pulp vascularization during tooth development, regeneration, and therapy[END_REF].

Pulp healing implies an initial inflammatory reaction followed by fibroblasts and dental pulp stem cell proliferation and migration to the injury site to replace the lost tissue. It has been well-demonstrated that many of these events are locally regulated. Many growth factors involved in these steps are either sequestered in the dentin and released because of the acidic environment during the carious progression or secreted by the underlying pulp cells [START_REF] Tran-Hung | Quantification of angiogenic growth factors released by human dental cells after injury[END_REF][START_REF] Roberts-Clark | Angiogenic growth factors in human dentine matrix[END_REF][START_REF] Cassidy | Comparative analysis of transforming growth factor-b isoforms 1-3 in human and rabbit dentine matrices[END_REF]. Among these, transforming growth factor b1 (TGF-b1) has been shown to play a key role in dental pulp stem cell migration and differentiation [START_REF] Laurent | Biodentine(TM) induces TGF-b1 release from human pulp cells and early dental pulp mineralization[END_REF][START_REF] Mathieu | Usefulness of controlled release of growth factors in investigating the early events of dentin-pulp regeneration[END_REF], whereas fibroblast growth factor 2 (FGF-2) has been shown to be involved in dentin-pulp complex regeneration inducing pulp cell proliferation and migration [START_REF] Shimabukuro | Fibroblast growth factor-2 regulates the cell function of human dental pulp cells[END_REF].

In addition, recent data have confirmed the involvement of the complement system in the balance between pulp inflammation and regeneration through secretion of active molecules such as C3a, C5a, and membrane attack complex within this inextensible environment [START_REF] Rufas | Complement C3a mobilizes dental pulp stem cells and specifically guides pulp fibroblast recruitment[END_REF][START_REF] Jeanneau | Can pulp fibroblasts kill cariogenic bacteria? role of complement activation[END_REF][START_REF] Chmilewsky | Pulp progenitor cell recruitment is selectively guided by a C5a gradient[END_REF]. Furthermore, it has been shown that pulp-capping material can modulate C5a secretion [START_REF] Giraud | Complement activation by pulp capping materials plays a significant role in both inflammatory and pulp stem cells' recruitment[END_REF].

Tricalcium silicates are now considered as the materials of choice for vital pulp therapy because histologic studies have demonstrated dentin-pulp regeneration without pulp inflammation in vivo [START_REF] Tran | Effect of a calcium-silicate-based restorative cement on pulp repair[END_REF] and in human teeth [START_REF] Nowicka | Response of human dental pulp capped with biodentine and mineral trioxide aggregate[END_REF][START_REF] Jeanneau | Light-cured tricalcium silicate toxicity to the dental pulp[END_REF]. Modifications of these types of materials aiming at reducing their setting time led to the development of light-cured tricalcium silicates such as TheraCal (Bisco, Schaumburg, IL). However, the presence of a high percentage of resins in this material raises questions about its possible effects on the pulp inflammatory reaction because of its reported toxicity [START_REF] Jeanneau | Light-cured tricalcium silicate toxicity to the dental pulp[END_REF][START_REF] Lee | Comparative study of pulpal responses to pulpotomy with ProRoot MTA, RetroMTA, and TheraCal in dogs' teeth[END_REF].

The therapeutic use of these materials in direct pulp-capping implies their direct application on the underlying fibroblasts that have been reported to play a significant role in initiating the pulp inflammatory response and regeneration. Indeed, these cells express and secrete complement active proteins such as C3a and C5a, known as anaphylatoxins, which induce vascular modifications and recruitment of inflammatory cells during the inflammatory reaction [START_REF] Klos | The role of the anaphylatoxins in health and disease[END_REF] and the recruitment of pulp stem cells during the regeneration [START_REF] Chmilewsky | Pulp progenitor cell recruitment is selectively guided by a C5a gradient[END_REF][START_REF] Chmilewsky | Sources of dentin-pulp regeneration signals and their modulation by the local microenvironment[END_REF]. Pulp fibroblasts also secrete VEGF, platelet-derived growth factor, and FGF-2, which directly affect pulp neoangiogenesis. Previous studies on cell injury or incubation with lipoteichoic acid (LTA), a component of grampositive bacteria, demonstrated a significant increase in the secretion of these molecules. This demonstrates a link between fibroblast stimulation and the initiation of the inflammatory reaction or regeneration process [START_REF] Tran-Hung | Quantification of angiogenic growth factors released by human dental cells after injury[END_REF][START_REF] Tran-Hung | Role of human pulp fibroblasts in angiogenesis[END_REF][START_REF] Chmilewsky | Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration[END_REF].

