Biology subjects

Haglund, L.

Publications and source records attributed to Haglund, L..

16 recordsLinked to original sources

bioRxiv2026

Multiscale fracture resistance of human annulus fibrosus and its deterioration with degeneration

Soft fibrous tissues must resist fracture under repeated loading, yet how degeneration compromises this resistance remains unclear. We investigated human annulus fibrosus using fracture and fatigue testing, proteoglycan depletion, microscopy, and X-ray scattering. Non-degenerate tissue exhibited pronounced fracture anisotropy, with axial toughness nearly fourfold higher than interlamellar delamination toughness. Degeneration reduced axial and radial toughness despite comparatively preserved tensile modulus and strength. Proteoglycan depletion reduced toughness most strongly in radial and delamination loading. Neither tissue state showed measurable macroscopic crack extension over 20,000 cycles at 70% of its respective monotonic failure strain. Nevertheless, fatigue produced localized structural changes, and degenerate tissue exhibited secondary delamination and diffuse collagen damage. X-ray scattering revealed fatigue- and degeneration-dependent changes in collagen periodicity and intermolecular packing. These findings connect tissue fracture resistance to collagen-matrix interactions across scales and inform the design of crack-resistant biomaterials.

bioengineering↗

bioRxiv2026

A Standardized In Vitro Platform for Senolytic Drug Discovery in Human Musculoskeletal Cells

Cellular senescence contributes to the progression of many age related musculoskeletal diseases. Cellular senescence is a biological state that arises from replicative exhaustion and various cellular stressors, including elevated oxidative stress, mitochondrial dysfunction, mechanical overload, and chronic exposure to pro-inflammatory cytokines and proteases. Although senolytic agents show promise for eliminating senescent cells, their translation has been hindered by the lack of physiologically relevant and scalable in vitro screening methods. In the present study, we developed a standardized, physiologically relevant senescence-induction model and validated a metabolic activity assay as a rapid, scalable method for screening senolytic compounds. We used primary human intervertebral disc cells (IVD) as an example, but the workflow applies to many other cell types. To mimic inflammatory and oxidative stress, we used a combination of TLR-2 activation (Pam2CSK4) and tert-butyl hydroperoxide (tBHP), a potent ROS generator. Senescence induction was validated by quantifying {beta}-galactosidase fluorescence intensity, {beta}-gal enzymatic activity, and the expression of the p16 senescence marker across 3 IVD cell types: nucleus pulposus (NP), inner annulus fibrosus (iAF), and outer annulus fibrosus (oAF) cells. The combined Pam2CSK4 + tBHP exposure generated a robust senescent phenotype across all 3 IVD cell types, with oAF cells exhibiting the strongest increases in {beta}-gal fluorescence, {beta}-gal enzymatic activity, and p16 expression. We then used oAF cells to evaluate if the metabolic activity assay (Alamar Blue) could be used to determine both cytotoxicity of senolytic drugs in non-senescent cells and senolytic activity in a mixed population of senescent and non-senescent cells. We validate the method by comparing metabolic activity results with {beta}-gal enzymatic activity and p16 expression in induced and noninduced cells following exposure to three known senolytics (o-Vanillin, RG-7112, and ABT-199). The metabolic activity assay reliably identified a therapeutic window in which the three senolytics were non-toxic to non-senescent cells while selectively reducing metabolic activity in a mixed population of senescent and non-senescent cells. The reductions in metabolic activity in the mixed population correlated with decreases in SA {beta}-gal enzymatic activity and p16 expression, validating metabolic activity as a sensitive and scalable senolytic readout.

cell biology↗

bioRxiv2026

3D-Printed Scaffolds for Local Chemotherapeutic Delivery in Resected Spine Metastases

