High-density Tissue Research Articles

Multidirectional Filamented Light Biofabrication Creates Aligned and Contractile Cardiac Tissues.

Biofabricating 3D cardiac tissues that mimic the native myocardial tissue is a pivotal challenge in tissue engineering. In this study, we fabricate 3D cardiac tissues with controlled, multidirectional cellular alignment and directedor twistingcontractility. We show that multidirectional filamented light can be used to biofabricate high-density (up to 60 ×106 cellsmL-1) tissues, with directed uniaxial contractility (3.8x) and improved cell-to-cell connectivity (1.6x gap junction expression). Furthermore, by using multidirectional light projection, we can partially overcome cell-induced light attenuation, and fabricatelarger tissues with multidirectional cellular alignment. For example, we fabricate a tri-layered myocardium-like tissue and a bi-layered tissue with torsional contractility. The approach provides a new strategy to rapidly fabricate aligned cardiac tissues relevant to regenerative medicine and biohybrid robotics.

Investigation and comparison on the interaction of laser and bio-tissue based on two classical models, the beam broaden model and diffusion approximation equation model, theoretically and experimentally

Three methods are used for a numerical solution, the Monte Carlo method, diffusion approximation equation model, and beam broaden model based on Beer–Lambert’s law equation. The comparison between the first two methods is reported theoretically, and the latter is a better choice in the high-density tissue. However, the comparison between the third method and the first or the second method is rarely reported. Two classical theoretical models describing the interaction between the laser and the bio-tissue are analyzed and compared to determine which is more suitable for analyzing the interaction, the beam broaden model or diffusion approximation equation model. Intensity distribution is simulated and compared for the two models. Temperature distribution and thermal damage are investigated theoretically and experimentally for both models. The differences and the reasons are analyzed. The diffusion approximation equation model is more suitable for analyzing the mechanism between the laser and the bio-tissue based on the degree of fitting between the simulated and experimental data. Theoretical analyses for the two models are carried out in detail. The comparison between the two models is rarely reported, and it is reported in this article for the first time, theoretically and experimentally. This report provides a better choice for quickly analyzing the interaction mechanism between the laser and the bio-tissue.

Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage

ObjectiveChondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in HIF-1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. MethodsPrimary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4 – 16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). ResultsIncreasing media volumes resulted in higher accumulation of cartilaginous extracellular matrix (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the TCA cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. ConclusionsElevated media availability upregulates extracellular matrix synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in extracellular matrix accumulation.

From acquisitive to conservative: The ontogenetic trajectory of root syndrome of annual and perennial co-generic species

Perennials has been proposed as new crops because their root syndrome offers more ecosystem services and a better use of resources than the annual one. Objective: To compare the ontogenetic trajectory of root syndromes of annual (P. gracilis and P. angustifolia) and perennial (P. pinetorum and P. mendocina) species of Physaria (Brassicaceae) and their root trait relationships. Results and ConclusionRoots became conservative as plants grew, independently of the life cycle of the species. However, perennials invested initially more resources in increasing storage capacity (bigger and thicker roots) while annuals favored increasing the exploration capacity (higher SRL). During first anthesis we found tradeoffs between acquisitive and conservative traits, as expected according to the Root Economic Spectrum (RES), but after the first year, those tradeoffs disappeared and perennials were able to combine a thick and deep taproot and high investment in C reserves with some well-developed acquisitive traits such as higher SRL and total length. This suggest that root traits should be selected during the second year, when no tradeoff exist. Differences between annuals and perennials in most structural taproot traits remained constant along the ontogenetic trajectory, thus the relative position of species in the RES did not change over time. We found a gradient of root strategies supported by the RES theory: from acquisitive in the annual P. gracilis (high SRL, STD and total length) to conservative in the perennial P. mendocina (high root biomass, root diameter, tissue density and storage) with intermediate strategies in P. angustifolia and P. pinetorum.

13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

A self-assembly and cellular migration based fabrication of high-density 3D tubular constructs of barrier forming membranes.

