INTRODUCTION

Soil organic matter (SOM) management is recognized as a cornerstone for successful farming in most tropical areas, with or without the application of mineral fertilizers [START_REF] Merckx | Soil organic matter and soil fertility[END_REF]. Several experiments have demonstrated the direct or indirect positive effects of SOM on chemical, physical and biological properties of soil related to plant response [START_REF] Sanchez | Properties and Management of Soils in the Tropics[END_REF][START_REF] Pieri | Fertility of Soils: A Future for Farming in the West African Savannah[END_REF]. Moreover, SOM is an essential reservoir of carbon (C), and SOM management can have significant implications on the global C balance and thus on climate change [START_REF] Craswell | The role and function of organic matter in tropical soils[END_REF]. In many rural areas of the tropics, the environmental challenge consists of reducing deforestation, increasing organic matter storage in cultivated soils, and reducing soil erosion. Therefore, under the economical conditions prevailing in developing countries, maintaining soil fertility and meeting the environmental challenge require land-use practices that include high levels of organic inputs and soil organic C sequestration [START_REF] Feller | Soil organic carbon sequestration in tropical areas. General considerations and analysis of some edaphic determinants for Lesser Antilles soils[END_REF].

Natural fallowing has long been the main practice to maintain soil fertility in tropical areas. However, as its effects only become significant after a period of at least 5 years, natural fallowing is no longer possible in the context of increasing population. Such is precisely the case in southern Benin, where the population density is 300 to 400 inhabitants km -2 [START_REF] Azontonde | Dégradation et restauration des terres de barre (sols ferrallitiques faiblement désaturés argilo-sableux) au Bénin[END_REF]. The benefits of legume-based cover crops in Africa (in regions with annual rainfall > 800 mm) as an alternative to natural fallow, to control weeds and soil erosion, and enrich soil organic matter and N are widely recognized [START_REF] Voelkner | Urgent needed: An ideal green mulch crop for the tropics[END_REF][START_REF] Raunet | Semis direct sur couverture végétale permanente du sol: De la technique au concept[END_REF][START_REF] Carsky | Mucuna cover crop fallow systems: potential and limitations[END_REF]). In southwestern Nigeria, higher maize ( Zea mays ) yields were obtained in live mulch plots under Centrosema pubescens or Psophocarpus palustris than in conventionally tilled and no-till plots for four consecutive seasons [START_REF] Akobundu | Live mulch: A new approach to weed control and crop production in the tropics[END_REF].

The effect of relay-cropping maize through Mucuna pruriens (var. utilis ) was assessed in southern Benin from 1988 to 1999 in terms of plant productivity and soil fertility [START_REF] Azontonde | Dégradation et restauration des terres de barre (sols ferrallitiques faiblement désaturés argilo-sableux) au Bénin[END_REF][START_REF] Azontonde | Le mucuna et la restauration des propriétés d'un sol ferrallitique au sud du Bénin[END_REF]. The relay-cropping system (M) was compared with traditional maize cropping system without any input (T), and with a maize cropping system with mineral fertilizers (NPK). This paper focuses on changes in soil C during the period of the experiment in relation to residue biomass C returned to the soil, runoff and soil erosion losses, and loss of C with erosion.

MATERIALS AND METHODS

Description of the Site and Treatments

The experiment was conducted from 1988 to 1999 at an experimental farm at Agonkanmey (6 ° 24'N, 2 ° 20' E), near Cotonou in southern Benin in an area of low plateaus. The climate is subhumid-tropical with two rainy seasons (March-July and September-November). Mean annual rainfall is 1200 mm and mean annual temperature is 27 ° C. The soils are classified as Typic Kandiustult (Soil Survey Staff 1994) or Dystric Nitisols (FAO-ISRIC-ISSS 1998), and have a sandy loam surface layer overlying a sandy clay loam layer at about 50 cm depth. Most of the land is cultivated to maize ( Zea mays ), beans ( Vigna sp.), cassava ( Manihot esculenta ), or peanuts ( Arachis hypogea ), often associated with oil palm ( Elaeis guineensis ).

The study was conducted on three 30 × 8 m plots on a 4% slope. These demonstration plots were not replicated, as it is usually difficult in long-term experiments [START_REF] Shang | Carbon turnover and carbon-13 natural abundance in organo-mineral fractions of a tropical dry forest soil under cultivation[END_REF], especially when these include runoff plots. Three cropping systems were compared: T (traditional), maize without any input; NPK, maize with mineral fertilizers (200 kg ha -1 of NPK 15-15-15, and 100 kg ha -1 of urea); M, relay-cropping of maize and a legume cover crop, Mucuna pruriens var. utilis , with no fertilizer. Maize (var. DMR ) was cropped during the first rainy season with shallow hoe tillage by hand (hoeing depth was about 5 cm). In M plot, maize was sown through the mucuna mulch from the previous year. Mucuna was sown one month later, and once maize had been harvested, its growth as a relay-crop continued until the end of the second (short) rainy season. During this short rainy season, the T and NPK treatments were maintained as natural fallow. Additional information on the site and soil properties has been provided by [START_REF] Azontonde | Dégradation et restauration des terres de barre (sols ferrallitiques faiblement désaturés argilo-sableux) au Bénin[END_REF] and [START_REF] Azontonde | Le mucuna et la restauration des propriétés d'un sol ferrallitique au sud du Bénin[END_REF].

Soil and Plant Sampling

Undisturbed soil profile samples were collected: (1) in March, June, August, andOctober 1988 and1995, at 18 locations per plot for 0 to 10, 10 to 20, and 20 to 40 cm depths, using 0.2-dm 3 soil cores, and (2) in November 1999 at three locations per plot for 0 to 10 and 10 to 20 cm depths in two replicates, and for 20 to 30, 30 to 40, and 50 to 60 cm depths in one replicate, using 0.5dm 3 soil cores. Soil samples were also obtained with a knife for different depths along the profile walls. Soil bulk density (Db) was determined after oven-drying core samples, whereas the other samples were air-dried, sieved (2 mm) and ground (< 0.2 mm) for C and N analyses.

Aboveground biomass of maize and mucuna was determined every year from five replicates (1 × 1 m) at maize harvest (August) and at mucuna maximum growth (October), respectively. In 1995, following the same pattern, roots of maize and mucuna were collected for 0 to 10, 10 to 20, and 20 to 40 cm depths, and hand-sorted [START_REF] Azontonde | Le mucuna et la restauration des propriétés d'un sol ferrallitique au sud du Bénin[END_REF]. Annual root biomass was calculated using the ratio of below-to aboveground biomass determined in 1995, and the annual aboveground biomass. Sampling of the aboveground biomass of weed was done in November 1999 at nine locations per plot, using a 0.25 × 0.25-m frame. Litter was simultaneously and similarly sampled. Root sampling was also carried out in November 1999 on six 0.25 × 0.25 × 0.30-m monoliths per plot: monoliths were cut into three layers (corresponding to 0-10, 10-20, and 20-30 cm depths), and visible roots were hand sorted. With respect to the vegetation cover, we assumed that roots and litter sampled in T and NPK originated from weeds, whereas those sampled in M originated from mucuna. All plant samples were dried at 70 ° C, weighed for biomass measurement, and finely ground for C determination.

Carbon and Nitrogen Determination, and Other Analyses

Total C content (Ct) of soil samples collected in 1988 and 1995 was determined by the Walkley and Black method (WB), and total N content (Nt) by the Kjehldahl method. Both Ct and Nt of soil samples collected in 1999 were determined by the dry combustion method (DC) using an Elemental Analyzer (Carlo Erba NA 1500). The Ct was analyzed on 60 samples using both WB and DC methods, leading to a relationship ( r = 0.971) that was used to convert WB data into DC data. All Ct data are thereafter expressed on a DC basis. The C content of plant samples was determined by dry combustion using an Elemental Analyzer (CHN LECO 600).

Particle-size analysis was performed by the pipette method after removal of organic matter with H 2 O 2 and dispersion by Na-hexametaphosphate. Soil pH in water was determined using a 1:2.5 volumetric soil:solution ratio.

Determinations of Runoff, Soil Losses, and Eroded Carbon

Each plot was surrounded by half-buried metal sheets and fitted out with a collector draining runoff and sediments toward two covered tanks set up in series. When the first tank was full, additional flow moved through a multi-divisor tank into the second tank, both with a capacity of 3-m 3 . Runoff and soil loss data were collected from 1993 to 1997.

Runoff amount (m 3 ) was assessed on every plot after each rainfall event or sequence of events, by measuring the volume of water in each tank and multiplying it by a coefficient depending on divisors. This runoff amount was converted to depth on the basis of the plot area. Annual runoff rate (mm mm -1 ) was defined as the ratio of annual runoff depth to annual rainfall, and mean annual runoff rate as the ratio of runoff depth to rainfall over five years.

The amount of dry coarse sediments (Mg) was deduced by weighing wet coarse sediments collected in the first tank, and oven-drying the aliquots. The quantity of suspended sediments (Mg) was assessed by flocculation and oven drying of aliquots collected from each tank. Annual soil losses (Mg ha -1 yr -1 ) were computed as the sum of dry-coarse and -suspended sediments over one year, and averaged over five years to calculate mean annual soil losses.

