Search

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1neurons coincident with lactation. After reducing CCN3 in ARCKISS1neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4β1 and αvβ3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro,the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4β1 and αvβ3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+cells, osteocalcin+cells, CD34+/CD45-cells and CD31+cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.

Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated β-galactosidase.We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent.Impact StatementThe accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.

To realize the promise of three-dimensional (3D) bioprinting, it is imperative to develop bioinks that possess the necessary biological and rheological characteristics for printing cell-laden tissue grafts. Alginate is widely used as a bioink because its rheological properties can be modified through precrosslinking or the addition of thickening agents to increase printing resolution. However, modification of alginate's physiochemical characteristics using common crosslinking agents can affect its cytocompatibility. Therefore, we evaluated the printability, physicochemical properties, and osteogenic potential of four common alginate bioinks: alginate-CaCl2(alg-CaCl2), alginate-CaSO4(alg-CaSO4), alginate-gelatin (alg-gel), and alginate-nanocellulose (alg-ncel) for the 3D bioprinting of anatomically accurate osteogenic grafts. While all bioinks possessed similar viscosity, printing fidelity was lower in the precrosslinked bioinks. When used to print geometrically defined constructs, alg-CaSO4and alg-ncel exhibited higher mechanical properties and lower mesh size than those printed with alg-CaCl2or alg-gel. The physical properties of these constructs affected the biological performance of encapsulated bone marrow-derived mesenchymal stromal cells (MSCs). Cell-laden constructs printed using alg-CaSO4and alg-ncel exhibited greater cell apoptosis and contained fewer living cells 7 days postprinting. In addition, effective cell–matrix interactions were only observed in alg-CaCl2printed constructs. When cultured in osteogenic media, MSCs in alg-CaCl2constructs exhibited increased osteogenic differentiation compared to the other three bioinks. This bioink was then used to 3D print anatomically accurate cell-laden scaphoid bones that were capable of partial mineralization after 14 days of in vitroculture. These results highlight the importance of bioink properties to modulate cell behavior and the biofabrication of clinically relevant bone tissues.Impact statementAlginate-based bioinks are widely used for three-dimensional (3D) bioprinting of bone tissues. However, a direct systematic comparison between alginate-based bioinks is needed to assess the optimal bioink properties for mesenchymal stromal cell survival and osteogenesis. This study evaluates the printability, physical properties, biocompatibility, and osteogenic potential of four commonly used alginate-based bioinks and establishes the importance of bioink properties for advancing toward the clinical translation of 3D bioprinted bone grafts.

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors (GFs) to influence neighboring cells and promote migration, proliferation, or differentiation. Despite promising results in preclinical models, the use of inductive biomacromolecules has achieved limited success in translation to the clinic. The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages, which commonly result in detrimental side effects. Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix (ECM), which acts as a reservoir for GFs viaelectrostatic interactions. Advances in the manipulation of extracellular proteins, decellularized tissues, and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs. Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules. This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone, cartilage, and muscle.

About 5% of the UK population are military veterans and have specific medical needs sometimes different from the general population. Veterans may be reluctant to seek help or talk about their problems, which means they do not always access care, support and treatments that are available. Others may face difficulty getting the right help. Veterans may think ‘civilians’do not understand military culture or know about options for help and services that are available. Experience has shown that general practitioners (GPs) would like more help and support when looking after veterans. The Royal College of General Practitioners has developed the ‘Veteran Friendly GP Practice Accreditation Programme’, which involves a simple online process of accreditation where practices are required to meet the specified criteria and provide evidence that they are supportive of veterans’ healthcare. The aim is to improve healthcare provided to veterans and their families by GPs working in primary healthcare.

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high‐passage numbers. However, the degree of cell senescence is dynamic, and cell‐cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin‐rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low‐passage, high‐passage, and mixtures of low‐ and high‐passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD‐treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high‐passage MSCs, the addition of low‐passage MSCs reduced p16expression, a known genetic marker of senescence. We observed a significant increase in p16expression in high‐passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low‐ and high‐passage cells and represent an exciting approach to use higher‐passage cells in cell‐based therapies. This study investigated the ability of low‐passage mesenchymal stem/stromal cells (MSCs), when formed into spheroids, to suppress the expression of senescence markers in high‐passage MSCs via tunneling nanotubes as a first step to retain MSC therapeutic potential.

