Biology subjects

Lammlin, L.

Publications and source records attributed to Lammlin, L..

11 recordsLinked to original sources

bioRxiv2026

PIEZO1 mediates mechanosensing and myofibroblastic activation of synovial fibroblasts in post-traumatic osteoarthritis

Fibrosis and inflammation of the synovium are common pathological features of joint diseases. In osteoarthritis (OA), extensive evidence supports that synovial pathology drives symptoms and disease progression, yet the mechanisms underpinning pathological synovial remodeling, including fibrosis, remain unclear. Here, we identify the mechanosensitive ion channel PIEZO1 as a critical regulator of myofibroblast-like activation of synovial fibroblasts (SFs) in post-traumatic osteoarthritis (PTOA). Using multi-omic assessments in human and murine synovium, a murine model of joint injury, histopathology, and functional in vitro phenotyping, we demonstrate that joint injury triggers expansion of myofibroblast-like SFs concurrent with fibrotic remodeling and mechanical stiffening of the synovium. Myofibroblastic commitment of synovial progenitors was associated with activation of the YAP1 regulon, and PIEZO1 agonism drove nuclear accumulation of YAP1 in SFs. PIEZO1 was enriched in synovia of injured joints, and in cultured SFs pharmacologic PIEZO1 activation promoted SMA enrichment and increased matrix contraction, which were both ablated by YAP1 inhibition. PIEZO1 silencing abrogated TGF-{beta}-induced contractility and markedly altered the transcriptomic response to mechanical loading in vitro, including ECM production and neuroinflammatory programs. In vivo, acute intra-articular pharmacologic PIEZO1 activation induced a pro-fibrotic, pro-inflammatory, and anti-adipogenic transcriptomic program in synovium. Collectively, these findings establish PIEZO1 as a key mediator of the pathogenic activation of SFs and identify aberrant mechanosensing as a mechanism underlying synovial fibrosis in PTOA.

cell biology↗

bioRxiv2026

Thrombospondin-2 deficiency primes the synovial joint for aberrant tissue remodeling and injury response

ObjectiveThis study investigates joint injury-induced angiogenesis and the effects of genetic deficiency of thrombospondin-2 (TSP2), an anti-angiogenic factor, in joint homeostasis and post-traumatic osteoarthritis (PTOA). MethodWe utilized a murine non-invasive anterior cruciate ligament rupture (ACLR) model of PTOA and mined published synovial transcriptomics datasets to investigate injury-induced synovial angiogenesis. Spatial transcriptomics and flow cytometry of TSP2-GFP reporter mice were used to assess injury-induced thrombospondin-2 and its cellular origins in synovium. Global TSP2 knockout mice (TSP2-KO) were used to assess the effect of TSP2 deficiency on early and late stages of PTOA development via molecular imaging of inflammation and angiogenesis, histopathology, micro-computed tomography, Raman spectroscopy, and synovium bulk RNA-sequencing. ResultsIntra-articular angiogenesis peaked at 7d post-ACLR and declined but remained elevated above baseline at 28d post-ACLR. We identified synovial crosstalk between endothelial cells and sublining fibroblasts as a key driver of angiogenesis and source of thrombospondin-2 signaling, with TSP2 primarily upregulated in sublining fibroblasts. TSP2-KO mice exhibited increased peri-articular inflammation at 7d post-ACLR and inferior bone quality. Histopathology revealed greater PTOA severity but paradoxically lower synovitis in TSP2-KOs. Additionally, aberrant structural remodeling of the entire knee joint was observed in uninjured and ACLR TSP2-KO limbs. The uninjured TSP2-KO synovial transcriptome demonstrated elevated immune, fibrotic, and angiogenic activation; however, TSP2-KO and WT synovial transcriptomes partially converged upon injury. ConclusionTSP2 is essential for joint homeostasis and trauma response. Global TSP2 deficiency causes premature OA and worsened PTOA, suggesting that therapeutic targeting with TSP2 mimetic could be used to prevent OA.

physiology↗

bioRxiv2026

Wnt/β-catenin signaling regulates fibrotic atrophy of intra-articular adipose tissue in post-traumatic osteoarthritis