Annual eroded C (Mg C ha -1 yr -1 ) was calculated as the product of annual soil losses by C content of sediments. Sediment C content was not measured, but was estimated as the product of soil Ct (at 0-10 cm depth, for the year under consideration) by an enrichment ratio (Starr et al. 2000). Soil Ct for the year under consideration was interpolated from soil Ct measurements carried out in 1988, 1995, and 1999. The enrichment ratio, defined as the ratio of Ct in sediments to that in the soil (0-10 cm depth), was estimated from the data in the literature: on light-textured Ultisols and Oxisols under maize cultivation (with mineral fertilizers) in southern and northern Ivory Coast, with 2100-and 1350-mm annual rainfall, respectively, C enrichment ratios measured in runoff plots by Roose (1980a[START_REF] Roose | Dynamique actuelle d'un sol ferrallitique gravillonnaire issu de granite sous culture et sous savane arbustive soudanienne du Nord de la Côte d'Ivoire -Korhogo: 1967-1975[END_REF] were 1.9 (7% slope) and 1.4 (3% slope), respectively. Thus, C enrichment ratio of 1.6 was assumed for maize plots (T and NPK). In the absence of literature data regarding cover crops, C enrichment ratio under maize-mucuna (M) was found similar to those measured in runoff plots having comparable soil cover conditions: for two light-textured Oxisols under bush savannas in northern Ivory Coast, with 1200-and 1350-mm annual rainfall, respectively, C enrichment ratio measured by [START_REF] Roose | Importance relative de l'erosion, du ruissellement et du drainage oblique et vertical sous une savane arbustive de moyenne Côte d'Ivoire -Bouaké: 1967-1971[END_REF] and [START_REF] Roose | Dynamique actuelle d'un sol ferrallitique gravillonnaire issu de granite sous culture et sous savane arbustive soudanienne du Nord de la Côte d'Ivoire -Korhogo: 1967-1975[END_REF] was 2.6 (4% slope) and 3.4 (3% slope), respectively; and for a sandy Ultisol under banana plantation in southern Ivory Coast (14% slope, 1800-mm annual rainfall), C enrichment ratio was 3 [START_REF] Roose | Pédogenèse actuelle d'un d'un sol ferrallitique remanié sur schiste sous forêt et sous bananeraie fertilisée de basse Côte d'Ivoire. Synthèse de huit années d'observations de l'erosion, du drainage et de l'activité des vers de terre à la station IRFA d'Azaguié et la Forêt du Téké[END_REF]. Averaging these data, C enrichment ratio of 3 was assumed for maize-mucuna (M) rotation. Dissolved C in runoff was neither measured nor taken into consideration.

Statistical Analyses

Differences in mean Ct and Ct stocks were tested by a Student unpaired t -test. Differences in mean annual runoff rates, soil losses, and eroded C were tested by a paired t -test. In both cases, no assumptions were made on normality and variance equality [START_REF] Dagnélie | Théorie et méthodes statistiques[END_REF].

RESULTS

General Properties of Soils (Table 10.1)

The clay (< 2 µ m) content of the soil ranged between 110 and 150 g kg -1 for 0 to 10 cm depth in 1988, and it increased between 1988 and 1999 in T (50%) but not in NPK and M treatments (increase < 15%). The clay content also increased with depth. Moreover, clay content in 1999 was higher at 0 to 10 cm in T than at 10 to 20 cm in NPK and M treatments. The sand (> 50 µ m) content was between 600 and 800 g kg -1 to 20 cm depth, mainly in the form of coarse sand (> 200 µ m) (data not shown). Soil pH was acidic (< 6) and decreased between 1988 and 1999, especially in T and NPK treatments (-0.5 over a decade).

Soil Carbon

Total soil carbon content Ct (g C kg -1 soil) was determined through 18-and 3-replicate sampling in March 1988 and November 1999, respectively (Table 10.1). The validity of the latter was assessed using 18-replicate sampling done in October 1995 as a reference: following [START_REF] Dagnélie | Théorie et méthodes statistiques[END_REF] and [START_REF] Shang | Carbon turnover and carbon-13 natural abundance in organo-mineral fractions of a tropical dry forest soil under cultivation[END_REF], at 95% confidence level, irrespective of the plot and the depth, threereplicate sampling in 1995 would have led to a less than 8% relative error in Ct estimation. Thus, Ct determined in 1999 by three-replicate sampling was representative of the mean value of the plot. Similarly, Ct stock (Mg C ha -1 ) estimated in November 1999 was representative of the large area.

Differences in Ct between plots were negligible (< 2% at 0-20 cm) in March 1988. Between March 1988 and November 1999, Ct increased considerably at 0 to 20 cm depth in M (90%, p < 0.01) but changed slightly in T (-8%) and NPK (3%), and for 20 to 40 cm depth (changes < 20%). In November 1999, and as a consequence, Ct at 0 to 20 cm depth was much greater in M than in T (100%, p < 0.01) and NPK (80%, p < 0.05) treatments. Differences between plots were rather small below this depth, as were differences between NPK and T (< 30% in general) treatments.

Changes in Ct stock (Mg C ha -1 ) for 0 to 40 cm depth were similar showing small initial differences between plots (< 7%); between March 1988 and November 1999, slight changes in T and NPK (< 15%) treatments but a considerable increase in M (50%, p < 0.01); higher final Ct stock in M than in T (70%, p < 0.01) and NPK (45%, p < 0.05) treatments. Stock of Ct for 0 to 40 cm depth finally attained the value of 24, 29, and 41 Mg C ha -1 in T, NPK, and M, respectively. Between 1988 and 1999, mean (± standard deviation) annual changes in Ct stock were 0.1 (±0.1), 0.2 (±0.4), and 1.4 (±0.4) Mg C ha -1 yr -1 in T, NPK, and M, respectively, for 0 to 20 cm depth; and -0.2 (±0.1), 0.2 (±0.5), and 1.3 (±0.5) Mg C ha -1 yr -1 , respectively, for 0 to 40 cm depth.

Residue Biomass

Average annual residue biomass (dry matter) returned to the soil in T, NPK, and M was 8, 13, and 19.9 Mg ha -1 yr -1 , with 35, 72, and 82% of aboveground biomass, respectively (Table 10.2). Mean annual residue C added was 3.5, 6.4, and 10 Mg C ha -1 yr -1 , with 39, 74, and 84% as aboveground biomass, respectively (aboveground biomass had a slightly more C content than roots). Returned C mainly originated from weeds in T (55% as roots and 17% as aboveground biomass), which represented 44 and 92% of aboveground and belowground residue C, respectively. In contrast, returned C in NPK was mainly from maize (61% as aboveground biomass and 14% as roots). In M, maize and mucuna accounted for similar amounts of residue C, either as aboveground biomass (about 40% each) or roots (8% each). Moreover, maize residue biomass C was of the same order of magnitude in NPK and M (ca. 5 Mg C ha -1 yr -1 ) treatments.

Runoff, Soil Losses, and Eroded Carbon

Annual rainfall ranged between 1000 and 1558 mm, and averaged 1200 mm between 1993 and 1997 (Table 10.3). Mean annual runoff rate in T, NPK, and M treatments was 0.28, 0.12, and 0.08 mm mm -1 , and mean annual soil losses was 34.0, 9.3, and 2.9 Mg ha -1 yr -1 , respectively. Using C enrichment ratios of sediments determined in similar soil and climate conditions (Roose, 1980a[START_REF] Roose | Dynamique actuelle d'un sol ferrallitique gravillonnaire issu de granite sous culture et sous savane arbustive soudanienne du Nord de la Côte d'Ivoire -Korhogo: 1967-1975[END_REF], mean eroded C was estimated at 0.3, 0.1, and 0.1 Mg C ha -1 yr -1 in T, NPK, and M treatments, respectively. In plots vulnerable to erosion, eroded C was thus of the same order of magnitude as changes in Ct stock for 0 to 40 cm depth: -0.3 vs. -0.2 Mg C ha -1 yr -1 in T, and -0.1 vs. 0.2 Mg C ha -1 yr -1 in NPK. In contrast, eroded C in M was negligible compared with changes in Ct stock: -0.1 vs. 1.3 Mg C ha -1 yr -1 . Moreover, mean annual runoff rate and soil losses were significantly more in T than in NPK and more in NPK than in M; and eroded C was more in T than in NPK and M (p < 0.01) treatments. Additionally, mean annual runoff rate, soil losses, and eroded C increased with the increase in annual rainfall.

DISCUSSION

Changes in Soil Carbon

At the end of our experiment, Ct stock for 0 to 40 cm depth was 24 Mg C ha -1 under unfertilized maize, 29 Mg C ha -1 under fertilized maize, and 41 Mg C ha -1 under maize-mucuna rotation. Elsewhere in southern Benin and in similar soil conditions, [START_REF] Djegui | Statut organique d'un sol ferrallitique du Sud-Bénin sous forêt et différents systèmes de culture[END_REF] reported Ct stocks for 0 to 35 cm depth at 27 Mg C ha -1 under oil palm plantation, 30 Mg C ha -1 under food crops (with fallow), and 48 Mg C ha -1 under forest.

The data on Ct stock presented herein are consistent with other published data (Table 10.4). For an Alfisol in southwestern Nigeria, rates of 0.2 Mg C ha -1 yr -1 were recorded for 0 to 10 cm depth for fertilized maize [START_REF] Lal | Land use and cropping systems effects on restoring soil carbon pools of degraded Alfisols in Western Nigeria[END_REF], vs. 0.3 Mg C ha -1 yr -1 for NPK; in Brazilian Ultisols and Oxisols, rates of around 1 Mg C ha -1 yr -1 were measured for 0 to 20 cm depth under long-term no-till cropping systems [START_REF] Bayer | Changes in organic matter fractions under subtropical no-till cropping systems[END_REF]Sá et al. 2001), vs. 1.4 Mg C ha -1 in M; in a Nigerian Alfisol, rates beyond 2 Mg C ha -1 yr -1 have even been measured for 0 to 20 cm depth under a 2year Pueraria cover [START_REF] Lal | Land use and soil management effects on soil organic matter dynamics on Alfisols in western Nigeria[END_REF]. These data confirm that residue mulching increases Ct stock in tropical soils, especially in cropping systems including legume cover crops.

Residue Biomass

The high rates of Ct increase in M resulted first from high residue biomass returned to the soil, which averaged 20 Mg ha -1 yr -1 (dry matter). The aboveground biomass of mucuna was 8 Mg ha -1 y -1 , within the range of published data: 6 to 7 Mg ha -1 yr -1 in 1-year mucuna fallows in Nigeria [START_REF] Vanlauwe | Utilization of rock phosphate by crops on a representative toposequence in the Northern Guinea savanna zone of Nigeria: Response by Mucuna pruriens, Lablab purpureus and maize[END_REF] and an average of 11 Mg ha -1 yr -1 in mucuna-maize systems in Honduras (> 2000-mm annual rainfall;Triomphe 1996b). The ratio of change in Ct stock to residue C measured in these plots also agreed with data in the literature: in a 12-year no-till maize-legume rotations on a sandy clay loam Ultisol in Brazil, Ct stock increase for 0 to 17.5 cm depth represented 11 to 15% of aboveground residue C (Bayer et al. 2001), vs. 15% in M (and 5% in NPK). In contrast, in long-term no-till cereal-legume rotations on clayey Oxisols also in Brazil, the increase in Ct stock for 0 to 40 cm depth represented 22 to 25% of total residue C (Sá et al. 2001), vs. 12% in M (and 3% in NPK). This difference confirms the role of clay content for C sequestration through the development of stable aggregates and hence organic matter protection [START_REF] Feller | Physical control of soil organic matter dynamics in the tropics[END_REF].