Spinal metastases often occur secondary to breast, lung and prostate cancer and lead to instability, pain and poor quality of life. Standard care for spine metastases includes a multidisciplinary approach with surgery playing a major role in tumor resection, stabilization and decompression. Surgical resection with adjuvant is an effective treatment, yet it is often accompanied by tumor recurrence from residual disease. Furthermore, acrylic cements applied to defect sites provide stability, but they do not promote bone repair and can become destabilized during recurrence events. Developing new tools to stabilize defect sites, promote bone repair and locally deliver therapeutics may circumvent these limitations. We have previously developed mechanically competent 3D printed lactide/mineral scaffolds conducive to bone repair in vivo. We have also developed 3D printed nanoporous scaffolds conducive to both bone repair and chemotherapeutic delivery. Here, we set out to assess doxorubicin and cisplatin uptake and release rates and efficacy of drug delivery in 2D and custom physiological 3D cultures of two human cancer cell lines associated with metastases, MDA-MB-231 (human breast) and C42B (human prostate). Composite scaffolds had a compressive modulus close to trabecular bone, and could sustainably and effectively release doxorubicin and cisplatin as measured against both breast and prostate cell lines in 2D and 3D custom physiological metastases models. As a proof-of-concept, doxorubicin loaded composite scaffolds were implanted into rat caudal vertebrae following MDA-MB-231 xenograft resection. Following 6 weeks of implantation, no adverse events were noted and microCT analysis revealed boney integration of the construct. Taken together, these data indicate that our composite scaffolds may be an appropriate alternate therapy to stabilize bone defects, promote bone repair and effectively inhibit cancer recurrence post-tumor resection. Future work will test composite scaffolds using in vivo bone metastases models.

bioengineering↗

bioRxiv2026

Targeting therapy-induced senescence across multiple breast cancer subtypes in a metastatic bone-like microenvironment

Chemotherapeutic treatment of breast cancer with Doxorubicin (DOX) can induce tumor and stromal cell senescence leading to therapy-resistance. Senescence-associated secretory phenotype (SASP) promotes secretion of pro-inflammatory and tumorigenic factors causing systemic inflammation. Combined, this can result in immune suppression, tumor growth and secondary spread of cancer. Targeting and removing senescent and cancerous cells using a combination of chemotherapeutic and senolytic drugs may reduce systemic inflammation, improve therapeutic efficacy, and prevent metastasis. Exposure of triple-negative breast cancer (MDA-MB-231), hormone-responsive (MCF-7) and HER2+ (MDA-MB-453) cells, and primary spine osteoblasts to DOX showed significant induction of p21-positive senescent cells. DOX and senolytics (RG-7112, o-Vanillin) treatment of co-culture spheroids showed a significant additive effect in reducing tumor sphere viability and growth, indicating reduced metastatic potential. This was correlated with reduced SASP in triple-negative and hormone responsive lines and decreased levels of senescent cells in all subtypes and primary stromal cells, while proliferation was decreased, and apoptosis increased across all breast cancer subtypes. Future chemotherapeutic treatment in breast cancer models may be optimized by adding senolytic drugs to more effectively clear senescent tumor and stromal cells, reducing risk for relapse and metastatic potential, while allowing for tissue regeneration in the bone metastatic environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724653v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@c4cb8forg.highwire.dtl.DTLVardef@105219org.highwire.dtl.DTLVardef@17e0517org.highwire.dtl.DTLVardef@802bd2_HPS_FORMAT_FIGEXP M_FIG C_FIG Senolytics selectively eliminate senescent cancer and stromal cells and enhance Doxorubicin efficacy in a 3D bone-like tumor microenvironment model.

cancer biology↗

bioRxiv2026

DNA Demethylation is Dispensable for Venetoclax-HMA Synergy in Acute Myeloid Leukaemia

