Search

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Despite the fact that the ability of MSCs to proliferate and differentiate has been studied extensively, there still remains much to learn about the signalling mechanisms and pathways which control proliferation and influence differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signalling systems and that acetylcholine (ACh) plays an important, ubiquitous, role in cell-to-cell communication. Indeed, cholinergic signalling is hypothesised to occur in stem cells and ACh synthesis, as well as ACh receptor (AChR) expression, has been identified in several stem cell populations; including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC will be summarised with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

The aim of this article was to study clinical and radiographic outcomes following treatment of bone healing disorders with a novel osteoinductive system that utilizes poly (ethyl acrylate), fibronectin and an ultra-low concentration of recombinant human bone morphogenetic protein-2. A case series of nine dogs and two cats were treated, and clinical records and radiographs were reviewed. Radiographs were scored by two blinded observers using the modified Radiographic Union Score for Tibial Fractures. Long-term follow-up was obtained using the Canine Orthopaedic Index and Feline Musculoskeletal Pain Index. Follow-up data were available for 11 treatments (10 cases). Complications: three minor, three major, one catastrophic (non-union requiring amputation). Lameness median 320 (range: 42–1,082) days postoperatively: ‘sound’ (three cases), ‘subtle’ (two), ‘mild’ (three), ‘moderate’ (one), and ‘non-weightbearing’ (one). The attending clinician judged 9 of 11 treatments achieved radiographic union; modified Radiographic Union Score for Tibial Fractures observers 1 and 2 agreed with the clinician in 8/9 and 5/9 treatments respectively. Long-term Canine Orthopaedic Index scores for five dogs median 650 (range: 544–1,724) days postoperatively: 15/64 (median) for four dogs with acceptable outcomes, 30/64 in one dog with a poor outcome. Feline Musculoskeletal Pain Index scores for two cats 433 and 751 days postoperatively: 48/60 and 60/60. Eight of 10 cases were sound or showed subtle or mild lameness in the short- or long-term, and radiographic union occurred in the majority of treatments.

Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologist grouped individuals by modified Bell’s staging (healthy, NEC1, NEC2/3) and faecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Bacillota (adj. p=0.0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of faecal cytokine levels revealed significantly higher concentrations of IL-1α (p=0.045), IL-5 (p=0.0074), and IL-10 (p=0.032) in Bell stage 1 NEC compared to healthy individuals. Conclusions: Differences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

ObjectivesNecrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed in NEC infants. New techniques, using biomarkers as diagnostic tool to improve diagnosis of NEC, are emerging. Here we investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers.Study designPremature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. All but two babies received a first course of antibiotics from birth onwards. Faecal samples from diapers were collected consecutively during the NICU stay.ResultsEvaluation of microbiota profiles between the study groups revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Firmicutes, withEnterococcusbeing the least abundant in Bell stage 2/3 NEC. Faecal cytokine levels were similar to those found in previous studies evaluating systemic cytokine concentrations in NEC settings, but measurement in faeces represents a non-invasive method to evaluate the early onset of the disease. For IL-1α, IL-5 and IL-10, a significantly rising gradient of levels were observed from healthy to NEC1 to NEC2/3.ConclusionsDifferences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles, contribute to improving knowledge about NEC pathogenesis.

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via allogeneic stem cell transplantation (alloSCT), to engraft and repopulate the blood system. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs (ST-HSC) and progenitor populations at the expense of naïve LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs or pericytes). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal ability and without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

SummaryPreterm infants with very low birthweight are at serious risk for necrotizing enterocolitis. To functionally analyse the principles of three successful preventive NEC regimens, we characterized faecal samples of 54 infants (< 1,500 g, n = 383) longitudinally (two weeks) with respect to gut microbiome profiles (bacteria, archaea, fungi, viruses), microbial function, virulence factors, antibiotic resistances and metabolic profiles, including human milk oligosaccharides (HMOs) and short-chain fatty acids. Probiotic Bifidobacterium longum ssp. infantis supplementation affected microbiome development globally, pointing toward the genomic potential to convert HMOs. Engraftment of Bifidobacterium substantially reduced microbiome-associated antibiotic resistance as compared to regimens using probiotic Lactobacillus rhamnosus or no supplementation. Crucially, the beneficial effects of Bifidobacterium supplementation depended on simultaneous feeding with HMOs. We demonstrate that preventive regimens have the highest impact on early maturation of the gastrointestinal microbiome, enabling the establishment of a resilient microbial ecosystem that reduces pathogenic threats in at-risk preterm infants.