To determine if preoperative leg pain and low back pain severity affected postoperative outcome.

Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links.

Cell-based therapies are a promising alternative to grafts and organ transplantation for treating tissue loss or damage due to trauma, malfunction, or disease. Over the past two decades, mesenchymal stem cells (MSCs) have attracted much attention as a potential cell population for use in regenerative medicine. Although the proliferative capacity and multilineage potential of MSCs provide an opportunity to generate clinically relevant numbers of transplantable cells, their use in tissue regenerative applications has met with relatively limited success to date apart from secreting paracrine-acting factors to modulate the defect microenvironment. Presently, there is significant effort to engineer the biophysical properties of biomaterials to direct MSC differentiation and further expand on the potential of MSCs in tissue engineering, regeneration, and repair. Biomaterials can dictate MSC differentiation by modulating features of the substrate including composition, mechanical properties, porosity, and topography. The purpose of this review is to highlight recent approaches for guiding MSC fate using biomaterials and provide a description of the underlying characteristics that promote differentiation toward a desired phenotype.

There is a critical need to develop noninvasive, nondestructive methods for assessing the quality of engineered constructs prior to implantation. Currently, the composition and maturity of engineered tissues are assessed using destructive, costly, and time-consuming biochemical and mechanical analyses. The goal of this study was to use noninvasive, multimodal imaging to monitor osteogenic differentiation and matrix deposition by human mesenchymal stem/stromal cells (MSCs) during in vitro culture. MSCs were encapsulated in alginate hydrogels and cultured in osteogenic conditions for 4 weeks. Samples were evaluated using fluorescence lifetime imaging (FLIm) and ultrasound backscatter microscopy (UBM) prior to traditional biochemical and mechanical testing. Using linear regression analysis, we identified strong correlations between imaging parameters (e.g., fluorescence lifetime and acoustic attenuation coefficient) and destructive mechanical and biochemical tests to assess the maturation of osteogenically induced constructs. These data demonstrate the promise of nondestructive label-free imaging techniques to noninvasively ascertain the progression and maturity of tissue engineered bone grafts.

Bone marrow aspirates provide a rich source of cells for use in tissue engineering of bone and other clinical indications. However, progenitor cells such as mesenchymal stem cells (MSCs) account for a small fraction of nucleated cells in bone marrow aspirate (BMA), requiring extensive culture expansion. Accessory cell populations such as endothelial or hematopoietic cells can potentiate the bone-forming potential of MSCs, and cell-secreted extracellular matrix (ECM) can increase cell seeding efficiency and osteogenic differentiation of heterogeneous cell populations. In this study, we hypothesized that cell-secreted ECM could be used to sequester MSCs and accessory cells from BMA for bone regeneration. To generate 3D implantable constructs, BMA was resuspended in media with or without type I collagen or ECM and injected into a perfusion bioreactor system. The addition of protein coatings increased cell seeding efficiency compared to uncoated scaffolds. Compared to fresh BMA, the culture of BMA on all scaffolds reduced the proportion of CD45+ myeloid cells and increased CD31+CD45− endothelial cells. Compared to uncoated scaffolds, we observed a 143- and 30-fold increase in MSCs when fresh BMA was cultured on ECM- or collagen-coated scaffolds, respectively. Upon subcutaneous implantation, ECM-coated scaffolds promoted cell survival and early vascularization. However, bone formation was comparable across all implant groups, suggesting additional osteogenic cues are necessary to increase the bone forming potential of fresh BMA. These results motivate further investigation into strategies which elicit more robust bone regeneration by tissue aspirates.