The aim of this in vitro study was to determine the influence of tricalcium silicate-based capping materials on pulp fibroblast stimulation and involvement in the initial steps of pulp inflammation and healing and to investigate the consequences of adding resins on these events.

Although Biodentine (Septodont, Saint-Maur-des-Foss es, France) and TheraCal are tricalcium silicate-based materials, TheraCal also contains resins to allow its light-curing. Xeno III (Densply Sirona, York, PA) is a resin bonding system used as a control. Eluates of the above mentioned materials were applied on LTA-stimulated and physically injured fibroblasts. We evaluated fibroblast secretion of cytokines involved in inflammation (IL-6, VEGF) and regeneration (TGF-b1, FGF-2). We also studied inflammatory cell adhesion on endothelial cells, their migration, and activation. Finally, we investigated fibroblast proliferation and colonization of the injury area.

Materials and Methods

Reagents

Media, reagents, and cell culture supplies were from Dutscher (Brumath, France).

Human Pulp Fibroblast Culture

Human primary pulp cells were prepared from immature third molars extracted for orthodontic reasons (male and female patients younger than 18 years) in compliance with French legislation (informed patients consent and institutional review board approval of the protocol used) by the explant outgrowth method as described previously [START_REF] About | Human dentin production in vitro[END_REF]. After sorting with the STRO-1 mesenchymal stem cell marker [START_REF] Chmilewsky | Pulp progenitor cell recruitment is selectively guided by a C5a gradient[END_REF], pulp stem cells were removed. The remaining STRO-1 negative cell population was characterized as fibroblasts. Briefly, they were positive for fibroblast surface protein and negative for Nanog, Oct3/4, Sox 2, Klf 4, CD 44, CD90, CD 105, CD 146, and CD 166 [START_REF] Rufas | Complement C3a mobilizes dental pulp stem cells and specifically guides pulp fibroblast recruitment[END_REF][START_REF] Chmilewsky | Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration[END_REF]. These fibroblasts were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B and used in this study.

Conditioned Medium Preparation

The materials were prepared according to manufacturers' instructions and inserted into standardized silicone molds. Samples of set materials were sterilized with UV (20 minutes on each side) and incubated in serum-free MEM (0.05 cm2 /mL) for 24 hours at 37 C, 5% CO 2. as described previously [START_REF] Giraud | Complement activation by pulp capping materials plays a significant role in both inflammatory and pulp stem cells' recruitment[END_REF]. These media will hereafter be called conditioned media. MEM culture medium was used as a control.

Fibroblast Treatment

To simulate bacterial infection in caries lesions, confluent fibroblast cultures (30,000 cells/cm 2 ) were stimulated with 1 mg/mL LTA for 4 hours. In addition, physical injuries were performed to simulate the cell injury during the cavity preparation process. This was done with sterile scalpels in vertical and horizontal directions (10 in each direction in 6-well plates and 5 on each in 12-well plates) in serum-free MEM. For clarity, these LTA-stimulated and injured fibroblasts will be hereafter referred to as treated fibroblasts.

Conditioned Medium Toxicity

Treated fibroblasts were incubated with the conditioned or MEM control medium. After 4, 24, and 48 hours the supernatants were removed, and MTT test (Sigma-Aldrich, St Louis, MO) was performed as described previously [START_REF] Mathieu | Usefulness of controlled release of growth factors in investigating the early events of dentin-pulp regeneration[END_REF] with the MTT substrate (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

Cytokine Secretion by Treated Fibroblasts

Treated fibroblasts were cultured in conditioned or MEM control medium in 12-well plates. After 4, 24, and 48 hours, supernatants were used for cytokine (IL-6, VEGF, TGF-b1, or FGF-2) quantification by the enzyme-linked immunosorbent assay using Duoset kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Results are expressed in pg/mL.