The BCL-2 inhibitor venetoclax (VEN) in combination with hypomethylating agents (HMAs) has improved treatment responses in acute myeloid leukaemia (AML), but the mechanisms underlying their synergy remain unclear. We investigated the role of DNA demethylation in the enhanced cytotoxicity of VEN-HMA combinations. Using AML cell lines, we compared the effects of azacitidine (AZA), decitabine (DAC), cytarabine (ARA-C) and the DNMT1-selective inhibitor GSK-3685032 (GSK5032) with VEN. As expected, VEN showed strong synergy with AZA, DAC, and the DNA-damaging agent ARA-C, but not with GSK5032, despite the latter inducing extensive DNA demethylation. Genome-wide methylation profiling confirmed that loss of DNA methylation did not correlate with increased cytotoxicity or synergy with VEN. Moreover, combining GSK5032 with ARA-C did not enhance cytotoxicity, indicating that DNA demethylation and DNA damage do not act additively. Instead, synergy was consistently associated with the DNA damage-inducing properties of AZA, DAC, and ARA-C. Extensive DNA demethylation tended to antagonize VEN activity, suggesting that the epigenetic effects of HMAs may limit their synergistic potential. Overall, our findings demonstrate that DNA damage-related cytotoxicity, rather than DNA demethylation, is the dominant mechanism driving VEN-HMA synergy and provide evidence that VEN-mediated cytotoxicity arises primarily from genotoxic stress, supporting refinement of treatment strategies.

cancer biology↗

bioRxiv2026

Tissue-to-Analysis Framework Enables Multiscale Mapping of the Architectural and Cellular Organization in the Human Dorsal Root Ganglion

Transcriptomic studies have helped us understand the dorsal root ganglias cellular milieu, yet our knowledge of protein expression and spatial organization/architecture remains less defined. Here we establish a comprehensive resource from processing through analysis of hDRG tissue. We optimize tissue-handling strategies and evaluate 114 antibodies targeting neuronal and non-neuronal cell types, identifying protocols that preserve neuronal morphology and antigen retain specificity. Integrating these workflows with our Deep Learning-assisted image analysis pipelines, we quantify size, expression, and spatial organization across 35,721 neurons from 15 donors. Female donors exhibited significantly larger neuronal somata, indicating sexual dimorphism. Neuronal subpopulations display clear spatial clustering. We further characterized the perineuronal niche, marked by dense vascularization, nuclear remodeling in perineuronal cells, and age-related increased turnover of neuron-associated macrophages. Together, this resource provides standardized methodologies and quantitative frameworks for reproducible protein-level interrogation of human sensory biology and pain mechanisms.

neuroscience↗

bioRxiv2025

Neutrophil-neuronal crosstalk drives arthritis-induced pain

Pain in rheumatoid arthritis (RA) often persists despite effective control of inflammation, suggesting distinct mechanisms driving nociception. In both patients and animal models, pain severity does not strongly correlate with the degree of inflammation1,2. Sensory neurons, with cell bodies located in the dorsal root ganglia (DRG), innervate peripheral tissues, including joints, and transmit pain signals to the central nervous system. Crosstalk between sensory neurons and immune cells occurs at all of these sites. While sensory neurons can be directly activated by immune mediators, it remains unclear whether pain-like behaviour in antibody-induced arthritis models arises independently of immune cell activity, or which immune cell populations and mediators are required to activate pronociceptive mechanisms. Through temporal profiling of the CAIA joint-DRG transcriptomic axis, we identified SEMA4D and OSM signalling as candidate molecular mediators of neutrophil-neuron communication and neuronal sprouting. The joint-DRG atlas also revealed persistent changes in the fibroblast-immune cellular composition of the joint, along with molecular changes in DRG neurons. We showed that mechanical and cold hypersensitivity, as well as sprouting of CGRP+ nociceptive fibers in synovial tissue of mice with collagen antibody-induced arthritis (CAIA), require neutrophils but not macrophages. Analysis of publicly available datasets showed that neutrophils from the synovium of RA patients express high levels of SEMA4D and OSM, and corresponding expression of their receptors, PLEXINB1 and OSMR, in human DRG neurons, underscoring the translational relevance of this axis. Both murine and human-derived DRG neurons sprout in response to OSM. Our findings demonstrate that neutrophils produce molecules that act as cues for nociceptor sensitization and structural remodelling. Targeting these molecules could improve the efficacy of RA treatments by reducing pain while simultaneously preventing disease progression.