Three-dimensional (3D) in vitro models, superior in simulating physiological conditions compared to 2D models, offer intricate cell-cell and cell-ECM interactions with diverse signaling cues like fluid shear stress and growth factor gradients. Yet, developing 3D tissue barrier models, specifically perfusable luminal structures with dense, multicellular constructs maintained for extended durations with oxygen and nutrients, remains a technical challenge. Here, we describe a molding-based approach for the fabrication of free-standing, perfusable, high cellular density tissue constructs using a self-assembly and migration process to form functional barriers. This technique utilizes a polytetrafluoroethylene (PTFE)-coated stainless-steel wire, held by stainless steel needles, as a template for a perfusable channel within an elongated PDMS well. Upon adding a bio-ink mix of cells and collagen, it self-assembles into a high cell density layer conformally around the wire. Removing the wire reveals a hollow construct, connectable to an inlet and outlet for perfusion. This scalable method allows creating varied dimensions and multicellular configurations. Notably, post-assembly, cells such as human umbilical vein endothelial cells (HUVECs) migrate to the surface and form functional barriers with adherens junctions. Permeability tests and fluorescence imaging confirm that these constructs closely mimic in vivo endothelial barrier permeability, exhibiting the lowest permeability among all in vitro models in the literature. Unlike traditional methods involving uneven post-seeding of endothelial cells leading to subpar barriers, our approach is a straightforward alternative for fabricating complex perfusable 3D tissue constructs and effective tissue barriers for use in various applications, including tissue engineering, drug screening, and disease modeling.

Changes of the facial soft tissue after mandibular reconstruction using vascularized iliac flap.

To reveal the change patterns of the facial soft tissue after applying mandibular reconstruction. 16 Patients with mandibular benign tumor were recruited in this retrospective study. For all patients, segmental mandibular osteotomy and concurrent reconstruction using vascularized iliac flap were conducted. The soft tissue thickness of patients' lower face was measured with CT scans before surgery, 1week, 6months and 1year after surgery. The time-dependent changes of tissue thickness were analyzed. The most significant tissue swelling was 28.86%, at 1week after the surgery. The average increase of tissue thickness was 4.78 ± 5.30mm across patient. After 1year of the surgery, tissue thickness decreased to the level before operation or the level of the healthy side. The thickness of the low-density tissue fluctuated mildly, while the thickness of the high-density tissue fluctuated significantly. The disuse atrophy of the masseter occurred 1week after the surgery, and was reversed after 1year. The removal of the submandibular gland caused depression in submandibular area, which intensified over time. Across patients, soft tissue thickness in the lower face after mandibular osteotomy and reconstruction increased significantly 1week after the surgery, and decreased over time. After 1year, tissue thickness went back to the pre-surgery level, where matched up with the healthy side. We documented the change patterns of the facial soft tissue after mandibular reconstruction. These results can help improve the planning of virtual surgeries and the timing for aesthetic assessment. ClinicalTrials.gov Identifier: ChiCTR2100054103.

The association of body composition and outcomes following autologous hematopoietic stem cell transplantation in patients with non-Hodgkin lymphoma.

Recently there has been a growing interest in evaluating body composition as a marker for prognosis in cancer patients. The association of body composition parameters and outcomes has not been deeply investigated in patients with autologous hematopoietic stem cell transplantation (HSCT) recipients with non-Hodgkin lymphoma (NHL). We conducted a retrospective cohort study of 264 NHL patients who received autologous HSCT. PreHSCT abdominal CT scans at the levels of L3 were assessed for body composition measures. We evaluated sarcopenia, myosteatosis, high visceral adipose tissue (VAT) and high visceral adipose tissue density (VATD). Using multivariable Cox proportional regression, we analyzed the association of clinical and transplant-related characteristics with overall survival (OS), relapse-free survival (RFS), and non-relapse mortality (NRM). In a multivariate regression model, patients with higher VATD had worse OS (HR 1.78; 95% confidence intervals CI 1.08-2.95, p = 0.02) and worse NRM (HR 2.31 95% CI 1.08-4.95, p = 0.02) than with lower VATD. Patients with lower levels of VAT also had worse RFS (HR 1.49 95% CI 1.03-2.15, p = 0.03). Sarcopenia and myosteatosis were not associated with outcomes. High pre-transplant VATD was associated with lower OS and higher NRM, and low pre-transplant VAT was associated with worse RFS in patients with NHL undergoing autologous HSCT.