In plots that were left under natural fallow during the short rainy season, weeds represented an important proportion of residue biomass, i.e., 77% in T and 29% in NPK. Weeds represented about 50% of the aboveground residue biomass in T, as was also the case in nonfertilized maize plots studied in Nigeria (Kirchhof and Salako 2000). These data underline the need for systematic measurements of weed biomass when it represents a noticeable proportion of biomass returned to the soil. In our experiment, weeds were sampled on one day only, and it is likely that it led to some uncertainties. Weed biomass was negligible in M: proportions of aboveground residue biomass for maize, mucuna, and weeds were 49, 51, and 0%, respectively. Similarly, these proportions were 49, 42, and 9%, respectively, in 1-year maize-mucuna plots studied in Nigeria (Kirchhof and Salako 2000). Indeed, [START_REF] Carsky | Mucuna cover crop fallow systems: potential and limitations[END_REF] reported that weed suppression was often cited as the reason for the adoption of mucuna fallow systems in Africa.

Nitrous Oxide Emissions

Use of nitrogenous fertilizers also impacts nitrous oxide (N 2 O) emissions, which can be roughly estimated using Equation 10.1 [START_REF] Bouwman | Direct emission of nitrous oxide from agricultural soils[END_REF]:

N-N 2 O emissions (kg ha -1 yr -1 ) = 1 + [0.0125 × N-fertilizer (kg ha -1 yr -1 )]
(10.1) a For 0-17.5 cm depth. b From +0.2 to +1.4 Mg C ha -1 yr -1 , depending on the site.

In NPK, N fertilizer was used at the rate of 76 kg N ha -1 yr -1 [START_REF] Azontonde | Le mucuna et la restauration des propriétés d'un sol ferrallitique au sud du Bénin[END_REF]. Following Equation 10.1, it resulted in 2-kg N-N 2 O ha -1 yr -1 emissions. As the global warming potential of N 2 O is about 300 times that of CO 2 (IPCC, 2001), these N 2 O emissions were equivalent to more than 0.2-Mg C-CO 2 ha -1 yr -1 emissions, and thus offset Ct increase (0.2 Mg C ha -1 yr -1 ).

In M, mucuna residues supplied the soil with more than 250 kg N ha -1 yr -1 [START_REF] Azontonde | Le mucuna et la restauration des propriétés d'un sol ferrallitique au sud du Bénin[END_REF]. In this case, Equation 10.1 led to an overestimation of N 2 O emissions, as it was established from a set of experiments excluding legume cover crops, which provide N that is less directly available than mineral fertilizers. However, it may give an order of magnitude: by following Equation 10.1, N supply by mucuna residues resulted in 4-kg N-N 2 O ha -1 yr -1 emissions, equivalent to 0.5-Mg C-CO 2 ha -1 yr -1 emissions (vs. 1.3 Mg C ha -1 yr -1 as Ct increase). Though overestimated, these data indicate that from an environmental point of view, Ct increase in soils under legume cover crops could be partly offset by N 2 O emissions.

Runoff, Soil Losses, and Eroded Carbon

As compared with T, mean annual runoff rate and soil losses were 57 and 73% less in NPK, respectively, and were 71 and 91% less in M, respectively. Protection of the soil surface by vegetation and residues dissipates kinetic energy of rainfall and has an important influence on the reduction of runoff and erosion [START_REF] Wischmeier | Predicting Rainfall Erosion Losses: A Guide to Erosion Planning[END_REF]. Thus, groundcover by mucuna mulch was probably the main reason for less runoff and soil losses in M than in T and NPK treatments. Similarly but to a lesser extent, it is likely that due to large biomass, fertilized maize provided a better groundcover than unfertilized maize. Additionally, residue return determines an increase in SOM that favors aggregate stability [START_REF] Feller | Aggregation and organic matter storage in kaolinitic and smectitic tropical soils[END_REF], thus preventing detachment of easily transportable particles, and thereby reducing surface clogging, runoff, and erosion (Le Bissonnais 1996) Therefore, higher Ct also resulted in less runoff and erosion in M than in NPK, and also less in NPK than in T treatment.

With respect to runoff plots from tropical areas cropped with maize (or sorghum), comparisons with published data show that annual runoff rate was high (> 0.25 mm mm -1 ) in T and under humid conditions (2100-mm annual rainfall); soil losses were high (> 20 Mg ha -1 yr -1 ) under humid or semiarid conditions (500-mm annual rainfall) and in nonfertilized plots (Table 10.5). In contrast, runoff rate was low (< 0.10 mm mm -1 ) on steep slopes with clayey soils (Kenya) and under maizemucuna (M); soil losses were low (< 5 Mg ha -1 yr -1 ) in M treatment. Thus, runoff and erosion increased with increase in annual rainfall and/or with a decrease in soil surface cover (absence of mulch, nonfertilized plots, semiarid conditions), in accordance with usual observations [START_REF] Wischmeier | Predicting Rainfall Erosion Losses: A Guide to Erosion Planning[END_REF][START_REF] Roose | Land husbandry, components and strategy[END_REF]. Under nonfertilized maize in Kenya, low runoff rates (0.02 mm mm -1 ) resulted in high soil losses (29 Mg ha -1 yr -1 ); assuming that the clayey Alfisol in this study had a stable structure with a high infiltration rate, steep slopes (30%) probably determined the nonselective transport of aggregates in the absence of adequate groundcover.

Mean annual C erosion was estimated at 0.3, 0.1, and 0.1 Mg C ha -1 yr -1 in T, NPK, and M treatments, respectively. Though mean soil losses were three times more in NPK than in M, eroded C was similar in both treatments probably because of high C content in surface soil (which supplies sediments) and higher C enrichment ratio of sediments in M than in NPK treatments. Indeed, several experiments have indicated that C enrichment ratio increases with decrease in soil losses (Roose 1980a[START_REF] Roose | Dynamique actuelle d'un sol ferrallitique gravillonnaire issu de granite sous culture et sous savane arbustive soudanienne du Nord de la Côte d'Ivoire -Korhogo: 1967-1975[END_REF]. Thus, mucuna mulch was less effective in reducing the amount of C erosion than in reducing runoff and soil losses; but it was very effective in reducing the proportion of topsoil C that was eroded, which was much lesser in M than in NPK treatments. This underlines the interest of referring C erosion to topsoil C (enrichment ratio), and to temporal changes in topsoil C.

These data are consistent with those reported in the literature, which showed that C erosion significantly increased with increase in the product of soil losses and soil Ct stock (r = 0.932, p < 0.01; Figure 10.1, drawn up from Table 10.5). The data reported herein show that either soil Ct stock (T and NPK) or soil losses (M) were rather small, thus C erosion was much smaller than in studies from Kenya (high soil Ct stocks on steep slopes) and Ivory Coast (humid conditions), where it ranged from 0.7 to 2.4 Mg C ha -1 yr -1 .

CONCLUSION

For this sandy loam Ultisol, relay-cropping of maize and mucuna (M) was very effective in enhancing C sequestration: change in Ct stock for 0 to 40 cm depth was 1.3 Mg C ha -1 yr -1 over the 12-year period of the experiment, ranging among the highest rates recorded for the eco-region. This increase resulted first from the high amount of residue biomass provided by mucuna, which amounted to 10 Mg DM ha -1 yr -1 (83% aboveground). Mucuna residues, supplying the soil with N, also favored the production of maize biomass, and total mucuna plus maize residue biomass returned to the soil was about 20 Mg ha -1 yr -1 . These results indicate the usefulness of mucuna for SOM management. In contrast, nonfertilized (T) and fertilized continuous maize cultivation (NPK) resulted in -0.2-and 0.2-Mg C ha -1 yr -1 change in Ct stock for 0 to 40 cm depth, respectively. Total residue biomass was 8 and 13 Mg ha -1 yr -1 , including 77 and 29% by weeds, respectively. These contributions demonstrate the need for weed biomass sampling, especially when noticeable rainfall occurs beside the cropping season. Weed biomass was negligible in M, underlining the potential of mucuna for weed control.

Moreover, the thick mulch produced by mucuna decreased losses by runoff and erosion, which were 0.28, 0.12, and 0.08 mm mm -1 , and 34, 9, and 3 Mg ha -1 yr -1 in T, NPK, and M treatments, respectively. Eroded C was estimated at 0.3, 0.1, and 0.1 Mg C ha -1 yr -1 in T, NPK, and M, respectively. Thus, C erosion was of the same order of magnitude as changes in soil Ct stock in treatments vulnerable to erosion (T and NPK). In contrast, C erosion under maize-mucuna was negligible as compared to changes in soil Ct stock.

Through its benefits on SOM management, weed suppression, and erosion control, cropping systems including a legume cover may have an adverse impact from a global change standpoint. Indeed, rough estimates show that N 2 O emissions resulting from N supply by mucuna may partly offset soil C storage in M treatment. In NPK, N 2 O fluxes consecutive to mineral N supply could even offset soil C storage completely. In order to characterize these adverse effects and establish greenhouse gas balances precisely, there is an urgent need for accurate field measurements of N 2 O fluxes, especially in cropping systems including legumes.

Figure 10 . 1

 101 Figure10.1 Relationship between mean annual C erosion (Mg C ha -1 yr -1 ) and the product of mean annual soil losses (Mg ha -1 yr -1 ) into Ct stock of bulk soil at 0-30 cm (Mg C ha -1 ) (data from Table10.5).