Tunneling nanotubes (TNTs) play an important role in cell–cell communication. TNTs have been predominantly reported among cells in monolayer culture. Using various imaging modalities, including scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM), this work reports the finding of TNTs between cells within human mesenchymal stem cell (MSC) spheroids. TNTs visualized by SEM are consistent in size and geometry with those observed in cellular monolayer culture. LSCM imaging of living spheroids confirms the presence of F-actin filaments within the TNTs, which are known to maintain nanotube integrity. In addition, LSCM revealed the distribution of F-actin fibers across the entire spheroid body instead of being confined within individual cells. Intracellular material transport by TNTs was tested in MSC spheroids treated with cytochalasin D (CytoD), a known actin polymerization inhibitor for disrupting TNT formation. CytoD treatment decreased the transport of cytosolic material by at least four-fold compared to untreated spheroids. To the best of our knowledge, this work represents the first direct observation of TNTs within MSC spheroids. These findings offer new physical insight into cellular interactions within spheroids, providing structural information for increasing interests in spheroid-based cell therapy.

Lung nodules are a diagnostic challenge, with an estimated yearly incidence of 1.6 million in the United States. This study evaluated the accuracy of an integrated proteomic classifier in identifying benign nodules in patients with a pretest probability of cancer (pCA) ≤ 50%.

Cell‐based approaches for musculoskeletal tissue repair are limited by poor cell survival and engraftment. Short‐term hypoxic preconditioning of mesenchymal stem cells (MSCs) can prolong cell viability in vivo, while the aggregation of MSCs into spheroids increases cell survival, trophic factor secretion, and tissue formation in vivo. We hypothesized that preconditioning MSCs in hypoxic culture before spheroid formation would increase cell viability, proangiogenic potential, and resultant bone repair compared with that of individual MSCs. Human MSCs were preconditioned in 1% O2in monolayer culture for 3 days (PC3) or kept in ambient air (PC0), formed into spheroids of increasing cell density, and then entrapped in alginate hydrogels. Hypoxia‐preconditioned MSC spheroids were more resistant to apoptosis than ambient air controls and this response correlated with duration of hypoxia exposure. Spheroids of the highest cell density exhibited the greatest osteogenic potential in vitro and vascular endothelial growth factor (VEGF) secretion was greatest in PC3 spheroids. PC3 spheroids were then transplanted into rat critical‐sized femoral segmental defects to evaluate their potential for bone healing. Spheroid‐containing gels induced significantly more bone healing compared with gels containing preconditioned individual MSCs or acellular gels. These data demonstrate that hypoxic preconditioning represents a simple approach for enhancing the therapeutic potential of MSC spheroids when used for bone healing. StemCells2018;36:1393–1403 Preconditioning MSCs in hypoxia prior to spheroid formation enhances their contribution towards bone regeneration.

Microgels are an emerging platform for in vitro models and guiding cell fate due to their inherent porosity and tunability. This work describes a light‐based technique for rapidly annealing microgels across a range of diameters. Utilizing 8‐arm poly(ethylene) glycol‐vinyl sulfone, the number of arms available for crosslinking, functionalization, and annealing is stoichiometrically controlled. Small and large microgels are fabricated to explore how microgel diameter impacts void space and the role of porosity on cell spreading, cell aggregation, and macrophage polarization. Mesenchymal stromal cells spread rapidly in both formulations, yet the smaller microgels permit a higher cell density. When seeded with macrophages, the smaller microgels promote an M1 phenotype, while larger microgels promote an M2 phenotype. As another application, the inherent porosity of annealed microgels is leveraged to induce cell aggregation. Finally, the microgels are implanted to examine how different size microgels influence endogenous cell invasion and macrophage polarization. The use of ultraviolet light allows for microgels to be noninvasively injected into a desired mold or wound defect before annealing, and microgels of different properties combined to create a heterogeneous scaffold. This approach is clinically relevant given its tunability and fast annealing time. Microgels are an emerging platform that have significant advantages over conventional bulk hydrogels including microporosity, modularity, and ability to decouple pore size from stiffness. Differences in microgel size directly correlate with porosity, which in turn influences how cells spread, interact, and ultimately their phenotype. Injectability and an annealing time of 1 min make microgels a promising therapeutic for the clinic.