[EN] Mesenchymal stem cells represent an important resource, for bone regenerative medicine and therapeutic applications. This review focuses on new advancements and biophysical tools which exploit different physical and chemical markers of mesenchymal stem cell populations, to finely characterize phenotype changes along their osteogenic differentiation process. Special attention is paid to recently developed label-free methods, which allow monitoring cell populations with minimal invasiveness. Among them, quantitative phase imaging, suitable for single-cell morphometric analysis, and nanoindentation, functional to cellular biomechanics investigation. Moreover, the pool of ion channels expressed in cells during differentiation is discussed, with particular interest for calcium homoeostasis.Altogether, a biophysical perspective of osteogenesis is proposed, offering a valuable tool for the assessment of the cell stage, but also suggesting potential physiological links between apparently independent phenomena.

Breakthroughs in our understanding of the complex interplay between cellular nanoenvironment and biomolecular signalling pathways are facilitating development of targeted osteogenic platforms. As critical biomolecules for osteogenesis, growth factors stimulate osteogenesis by activating key genes and transcription factors. The first half of this review presents emerging interconnectedness and recent discoveries of osteogenic signalling pathways initiating from growth factors for example, bone morphogenetic protein 2 (BMP-2). To complement this, the second half of review proposes a number of strategies to induce osteogenesis which include metallic, organic implants, nanotopological environments as well as growth factor immobilization techniques. The drawbacks of traditional osteogenic implants and how these have been overcome by biomedical engineers in the recent years without producing side-effects have also been summarized.

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features also significantly reduce MSC adhesion and activity. Here, we use a poly (ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. Using a novel MSC–Pseudomonas aeruginosa co-culture, we show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules, enhance MSC adhesion and osteoblast differentiation and reduce biofilm formation. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection, and supporting osteogenic repair.

Twenty-five years have passed since the first clinical trial utilising mesenchymal stomal/stem cells (MSCs) in 1995. In this time academic research has grown our understanding of MSC biochemistry and our ability to manipulate these cells in vitro using chemical, biomaterial, and mechanical methods. Research has been emboldened by the promise that MSCs can treat illness and repair damaged tissues through their capacity for immunomodulation and differentiation. Since 1995, 31 therapeutic products containing MSCs and/or progenitors have reached the market with the level of in vitro manipulation varying significantly. In this review, we summarise existing therapeutic products containing MSCs or mesenchymal progenitor cells and examine the challenges faced when developing new therapeutic products. Successful progression to clinical trial, and ultimately market, requires a thorough understanding of these hurdles at the earliest stages of in vitro pre-clinical development. It is beneficial to understand the health economic benefit for a new product and the reimbursement potential within various healthcare systems. Pre-clinical studies should be selected to demonstrate efficacy and safety for the specific clinical indication in humans, to avoid duplication of effort and minimise animal usage. Early consideration should also be given to manufacturing: how cell manipulation methods will integrate into highly controlled workflows and how they will be scaled up to produce clinically relevant quantities of cells. Finally, we summarise the main regulatory pathways for these clinical products, which can help shape early therapeutic design and testing.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs' responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells' mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