immunology↗

bioRxiv2025

A novel method to sort and enrich sensory neurons

Peripheral sensory neurons, residing in the dorsal root ganglia (DRG), relay sensory information from the periphery to the central nervous system. Although single-cell transcriptomic studies have identified over 20 distinct sensory neuron subtypes, functional analysis and assessment of subtype-specific pathological changes remain difficult. Effective isolation and enrichment of sensory neurons are challenging yet essential for functional studies. Therefore, we used single-cell transcriptomic data from DRG to identify a panel of neuronal surface markers, including Nrxn2 and Pirt. Using these markers, we developed a fluorescence-activated cell sorting (FACS) panel for neuronal enrichment and analysis that does not rely on transgenic mouse strains and can be broadly applied. The panel was validated by microscopy and single-cell RNA (scRNA) sequencing, which also revealed broad representation of neuronal subtypes. Expression of these markers in human DRG underscores the translational value of this isolation method for sensory and pain studies. Overall, this study provides a valuable tool for isolating DRG neurons, advancing research on sensory neuron function and pain biology, and facilitating neuroimmune studies.

neuroscience↗

bioRxiv2025

Senolytic Therapy as a Preventive Strategy for Low Back Pain

Cell senescence drives inflammation and tissue breakdown and is a key hallmark of aging. Low back pain is strongly linked to age-related degeneration of spine tissues, and with an accumulation of senescent. Here we show that preventive administration of the senolytic agents o-vanillin and RG-7112 prevent the development of pain-related behaviour in young sparc-/- mice. Treated mice exhibit a reduction of senescence markers in the intervertebral discs, vertebral endplates, vertebral bone, and spinal cord, alongside a dampening of pro-inflammatory senescence-associated secretory factors in these tissues. This early senolytic intervention also preserves intervertebral disc volume and vertebral bone microarchitecture, indicating protection against structural degeneration of the spine. These findings demonstrate that targeting cellular senescence at an early stage can mitigate degenerative changes and pain, supporting senolytic therapy as a promising preventive strategy for musculoskeletal decline.

cell biology↗

bioRxiv2025

Beyond miRNA cargo profiles: anti-inflammatory roles of extracellular vesicle-enriched miRNAs derived from human intervertebral disc cells unveiled by functional testing

Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain and a major contributor to global disability. Understanding the molecular mechanisms underlying this condition is essential for developing targeted therapies. Among these mechanisms, microRNAs (miRNAs) have emerged as critical post-transcriptional regulators of gene expression in IVD cells, influencing key processes such as extracellular matrix (ECM) homeostasis, inflammatory signalling, and cellular senescence. Extracellular vesicles (EVs), which transport miRNAs between cells, represent a promising avenue for therapeutic intervention. However, the composition of their miRNA cargo across different stages of disc degeneration remains inadequately characterized. We isolated EVs from primary human IVD cells derived from non-degenerate, mildly-degenerate, and severely degenerate tissues, and performed small RNA sequencing to profile their miRNA content. Bioinformatic analyses revealed enrichment in pathways related to ECM-receptor interaction, focal adhesion, inflammation, and cell cycle regulation. Notably, let-7b-5p and miR-100-5p were among the most abundant miRNAs and were significantly lower in EVs from degenerate discs. Functional assays demonstrated that transfection of IVD cells with let-7b-5p or miR-100-5p mimics individually suppressed IL-1{beta} expression at both mRNA and protein levels, confirming their anti-inflammatory roles. Strikingly, co-delivery of both miRNAs enhanced suppression of pro-inflammatory mediators, reduced senescence-associated p16 expression, and upregulated TIE2 mRNA, indicating synergistic effects in promoting a regenerative cell phenotype. These findings highlight the regulatory roles of EV-enriched let-7b-5p and miR-100-5p in modulating inflammation and senescence in IVD cells, and underscore the potential of miRNA-loaded EVs as cell-free regenerative therapies for disc degeneration.