Within the last two centuries, the Mississippi River basin has been transformed by changes in land use practices, dam construction, and training of the rivers for navigation. Here we analyze the contemporary patterns of fluvial sediment yield in the Mississippi River basin using all available data in order to assess the influence of regional land condition on the variation of sediment yield within the basin. We develop regional‐scale relations between specific sediment yield (yield per unit area) and drainage area to reveal contemporary regional sediment yield patterns and source areas of riverine sediments. Extensive upland erosion before the development of soil conservation practices exported large amounts of sediment to the valleys and floodplains. We show that sediment today is sourced primarily along the river valleys from arable land, and from stream bank and channel erosion, with sediment yields from areas dominated by arable land 2 orders of magnitude greater than that of grassland dominated areas. Comparison with the “Tfactor,” a commonly quoted measure of agricultural soil resilience suggests that the latter may not reflect contemporary soil loss from the landscape. We use an unexploited data set to study spatial patterns of sediment dynamics along the Mississippi basinOur analysis shows that river sediments today are derived primarily from the major valleysThe results have implication toward soil security and management

Groundwater discharge along streams exerts an important influence on biogeochemistry and thermal regimes of aquatic ecosystems. A common approach for predicting locations of shallow lateral groundwater discharge is to use digital elevation models (DEMs) combined with upslope contributing area algorithms. We evaluated a topography‐based prediction of subsurface discharge zones along a 1500 m headwater stream reach using temperature and water isotope tracers. We deployed fiber‐optic distributed temperature sensing instrumentation to monitor stream temperature at 0.25 m intervals along the reach. We also collected samples of stream water for the analysis of its water isotope composition at 50 m intervals on five occasions representing distinct streamflow conditions before, during, and after a major rain event. The combined tracer evaluation showed that topography‐predicted locations of groundwater discharge were generally accurate; however, predicted magnitude of groundwater inflows estimated from upslope contributing area did not always agree with tracer estimates. At the catchment scale, lateral inflows were an important source of streamflow at base flow and peak flow during a major rain event; however, water from a headwater lake was the dominant water source during the event hydrograph recession. Overall, this study highlights potential utility and limitations of predicting locations and contributions of lateral groundwater discharge zones using topography‐based approaches in humid boreal regions. Topography‐based predicted locations of discrete lateral subsurface inflow along a boreal stream agreed with tracer estimatesTopography‐based predicted magnitude of lateral subsurface inflow did not always agree with tracer estimatesFlow from a headwater lake was much greater than lateral subsurface inflow contributions to streamflow following a rain event

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three‐dimensional spheroids increases cell survival and augments their anti‐inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti‐inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2(PGE2) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE2and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model—demonstrating efficacy on three key cell types that are dysfunctional in chronic non‐healing wounds. We conclude that DOE‐based analysis effectively identifies optimal culture conditions to enhance the anti‐inflammatory and proangiogenic potential of MSC spheroids. StemCells2017;35:1493–1504 Multivariable analysis was used to determine culture conditions that optimize the anti‐inflammatory and proangiogenic potential of mesenchymal stem cell spheroids for potential use in wound healing applications.

Spheroids exhibit enhanced cell–cell interactions that facilitate improved survival and mimic the physiological cellular environment in vivo. Cell spheroids have been successfully used as building blocks for engineered tissues, yet the viability of this approach with skeletal muscle spheroids is poorly understood, particularly when incorporated into three-dimensional (3D) constructs. Bioprinting is a promising strategy to recapitulate the hierarchical organization of native tissue that is fundamental to its function. However, the influence of bioprinting on skeletal muscle cell spheroids and their function are yet to be interrogated. Using C2C12 mouse myoblasts and primary bovine muscle stem cells (MuSCs), we characterized spheroid formation as a function of duration and cell seeding density. We then investigated the potential of skeletal muscle spheroids entrapped in alginate bioink as tissue building blocks for bioprinting myogenic tissue. Both C2C12 and primary bovine MuSCs formed spheroids of similar sizes and remained viable after bioprinting. Spheroids of both cell types fused into larger tissue clusters over time within alginate and exhibited tissue formation comparable to monodisperse cells. Compared to monodisperse cells in alginate gels, C2C12 spheroids exhibited greater MyHC expression after 2 weeks, while cells within bovine MuSC spheroids displayed increased cell spreading. Both monodisperse and MuSC spheroids exhibited increased expression of genes denoting mid- and late-stage myogenic differentiation. Together, these data suggest that skeletal muscle spheroids have the potential for generating myogenic tissue via 3D bioprinting and reveal areas of research that could enhance myogenesis and myogenic differentiation in future studies.