Synovial joints like the knee are home to adipose tissue depots whose anatomy and functions are closely intertwined with that of other intra-articular soft tissues such as synovium, underscoring the growing understanding that joints are multi-tissue organs. Traumatic joint injury and the onset of osteoarthritis (OA) dramatically remodel the intra-articular adipose niche, marked by infiltration of fibrotic tissue postulated to underpin OA-associated joint stiffness and pain, yet we know very little about the disease-associated dynamics of joint adipose remodeling nor the mechanisms driving these phenomena. Here, we employed 2D histomorphometry and spatial transcriptomics, alongside 3D osmium tetroxide-enhanced micro-computed tomography to comprehensively define the spatiotemporal, structural, and transcriptional rewiring of joint adipose tissue in a non-invasive mouse model of post-traumatic osteoarthritis (PTOA). These revealed marked loss of intra-articular adiposity accompanied by expansion of fibroblast-rich, collagen-dense tissue with pro-fibrotic hallmarks and Wnt/{beta}-catenin-enriched gene programs. Joint adipose exhibited a distinct transcriptional signature compared to subcutaneous white adipose tissue, pointing to unique, depot-specific functions. Stromal cells isolated from PTOA joints had heightened baseline expression of fibrotic and Wnt pathway genes and exhibited impaired de novo adipogenesis, in contrast to cells derived from healthy joints. In accordance with the destabilized biomechanics of PTOA joints, in vitro modeling demonstrated that prolonged, injurious loading and perturbed Wnt/{beta}-catenin signaling were convergent anti-adipogenic cues that suppressed lipid droplet formation and adipogenic gene induction, while promoting markers of fibrosis in joint-derived stromal cells. Complementary gain-of-function studies using ex vivo joint adipose explants and in vivo joint injections demonstrated that chronic Wnt/{beta}-catenin activation, as seen in OA joints, is sufficient to diminish the intra-articular adipogenic program and shift adipose to a more fibrotic phenotype, independent of joint injury. Collectively, these findings establish a multi-modal framework for quantifying joint adipose atrophy and implicate aberrant Wnt/{beta}-catenin signaling and pathological mechanical loading as key factors impairing de novo adipogenesis and driving fibrotic remodeling of intra-articular adipose tissue in PTOA.

cell biology↗

bioRxiv2025

Spatial transcriptomic profiling of decalcified murine musculoskeletal samples via Xenium Prime 5K

Successful generation of high-quality spatial transcriptomics data from murine musculoskeletal tissues has been impeded by the challenge of preserving RNA integrity through the harsh tissue processing steps required for histological sectioning. In particular, the need to thoroughly fix and decalcify mineralized tissues has proven problematic. We detail a comprehensive sample processing pipeline for three common murine musculoskeletal tissue samples, enabling high-quality transcript detection via imaging-based spatial transcriptomics using the Xenium Prime 5K platform from 10x Genomics. Our protocol outlines methodological details for transcardiac perfusion, fixation, decalcification, paraffin processing, and a sample co-embedding strategy facilitating anatomically consistent and simultaneous sectioning of multiple samples onto the spatial transcriptomics slide. Rigorous quality control demonstrates high-quality tissue-specific outcomes across intact knee joints, tibiae, and lumbar spines from adult mice. Our pipeline enabled 70-91% high-quality transcripts across synovium, meniscus, patellar tendon, articular cartilage, subchondral bone, cortical bone, bone marrow, muscle, fracture callus, and dorsal root ganglion tissues. The average number of detected transcripts varied markedly between tissue types - soft tissues such as synovium, patellar tendon, muscle, bone marrow, and callus exhibited ~200 - 400 transcript per cell; mineralized tissues such as subchondral bone, meniscus, and cortical bone exhibited ~ 13 - 150 transcripts per cell; highly active neuronal tissues such as dorsal root ganglion neurons yielded 750 - 1100 transcripts per cell. Canonical cell markers within each tissue confirmed successful identification and representation of key cell types. Through rigorous sample quality assessment at multiple stages of processing, this protocol yields high-quality RNA transcript detection while preserving critical anatomical context and will serve as a valuable tool enabling spatial transcriptomic profiling of intact musculoskeletal tissue samples. Lay summarySpatial transcriptomics is a powerful scientific tool that characterizes genetic coding material ("transcripts") of specific cell types in their native anatomical context. However, successfully applying this tool to musculoskeletal tissues has been challenging because preserving transcript integrity in these tissues requires additional care. The authors of this publication developed a specialized method for preparing tissue samples from mice that works with the spatial transcriptomics platform, 10x Genomics Xenium Prime 5K. This protocol improves sample quality while preserving high-quality genetic information in various mouse musculoskeletal tissues, making it easier to explore their functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/693132v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@4c4389org.highwire.dtl.DTLVardef@e607bcorg.highwire.dtl.DTLVardef@5f957org.highwire.dtl.DTLVardef@34ce27_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Acute immune modulation with poly-salicylic acid particles ameliorates pain and structural damage in post-traumatic osteoarthritis