[EN] Biomaterials have evolved from inert materials that lack interaction with the body to biologically active, instructive materials that host and provide signals to surrounding cells and tissues. Engineered living materials contain living cells (responsive function) and polymeric matrices (scaffolding function) and, thus, can be designed as active and response biomaterials. In this Review, we discuss engineered living materials that incorporate microorganisms as the living, bioactive component. Microorganisms can provide complex responses to environmental stimuli, and they can be genetically engineered to allow user control over responses and integration of numerous inputs. The engineered microorganisms can either generate their own matrix, such as in biofilms, or they can be incorporated in matrices using various technologies, such as coating, 3D printing, spinning and microencapsulation. We highlight biomedical applications of such engineered living materials, including biosensing, wound healing, stem-cell-based tissue engineering and drug delivery, and provide an outlook to the challenges and future applications of engineered living materials., Support from EPSRC through a programme grant (EP/P001114/1) is acknowledged. M.S.-S. and M.J.D acknowledge support from a grant from the UK Regenerative Medicine Platform "Acellular/Smart Materials - 3D Architecture" (MR/R015651/1). S.S. and A.d.C. acknowledge support from the Deutsche Forschungsgemeinschaft's Collaborative Research Centre, SFB 1027 and the Leibniz Science Campus on Living Therapeutic Materials, LifeMat.

Models of bone remodelling could be useful in drug discovery, particularly if the model is one that replicates bone regeneration with reduction in osteoclast activity. Here we use nanovibrational stimulation to achieve this in a 3D co-culture of primary human osteoprogenitor and osteoclast progenitor cells. We show that 1000 Hz frequency, 40 nm amplitude vibration reduces osteoclast formation and activity in human mononuclear CD14+ blood cells. Additionally, this nanoscale vibration both enhances osteogenesis and reduces osteoclastogenesis in a co-culture of primary human bone marrow stromal cells and bone marrow hematopoietic cells. Further, we use metabolomics to identify Akt (protein kinase C) as a potential mediator. Akt is known to be involved in bone differentiation via transforming growth factor beta 1 (TGFβ1) and bone morphogenetic protein 2 (BMP2) and it has been implicated in reduced osteoclast activity via Guanine nucleotide-binding protein subunit α13 (Gα13). With further validation, our nanovibrational bioreactor could be used to help provide humanised 3D models for drug screening.

Summary The gut microbiota’s function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development., Graphical abstract, Highlights • Microbiota disturbances promote tumor growth in several breast cancer models • Increased tumor volume positively correlates with stromal mast cell density • Supplementation with a commensal restores tumor growth to normal, Pathophysiology; Microbiology; Cancer

It is well known that extracellular matrix stiffness can affect cell fate and change dynamically during many biological processes. Existing experimental means for in situ matrix stiffness modulation often alters its structure, which could induce additional undesirable effects on cells. Inspired by the phenomenon of depth sensing by cells, we introduce here core–shell microfibers with a thin collagen core for cell growth and an alginate shell that can be dynamically stiffened to deliver mechanical stimuli. This allows for the maintenance of biochemical properties and structure of the surrounding microenvironment, while dynamically modulating the effective modulus “felt” by cells. We show that simple addition of Sr2+ in media can easily increase the stiffness of initially Ca2+ cross-linked alginate shells. Thus, despite the low stiffness of collagen cores (

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

[EN] Central nervous system (CNS) pathologies are a prevalent problem in aging populations, creating a need to understand the underlying events in these diseases and develop efficient CNS-targeting drugs. The importance of the blood-brain barrier (BBB) is evident, acting both as a physical barrier to drug entry into the CNS and potentially as the cause or aggravator of CNS diseases. The development of a biomimetic BBB invitro model is required for the understanding of BBB-related pathologies and in the screening of drugs targeting the CNS. There is currently great interest in understanding the influence of biochemical and biophysical factors, as these have the potential to greatly improve the barrier function of brain microvascular endothelial cells (BMECs). Recent advances in understanding how these may regulate barriergenesis in BMECs help promote the development of improved BBB invitro models and therefore novel interventional therapies for pathologies related to its disruption. Herein, an overview of specific biochemical and biomechanical cues in the formation of the BBB, with a focus on invitro models and how these might recapitulate the BBB function, is provided., The authors acknowledge support via the EPSRC program grant (EP/P001114/1) and funding from MRC (MR/R005567-1), Stroke Association (PPA 2016/02) and Heart Research UK (RG 2664/17/20) to TVA. All the figures were created with BioRender.com. The authors would like to apologize to their colleagues whose work they could not cite due to space constraints.

