Biology subjects

Grinstaff, M. W.

Publications and source records attributed to Grinstaff, M. W..

10 recordsLinked to original sources

bioRxiv2026

Raman-probe-derived biomarkers account for cartilage composition, structure, and viscoelastic properties

Raman spectroscopy is an inelastic light scattering technique that reflects the vibrational modes of the biochemical building blocks of the major constituents that comprise the cartilage extracellular matrix (ECM): glycosaminoglycans (GAG), type-II collagen (COL), and water. We investigated whether ECM-specific biomarkers measured with our recently developed arthroscopy-compatible Raman needle probe characterize the functional viscoelastic properties of articular cartilage explants. Equine osteochondral explants were harvested from high-load (femoral condyle) and low-load (intertrochlear groove) sites to evaluate Raman-derived biomarkers of cartilage structure and function. Viscoelastic material properties (equilibrium/dynamic modulus, phase shift) were determined by mechanical indentation. A computational fibril-reinforced biphasic material model was used to estimate the glycosaminoglycan-associated non-fibrillar-matrix modulus, the collagen-associated fibril-network modulus, and hydraulic permeability. Raman-derived biomarkers enabled evaluation of cartilage tissue composition and structure, accounting for 81% of the variation in GAG content, 53% of the variation in COL content, and 58% of the variation in COL anisotropy. Biomarkers also accounted for 81% of the variation in equilibrium modulus, 58% in dynamic modulus, and 51% in phase shift. For poroelastic properties, biomarkers accounted for 83% of the variation in non-fibrillar-matrix modulus, 25% of the variation in fibril-network modulus, and 34% of the variation in hydraulic permeability. Clinical Significance: Raman spectroscopy provides objective compositional biomarkers that account for viscoelastic material properties underlying articular cartilage function. These findings support its future use as an arthroscopic platform for objective assessment of cartilage injury, monitoring of disease progression, and evaluation of therapeutic interventions.

bioengineering↗

bioRxiv2026

A nicotine biosensor derived from microbial screening

Physiologically relevant biosensors are in increasingly high demand, yet existing ones are severely limited in the number and type of biomarkers that are detected. The lack of biorecognition elements for most medically relevant biomarkers restricts the development of next generation single and continuous use monitors. Over billions of years, microbes have evolved a vast array of proteins to sense and metabolize small molecules, including those pertinent to human health. Of particular interest to us is the identification and subsequent integration of new microbial redox enzymes into electronic biosensors building off the established electrochemical technology of the continuous glucose monitor. Here we deploy genomic screening to identify analyte specific redox enzymes for biosensor development. As a proof of concept, we report the first electrochemical enzyme-based nicotine biosensor from a novel microbial enzyme, and use a variant with improved catalytic performance to enhance sensor performance. The biosensor detects nicotine over 0.4-100 M, a range relevant to nicotine concentrations present in active smoker sweat, saliva, gastric juice, and urine. This microbial mining approach for discovering redox enzymes expands the sensing parts toolbox available over conventional antibodies and aptamers.

bioengineering↗

bioRxiv2025

Wearable sweat nicotine biosensor based on a nicotineoxidoreductase and its natural electron acceptor

The advent of wearable biosensors is empowering clinical and lifestyle decision making. Nicotine is a drug of significant negative impact on public health given the prevalence of smoking and vaping. Yet noninvasive, continuous monitoring of nicotine is challenging due to lack of specific and sensitive biosensing elements. Here, we report a wearable highly sensitive nicotine biosensor and demonstrate on-body deployment of the sensor in a first-in-human study. The biosensor comprises nicotine oxidoreductase (NicA2) with its natural cytochrome c electron acceptor (CycN) as mediator: both proteins play a key role in the nicotine-degrading bacterium, Pseudomonas putida S16. The biosensor detects nicotine over four-orders of magnitude (0.1-100 M) with nanomolar sensitivity (LOD [~]33.6 nM), performs in the physiological pH range (pH 6-9), and is highly selective against common interferants and the major human nicotine metabolite, cotinine. Incorporation of the biosensor in a custom wearable device enables real-time measurement of nicotine from locally induced sweat on volunteer subjects and demonstrates higher analytical accuracy than gold-standard mass spectrometry.