cell biology↗

bioRxiv2025

Targeting breast cancer senescence in 3D models of bone metastasis

Chemotherapeutic treatment of breast cancer with Doxorubicin can induce tumor and stromal cell senescence leading to therapy-resistance. Senescence-associated secretory phenotype (SASP) promotes secretion of pro-inflammatory and tumorigenic factors causing systemic inflammation. Combined, this can result in immune suppression, tumor growth and secondary spread of cancer. Targeting and removing senescent and cancerous cells using a combination of chemotherapeutic and senolytic drugs may reduce systemic inflammation, improve therapeutic efficacy, and prevent metastasis. Treatment of both triple-negative breast cancer (MDA-MB-231) cells, and primary spine osteoblasts 0.25 {micro}M Doxorubicin showed significant induction of senescence indicated by p21 positive cells. Doxorubicin and senolytics (RG-7112, o-Vanillin) treatment of mono-culture and co-culture spheroids showed a significant additive effect on decreased tumor sphere viability and growth. This was correlated with decreased p21 and Ki67 proliferation marker in both the breast cancer and osteoblast cells. In all cases, combined Doxorubicin and senolytics significantly reduced sphere size and cancer cell outgrowth, indicating reduced metastatic potential. Future chemotherapeutic treatment of breast cancer patients may be optimized by adding senolytic drugs to more effectively clear tumors and help regenerate surrounding stroma tissue such as in the bone metastatic environment.

cancer biology↗

bioRxiv2024

Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc

Cellular senescence, characterized by a permanent state of cell cycle arrest and a secretory phenotype contributing to inflammation and tissue deterioration, has emerged as a target for age-related interventions. Accumulation of senescent cells is closely linked with intervertebral disc (IVD) degeneration, a prevalent age-dependent chronic disorder causing low back pain. Previous studies have highlighted that platelet-derived growth factor (PDGF) mitigated IVD degeneration through anti-apoptosis, anti-inflammation, and pro-anabolism. However, its impact on IVD cell senescence remains elusive. In this study, human NP and AF cells derived from aged, degenerated IVDs were treated with recombinant human (rh) PDGF-AB/BB for 5 days and changes of transcriptome profiling were examined through mRNA sequencing. NP and AF cells demonstrated similar but distinct responses to the treatment. However, the effects of PDGF-AB and BB on human IVD cells were comparable. Specifically, PDGF-AB/BB treatment resulted in downregulation of gene clusters related to neurogenesis and response to mechanical stimulus in AF cells while the downregulated genes in NP cells were mainly associated with metabolic pathways. In both NP and AF cells, PDGF-AB and BB treatment upregulated the expression of genes involved in cell cycle regulation, mesenchymal cell differentiation, and response to reduced oxygen levels, while downregulating the expression of genes related to senescence associated phenotype, including oxidative stress, reactive oxygen species (ROS), and mitochondria dysfunction. Network analysis revealed that PDGFRA and IL6 were the top hub genes in treated NP cells. Furthermore, in irradiation-induced senescent NP cells, PDGFRA gene expression was significantly reduced compared to non-irradiated cells. However, rhPDGF-AB/BB treatment increased PDGFRA expression and mitigated the senescence progression through increased cell population in the S phase, reduced SA-{beta}-Gal activity, and decreased expression of senescence related regulators including P21, P16, IL6, and NF-{kappa}B. Our findings reveal a novel anti-senescence role of PDGF in the IVD, making it a promising potential candidate to delay aging-induced IVD degeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/617862v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f151d0org.highwire.dtl.DTLVardef@324747org.highwire.dtl.DTLVardef@e58c92org.highwire.dtl.DTLVardef@6fe26c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Senolytic Treatment for Low Back Pain.