[EN] Collagen hydrogels are among the most well-studied platforms for drug delivery and in situ tissue engineering, thanks to their low cost, low immunogenicity, versatility, biocompatibility, and similarity to the natural extracellular matrix (ECM). Despite collagen being largely responsible for the tensile properties of native connective tissues, collagen hydrogels have relatively low mechanical properties in the absence of covalent cross-linking. This is particularly problematic when attempting to regenerate stiffer and stronger native tissues such as bone. Furthermore, in contrast to hydrogels based on ECM proteins such as fibronectin, collagen hydrogels do not have any growth factor (GF)-specific binding sites and often cannot sequester physiological (small) amounts of the protein. GF binding and in situ presentation are properties that can aid significantly in the tissue regeneration process by dictating cell fate without causing adverse effects such as malignant tumorigenic tissue growth. To alleviate these issues, researchers have developed several strategies to increase the mechanical properties of collagen hydrogels using physical or chemical modifications. This can expand the applicability of collagen hydrogels to tissues subject to a continuous load. GF delivery has also been explored, mathematically and experimentally, through the development of direct loading, chemical cross-linking, electrostatic interaction, and other carrier systems. This comprehensive article explores the ways in which these parameters, mechanical properties and GF delivery, have been optimized in collagen hydrogel systems and examines their in vitro or in vivo biological effect. This article can, therefore, be a useful tool to streamline future studies in the field, by pointing researchers into the appropriate direction according to their collagen hydrogel design requirements., This work was supported by Medical Research Scotland, EPSRC (through a programme grant EP/P001114/1) and a programme of research funded by the Sir Bobby Charlton Foundation. M.S.S. acknowledges support from a grant from the UK Regenerative Medicine Platform 'Acellular/Smart Materials - 3D Architecture' (MR/R015651/1). The graphical abstract was created using BioRender.com.

Design of bioinspired materials that mimic the extracellular matrix (ECM) at the nanoscale is a challenge in tissue engineering. While nanofibrillar gelatin materials mimic chemical composition and nano-architecture of natural ECM collagen components, it lacks the characteristic D-staggered array (D-periodicity) of 67 nm, which is an important cue in terms of cell recognition and adhesion properties. In this study, a nanofibrous gelatin matrix with improved biomimicry is achieved using a formulation including a minimal content of D-periodic self-assembled atelocollagen. We suggest a processing route approach consisting of the thermally induced phase separation of the gelatin based biopolymeric mixture precursor followed by chemical-free material cross-linking. The matrix nanostructure is characterized using field emission gun scanning electron microscopy (FEG-SEM), transmission electron microscopy (TEM), wide angle X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). The cell culture assays indicate that incorporation of 2.6 wt.% content of D-periodic atelocollagen to the gelatin material, produces a significant increase of MC3T3-E1 mouse preosteoblast cells attachment and human mesenchymal stem cells (hMSCs) proliferation, in comparison with related bare gelatin matrices. The presented results demonstrate the achievement of an efficient route to produce a cost-effective, compositionally defined and low immunogenic “collagen-like” instructive biomaterial, based on gelatin

We report the use of self-assembled peptide (F

We report the use of self-assembled peptide (F2/S) hydrogels and cellular metabolomics to identify a number of innate molecules that are integral to the metabolic processes which drive cellular differentiation of multipotent pericyte stem cells. The culture system relies solely on substrate mechanics to induce differentiation in the absence of traditional differentiation media and therefore is a non-invasive approach to assessing cellular behavior at the molecular level and identifying key metabolites in this process. This novel approach demonstrates that simple metabolites can provide an alternative means to direct stem cell differentiation and that biomaterials can be used to identify them simply and quickly.

Osteogenic activity metabolites can be identified in nanovibrational culture and have their specificity and potency enhanced., Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce osteogenic differentiation of mesenchymal stem cells, in the absence of off-target, nonosteogenic differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to culture media, provides an artifact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite, cholesterol sulfate, an endogenous steroid. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate with both specific and highly potent osteogenic-inducing activity. Further, we implicate cytoskeletal contractility as a measure of osteogenic potency and cell stiffness as a measure of specificity. These findings demonstrate that physical principles can be used to identify bioactive metabolites and then enable optimization of metabolite potency can be optimized by examining structure-function relationships.