Deep dives and high tissue density increase mean dive costs in California sea lions (Zalophus californianus).

Diving is central to the foraging strategies of many marine mammals and seabirds. Still, the effect of dive depth on foraging cost remains elusive because energy expenditure is difficult to measure at fine temporal scales in wild animals. We used depth and acceleration data from eight lactating California sea lions (Zalophus californianus) to model body density and investigate the effect of dive depth and tissue density on rates of energy expenditure. We calculated body density in 5 s intervals from the rate of gliding descent. We modeled body density across depth in each dive, revealing high tissue densities and diving lung volumes (DLVs). DLV increased with dive depth in four individuals. We used the buoyancy calculated from dive-specific body-density models and drag calculated from swim speed to estimate metabolic power and cost of transport in 5 s intervals during descents and ascents. Deeper dives required greater mean power for round-trip vertical transit, especially in individuals with higher tissue density. These trends likely follow from increased mean swim speed and buoyant hinderance that increasingly outweighs buoyant aid in deeper dives. This suggests that deep diving is either a 'high-cost, high-reward' strategy or an energetically expensive option to access prey when prey in shallow waters are limited, and that poor body condition may increase the energetic costs of deep diving. These results add to our mechanistic understanding of how foraging strategy and body condition affect energy expenditure in wild breath-hold divers.

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine.

The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2µL), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and ∼2000 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. STATEMENT OF SIGNIFICANCE: A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.

Constructing high-density tissue microarrays with a novel method and a self-made tissue-arraying instrument.

Tissue microarrays (TMAs), also called tissue chips, contain hundreds to thousands of tissue cores obtained from different tissue donor blocks. By using TMA technology, a molecular marker, such as protein, RNA or DNA, can be simultaneously examined in hundreds of different specimens under the same experimental conditions. A growing number of previous studies have introduced different methods for constructing TMAs. Many authors tried to use various methods to implant more tissue cores in a single recipient block, and most of these methods involved reducing the diameter of the tissue cores and/or the spacing between adjacent tissue cores. However, when creating TMAs, it is difficult to reduce the distance between tissue cores to zero except with extremely expensive automatic TMA arrayers. Here, we introduce a novel method to construct a high-density TMA that does not have spacing between the tissue cores. We also introduce a method for preparing a self-made tissue-arraying instrument. With this method and the tissue-arraying instrument, we successfully created a TMA containing 126 tissue cores that were 2mm in diameter. H&E staining and immunohistochemical staining were performed on the sections cut from the TMA without any tissue spot loss. This method is easy to operate, and the materials for creating the tissue-arraying instrument are inexpensive and can be purchased anywhere. Therefore, this method can be applied in all laboratories.

Magnetic field induced dose effects in radiation therapy using MR-linacs.