Culture of Human Umbilical Vein Endothelial Cells and Inflammatory THP-1 Cells

Human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth medium 2 (PromoCell). THP-1 cells, a human monocytic cell line (Sigma, St Quentin Fallavier, France), were cultured in RPMI (supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, 2 mmol/L L-glutamine, and 1 mmol/L sodium pyruvate), as described [START_REF] Tsuchiya | Establishment and characterization of a human acute monocytic leukemia cell line (THP-1)[END_REF].

THP-1 Cell Recruitment Sequence

THP-1 Cell Adhesion on Endothelial Cells. Confluent HUVECs (80,000 cells/cm 2 ) were cultured for 4 hours in 6-well plates with supernatants from treated fibroblasts cultured in conditioned or MEM control medium for 24 hours. THP-1 cells were first incubated with 0.1 mmol/L BCECF acid (2 0 ,7 0 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) for 1 hour. Suspension of BCECF labeled THP-1 (25,000 cells/mL) was applied on the HUVEC monolayer for 30 minutes under gentle orbital agitation. Media were then removed, and HUVEC monolayers were washed 4 times with phosphate-buffered saline with calcium and magnesium (PBS). The THP-1 adherent cells were counted in 5 random fields by using a fluorescent microscope (Â100). Results are expressed as percentage of the control. THP-1 Cell Migration. Migration assays were performed in Boyden chambers. Treated fibroblasts were plated in the lower chambers (12-well plates) with 1 mL conditioned or MEM control medium. The upper chambers were seeded with 25,000 THP-1 cells in 100 mL. THP-1 cells were activated with phorbol myristate acetate (400 ng/mL, 24 hours) before migration. After 24 hours, migrating cells on the lower side of the insert membrane were fixed (15 minutes, cold ethanol 70%) and stained with eosin (20 minutes). Migrating cells were counted in 5 random fields by using light microscopy (Â100). Results are expressed as percentage of control. THP-1 Cell Activation. THP-1 suspension (25,000 cells/mL) was cultured 24 hours in 12-well plates with supernatants from treated fibroblasts cultured for 24 hours in conditioned or MEM control medium. Then, the wells were washed 4 times with PBS. This ensures removal of dead cells and allows for adhesion of living THP-1 cells only. Adherent cell nuclei were stained (20 minutes) with DAPI (4 0 ,6-diamidino-2-phenylindole), and the number of activated (adherent) cells was counted in 5 random fields by using a fluorescent microscope (Â100). Results are expressed as percentage of the control.

Human Pulp Fibroblast Proliferation

Pulp fibroblasts were cultured at a low density (1000 cells/cm 2 ) in 12-well plates for 24 hours. After this period, media were replaced by supernatants from treated fibroblasts cultured for 24 hours with conditioned or MEM control medium. After 3, 6, and 9 days the supernatants were removed, and a MTT test was performed to measure cell proliferation [START_REF] Jia | Insulin and heparin-binding epidermal growth factor-like growth factor synergistically promote astrocyte survival and proliferation in serumfree medium[END_REF]. Results are expressed as percentage of control at day 3 considered as the baseline.

Fibroblasts Scratch Wound Healing Assays

Confluent fibroblasts (30,000 cells/cm 2 ) in 6-well plates were injured with a pipette tip and incubated with supernatants from treated fibroblasts cultured 24 hours with conditioned or MEM control medium. After 12 hours of incubation, cell nuclei were stained with 1 mg/mL DAPI (20 minutes), and migrating cells to the injured zone were counted in 5 random fields under a fluorescence microscope (Â100). Results are expressed as percentage of control.

Statistical Analysis

All experiments were done in triplicate and repeated 3 times. Data are expressed as mean AE standard error of mean. Student t was used to determine statistical significance (P < .05).

Results

Pulp-Capping Materials Conditioned Media Were Not Toxic to Treated Fibroblasts

There was no statistically significant difference in the viability of treated fibroblasts cultured with pulp-capping materials conditioned media compared with the control regardless of the incubation time (data not shown).