Senescent cells (SnCs) accumulate due to aging and external cellular stress throughout the body. They adopt a senescence-associated secretory phenotype (SASP) and release inflammatory, and degenerative factors that actively contribute to age-related diseases such as low back pain (LBP). The senolytics, o-Vanillin and RG-7112, remove senescent human intervertebral (IVD) cells and reduce SASP release, but it is not known if they can treat LBP. sparc-/- mice, with LBP, were treated orally with o-Vanillin and RG-7112 as single or combination treatments. Treatment reduced LBP and SASP factor release and removed SnCs from the IVD and spinal cord. Treatment also lowered degeneration score in the IVDs, improved vertebral bone quality, and reduced the expression of pain markers in the spinal cord. The result indicates that RG-7112 and o-Vanillin with the combination treatment providing the strongest effect are potential disease-modifying drugs for LBP and other painful disorders where cell senescence is implicated. One Sentence Summary: Senolytics drugs can reduce back pain

cell biology↗

bioRxiv2023

A network of CD163+ macrophages monitors enhanced permeability at the blood-dorsal root ganglion barrier

In dorsal root ganglia (DRG), macrophages reside in close proximity to sensory neurons, and their functions have largely been explored in the context of pain, nerve injury and repair. In this study, however, we discovered that the majority of macrophages in DRGs are in direct contact with the vasculature where they constantly monitor the circulation, efficiently phagocytosing proteins and macromolecules from the blood. Characterization of the DRG endothelium revealed a specialized vascular network spanning the arteriovenous axis, which gradually transformed from a barrier type endothelium in arteries to a highly permeable endothelium in veins. Macrophage phagocytosis spatially aligned with peak endothelial permeability and we identified caveolar transcytosis as a mechanism regulating endothelial permeability. Profiling of the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover and function. CD163 expressing macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation and were conserved in mouse and Man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.

immunology↗

bioRxiv2022

Fibromyalgia patients with high levels of anti-satellite glia cell IgG antibodies present with more severe symptoms

ObjectiveTransferring fibromyalgia patient IgG to mice induces pain-like behaviour and fibromyalgia IgG binds mouse and human satellite glia cells (SGCs). These findings suggest that autoantibodies could be part of fibromyalgia pathology. However, it is unknown how frequently fibromyalgia patients have anti-SGC antibodies and how anti-SGC antibodies associate with disease severity. MethodsWe quantified serum or plasma anti-SGC IgG levels in two fibromyalgia cohorts from Sweden and Canada using an indirect immunofluorescence murine cell culture assay. Fibromyalgia serum IgG binding to human SGCs in human dorsal root ganglia tissue sections was assessed by immunofluorescence (n=14/group). ResultsIn the cell culture assay anti-SGC IgG levels were increased in both fibromyalgia cohorts compared to controls. Elevated anti-SGC IgG was associated with higher levels of self-reported pain in both cohorts, and higher fibromyalgia impact questionnaire scores and increased pressure sensitivity in the Swedish cohort. Anti-SGC IgG levels were not associated with fibromyalgia duration. Swedish FM patients were clustered into FM-severe and FM-mild groups and the FM-severe group had elevated anti-SGC IgG compared to the FM-mild and controls. Anti-SGC IgG levels detected in culture were positively correlated with increased binding to human SGCs. Moreover, the FM-severe group had elevated IgG binding to human SGCs compared to the FM-mild and control groups. ConclusionsA subset of fibromyalgia patients have elevated levels of anti-SGC antibodies, and the antibodies are associated with more severe fibromyalgia severity. Screening fibromyalgia patients for anti-SGC antibodies could provide a path to personalized treatment options that target autoantibodies and autoantibody production.

neuroscience↗

bioRxiv2020

Single-cell RNA sequencing reveals time- and sex-specific responses of spinal cord microglia to peripheral nerve injury and links ApoE to neuropathic pain

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unknown whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. We show, using single-cell RNA-sequencing, that nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in males as compared to females. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following injury. Apolipoprotein E (Apoe) is the top upregulated gene in microglia at chronic time points after nerve injury in mice and polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a previously unrecognized role for ApoE in neuropathic pain.

neuroscience↗