Table 10 .1 Soil Clay Content, pH in Water, Total Carbon Content Ct, C:N Ratio, and Total Carbon Stock in 1988 and 1999 (mean ± standard deviation when available)

 10

		Depth	T		NPK	M
		(cm)	1988	1999	1988	1999	1988	1999
	Clay (g kg -1 )	0-10	147 ± 1	216	111 ± 6	128	127 ± 6	136
		10-20	nd	339	nd	198	nd	179
	pH	0-10	5.6 ± 0.1	5.1	5.6 ± 0.1	5.2	5.2 ± 0.1	5.0
		10-20	5.4 ± 0.2	4.7	5.4 ± 0.2	5.0	5.1 ± 0.2	5.0
	Ct (g kg -1 )	0-10	5.5 ± 0.2	5.3 ± 0.1	5.4 ± 0.1	6.7 ± 1.8	5.2 ± 0.1	11.5 ± 2.0
		10-20	4.6 ± 0.3	4.0 ± 0.7	4.8 ± 0.4	3.8 ± 1.2	4.8 ± 0.4	7.3 ± 0.9
		20-30 a	4.1 ± 0.2	3.5 ± 0.5	4.0 ± 0.4	3.6 ± 1.1	4.6 ± 0.3	4.4 ± 0.1
		30-40 a		3.2 ± 0.1		4.1 ± 0.7		4.2 ± 0.2
		50-60	nd	2.4 ± 0.1	nd	3.5 ± 1.8	nd	3.3 ± 0.5
	C:N	0-10	10.2 ± 1.0	12.2 ± 0.4	10.8 ± 0.5	11.3 ± 0.1	11.5 ± 0.5	11.9 ± 0.8
		10-20	10.9 ± 1.4	10.1 ± 0.6	10.7 ± 1.8	9.9 ± 0.7	12.0 ± 1.8	11.6 ± 0.8
		20-30 a	11.4 ± 1.2	8.7 ± 0.5	10.6 ± 1.9	9.3 ± 1.0	12.8 ± 1.7	10.0 ± 1.2
		30-40 a		8.2 ± 0.8		8.8 ± 1.4		8.9 ± 1.3
		50-60	nd	7.0 ± 0.4	nd	8.8 ± 3.2	nd	8.1 ± 1.4
	Ct stock	0-10	7.7 ± 0.7	8.4 ± 0.3	7.3 ± 0.5	10.6 ± 3.4	6.8 ± 0.3	17.4 ± 3.3
	(Mg C ha -1 )	0-20	13.6 ± 0.9	14.5 ± 0.4	14.6 ± 1.0	17.0 ± 3.9	13.8 ± 0.8	28.7 ± 3.9
		0-40	25.9 ± 1.5	24.2 ± 0.5	27.0 ± 1.8	28.8 ± 5.7	27.7 ± 1.7	41.4 ± 4.9
		0-60	nd	32.0 ± 0.3	nd	39.7 ± 3.6	nd	51.7 ± 4.1

Note: nd: not determined. a 20-40 cm in 1988.

Table 10 .2 Residue Biomass Returned to the Soil (mean ± standard deviation) Origin Residue Biomass (dry matter) (Mg ha -1

 10

			M		84	91	86		18	22	19		-	-	-		51	55	52
	C:N of	Residues	NPK		75	78	75		-	-	-		25	nd	nd		65	nd	nd
			T		118	121	118		-	-	-		35	nd	nd		78	nd	nd
	Residue Biomass C	(Mg C ha -1 yr -1 )	T NPK M		0.77 ± 0.03 3.91 ± 0.12 4.33 ± 0.12	0.20 ± 0.01 0.89 ± 0.04 0.76 ± 0.03	0.97 ± 0.03 4.80 ± 0.12 5.10 ± 0.13		0.00 0.00 4.07 ± 0.12	0.00 0.00 0.85 ± 0.03	0.00 0.00 4.93 ± 0.13		0.60 ± 0.18 0.82 ± 0.14 0.00	1.91 ± 0.72 0.75 ± 0.37 0.00	2.51 ± 0.74 1.57 ± 0.40 0.00		1.37 ± 0.18 4.73 ± 0.18 8.41 ± 0.17	2.11 ± 0.72 1.64 ± 0.37 1.61 ± 0.04	3.48 ± 0.74 6.37 ± 0.41 10.02 ± 0.18
	C Content of Residues	(g C kg -1 )	T NPK M		533 ± 26 524 ± 16 538 ± 10	474 ± 20 508 ± 20 456 ± 21	519 ± 33 521 ± 26 524 ± 23		--488 ± 25	--455 ± 20	--482 ± 32		440 ± 12 430 ± 10 -	400 400 -	409 415 -		488 ± 29 505 ± 19 513 ± 27	406 452 455 ± 29	435 490 503 ± 40
			M		8.05 ± 0.20	1.67 ± 0.07	9.72 ± 0.21		8.34 ± 0.24	1.88 ± 0.06	10.22 ± 0.25		0.00	0.00	0.00		16.39 ± 0.32	3.55 ± 0.09	19.94 ± 0.33
		) yr -1	NPK		7.46 ± 0.19	1.76 ± 0.05	9.22 ± 0.19		0.00	0.00	0.00		1.89 ± 0.29	1.89 ± 0.92	3.78 ± 0.96		9.35 ± 0.34	3.65 ± 0.92	13.00 ± 0.98
			T	Maize	Aboveground 1.44 ± 0.06	Roots 0.42 ± 0.01	Subtotal 1.86 ± 0.07	Mucuna	Aboveground 0.00	Roots 0.00	Subtotal 0.00	Weeds	Aboveground 1.36 ± 0.39	Roots a 4.77 ± 1.81	Subtotal 6.13 ± 1.85	Subtotal	Aboveground 2.80 ± 0.40	Roots 5.19 ± 1.81	Total 7.99 ± 1.85	Note: nd: not determined.

a 0-30 cm; data resulting from sampling carried out in November 1999, assuming that roots collected in T and NPK were weed roots only and had a C content of 400 g C kg -1 .

Table 10 .3 Annual Runoff Rates, Soil Losses, and C Erosion Year Rainfall (mm yr -1 ) Runoff Rate (mm mm -1 ) Soil Losses (Mg ha -1 yr -1 )

 10

	C Erosion
	(Mg C ha -1 yr -1 )

a SD: standard deviation.

Table 10 .4 Compared Values of Annual Changes in Ct Stock under Various Tropical Cropping Systems Including Reduced or No Tillage Country and Soil Type Cropping System (and duration, in yr) Change in Ct Stock (Mg C ha -1 yr -1 ) Reference For 0-20 cm Depth

 10

	Nigeria, Alfisol	Pueraria sp. (2)	+2.1	Lal (1998)
	Benin, Ultisol	maize-mucuna (11)	+1.4	this chapter
	Brazil, clayey Oxisol	cereals and soybean (10, 22)	+1.0	Sá et al. (2001)
	Brazil, clay loam Ultisol	Cajanus cajan-maize (12)	+0.9 a	Bayer et al. (2001)
	Nigeria, Alfisol	Stylosanthes sp. (2)	+0.4	Lal (1998)
	Benin, Ultisol	fertilized maize (11)	+0.2	this chapter
	Nigeria, Alfisol	Centrosema sp. (2)	+0.1	Lal (1998)
	Benin, Ultisol	nonfertilized maize (11)	+0.1	this chapter
		For 0-10 cm Depth
	Benin, Ultisol	maize-mucuna (11)	+1.0	this chapter
	Nigeria, Alfisol	Cajanus cajan-maize (3)	+0.7	Lal (2000)
	Honduras, various soils	mucuna-maize (1 to 15)	+0.5 b	Triomphe (1996a)
	Benin, Ultisol	fertilized maize (11)	+0.3	this chapter
	Nigeria, Alfisol	fertilized maize (3)	+0.2	Lal (2000)
	Benin, Ultisol	nonfertilized maize (11)	+0.1	this chapter

Table 10 .5 Compared Values of Annual Runoff Rate, Soil Losses, and C Erosion from Runoff Plots Cropped with Maize (or sorghum) in Tropical Areas Country

 10

		Reference		Gachene et al. (1997)	this chapter		Roose (1980a)	Gachene et al. (1997)	Roose (1978)	Moyo (1998)	Roose (1980b)	this chapter		this chapter
	C Erosion	) yr -1 (Mg C ha -1		2.4	0.3		1.8	0.7	0.2	0.2	0.1	0.1		0.1
	Soil Losses	) yr -1 (Mg ha -1		29.0	34.0		89.4	8.4	7.3	20.6	5.5	9.3		2.9
	Ct Stock a Runoff Rate	(Mg C ha -1 ) (mm mm -1 )	Nonfertilized Maize	80 0.02	20 0.28	Fertilized Maize (or Sorghum)	34 0.27	80 0.01	13 0.25	15? 0.17	21 0.20	22 0.12	Maize-Mucuna	35 0.08
		Soil Type		Clayey Alfisol	Sandy loam Ultisol		Sandy loam Ultisol	Clayey Alfisol	Sandy Alfisol	Sandy Alfisol	Sandy (gravely) Oxisol	Sandy loam Ultisol		Sandy loam Ultisol
	Slope	(%)		30	4		7	30	1	5	3	4		4
	Rainfall	) (mm yr -1		1000	1200		2100	1000	800	500	1350	1200		1200
				Kenya	Benin		Ivory Coast	Kenya	Burkina Faso	Zimbabwe	Ivory Coast	Benin		Benin

a At 0-30 cm.

ACKNOWLEDGMENTS

We thank Barthélémy Ahossi, Maurice Dakpogan, Charles de Gaulle Gbehi, Nestor Gbehi, Branco Sadoyetin, and André Zossou for fieldwork, Jean-Yves Laurent and Jean-Claude Marcourel for technical assistance, and Gerard Bourgeon for his comments on a previous version of the paper. This work was financially supported by a French PROSE grant (CNRS-ORSTOM program on soil and erosion).


E Potier

N C Rivron

C A Van Blitterswijk

K Ito
email: k.ito@tue.nl

PhD Dr K Ito

Micro-aggregates do not influence bone marrow stromal cell chondrogenesis

Keywords: bone marrow stromal cells, mesenchymal stem cells, chondrogenesis, cell-cell interactions, micro-aggregates, hydrogel

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC-based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro-aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro-aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro-aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro-aggregates.

Introduction

Articular hyaline cartilage only possesses a limited self-repair capacity. Most tissue damage, caused either by wear and tear or trauma, will not be healed but replaced by fibrocartilage. This tissue has inferior biochemical and biomechanical properties compared to native hyaline cartilage, altering the function of the joint and ultimately leading to severe pain [START_REF] Ahmed | Strategies for articular cartilage lesion repair and functional restoration[END_REF][START_REF] Nesic | Cartilage tissue engineering for degenerative joint disease[END_REF]. Surgical approaches are commonly proposed to promote the healing of cartilage damage. They present, however, several limitations linked to the cell/tissue source and may lead to the formation of fibrocartilage rather than hyaline cartilage [START_REF] Ahmed | Strategies for articular cartilage lesion repair and functional restoration[END_REF]Khan et al., 2010). Alternative sources of cells/tissues are therefore needed to regenerate cartilage damage.