Biomaterials Modified Cytokine Secretion

Applying all biomaterial conditioned media on treated fibroblasts led to a significant increase of IL-6 secretion. However, this increase was significantly lower with Biodentine regardless of the incubation period (Fig. 1A). VEGF was secreted in all culture conditions. This secretion increased with all materials but was significantly higher with resincontaining materials (TheraCal and Xeno III) (Fig. 1B). Biodentine induced a significantly higher secretion of TGF-b1 after 24 and 48 hours, whereas a decrease was observed with TheraCal and Xeno III after 24 hours (Fig. 1C). Finally, a rapid increase in FGF-2 secretion was observed after 4 hours with all materials. However, a significantly higher After 24 and 48 hours, the secretion was statistically higher in all conditioned media compared with the control but to a lesser extent with Biodentine. (C) Treated fibroblasts secreted significantly more TGF-b1 after 24 and 48 hours only when incubated with Biodentine. (D) Treated fibroblasts incubated with all conditioned media showed significant increase in FGF-2 compared with the control, but this level was higher with Biodentine compared with TheraCal and Xeno III. Results are expressed in pg/mL. *Significant difference as compared with the control; **significant differences between 2 conditions (P < .05).

Basic Research-Biology

JOE -Volume -, Number -, -2018
Tricalcium Silicates and Pulp Inflammation level was obtained with Biodentine as compared with TheraCal and Xeno III regardless of the incubation period (Fig. 1D).

Inflammatory Cell Recruitment Modulation

Inflammatory THP-1 cell adhesion on HUVEC monolayer assay protocol is represented (Fig. 2A) and illustrated with representative pictures used for cell counts (Fig. 2Ba-d). THP-1 cell adhesion significantly decreased when the HUVECs were cultured with Biodentine and TheraCal conditioned media. Adhesion with Biodentine was significantly lower than with TheraCal. By contrast, Xeno III significantly increased this adhesion as compared with the control and with the other materials (Fig. 2C). THP-1 cell migration significantly decreased only with Biodentine conditioned medium (Fig. 3A). Activation of THP-1 cells representative pictures were presented (Fig. 3Ba-d) and used for cell count. This activation decreased with silicates-containing materials conditioned media (Biodentine and TheraCal), whereas it increased with Xeno III (Fig. 3Be).

Materials Modulate Human Pulp Fibroblast Proliferation and Migration

Biodentine conditioned medium significantly increased fibroblast proliferation at all incubation periods (Fig. 4A) and significantly increased fibroblast colonization of the injury area (Fig. 4Ba-e). By contrast, Xeno III decreased fibroblast proliferation at 6 and 9 days, whereas TheraCal significantly increased fibroblast proliferation only at 6 days but to a lesser extent than Biodentine (Fig. 4A). Finally, Ther-aCal and Xeno III did not affect fibroblast colonization of the injury area (Fig. 4Ba-e).

Discussion

This work shows that after simulating pulp fibroblast injury and bacterial infection, applying pulp-capping material eluates on these cells affects pulp inflammation and healing processes. Both events are crucial because their balance is a pre-requisite for the regeneration process within the pulp's inextensible environment. This study shows that although tricalcium silicates shift the balance toward the healing process, resin-containing materials drive this balance toward an inflammatory reaction.

The influence of these materials on pulp fibroblast response was investigated on LTA-stimulated and injured fibroblasts. This simulated some of the clinical aspects of pulp-capping procedure that is performed after carious decays on pulps subjected to injuries with dental burs during the cavity preparation. The physical injury protocol has been reported in multiple studies in different tissues using pipette tips or scalpels [START_REF] Rajan | Conditioned medium from human gingival mesenchymal stem cells protects motor-neuron-like NSC-34 cells against scratch-injury-induced cell death[END_REF][START_REF] L€ O€ Ov | Identification of injury specific proteins in a cell culture model of traumatic brain injury[END_REF]. Because the majority of bacterial species involved in the cariogenic process are gram positive, the presence of these bacteria is simulated in this work by incubating the fibroblasts with LTA [START_REF] Jeanneau | Can pulp fibroblasts kill cariogenic bacteria? role of complement activation[END_REF][START_REF] Chmilewsky | Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration[END_REF][START_REF] Keller | Toll-like receptor 2 activation by lipoteichoic acid induces differential production of pro-inflammatory cytokines in human odontoblasts, dental pulp fibroblasts and immature dendritic cells[END_REF].

Here, materials were already set and were not placed in direct contact with the cells. Instead, eluates of these materials were applied on the cells. Indeed, a necrotic zone is formed in vivo because of the alkaline pH of calcium hydroxide or tricalcium silicate-based materials [START_REF] Laurent | Human tooth culture: a study model for reparative dentinogenesis and direct pulp capping materials biocompatibility[END_REF][START_REF] Aeinehchi | Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report[END_REF], whereas only eluates of set capping materials reach the underlying viable cells.