The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone‐forming potential. Spheroids formed from induced cells exhibited improved retention of osteogenic markers as a function of integrin binding to cell‐secreted extracellular matrix (ECM). These results demonstrate the capacity of spheroidal culture to sustain the mineral‐producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone‐forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone‐forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone‐forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer‐cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1integrin binding to cell‐secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral‐producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Despite the promise of mesenchymal stem cells (MSCs) for cell‐based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell‐secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair.

Mesenchymal stem cell (MSC)‐based therapies are under investigation for tissue repair but suffer from poor cell persistence and engraftment upon transplantation. When entrapped in an adhesive biomaterial, MSC spheroids exhibited improved survival and proangiogenic growth factor secretion in vitro and bone formation in vivo compared with cells in nonadhesive hydrogels. These findings demonstrate the value of deploying MSC spheroids in instructive biomaterials to improve cell function. Mesenchymal stem cell (MSC)‐based therapies are under broad investigation for applications in tissue repair but suffer from poor cell persistence and engraftment upon transplantation. MSC spheroids exhibit improved survival, anti‐inflammatory, and angiogenic potential in vitro, while also promoting vascularization when implanted in vivo. However, these benefits are lost once cells engage the tissue extracellular matrix and migrate from the aggregate. The efficacy of cell therapy is consistently improved when using engineered materials, motivating the need to investigate the role of biomaterials to instruct spheroid function. In order to assess the contribution of adhesivity on spheroid activity in engineered materials and promote the bone‐forming potential of MSCs, we compared the function of MSC spheroids when entrapped in Arg‐Gly‐Asp (RGD)‐modified alginate hydrogels to nonfouling unmodified alginate. Regardless of material, MSC spheroids exhibited reduced caspase activity and greater vascular endothelial growth factor (VEGF) secretion compared with equal numbers of dissociated cells. MSC spheroids in RGD‐modified hydrogels demonstrated significantly greater cell survival than spheroids in unmodified alginate. After 5 days in culture, spheroids in RGD‐modified gels had similar levels of apoptosis, but more than a twofold increase in VEGF secretion compared with spheroids in unmodified gels. All gels contained mineralized tissue 8 weeks after subcutaneous implantation, and cells entrapped in RGD‐modified alginate exhibited greater mineralization versus cells in unmodified gels. Immunohistochemistry confirmed more diffuse osteocalcin staining in gels containing spheroids compared with dissociated controls. This study demonstrates the promise of cell‐instructive biomaterials to direct survival and function of MSC spheroids for bone tissue engineering applications. Mesenchymal stem cell (MSC) spheroids exhibit improved therapeutic potential in vitro compared with dissociated MSCs, yet spheroids are directly injected into tissues, ceding control of cell function to the extracellular matrix and potentially limiting the duration of improvement. Cell delivery using adhesive biomaterials promotes cell retention and function. These studies explored the role of adhesion to the surrounding matrix on spheroid function. When entrapped in an adhesive biomaterial, MSC spheroids exhibited improved survival and proangiogenic growth factor secretion in vitro and bone formation in vivo compared with cells in nonadhesive hydrogels. These findings demonstrate the value of deploying MSC spheroids in instructive biomaterials to improve cell function.