One of the most promising sources is bone marrow stromal cells (BMSCs) [START_REF] Gregory | Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation[END_REF]Khan et al., 2010;[START_REF] Krampera | Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair[END_REF][START_REF] Prockop | Marrow stromal cells as stem cells for nonhematopoietic tissues[END_REF]. As these cells are isolated from the bone marrow, no cartilage tissue harvesting is required and the tissue source is exempt from degenerative cartilage disease. BMSCs also possess a high proliferative rate, allowing the regeneration of large defects. Many studies have established that BMSCs can differentiate in vitro into chondrocytes (Muraglia et al., 2000;[START_REF] Halleux | Multi-lineage potential of human mesenchymal stem cells following clonal expansion[END_REF][START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF]. The patient's condition can affect BMSC proliferation and differentiation: age and osteoarthritis have been reported to reduce the chondrogenic potential of BMSCs [START_REF] Murphy | Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis[END_REF]; although other studies report that these factors do not influence BMSC chondrogenesis [START_REF] Dudics | Chondrogenic potential of mesenchymal stem cells from patients with rheumatoid arthritis and osteoarthritis: measurements in a microculture system[END_REF][START_REF] Scharstuhl | Chondrogenic potential of human adult mesenchymal stem cells is independent of age or osteoarthritis etiology[END_REF]. BMSCs have been used to repair cartilage lesions in numerous animal models [START_REF] Guo | Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into β-tricalcium phosphate in a sheep model[END_REF][START_REF] Uematsu | Cartilage regeneration using mesenchymal stem cells and a three-dimensional polylactic-glycolic acid (PLGA) scaffold[END_REF] but also in humans [START_REF] Wakitani | Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees[END_REF][START_REF] Nejadnik | Autologous bone marrow-derived mesenchymal stem cells versus autologous chondrocyte implantation: an observational cohort study[END_REF]. Although the results are promising, the repair tissues were not completely composed of hyaline cartilage [START_REF] Matsumoto | Articular cartilage repair with autologous bone marrow mesenchymal cells[END_REF]. However, it is the general belief that, with further advancements, BMSC-based therapies will eventually be helpful in clinics.

One important direction for improvement is to better understand and control the differentiation pathway leading BMSCs to fully differentiated and functional chondrocytes. For many years, BMSC differentiation has been induced by various cocktails of biochemical factors [START_REF] Augello | The regulation of differentiation in mesenchymal stem cells[END_REF] and more and more evidence indicates that their biomechanical environment can control their differentiation [START_REF] Potier | Directing bone marrow-derived stromal cell function with mechanics[END_REF]. Other than applied exogenous stimulation, direct communication of cells with their environment can also affect their behaviour. For example, cells can respond to different substrate stiffness, for BMSCs, by adapting their differentiation pathways [START_REF] Engler | Matrix elasticity directs stem cell lineage specification[END_REF][START_REF] Pek | The effect of matrix stiffness on mesenchymal stem cell differentiation in a 3D thixotropic gel[END_REF] or, for chondrocytes, their chondrogenic phenotype [START_REF] Sanz-Ramos | Response of sheep chondrocytes to changes in substrate stiffness from2 to 20 Pa: effect of cell passaging[END_REF][START_REF] Schuh | Effect of matrix elasticity on the maintenance of the chondrogenic phenotype[END_REF]. However, so far, few studies have focused on the relationship between cell-cell communication and BMSC differentiation, when adhesion of cells to each other may also provide important clues to control BMSCs, as shown with the osteogenic differentiation pathway [START_REF] Tang | The regulation of stem cell differentiation by cell-cell contact on micropatterned material surfaces[END_REF].

The aim of this study was, therefore, to modulate the cell-cell interactions between BMSCs and evaluate the impact on BMSC in vitro chondrogenesis. In order to create different cell-cell interactions, BMSCs were seeded into hydrogel either as dispersed cells, where interactions rely on paracrine signalling, or as microaggregates, where interactions rely on paracrine and juxtacrine signalling. Micro-aggregates, rather than micromass, were used to promote cell-cell contact locally. Indeed, it has been shown that micromass culture, used to mimic the condensation phenomenon of mesenchymal cells during development [START_REF] Bobick | Regulation of the chondrogenic phenotype in culture[END_REF], leads to heterogeneous distribution of the cartilaginous matrix [START_REF] Barry | Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components[END_REF][START_REF] Mackay | Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[END_REF][START_REF] Schmitt | BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in highdensity culture[END_REF][START_REF] Murdoch | Chondrogenic differentiation of human bone marrow stem cells in Transwell cultures: generation of scaffold-free cartilage[END_REF], most likely due to mass transport limitations within the micromass. Downscaling from micromass (200 000-250 000 cells) to micro-aggregates (50-300 cells) should overcome these mass transport issues. In fact, micro-aggregate culture has already been shown to be superior to micromass for BMSC chondrogenesis, with a more homogeneous differentiation and matrix deposition observed [START_REF] Markway | Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures[END_REF]. Finally, the effects of cell-cell interactions on BMSC chondrogenesis could be attenuated by the presence of exogenous growth factors (e.g. TGFβ3) in the culture medium. We therefore used different patterns of TGFβ stimulation (0, 7 or 21 days) to assess the influence of different cellular environments [dispersed cells (DC) vs micro-aggregates (MA)] on BMSC chondrogenesis.

Materials and Methods

Bovine BMSC isolation and expansion

Bovine BMSCs were isolated from three cows (8-12 months old, all skeletally immature), in accordance with local regulations. Bone marrow was aspirated from the pelvis and immediately mixed 1:1 with high-glucose (4.5 g/l) Dulbecco's modified Eagle's medium (hgDMEM; Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/ml heparin (Sigma, Zwijndrecht, The Netherlands) and 3% penicillin-streptomycin (Lonza, Basel, Switzerland). Bone marrow samples were then centrifuged (300 × g, 5 min) and resuspended in growth medium: hgDMEM +10% fetal bovine serum (FBS; Gibco Invitrogen; batch selected for BMSC growth and differentiation) +1% penicillin-streptomycin. BMSCs were isolated by adhesion [START_REF] Friedenstein | The development of fibroblast colonies in monolayer cultures of guineapig bone marrow and spleen cells[END_REF][START_REF] Kon | Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical size defects of sheep long bones[END_REF][START_REF] Potier | Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression[END_REF]. Cells were seeded in flasks (using 7-10 ml medium:bone marrow mix per 75 cm2) and, after 4 days, the medium was changed. BMSCs were then expanded up to P1 (passage at 5,000 cells/cm2) before freezing [70-80% confluence; in 90% FBS/10% dimethylsulphoxide (Sigma)]. A fresh batch of BMSCs was thawed and cultured up to P4 for each experiment (each passage at 5000 cells/cm2). Cells from each donor were cultured separately. Bovine BMSCs (n = 4) isolated and expanded following these protocols showed successful chondrogenesis using the micromass approach [START_REF] Johnstone | In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[END_REF]) (as shown with safranin O staining).

Production of agarose chips

Custom-made PDMS stamps, with a microstructured surface consisting of 2865 rounded pins with a diameter of 200 μm and a spacing of 100 μm, were produced. The stamps were sterilized with alcohol and placed in a sixwell plate, microstructured surface up. Warm ultra-pure agarose solution [Gibco Invitrogen; 3% in phosphate-buffered saline (PBS)] was poured on the stamps, centrifuged for 1 min at 2,500 rpm and incubated for 30 min at 4°C. The agarose chips were then separated from the stamps, cut to size to fit in a well of a 12-well plate, covered with PBS and kept at 4°C until use [START_REF] Rivron | Tissue deformation spatially modulates VEGF signaling and angiogenesis[END_REF].

Formation of micro-aggregates and alginate seeding

At passage 5, BMSCs were used to seed: (a) alginate beads (dispersed cells; DC); or (b) agarose chips (microaggregates; MA) (Figure 1). For the DC condition, BMSCs were resuspended in 1.2% sodium alginate (Sigma) solution (in 0.9% NaCl; Merck, Darmstadt, Germany) at a concentration of 7 × 106 cells/ml. The cell + alginate suspension was slowly forced through a 22G needle and added dropwise to a 102 mM CaCl2 (Merck) solution [START_REF] Guo | Culture and growth characteristics of chondrocytes encapsulated in alginate beads[END_REF][START_REF] Jonitz | Differentiation capacity of human chondrocytes embedded in alginate matrix[END_REF]. Beads were incubated for 10 min at 37°C to polymerize and were then rinsed three times in NaCl 0.9% and twice in hgDMEM + 1% penicillin-streptomycin. For the MA condition, BMSCs were resuspended in growth medium at 2 × 10 5 cells/ml and 750 μl cell suspension was used per agarose chip (with PBS previously removed) to produce the microaggregates. Seeded chips were centrifuged for 1 min at 200 × g to force the cells to the bottom of the microwells; 3 ml growth medium was then slowly added and the cells were cultured for an additional 3 days in growth medium to allow cell aggregation. Microaggregates were then collected, flushing the agarose chips with growth medium, and used to seed alginate beads at a final concentration of 7 × 10 6 cells/ml, as described for the DC condition.

Culture

Seeded beads (with either DC or MA) were cultured for 3 weeks in Ch-medium [hgDMEM + 1% penicillin-streptomycin + 0.1 μM dexamethasone (Sigma) + 1% ITS-1+ (Sigma) + 1.25 mg/ml bovine serum albumin (BSA; Sigma) + 50 μg/ml ascorbic acid 2-phosphate (Sigma) + 40 μg/ml L-proline (Sigma) + 100 μg/ml sodium pyruvate (Gibco Invitrogen)] [START_REF] Mackay | Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[END_REF]. This medium was supplemented with 10 ng/ml (TGFβ3; (Peprotech, Rocky Hill, NJ, USA) [START_REF] Barry | Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components[END_REF] (Ch(+) medium) for 0, 7 or 21 days (Figure 1). BMSCs were cultured under 5% CO2 and 2% O2 [START_REF] Markway | Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures[END_REF]; six beads/well, of a six-well plate containing 3 ml medium, were cultured.

Cell viability

At days 0 and 21, beads (n = 3 beads/donor/group) were washed in PBS and incubated in 10 μM calcein AM (Sigma)/10 μM propidium iodide (Gibco Invitrogen) solution (in PBS) for 1 h at 37°C. Cells were then imaged in the centres of the beads at a depth of 200 μm, using a confocal microscope (CLSM 510 Meta, Zeiss, Sliedrecht, The Netherlands).