The proinflammatory activity was studied here by investigating the effects of these materials on the pulp fibroblasts in the early steps of the inflammation including secretion of proinflammatory cytokines, adhesion of inflammatory THP-1 cells on blood vessel endothelial cells, their migration, and activation at the injury site. Indeed, although injured cells initiate the inflammatory reaction by secreting proinflammatory cytokines, our study demonstrates that applying pulp-capping materials conditioned media on these cells modulate cytokine secretion. All materials induced a significant release of IL-6 and VEGF. IL-6 is considered a potent cytokine playing a major role in the acute inflammation process [START_REF] Heinrich | Interleukin-6 and the acute phase response[END_REF], and VEGF is known to increase vascular permeability [START_REF] Hippenstiel | VEGF induces hyperpermeability by a direct action on endothelial cells[END_REF]. This allows inflammatory cells to adhere to blood vessel endothelial cells and their migration to the injury site where these cells are activated. The secretion level of IL-6 and VEGF was significantly higher with resin-containing materials than with Biodentine. This is confirmed by the fact that Xeno III significantly induced THP-1 adhesion to endothelial cells and their activation. This reflects a stimulation of the inflammatory reaction and confirms the resin-containing materials' inflammatory potential when applied directly to the dentin-pulp complex. By contrast, our results show that the secretion level of proinflammatory IL-6 and VEGF with Biodentine was significantly lower than that with resincontaining materials. This can be related to the significant decrease of THP-1 adhesion to endothelial cells and their decreased migration and activation. These results suggest an inflammatory inhibiting potential with Biodentine.

On the other hand, our study demonstrated that Biodentine increased FGF-2 and TGF-b1 secretion. This can be correlated to the observed stimulation of fibroblast proliferation and the induction of their migration to the injury site in the scratch assay. By contrast, Xeno III decreased TGF-b1 secretion and decreased fibroblast proliferation. This is in line with previous works that reported the involvement of FGF-2 and TGF-b1 in the neoangiogenesis during the healing process (4) and their involvement in pulp fibroblast and stem cell proliferation and their recruitment [START_REF] Chmilewsky | Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration[END_REF].

TheraCal, which contains resins and silicates, increased the proinflammatory IL-6 and VEGF cytokine secretion as observed with Xeno III. But unlike Xeno III, TheraCal reduced inflammatory cell adhesion on endothelial cells and their activation but to a lesser extent than Biodentine. TheraCal did not affect fibroblast proliferation (except after 6 days) and did not induce their migration. These results are very close to those obtained with Xeno III and suggest that at a cellular level, the addition of resins alters the bioactive silicate properties in the context of the pulp inflammation and healing balance. Taken together, these results show that while Biodentine decreases the inflammation, it induces the secretion of growth factors involved in the healing process. These results are in line with previously published investigations on human tooth cultures where Biodentine induced a high mineralization potential in well-organized pulp tissue with increased expression of dentin sialoprotein and nestin, indicating odontoblastic differentiation and mineralization potential. By contrast, decreased expression of these markers was obtained with TheraCal with disorganized and scattered mineralization within the pulp tissue [START_REF] Jeanneau | Light-cured tricalcium silicate toxicity to the dental pulp[END_REF].

Moreover, this study provides an explanation of the consequences of adding resins to tricalcium silicates at the cellular level. It appears that resin toxicity initiates an inflammatory reaction within the dental pulp that is located within rigid dentinal walls. This inflammation may be deleterious to the pulp regeneration process. Indeed, partial pulpotomy of human teeth with TheraCal revealed pulp tissue disorganization without formation of an organized dentin bridge, whereas a wellorganized and thick bridge was formed with Biodentine [START_REF] Bakhtiar | Human pulp responses to partial pulpotomy treatment with TheraCal as compared with Biodentine and ProRoot MTA: a clinical trial[END_REF]. The absence of mineralization may be due to the release of monomers that, in addition to their toxicity [START_REF] Hume | Bioavailability of components of resin-based materials which are applied to teeth[END_REF][START_REF] Ratanasathien | Cytotoxic interactive effects of dentin bonding components on mouse fibroblasts[END_REF], have been shown to inhibit pulp cell differentiation and mineralization [START_REF] About | Influence of resinous monomers on the differentiation in vitro of human pulp cells into odontoblasts[END_REF].