The presentation and controlled release of bioactive signals to direct cellular growth and differentiation represents a widely used strategy in tissue engineering. Historically, work in this field has primarily focused on the delivery of large cytokines and growth factors, which can be costly to manufacture and difficult to deliver in a sustained manner. There has been a marked increase over the past decade in the pursuit of lipid mediators due to their wide range of effects over multiple cell types, low cost, and ease of scale-up. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are two bioactive lysophospholipids (LPLs) that have gained attention for use as pharmacological agents in tissue engineering applications. While these lipids can have similar effects on cellular response, they possess distinct chemical backbones, mechanisms of synthesis and degradation, and signaling pathways using a discrete set of G-protein-coupled receptors (GPCRs). LPA and S1P predominantly act extracellularly on their GPCRs and can directly regulate cell survival, differentiation, cytokine secretion, proliferation, and migration—each of the important functions that must be considered in regenerative medicine. In addition to these potent physiological functions, these LPLs play pivotal roles in a number of pathophysiological processes. To capitalize on the promise of these molecules in tissue engineering, these lipids have been incorporated into biomaterials for in vivodelivery. Here, we survey the effects of LPA and S1P on both cellular- and tissue-level phenotypes, with an eye toward regulating stem/progenitor cell growth and differentiation. In particular, we examine work that has translational applications for cell-based tissue engineering strategies in promoting cell survival, bone and cartilage engineering, and therapeutic angiogenesis.

A growing body of research on stream thermal regimes has highlighted the importance of heat advection associated with surface water and groundwater interactions, such as hyporheic exchange, groundwater discharge, and hillslope throughflow inputs. Existing catchment models that predict stream temperature use a variety of approaches to estimate throughflow temperatures, but none has been evaluated against field measurements of throughflow temperature. In this study, throughflow temperatures were monitored over two winters at 50 locations adjacent to a headwater stream (11 ha catchment area) located in the rain‐on‐snow zone of the Pacific Northwest. Existing approaches to estimate throughflow temperature under or overpredicted throughflow temperatures by up to 5°C, or were unable to represent the influence of transient snow cover. Therefore, a conceptual‐parametric model that is computationally efficient was developed that simulates hillslope hydrology and throughflow temperatures. The model structure includes an upslope reservoir that drains into a downslope reservoir that, in turn, drains into the stream. Vertical and lateral energy and water fluxes are simulated using simplified process representations. The model successfully predicts throughflow temperatures and highlights the dominant role of throughflow advection and the influence of snow cover on stream thermal regimes during high flow periods and rain‐on‐snow events. Throughflow temperatures varied between 2 and 12°C during the study periodThroughflow temperature models under or overpredict or do not represent snowA new throughflow temperature model is developed that includes the role of snow

The clinical translation of mesenchymal stromal cell (MSC)‐based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue‐forming potential by promoting cell–cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell‐secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell‐secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell–matrix and cell–cell interactions. ECM‐loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes‐associated protein (YAP) activation via integrin α2β1 binding. ECM‐loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage‐specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM‐loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM‐loaded spheroids as building blocks for the repair of musculoskeletal tissues. Mesenchymal stromal cell (MSC) aggregation into spheroids increases their tissue‐forming potential by promoting cell–cell interactions. However, MSC spheroids initially lack engagement with an extracellular matrix (ECM). Cell‐secreted ECM incorporated into spheroids promotes cell–matrix interactions, increasing MSC mechanosensing, survival, and response to soluble stimuli. ECM‐loaded MSC spheroids are a promising strategy to produce clinically relevant tissues for musculoskeletal regeneration.

Mesenchymal Stromal Cells In article number 2102337by J. Kent Leach and co‐workers, an engineered cell‐secreted extracellular matrix (ECM) is incorporated into mesenchymal stromal cell (MSC) spheroids to promote cell–cell and cell–matrix interactions and enhance MSC survival and differentiation. Activation of YAP through integrin binding to the incorporated ECM promotes MSC proliferation and osteogenic differentiation, highlighting the therapeutic potential of ECM‐loaded spheroids as building blocks for bone repair.