MR-guided radiotherapy (MRgRT) is one of the most significant advances in radiotherapy in recent years. The hybrid systems were designed to visualize patient anatomical and physiological changes during the course of radiotherapy, enabling more precise treatment. However, before MR-linacs reach their full potential in delivering safe and accurate treatments to patients, the radiotherapy team must understand how a magnetic field alters the dosimetric properties of the radiation beam and its potential impact on treatment quality and clinical outcomes. This review aims to provide an in-depth description of the magnetic field induced dose effects for the two widely available systems, the 0.35 T and the 1.5 T MR-linacs. In MR-linac treatments, the primary photon beam passes through MR components that never exist in conventional linacs, which alter both in-field and out-of-field doses. More importantly, the interplay between the always-on magnetic field and the secondary electrons is not negligible. This interplay affects dose deposition in the patient, resulting in reduced in-field skin dose due to purged-out contaminant electrons, shortened build-up distance and a shifted crossline profile owing to asymmetric dose kernel. Especially two effects, namely, electron return effect (ERE) and electron stream effect (ESE), are not seen in conventional radiotherapy. This review also summarizes the clinical observations on the site-specific treatments influenced mostly by the magnetic field. In MR-linac treatment, the head and neck region is one of the most challenging sites as ERE occurs at low and high density tissue interfaces and around air cavities, generating hot and cold spots. In breast cancer treatment, consideration should be given to the increased in-field skin dose induced by ERE and the increased out-of-field dose caused by ESE for regions such as the ears, chin, and neck. In lung cancer treatments, tissue inhomogeneity combined with ERE will exacerbate target dose heterogeneity and increase or decrease interface dose. Lastly, treatment in the abdomen and pelvic region will be affected by the presence of gas pockets near the target. The review provides practical recommendations to mitigate these effects.

Intravenous-Therapy-Associated Circulatory Overload: A Retrospective Study of Forensic Cases

Background and Objective: Intravenous therapy is widely used in clinics to deliver medications and fluids to patients, and overuse may cause intravenous-infusion-associated circulatory overload (IACO) and death. However, forensic data on deaths from the overuse of intravenous therapy are limited. We performed a retrospective study to identify whether causes of death in forensic practice were associated with IACO. Methods: A total of 572 medical-related cases with a history of intravenous infusion who suffered from injuries or illnesses and died after treatment in hospitals were recruited from two centers of forensic medicine between 2002 and 2018. Results: The results demonstrated that 6.47% of cases (37/572) were exposed to an infusion overdose that resulted in deaths related to IACO, and 43.24% of cases (16/37) had a net fluid retention ranging from 3.0 L/d to 13.8 L/d. The highest case was administered 1.4 L blood products and 13.6 L fluids within 25 h. We observed significant decreases in red blood cells, hematocrit, hemoglobin, and platelets from the time of “on admission” to “before death”, except for white blood cells. Autopsy findings of 16 cases revealed some organ or tissue pathological alterations related to IACO, including pale-yellow liquid overflow under the abdominal epidermis, much transudate in body cavities, and lung edema with a high tissue density and no obvious slurry deposition in the alveolar lumen. Another 21 cases died after a rapid infusion rate ranging from 111 to 293 drops/min, which resulted from viral myocarditis on autopsy. Conclusions: Our data on excessive or irrational use of intravenous therapy indicate a severe circulatory overload, which may eventually result in lethal outcomes. Therefore, the use of improper intravenous therapy should be reduced to ameliorate adverse health consequences during clinical treatment.

Bioengineering of High Cell Density Tissues with Hierarchical Vascular Networks for ex vivo Whole Organs.

The development of tissue-like structures such as cell sheets, spheroids, and organoids has contributed to progress in regenerative medicine. Simultaneous achievement of scale up and high cell density of these tissues is challenging because sufficient oxygen cannot be supplied to the inside of large, high cell density tissues. Here, we show in vitro fabrication of vessels to supply oxygen to the inside of millimetre-sized scaffold-free tissues whose cell density is approximately 200 million cells/mL, corresponding to those of native tissues. Hierarchical vascular networks by anastomosis of capillaries and a large vessel were essential for oxygen supply, whereas a large vessel or capillary networks alone made negligible contributions to the supply. The hierarchical vascular networks are formed by a top-down approach, which offers a new option for ex vivo whole organs without decellularization and 3D-bioprinting. This article is protected by copyright. All rights reserved.

Bone Density Variation in Rattails (Macrouridae, Gadiformes): Buoyancy, Depth, Body Size, and Feeding.