bioengineering↗

bioRxiv2025

A generalized and efficient approach for complete mRNA design improves translation, stability and specificity

End-to-end, machine-learning-based design of mRNA molecules offers a powerful means to tailor their properties for specific tasks. mRNA expression level, immunogenicity, tissue specificity, stability, and localization strongly depend on sequence, providing a rich set of properties amenable to optimization. Despite this potential, the components of mRNA are governed by distinct grammatical and functional rules that hinder a unified approach to complete mRNA design. While machine learning and generative AI techniques can excel on individual sequence design tasks, out-of-distribution design, where the biological objective shifts substantially from the original training data, remains difficult. Moreover, there is a disconnect between available sequence generation technologies and the diverse body of biological datasets needed to form and test mechanistic hypotheses. In this work, we describe a simple and powerful alteration to integrated gradients (Design by Integrated Gradients or DIGs) that serves as the foundation for several mRNA design tasks and an agentic hypothesis engine, the Structured RNA Evidence Aggregation Module (STREAM), which enables rapid adaptation of this technique to new contexts. Using this framework, we demonstrate complete model-informed mRNA design and reveal the underexplored rules governing the assembly of mRNA components into high-performance transcripts. By linking neural-network-based design to independent datasets, we design complete mRNA sequences in shifted settings, culminating in up to 6-fold increases in intramuscular expression compared to state-of-the-art methods in vivo. Together, DIGs and STREAM enable automated mRNA design in increasingly complex settings.

synthetic biology↗

bioRxiv2025

A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection

Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain manufacturing of traditional diagnostic platforms in low-resource settings. To address these challenges, we describe the Rapid and Modular Nanobody Assay (RAMONA) as a versatile antigen detection platform that leverages nanobody-coiled coil fusion proteins for modular integration with downstream readout methods. RAMONA merges the portability of LFAs with the benefits of nanobodies, such as their smaller size, improved solubility, and compatibility with cell-free protein synthesis systems, enabling on-demand biomanufacturing and rapid adaptation for diverse targets. We demonstrate assay generalizability through the detection of three distinct protein targets, robustness across various temperatures and incubation periods, and compatibility with saliva samples and cell-free synthesis. Detection occurs in under 30 minutes, with results strongly and positively correlating to ELISA data while requiring minimal resources. Moreover, RAMONA supports multiplexed detection of three antigens simultaneously using orthogonal capture probes. By overcoming several limitations of traditional immunoassays, RAMONA represents a significant advancement in rapid, adaptable, and field-deployable antigen detection technologies.

bioengineering↗

bioRxiv2025

A Bio-inspired Latent TGF-β Conjugated Scaffold Improves Neocartilage Development

In cartilage tissue engineering, active TGF-{beta} is conventionally supplemented in culture medium at highly supraphysiologic doses to accelerate neocartilage development. While this approach enhances cartilage extracellular matrix (ECM) biosynthesis, it further promotes tissue features detrimental to hyaline cartilage function, including the induction of tissue swelling, hyperplasia, hypertrophy, and ECM heterogeneities. In contrast, during native cartilage development, chondrocytes are surrounded by TGF-{beta} configured in a latent complex (LTGF-{beta}), which undergoes cell-mediated activation, giving rise to moderated, physiologic dosing regimens that enhance ECM biosynthesis while avoiding detrimental features associated with TGF-{beta} excesses. Here, we explore a bio-inspired strategy, consisting of LTGF-{beta}-conjugated scaffolds, providing TGF-{beta} exposure regimens that are moderated and uniformly administered throughout the construct. Specifically, we evaluate the performance of LTGF-{beta} scaffolds to improve neocartilage development with bovine chondrocyte-seeded agarose constructs compared to outcomes from active TGF-{beta} media supplementation (MS) at a physiologic 0.3 ng/mL dose (MS-0.3), supraphysiologic 10 ng/mL dose (MS-10), or TGF-{beta} free. For small-size constructs ({emptyset}3x2 mm), LTGF-{beta} scaffolds yield neocartilage that achieves native-matched mechanical properties (800-925 kPa) and sGAG content (6.6%-7.1%), while providing a cell morphology and collagen distribution more reminiscent of hyaline cartilage. LTGF-{beta} scaffolds further afford an optimal chondrogenic phenotype, marked by a 12-to 28-fold reduction of COL-I expression relative to TGF-{beta}-free and a 7-to 17-fold reduction of COL-X expression relative to MS-10. Further, for large-size constructs, which approach the dimensions needed for clinical cartilage repair, LTGF-{beta} scaffolds significantly reduce mechanical and biochemical heterogeneities relative to MS-0.3 and MS-10. Overall, the use of LTGF-{beta} scaffolds improves the composition, structure, material properties, and cell phenotype of neocartilage.