Cell morphology and adhesion

At days 0 and 21, beads (n = 3 beads/donor/group) were embedded in cryo-compound (Tissue-Tek® OCT™; Sakura, Alphen aan den Rijn, The Netherlands) and snap-frozen in liquid nitrogen; 50 μm-thick cryosections were cut in the middle of the beads. The sections were then thawed, fixed for 30 min at room temperature (RT) in buffered formalin 3.7% (Merck), rinsed in PBS and incubated for 5 min at RT in Triton 1.5% in PBS. The sections were rinsed in PBS and stained with TRITC-phalloidin (Sigma; 1 μM in PBS + 1% BSA) for 2 h at RT. The sections were then rinsed in PBS, counterstained with DAPI for 15 min at RT (Sigma; 100 ng/ml in PBS), rinsed in PBS and MilliQ water, airdried and mounted in Entellan (Merck). The stained sections were observed using a confocal microscope. Morphometric analyses to determine cluster areas and numbers of cells/cluster were conducted on these images, using Zen 2012 software (Zeiss). For each group, 25 clusters or cells were analysed. Stained clusters or cells were manually outlined and the corresponding area determined. Cell numbers/cluster were also counted manually. Immunostaining for vinculin and pan-cadherin was conducted on 10 μm-thick cryosections. The sections were thawed, fixed for 10min at RT in buffered formalin 3.7%, rinsed in PBS and incubated for 10 min at RT in Triton 0.5% in PBS. After blocking in 3% BSA for 1 h, the sections were incubated for 1 h at RT with monoclonal mouse anti-vinculin antibodies (Sigma), diluted at 1:400, or with monoclonal mouse anti-cadherin antibodies (Abcam; Cambridge, UK), diluted at 1:100, in 3% BSA. The sections were then washed three times in PBS and incubated for 1 h at 38°C with Alexa 488-conjugated goat anti-mouse antibodies (Molecular Probes; Bleiswijk, The Netherlands), diluted 1:300 in PBS. The stained sections were then rinsed three times and mounted with Mowiol. For both stainings, human cardiomyocyte progenitor cells grown on coverslips were used as a positive control. Both antibodies are known to work with bovine material.

Cartilaginous matrix formation and cell proliferation

At days 0 and 21, five beads/donor and group were pooled and digested in papain solution [150 mM NaCl (Merck), 789 μg/ml Lcysteine (Sigma), 5 mM Na2EDTA.2H2O (Sigma), 55 mM Na3citrate.2H2O (Sigma) and 125 μg/ml papain (Sigma)] at 60°C for 16 h. Digested samples were then used to determine their content of sulphated glycosaminoglycans (sGAG), as a measure of proteoglycans, and DNA. sGAG content was determined using the dimethyl methylene blue (DMMB) assay, adapted for alginate presence [START_REF] Enobakhare | Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9dimethylmethylene blue[END_REF]. Shark cartilage chondroitin sulphate (Sigma) was used as a reference and digested with empty alginate beads (i.e. alginate concentration identical for references and experimental samples). DNA content was measured using the Hoechst dye method [START_REF] Cesarone | Improved microfluorometric DNA determination in biological material using 33258 Hoechst[END_REF], with a calf thymus DNA reference (Sigma). For the 7 days of TGFβ3-treatment group, the beads were also analysed at day 7. At days 0 and 21, beads (n = 3 beads/donor/group) were also embedded in cryo-compound and snap-frozen in liquid nitrogen. 10 μm thick cryosections were cut in the middle of the beads. The sections were then thawed, incubated for 5 min in 0.1 M CaCl2 at RT and fixed in buffered formalin 3.7% for 3 min at RT. The sections were then rinsed in 3% glacial acetic acid (Merck) and stained in Alcian blue solution (Sigma; 1%, pH 1.0, for alginate presence) for 30 min at 37°C. The sections were then rinsed in 0.05 M CaCl2 and counterstained with nuclear fast red solution (Sigma) for 7 min at RT. The stained sections were rinsed in 0.05 M CaCl2 before mounting in Mowiol (Merck) and were observed using a brightfield microscope (Observer Z1, Zeiss).

Gene expression

At days 0 and 21, nine beads/donor and group were pooled, snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation. Frozen beads were placed in between a 316 SS 8 mm bead and a custom-made lid, placed in a 2 ml Eppendorf tube, and were disrupted for 30 s at 1,500 rpm (Micro-dismembrator; Sartorius, Göttingen, Germany). RNA was then extracted using TRIzol® (Gibco Invitrogen) and purified using an RNeasy mini-kit (Qiagen, Venlo, The Netherlands). The quantity and purity of the isolated RNA were measured by spectrophotometry (ND-1000, Isogen, De Meern, The Netherlands) and integrity by gel electrophoresis. Absence of genomic DNA was validated by endpoint PCR and gel electrophoresis using primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Total RNA (300 ng) was then reverse-transcribed (M-MLV; Gibco Invitrogen) and the gene expression levels of sox9, aggrecan, type II collagen, TGFβ, type X collagen and sp7 (also known as Osterix) were assessed with SYBR green qPCR (iCycler; Biorad, Hercules, CA, USA) (see Table 1 for primer list). 18S (PrimerDesign Ltd, Southampton, UK) was selected as a reference gene from three genes (RPL13A, GAPDH and 18S) as the most stable gene throughout our experimental conditions. Expression of the gene of interest is reported as relative to 18S expression (2 -ΔCT method). When gene expression was not detected, the 2 -ΔCT value was set to 0 to conduct the statistical analysis. For the 7 days of TGFβ3 treatment group, beads were also analyzed at day 7.

Statistical analysis

General linear regression models based on ANOVAs were used to examine the effects of seeding (DC and MA), TGFβ treatment (0, 7 and 21 days) and days of culture (days 0, 7 and 21) and their interactions on the variables DNA and GAG/DNA contents, and sox9, type II collagen, aggrecan, TGFβ, type X collagen and sp7 gene expression. In all analyses, full factorial models were fitted to the data and then a backwards stepwise procedure was used to remove the non-significant effects. For each significant effect, a Tukey-HSD post hoc test was conducted; p < 0.05 was considered significant. All data analyses were performed in R v. 2.9.0 (R Development Core Team, 2009).

Results

Physical properties of the granule surfaces

BMSCs showed high cell viability after seeding for all conditions (Figure 2A,E). At day 0, cells appeared as a welldispersed cell population for DC conditions (Figure 2A) or as dense micro-aggregates for MA conditions (Figure 2E). However, some dead cells could be observed around the micro-aggregates (Figure 2E), most likely due to a higher shear stress exerted on micro-aggregates than dispersed cells when producing the alginate beads. At day 21, cell viability remained high for all conditions (Figure 2B-D, F-H). DNA content confirmed that cells proliferated (Figure 2I). DC conditions, however, led to higher proliferation than MA conditions.

Cell morphology and adhesion

At day 0, the DC condition led to single dispersed cells (Figure 3A,I), while MA resulted in large clusters (Figure 3E,I) containing, on average, 14 cells (Figure 3J). In DC conditions, after 21 days of culture BMSCs proliferated (Figure 3J) and formed clusters (Figure 3B-D) whose size increased when TGFβ was added, although not significantly (Figure 3I). In MA conditions (Figure 3F-H), micro-aggregates grew during culture, with the bigger clusters observed for 7 days of TGFβ (Figure 3I), although cell proliferation was limited (Figure 3J). At day 0, cell-cell interactions were more developed in MA than in DC, as shown by the immunostaining of pancadherins (Figure 4D andA, respectively), which are glycoproteins involved in cell-cell adhesion. These improved cell-cell interactions, however, disappeared after 3 weeks of culture (Figure 4E). TGFβ treatment had no effects on cadherin expression for either DC or MA (data not shown). Regarding vinculin, a membrane-cytoskeletal protein involved in cell-matrix adhesion, its expression was similar for DC and MA (Figure 4G and J, respectively), with a dispersed localization of vinculin through the cell surface. At day 21, vinculin condensed in focal adhesions. Distribution seemed similar for DC and MA (Figure 4H and K, respectively), and was not influenced by the different TGFβ stimulation patterns (data not shown).

Matrix production

In all conditions, proteoglycans (PGs) were deposited (Figure 5A-H), demonstrating successful chondrogenic differentiation of the BMSCs. For both DC and MA conditions, prolonging exposure to TGFβ increased the PG production, as confirmed by a quantitative assay (Figure 5I). However, no differences between DC and MA could be detected. In both conditions, PGs appeared to be concentrated within the clusters (for DC) or the microaggregates (for MA), filling the void spaces previously observed.

Gene expression

Levels of gene expression of chondrogenic markers (sox9, a transcription factor involved in early chondrogenesis; type II collagen and aggrecan, both main components of cartilage matrix) increased at day 21 for all conditions (Figure 6A-C). In MA conditions, however, type II collagen and aggrecan mRNA expression were inhibited compared to DC conditions at day 21 (Figure 6B,C). Seven days of TGFβ treatment led to the highest levels of expression of all chondrogenic markers for both MA and DC conditions (Figure 6A-C). At day 0, MA upregulated TGFβ gene expression compared to DC, but this high level of expression disappeared at day 21 under all TGFβ stimulation conditions (Figure 6D).

Transient TGFβ stimulation

For the 7 days of TGFβ treatment, chondrogenic marker gene expression and matrix production were also evaluated at day 7. The results showed that BMSCs had already started to differentiate at that point. All chondrogenic markers (sox9, aggrecan, type II collagen) were already highly upregulated at the gene levels (Figure 7B-D). PGs were also produced at day 7 (Figure 7A), but to a limited amount. PG content and type II collagen expression were significantly higher at day 21 than at day 7, indicating that the cells were still going along the chondrogenic pathway although TGFβ was withdrawn.

Hypertrophy and osteogenic differentiation

Both type X collagen (Figure 8A), an indicator of chondrocyte hypertrophy, and sp7 (Figure 8B), a transduction factor involved in early osteogenesis, mRNA expressions increased at day 21 for the DC condition, while only type X collagen expression increased in the MA condition. For both conditions and genes, the highest level of expression at day 21 was for the 7 days of TGFβ treatment. When type X collagen levels of expression were similar for MA and DC conditions, sp7 mRNA expression was upregulated at day 0 for the MA condition (Figure 8B).

Discussion

In summary, these results show that BMSCs underwent (partial) chondrogenic differentiation for all conditions tested (PG production and increased chondrogenic marker expression). MA, however, did not perform better (matrix production) or even as well (gene expression) as DC under all the TGFβ stimulation patterns tested. Nonetheless, these data show that small microaggregates can be successfully integrated into hydrogel (alginate). Although cell death was slightly upregulated at day 0 (Figure 2E), BMSCs in MA survived up to 21 days (Figure 2) and differentiated into chondrocytes, with the deposition of a PG-rich matrix within the MA (Figure 5H) and substantial upregulation of sox9, type II collagen and aggrecan gene expression (Figure 6A-C).