Extreme abiotic factors in deep-sea environments, such as near-freezing temperatures, low light, and high hydrostatic pressure, drive the evolution of adaptations that allow organisms to survive under these conditions. Pelagic and benthopelagic fishes that have invaded the deep sea face physiological challenges from increased compression of gasses at depth, which limits the use of gas cavities as a buoyancy aid. One adaptation observed in deep-sea fishes to increase buoyancy is a decrease of high-density tissues. In this study, we analyze mineralization of high-density skeletal tissue in rattails (family Macrouridae), a group of widespread benthopelagic fishes that occur from surface waters to greater than 7000 m depth. We test the hypothesis that rattail species decrease bone density with increasing habitat depth as an adaptation to maintaining buoyancy while living under high hydrostatic pressures. We performed micro-computed tomography (micro-CT) scans on 15 species and 20 specimens of rattails and included two standards of known hydroxyapatite concentration (phantoms) to approximate voxel brightness to bone density. Bone density was compared across four bones (eleventh vertebra, lower jaw, pelvic girdle, and first dorsal-fin pterygiophore). On average, the lower jaw was significantly denser than the other bones. We found no correlation between bone density and depth or between bone density and phylogenetic relationships. Instead, we observed that bone density increases with increasing specimen length within and between species. This study adds to the growing body of work that suggests bone density can increase with growth in fishes, and that bone density does not vary in a straightforward way with depth.

Breast Tissue Chemistry Measured In Vivo In Healthy Women Correlate with Breast Density and Breast Cancer Risk

Representative two-dimensional correlated spectroscopy (2D COSY) spectra of the region F2/F1: 0.00 ppm to 6.00 ppm in participants at low risk. (a) low dense tissue and premenopausal status (30 years old), (b) low dense tissue and postmenopausal (54 years old), (c) high dense tissue and premenopausal (36 years old), and (d) high dense tissue and postmenopausal (66 years old). Note the increase in intensity for cross peaks F, D, G, and G′ across categories. By Gorane Santamaría et al. (1355–1369)

IL-33 Participates in the Development of Esophageal Adenocarcinoma.

Background: The progression from chronic gastroesophageal reflux disease (GERD) to Barrett esophagus (BE) and esophageal adenocarcinoma (EAC) is an inflammatory-driven neoplastic change. Interleukin-33 (IL-33) has identified as a crucial factor in several inflammatory disorders and malignancies. Methods: The high-density tissue microarray of the human EAC was analyzed with IL-33 immunohistochemistry staining (IHC). By anastomosing the jejunum with the esophagus, the rat model of EAC with mixed gastroduodenal reflux was established. The expression of IL-33 was determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blot (WB), IHC and enzyme-linked immunosorbent assay (ELISA). Esophageal adenocarcinoma cells (OE19 and OE33) and human esophageal epithelial cells (HEECs) were used. Results: In the cytoplasm of human EAC tissue, IL-33 expression was substantially greater than in adjacent normal tissue. In rat model, the expression of IL-33 in the EAC group was considerably greater than in the control group, and this expression increased with the upgrade of pathological stage. In in vitro experiment, the mRNA and protein levels of IL-33 were considerably greater in OE19 and OE33 than in HEECs. The stimulation of IL-33 enhanced the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OE19 and OE33, but soluble ST2 (sST2) inhibited these effects. IL-33 stimulated the release of IL-6 by OE19 and OE33 cells. Conclusion: This study demonstrated the overexpression of IL-33 in the transition from GERD to EAC and that IL-33 promoted carcinogenesis in EAC cells through ST2. IL-33 might be a possible preventive target for EAC.

Elevational trends of tree fine root traits in species‐rich tropical Andean forests

With increasing elevation, trees in tropical montane forests have to invest larger fractions of their resources into their fine roots in order to compensate for increasingly unfavorable soil conditions. It is unclear how elevation and related edaphic changes influence the variability in tree fine root traits and belowground functional diversity. We measured six fine root traits related to resource acquisition on absorptive fine roots of 288 trees from 145 species along an elevational gradient from 1000 m to 3000 m a.s.l. in tropical montane forests of the Ecuadorian Andes. We analyzed trait relationships with elevation and soil nutrient availability, and tested whether root functional diversity varied along these gradients. Fine roots at higher elevations and at more nutrient‐poor sites were thicker, had higher tissue densities, and lower specific root length and nutrient concentrations than at lower elevations. These trends were diluted by the coexistence of tree species with a broad range of different root traits within communities particularly towards lower elevations, where root functional diversity was significantly higher. We conclude that nutrient limitation and potentially further adverse conditions at higher elevations are strong environmental filters that lead to trait convergence towards a conservative resource use strategy, whereas different trait syndromes are equally successful at lower elevations.