bioengineering↗

bioRxiv2023

Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1m{Psi}), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo. A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

bioengineering↗

bioRxiv2023

Acidic nanoparticles restore lysosomal acidification and rescue metabolic dysfunction in pancreatic β-cells under lipotoxic condition

Type 2 diabetes (T2D), a prevalent metabolic disorder lacking effective treatments, is associated with lysosomal acidification dysfunction as well as autophagic and mitochondrial impairments. Here, we report a series of biodegradable poly(butylene tetrafluorosuccinate-co-succinate) (PBFSU) polyesters, comprising an 1,4-butanediol linker and varying ratios of tetrafluorosuccinic acid (TFSA) and succinic acid as components, to engineer new lysosome acidifying nanoparticles (NPs). Notably, TFSA NPs, which composed entirely of TFSA, exhibit the strongest degradation capability and superior acidifying property. We further reveal significant downregulation of lysosomal vacuolar (H+)-ATPase (V-ATPase) subunits, which are responsible for maintaining lysosomal acidification, in human T2D pancreatic islets and INS-1 {beta}-cells under lipotoxic condition. Treatment of TFSA NPs counteracts lipotoxicity in INS-1 {beta}-cells by restoring lysosomal acidification, autophagic function, and mitochondrial activity, along with promoting glucose-stimulated insulin secretion. Administration of TFSA NPs to high-fat diet T2D mice improves glucose clearance and reduces insulin resistance. These findings highlight the therapeutic potential of lysosome acidifying TFSA NPs for T2D. Graphical Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/548395v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@faec9eorg.highwire.dtl.DTLVardef@1c4a6b5org.highwire.dtl.DTLVardef@19cd6ceorg.highwire.dtl.DTLVardef@1da9a4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Raman Needle Arthroscopy for In Vivo Molecular Assessment of Cartilage

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key ECM biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include: 1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2O] scores), and 2) multiplexed polarized Raman spectroscopy to quantify superficial zone collagen anisotropy via a PLS-DA-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: 1) chemically GAG-depleted bovine cartilage explants (n=40), 2) mechanically abraded bovine cartilage explants (n=30), 3) aging human cartilage explants (n=14), and 4) anatomical-site-varied ovine osteochondral explants (n=6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p<0.001). RCAF values were significantly different for explants with abrasion-induced superficial zone collagen loss (p<0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2O scores) predicted 94% of the variation in elastic modulus of ovine explants (p<0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This work advances Raman arthroscopy towards a transformative low cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

bioengineering↗

bioRxiv2017

Recapitulating bone development for tissue regeneration through engineered mesenchymal condensations and mechanical cues

Large bone defects cannot heal without intervention and have high complication rates even with the best treatments available. In contrast, bone fractures naturally healing with high success rates by recapitulating the process of bone development through endochondral ossification.1 Endochondral tissue engineering may represent a promising paradigm, but large bone defects are unable to naturally form a callus. We engineered mesenchymal condensations featuring local morphogen presentation (TGF-{beta}1) to mimic the cellular organization and lineage progression of the early limb bud. As mechanical forces are 2,3 critical for proper endochondral ossification during bone morphogenesis2,3 and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5 Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found that in vivo mechanical loading was necessary to restore bone function to large bone defects through endochondral ossification. Endochondral regeneration produced zonal cartilage and primary spongiosa mimetic of the native growth plate. Live human chondrocytes contributed to endochondral regeneration in vivo, while cell devitalization prior to condensation transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose BMP-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity.6-8 In vitro, mechanical loading promoted chondrogenesis, and upregulated pericellular collagen 6 deposition and angiogenic gene expression. Consistently, in vivo mechanical loading regulated cartilage formation and neovascular invasion dependent on load timing. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration, and provides a new template for recapitulating developmental programs for tissue engineering.

bioengineering↗