Increase of TGFβ gene expression at day 0 in MA compared to DC (Figure 6D) suggests an early stimulant effect of MA, maybe due to improved juxtacrine signaling (cell-cell contact) rather than paracrine signaling (limited distance between neighbouring cells), as cell-cell contact was improved at day 0 in the MA condition, as shown by pan-cadherin staining. This upregulation, however, was lost at day 21 and, more importantly, was not translated into enhanced chondrogenic matrix production (Figure 5) or gene expression (Figure 6), even if no exogenous TGFβ was added. One explanation for this absence of effects may be the disappearance, during the 3 weeks of culture, of the cell-cell interactions observed at day 0 (Figure 4). BMSCs in MA most likely lost contact with each other (as shown by pan-cadherin staining) due to extracellular matrix production between the cells, but formed new junctions (as shown by vinculin staining) with this matrix (Figure 5). The lack of effects of TGFβ gene expression upregulation may also been explained by a low translation efficiency or by post-transcriptional regulatory mechanisms. Several studies comparing genomic and proteomic analyses report, indeed, moderate correlation between mRNA and protein expression [START_REF] Chen | Discordant protein and mRNA expression in lung adenocarcinomas[END_REF][START_REF] Huber | Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the anti-estrogen-resistant derivative T47D-r[END_REF][START_REF] Oberemm | Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters[END_REF][START_REF] Tian | Integrated genomic and proteomic analyses of gene expression in mammalian cells[END_REF].

Another explanation for the absence of effects of upregulated TGFβ expression might be that BMSCs are not sensitive to the levels of TGFβ they are producing, either because these levels are too low or because BMSCs are less sensitive in MA. Cytoskeleton organization, indeed, has been shown to modulate cell sensitivity to TGFβ. Disorganization of the microfilaments in rabbit articular chondrocytes after treatment with dihydrocytochalasin B enhanced the sensitivity of the cells to TGFβ (increased PG and collagen synthesis) [START_REF] Benya | Dihydrocytochalasin B enhances transforming growth factor-β-induced re-expression of the differentiated chondrocyte phenotype without stimulation of collagen synthesis[END_REF]. In the present study, BMSCs at day 0 displayed more organized microfilaments in MA cells than in the round cells of the DC condition (Figure 3A/E). This difference in cytoskeleton organization may also explain why MA are not upregulating chondrogenic gene expression as well as DC under transient and continuous TGFβ treatment (Figure 6). Although no significant differences were observed at the matrix level (Figure 5), our data support results observed with bovine articular chondrocytes in hydrogel, where small micro-aggregates (5-18 cells) inhibit chondrocyte biosynthesis compared to dispersed cells [START_REF] Albrecht | Probing the role of multicellular organization in threedimensional microenvironments[END_REF]. Distribution of PGs, however, was quite distinct between the two conditions, with a more evenly distributed matrix for DC (Figure 5). As the cell concentration was similar at day 0 for both conditions, DC resulted in a more dispersed and homogeneous distribution of cells (Figure 2), which could account for a more even distribution of the matrix produced by the BMSCs.

Still, MA might be more potent for the osteogenic differentiation of BMSCs. In fact, MA (not embedded into a hydrogel) have already been shown to promote osteogenic differentiation of human BMSCs compared to 2D culture (increased calcium deposition and osteogenic gene expression) [START_REF] Kabiri | 3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling[END_REF]. In the present study, we observed an upregulation of sp7, a transcription factor involved in early osteogenic differentiation, in MA at day 0 compared to DC (Figure 8). This suggests a positive influence of the MA on the osteogenic differentiation pathway. The absence of factors required for osteogenic differentiation, such as FBS or β-glycerophosphate, during culture, however, probably nullifies this influence, and additional experiments need to be conducted to assess the potential of MA to promote BMSC osteogenesis.

Contrary to MA, BMSCs in the DC condition proliferated during the 3 weeks of culture. This absence of significant proliferation in MA already containing several cells (Figure 3) may be explained by contact inhibition present in the MA but not in the DC. At day 21, cloned DC spontaneously formed clusters. Although these structures appeared similar to the MA, they were smaller and contained fewer cells (Figure 3). Recreating and amplifying this natural process of cloning and clustering in the MA, however, did not exert any substantial effect on BMSC differentiation, suggesting that cell-cell interactions are not required for initiating chondrogenesis.

These results also confirm that bovine BMSCs can spontaneously differentiate toward the chondrogenic lineage without the presence of TGFβ (PG production and increased sox9, type II collagen and aggrecan gene expression; Figure 5, Figure 6, Figure 7) when cultured in hydrogel and serum-free conditions, as previously reported for bovine BMSCs in micromass culture [START_REF] Bosnakovski | Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system[END_REF]. Seven days of TGFβ treatment were enough to enhance the production of cartilaginous matrix, as shown previously with human BMSCs [START_REF] Buxton | Temporal exposure to chondrogenic factors modulates human mesenchymal stem cell chondrogenesis in hydrogels[END_REF], but, surprisingly, gave the highest upregulation of chondrogenic marker expression (Figure 6) for both MA and DC. However, the transient TGFβ stimulation also led to higher expression of type X collagen (a marker of chondrocyte hypertrophy). Hence, continuous stimulation with TGFβ resulted in a more stable chondrogenic phenotype; it also led to the highest matrix production (Figure 5).

Conclusions on the (absence of) effects of MA on BMSC chondrogenesis, however, are only valid for the cell concentration and hydrogel tested here. Using a lower concentration may dilute paracrine signaling in the DC condition and, therefore, diminish the chondrogenic differentiation of BMSCs. [START_REF] Buxton | Temporal exposure to chondrogenic factors modulates human mesenchymal stem cell chondrogenesis in hydrogels[END_REF] have already evaluated the influence of cell concentration on the chondrogenesis of BMSCs seeded into a hydrogel. They reported a maximal PG/collagen synthesis/cell for concentrations in the range 12.5-25 million cells/ml. Lower concentrations led to lower matrix production, indicating the involvement of paracrine signaling in BMSC chondrogenesis. With the concentration used here (7 million cells/ml), paracrine signaling should be diluted in the DC condition and so MA could have a beneficial effect by locally increasing this paracrine signaling. As no positive effect was found for the MA, it seems that cell-cell contact or cytoskeleton organization have a stronger negative effect than paracrine signaling, a positive one for BMSC chondrogenesis. Moreover, the effect of MA on BMSC chondrogenesis has only been tested here in alginate and could, therefore, be an artifact of that system. The previous observation, that micro-aggregates inhibit chondrogenesis of bovine chondrocytes seeded in photo-polymerizable hydrogel [START_REF] Albrecht | Probing the role of multicellular organization in threedimensional microenvironments[END_REF] when compared to dispersed cells, tends to indicate, however, that the negative effects observed here were not an artifact of alginate.

Another limitation of the study may be the use of exogenous TGFβ if it is the endogenous molecular agent involved in juxtacrine signaling. In this case, adding TGFβ to the culture medium may have overpowered any increase of TGFβ expression present in MA, but not in DC, conditions. Such a beneficial effect, however, should have been observed when the BMSCs were cultured without exogenous TGFβ, when no differences between MA and DC were observed (Figure 5, Figure 6, 0 days TGFβ group). Nonetheless, if TGFβ had been involved in cellular signaling after BMSC differentiation, bone morphogenic protein 2 (BMP2) could have been used to induce BMSC chondrogenic differentiation instead [START_REF] Schmitt | BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in highdensity culture[END_REF].

This study provides important clues about the communication of BMSCs with their environment, where cell-cell interaction seems to have a limited involvement in their (chondrogenic) differentiation. Although DC cloned and spontaneously formed clusters, accelerating and amplifying this process with the MA did not provide beneficial effects. This suggests that influencing cellmatrix, rather than cell-cell, interactions may be a more potent tool to control BMSC differentiation, at least for the chondrogenic pathway.

To conclude, this study shows that micro-aggregates, although potentially promoting cell-cell contacts and improving paracrine signaling, have no beneficial effects on bovine BMSC chondrogenesis in alginate. Bovine BMSCs (n = 3) were expanded up to P4 in hgDMEM + 10% FBS + 1% P/S (growth medium). Cells were then used to seed either alginate beads at 7 million cells/ml (dispersed cells; DC) or agarose chips cast on PDMS stamps (micro-aggregates; MA). BMSCs on agarose chips were cultured for 3 additional days in growth medium to allow the cells to form micro-aggregates. Those were then used to seed alginate beads at 7 million cells/ml. After seeding in alginate, BMSCs (DC or MA) were cultured for 3 weeks in hgDMEM + 1%P/S + 0.1 μM dexamethasone + 1% ITS-1+ + 1.25 mg/ml BSA + 50 μg/ml ascorbic acid 2-phosphate + 40 μg/ml L-proline + 100 μg/ml sodium pyruvate (Ch-medium). This medium was supplemented with 10 ng/ml TGFβ3 (Ch+ medium) for 0, 7 or 21 days. At D0 and D21, cell viability was characterized by live/dead staining; cell morphology by histology (phalloidin, antivinculin and anti-pan-cadherin staining); produced matrix by histology (Alcian blue staining); biochemical assays [glycosaminoglycan (GAG) and DNA content]; and cell phenotype was characterized by qRT-PCR (types II and X collagens, sox9, aggrecan, TGFβ and sp7). Gene expression of type X collagen (A) and sp7 (B), as determined by qRT-PCR; expression is relative to 18S reference gene (2 -ΔCT method). Values are mean+ SD (NB: logarithmic y axis, and error bars are also logarithmic); * p < 0.05 vs D0; # p < 0.05 vs 7 days of TGFβ3 treatment; @ p < 0.05 vs dispersed cells.

Table 1. Primer sequences for target and reference genes used in RT-qPCR assays

  RPL13a, ribosomal protein L13a; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SOX9, SRY (sex determining region Y)-box 9; COL2A1, collagen type II α1; ACAN, aggrecan; TGFB1, transforming growth factor-β1; COL10A1, collagen type X α1; SP7, Sp7 transcription factor. *GenBank™ accession number. **BD, primers designed with Beacon designer software (Premier Biosoft, Palo Alto, CA, USA) and ordered from Sigma.

Figure 1 .

 1 Figure 1. Experimental design.

Figure 2 .