Overall, this work highlights the fact that the choice of pulpcapping material is of prime importance in direct pulp-capping procedures. Indeed, increasing the inflammatory reaction in a closed environment may lead to pulp destruction and disorganization and inhibition of the healing and regeneration processes.

Thus, in spite of decreasing the setting time of resin-based tricalcium silicates by light-curing and within the limits of this work performed in vitro, our work does not support using resin-containing materials for direct pulp-capping. Instead, it shows that adding resins alters the bioactive properties of tricalcium silicates.

Figure 1 .

 1 Figure1. Effects of biomaterials on cytokine secretion. (A) IL-6 secretion by treated fibroblasts significantly increased with all biomaterials conditioned media regardless of incubation time. This secretion was significantly lower with Biodentine. (B) All incubation conditions led to significant increase of VEGF secretion. After 24 and 48 hours, the secretion was statistically higher in all conditioned media compared with the control but to a lesser extent with Biodentine. (C) Treated fibroblasts secreted significantly more TGF-b1 after 24 and 48 hours only when incubated with Biodentine. (D) Treated fibroblasts incubated with all conditioned media showed significant increase in FGF-2 compared with the control, but this level was higher with Biodentine compared with TheraCal and Xeno III. Results are expressed in pg/mL. *Significant difference as compared with the control; **significant differences between 2 conditions (P < .05).

Figure 2 .

 2 Figure 2. Inflammatory THP-1 adhesion on endothelial cells (HUVECs). (A)Schematic representation of THP-1 cell adhesion on HUVEC assay. A monolayer of HUVECs was stimulated for 4 hours with supernatants from treated fibroblasts cultured 24 hours with conditioned or MEM control media. In parallel, THP-1 cells were labelled with BCECF acid for 1 hour. Both cells were then co-cultured for 30 minutes under gentle agitation to avoid spontaneous cell attachment. After this incubation time, media were removed, and adherent THP-1 cells to the endothelial cell layer were counted with fluorescent microscope (Â100). (B) Representative pictures on fluorescent microscopy of the adhesion assay showing (a) control, (b) Biodentine, (c) TheraCal, and (d) Xeno III. Framed areas show adherent fluorescent THP-1 cells on endothelial cell monolayer. Scale bars, 50 mm. (C) Quantification of THP-1 adhesion on endothelial cells. Biodentine and TheraCal significantly decreased THP-1 cell adhesion on HUVECs compared with the control and with Xeno III, which induced significantly more adhesion. Number of adhering cells with Biodentine was significantly lower than with TheraCal. Results are expressed in percentage of the control. *Significant differences as compared with the control; **significant differences between 2 conditions (P < .05).

Figure 3 .Figure 4 .

 34 Figure 3. THP-1 cell migration and activation. (A) THP-1 cell migration assay in Boyden chambers showed that Biodentine significantly reduced this migration, which was not affected by TheraCal or Xeno III. (B) THP-1 activation assay. THP-1 cells were stimulated for 24 hours with supernatants from treated fibroblasts incubated with conditioned or MEM control media for 24 hours. (a-d) Representative pictures on fluorescent microscopy showing (a) control, (b) Biodentine, (c) TheraCal, and (d) Xeno III. Framed areas show superimposition phase-contrast and fluorescent images of adherent cells, indicating their activation into macrophage-like cells. Scale bars, 200 mm. (e) THP-1 cell activation was significantly reduced by TheraCal and Biodentine and significantly increased with Xeno III. Results are expressed in percentage of control. *Significant difference as compared with the control; **significant differences between 2 conditions (P < .05).

JOE -Volume -, Number -, -2018Tricalcium Silicates and Pulp Inflammation

Giraud et al. JOE -Volume -, Number -, -2018

Giraud et al. JOE -Volume -, Number -, -2018

Giraud et al. JOE -Volume -, Number -, -2018

Acknowledgments

The authors thank Dr Jean-Charles Gardon for providing the teeth.

This work was supported by Aix-Marseille University and CNRS.

The authors deny any conflicts of interest related to this study.