Soil conditions drive below‐ground trait space in temperate agricultural grasslands

Abstract Plant below‐ground organs perform essential functions, including water and nutrient uptake, anchorage, vegetative reproduction and recruitment of mutualistic soil microbiota. Recently, multivariate analyses showed that root traits of species can largely be linked to a ‘conservation’ and a ‘collaboration’ gradient. Here, we tested whether this species‐level bidimensional below‐ground trait space also exists at the community level in grasslands. Furthermore, we tested whether the position of grassland communities in below‐ground trait space relates to environmental variables. For a total of 313 species, we collected data on eight below‐ground traits in greenhouse and common garden experiments and supplemented it with data on bud‐bank size and specific leaf area from databases. We calculated community weighted means (CWMs) of these 10 traits for 150 temperate grassland plots to investigate below‐ground plant‐trait dimensionality and its variation along 10 soil and land‐use parameters. Using PCA, we found that about 55% of variance in CWMs was explained by two main dimensions, corresponding to a mycorrhizal ‘collaboration’ and a resource ‘conservation’ gradient. Frequently overlooked traits such as rooting depth, bud‐bank size and root‐branching intensity were largely integrated in this trait space. The two plant‐strategy gradients were partially dependent on each other, with communities that do ‘outsourcing’ of resource uptake to mycorrhizal fungi along the collaboration gradient also being more ‘slow’ along the conservation gradient. (i.e. high root tissue density and high root weight ratio). ‘Outsourcing’ communities were also more often deep rooting and associated with soil parameters, such as low moisture and sand content, high topsoil pH, high C:N and low δ15N. ‘Slow’ communities had large bud banks and were associated with low land‐use intensity, high topsoil pH and low nitrate but high ammonium concentration in the soil. Surprisingly, we did not find an association of phosphorus availability with the mycorrhizal ‘collaboration’ gradient. Synthesis. The ‘collaboration’ and ‘conservation’ gradients previously identified among species scale up to the community level in grasslands, encompass more traits than previously described, and vary with the environment.

Adaptation strategy of karst forests: Evidence from the community-weighted mean of plant functional traits.

Conservative survival strategy of plants growing in harsh karst habitats is observed from the view of plant functional traits, such as morphological traits and ecological stoichiometry. However, whether the plant communities in karst forests with high species turnover adopt a conservative strategy remains undetermined. This study comprehensively investigated the characteristics of functional traits of dominant plant species in four forests (i.e. Platycarya strobilacea, Quercus fabri, Quercus variabilis, and Pinus massoniana forests) in a trough‐valley karst watershed in Northern Guizhou Province, Southwestern China to explore the adaptation strategy of karst forests at the community level. At the organ and the species levels, traits differed among species, and the leaf and the bark morphological traits and root C:N:P ecological stoichiometry presented large interspecific variations. At the community level, the P. massoniana forest presented the lowest specific root length and dry matter content and tissue density of roots, branch, twig, and bark; the Q. fabri and the Q. variabilis forests displayed low specific leaf area and high dry matter content and tissue density of roots, branch, and twig; and the Platycarya strobilacea forest exhibited high specific leaf area. The P. massoniana forest was subjected to N and P colimitation, and the three other broad‐leaved forests were limited by P supply. The community‐weighted means rather than the arithmetic means of traits were preferential to represent the trait characteristics at the community level. From the view of plant functional traits at the community level, karst forests develop multiple functional traits like low specific leaf area, high dry matter content and tissue density of leaf, roots, branch, and twig, and decrease N and P investments in leaf for a conservative survival strategy to adapt to harsh habitats.