 2 Figure 2. Cell viability and proliferation.(A-H) Bovine BMSCs seeded in alginate beads as dispersed cells (A-D) or as micro-aggregates (E-H) at days 0 (A, E) and 21 after exposure to TGFβ3 for 0 (B, F), 7 (C, G), and 21 (D, H) days. Cells were stained with calcein AM (green fluorescence) for living cells and propidium iodide (red fluorescence) for dead cells. White frames are ×2.5 digital magnification; representative of three donors/group; scale bar = 100 μm; colour images are available online. (I) DNA content/bead, as determined with Hoechst dye assay; values are mean ± SD; n = 3/group. * p < 0.05 vs D0; @ p < 0.05 vs dispersed cells.

Figure 3 .

 3 Figure 3. Cell morphology. (A-H) Bovine BMSCs seeded in alginate beads as dispersed cells (A-D) or micro-aggregates (E-H) at day 0 (A, E) and day 21 after exposure to TGFβ3 for 0 (B, F), 7 (C, G) and 21 (D, H) days. The beads were cryosectioned, fixed and stained with phalloidin (red fluorescence) for F-actin filaments and counterstained with DAPI (blue fluorescence) for cell nuclei; representative of three donors/group; scale bar = 20 μm; colour images available online. (I, J) Morphometric analysis: area covered by cells or clusters (I) and cell number/cluster (J) were determined by image analysis; values are mean ± SD; n = 25 clusters/cells analysed/ group. * p < 0.05 vs D0; # p < 0.05 vs 7 days of TGFβ3 treatment; $ p < 0.05 vs 21 days of TGFβ3 treatment; @ p < 0.05 vs dispersed cells.

Figure 4 .

 4 Figure 4. Cell adhesion.(A-F) Bovine BMSCs seeded in alginate beads as dispersed cells (A, B) or as micro-aggregates (D, E) at day 0 (A, D) and after 21 days of exposure to TGFβ3 (B, E). The beads were cryosectioned, fixed and stained with anti-pan-cadherin (green and counterstained with DAPI (blue fluorescence) for cell nuclei. Human cardiomyocyte progenitor cells were used as positive controls (C) and experimental samples for secondary antibody negative control (F). (G-L) Bovine BMSCs seeded in alginate beads as dispersed cells (G, H) or as micro-aggregates (J, K) at day 0 (G, I) and after 21 days of exposure to TGFβ3 (H, K). Beads were cryosectioned, fixed and stained with anti-vinculin (green fluorescence) and counterstained with DAPI (blue fluorescence) for cell nuclei. Human cardiomyocyte progenitor cells were used as positive controls (I) and experimental samples for secondary antibody negative control (L); representative of three donors/group; scale bar = 10 μm.

Figure 5 .

 5 Figure 5. Matrix production. (A-F) Bovine BMSCs seeded in alginate beads as dispersed cells (A-C) or as micro-aggregates (D-F) at day 21 after exposure to TGFβ3 for 0 (A, D), 7 (B, E) and 21 (C, F) days. Beads were cryosectioned, fixed and stained with Alcian blue for proteoglycans (note that light blue is alginate); representative of three donors/group; scale bar = 200 μm. (G, H) Higher magnifications of (B, E), respectively; scale bar = 50 μm; colour images available online. (I) GAG/DNA content after 21 days of culture, as determined with DMMB and Hoechst dye assays, respectively. Values are mean ± SD (NB: logarithmic y axis, and error bars are also logarithmic); n = 3/group; * p < 0.05 vs D0; $ p < 0.05 vs 21 days of TGFβ3 treatment; ND, not detected.

Figure 6 .

 6 Figure 6. Gene expression -chondrogenesis markers.Gene expression of sox9 (A), type II collagen (B), aggrecan (C) and TGFβ (D), as determined by qRT-PCR. Expression is relative to 18S reference gene (2 -ΔCT method). Values are mean + SD (NB: logarithmic y axis, and error bars are also logarithmic);n=3/group; * p < 0.05 vs D0; # p < 0.05 vs 7 days of TGFβ3 treatment; $ p < 0.05 vs 21 days of TGFβ3 treatment; @ p < 0.05 vs dispersed cells.

Figure 7 .

 7 Figure 7. Transient TGFβ stimulation.(A) GAG/DNA content at days 0, 7 and 21, as determined with DMMB and Hoechst dye assays, respectively; values are mean ± SD (NB: logarithmic y axis, and error bars are also logarithmic). (B-D) Gene expression of sox9 (B), type II collagen (C) and aggrecan (D), as determined by qRT-PCR; expression is relative to 18S reference gene (2-ΔCT method). Values are mean + SD (NB: logarithmic y axis, and error bars are also logarithmic); n = 3/group; * p < 0.05 vs D0; # p < 0.05 vs D21; @ p < 0.05 vs dispersed cells; ND, not detected.

Figure 8 .

 8 Figure 8. Gene expression -Hypertrophy and osteogenesis markers.

Acknowledgments

The authors would like to thank R. R. Delcher for his help with statistical analysis and Marina van Doeselaar for immunostainings. The authors did not receive financial support or benefits from any commercial source related to the scientific work reported in this manuscript.

Disclosure Statement

The authors declare no competing financial interests.


Thomas ' Pierret

Salafis'at'War'in'Syria:'Logics'of'Fragmentation'and'Realignment' '

Introduction' '

By 'March'2011,'Salafism'was'a'very'marginal'force'on'Syria's'religious'scene,'as' its' growing' popular' appeal' during' the' previous' decade' had' been' matched' by' increasing' state' repression. 1 'Following' the' outbreak' of' the' civil' war,' however,' Salafi'factions'rapidly'became'the'dominant'force'among'the'insurgency,'to'the' extent' that' by' October' 2013' five' of' them' featured' among' the' seven' most' powerful' rebel' groups' in' the' country,' totalling' several' dozens' of' thousands' of' fighters:'two'of'them'were'offspring'of'the'Jihadi'Islamic'State'in'Iraq'(Jabhat&al( Nusra,'hereafter'al(Nusra,'and'the'Islamic'State'in'Iraq'and'Sham,'ISIS),'and'the' three' others' would' soon' become' the' founding' factions' of' the' more' pragmatic' Islamic'Front'(Ahrar&al(Sham,'the'Army'of'Islam,'and'Suqur&al(Sham),'along'with' the'Islamist,'but'nonRSalafi'Tawhid&Brigade. 2 ' ' To'a'certain'extent,'the'spectacular'rise'of'Salafism'in'Syria'was'function'of'this' doctrine's'inherent'features:'in'particular,'an'exclusive'definition'of'the'Islamic'

allegiance ' of' entire' preRexisting' local' rural' battalions' (kata'ib)' that' kept' their' names 'and,'initially'at'least,'only'superficially'embraced'the'Salafi'orientation'of' their'umbrella'organisation. 32 '' ' Third,' Ahrar' alRSham's' posture' towards' nonRJihadi' partners' was' far' more' flexible' than' that' of' alRNusra.' Although' it' expressed' no' sympathy' for' the' WesternRbacked'opposition'in'exile,'it'joined'the'Syria'Revolutionaries'Front, 33 'a' shortRlived' rebel' coalition' whose' politburo' included' MuslimRBrotherhood' affiliated' members' of' the' Syrian' National' Council,' the' National' Coalition's' ancestor. 34 'The' group' also' distanced' itself' from' alRNusra's' aforementioned' rhetorical' onslaught' against' the' National' Coalition, 35 'and' although' Ahrar' alR Sham'never'integrated'FSA'structures,'it'cooperated'with'the'latter's'provincial' Military' Councils,' and' representatives' of' the' movement' attended' a' highRprofile' meeting' convened' by' FSA' Chief' of' staff' Salim' Idris' in' Ankara' in' June' 2013. 36 ' Even' more' remarkably,' Ahrar' alRSham' rapidly' opened' channels' of' ! communication'with'Western'states, 37 'arguably'as'a'means'to'reassure'the'latter' about'its'agenda'and'consequently'avoid'being'blacklisted.'' ' Fourth,' Ahrar' alRSham's' political' platform' was' consistently' presented' in' an' ambiguous'manner'that'did'not'reflect'ideological'flexibility,'but'testified'to'the' movement's' feeling' of' embarrassment' towards' its' own' doctrinaire' agenda.' Jihadi'ideologues'who'scrutinised'the'charter'of'the'SIF'formulated'polite'regrets' at' the' fact' that' although' not' explicitly' compromising' on' key' issues' such' as' the' Islamic' state' and' the' legal' inferiority' of' nonRMuslims,' the' document' featured' vague' phrasing' that' left' the' door' open' for' various' interpretations. 38 'In' an' Al' Jazeera'interview,'Ahrar'alRSham's'leader''Abbud'showed'utterly'uncomfortable' -'and'eventually'silent'-'about'the'specifics'of'the'state'his'movement'aimed'to' establish. 39 ' ' Ahrar' alRSham's' relative' pragmatism' must' be' understood' in' the' light' of' the' unique' context' in' which' it' emerged,' that' is,' the' Arab' Awakening.' Starting' with' Sayyid'Qutb'in'the'1960s,'the'militant'Islamists''vision'of'politics'was'shaped'by' a' highly' elitist,' 'vanguardist'' model' well' illustrated' by' the' name' of' the' Syrian' group' in' which' the' elders' among' Ahrar' alRSham's' cadre' had' fought' during' 39 Al Jazeera, 9 June 2013, https://www.youtube.com/watch?v=GRPb4nFU2UA.

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 32 Al-
the' !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!

37

  Lund, Syria's Salafi Insurgents, p. 18. 38 Abu Basir al-Tartusi, 'A Note on the Charter of the Syrian Islamic Front' (in Arabic), Jihadology, 22 January 2013; Iyad Qunaybi, 'A Look at the Charter of the Syrian Islamic Front' (in Arabic), Jihadology, 22 January 2013.

  Hayat, 21 August 2012, http://alhayat.com/Details/427912; on the parochial model, see Staniland, Not to be confused with the coalition of the same name established in late 2013 by FSA commander Syria's Salafi Insurgents, p. 29; on the Muslim Brotherhood's occasional funding of Ahrar al-Sham, see Raphaël Lefèvre, 'The Syrian Brotherhood's armed struggle', Carnegie Middle East Center,

	Networks of Rebellion.
	Jamal Ma'ruf.
	14 December 2012.
	35 Lund, 'Aleppo and the battle'.
	36 Al Jazeera, 20 June 2013,

33

34

Lund, https://www.youtube.com/watch?v=simuv4yIVgU&feature=youtube_gdata_player.