Joint inflammation is a hallmark of post-traumatic osteoarthritis (PTOA) progression and a recognized driver of articular destruction and symptoms. Despite its known pathological role, inflammation has not been successfully targeted to treat PTOA. With the hypothesis that blocking the acute influx of systemically-derived immune cells can mitigate injury-induced inflammation and downstream PTOA disease severity, we targeted immune cell recruitment via systemically-administered poly salicylic acid (PolySA) particles. This formulation targets immune cells in circulation, namely neutrophils and monocytes, to inhibit their vascular extravasation into injured tissue. Employing a murine joint injury model, we show that PolySA particles reduced neutrophil and monocyte recruitment to the synovium by >50% when administered acutely after injury. Sex-specific therapeutic effects of PolySA emerged 7d post-ACLR, whereby female knee joints exhibited increased cathepsin activity and alleviation of knee hyperalgesia. Despite also observing reduced immune cell recruitment in male mice treated with PolySA, therapeutic effects were entirely absent in males. RNAseq of female synovium revealed a transcriptomic signature indicative of accelerated immune resolution and matrix remodeling in PolySA-treated female mice. Analyses at a timepoint of established disease showed that PolySA-treated female mice exhibited sustained pain alleviation, reduced osteophyte formation, and decreased histopathological PTOA and synovitis severity scores. Together, these findings indicate that blocking acutely-recruited immune cells to the local joint microenvironment via systemic PolySA particle treatment is a promising therapeutic for PTOA prevention by reprogramming early injury-induced inflammation.

bioengineering↗

bioRxiv2025

NERVE GROWTH FACTOR IS SUFFICIENT TO CAUSE MULTIPLE OSTEOARTHRITIS-RELEVANT PATHOLOGICAL FEATURES IN NAIVE MURINE KNEE JOINTS

BACKGROUNDNerve growth factor (NGF), a key mediator of pain and inflammation, is increased in joints with osteoarthritis (OA). Neutralizing NGF with monoclonal antibodies has shown analgesic effects in painful knee OA, but clinical development was stopped due to side effects in the joints. Knowledge about the biological effects of long-term exposure of joint tissues to NGF is limited. Therefore, we aimed to explore the effects of repeated intra-articular (IA) injections of NGF into the knee joints of healthy mice on pain and sensitization, as well as joint innervation and structure. METHODSWe conducted five experiments in male C57BL/6 mice. In Experiment 1, NGF (50ng or 500ng) or vehicle was injected IA into the knee of naive wildtype (WT) mice, twice a week for 4 weeks. We assessed knee swelling, knee hyperalgesia and histopathology. In Experiment 2, mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks and microCT of the knee was performed. In Experiment 3, NaV1.8-tdTomato reporter mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks, and joint innervation was assessed. In Experiment 4, WT mice received 500ng NGF or vehicle twice a week for 4 weeks and were used for single cell RNA sequencing (scRNAseq) of the synovium. In Experiment 5, L3-L5 DRGs of mice that received 3 IA injections of 500ng NGF or vehicle twice a week were used for bulk RNA sequencing. RESULTSRepeated bi-weekly IA injections of NGF caused knee hyperalgesia in naive mice. NGF caused dose-dependent knee swelling, synovial pathology, increased bone mineral density and trabecular bone thickness in the medial subchondral bone, growth of pre-osteophytes in the medial compartment, but no cartilage degeneration. NGF injection caused sprouting of NaV1.8+ neurons in the medial but not the lateral synovium. ScRNAseq of the synovium revealed upregulated genes related to neuronal sprouting, synovial fibrosis and ossification, confirming histopathological findings. Bulk RNA seq of DRG showed upregulated pathways related to axonal growth. CONCLUSIONSIn healthy mouse knees, NGF induced mechanical sensitization, synovitis, neoinnervation in the medial synovium, subchondral bone changes and pre-osteophyte growth in the medial compartment, thus capturing many pathological changes observed in OA, except cartilage damage.

neuroscience↗

bioRxiv2025

CXCL16 mediates nociception and inflammation in murine post-traumatic osteoarthritis

This study investigates the role of the chemokine CXCL16 and its receptor, CXCR6, in post-traumatic osteoarthritis (PTOA) and joint nociception, highlighting the potential of targeting the CXCL16-CXCR6 axis for therapeutically managing joint inflammation and pain. Following joint injury in mice, the CXCL16-CXCR6 signaling axis is activated in synovium, driven by synovial fibroblasts and macrophages. Human OA synovium also exhibited increased CXCL16 and CXCR6 gene expression. CXCL16 stimulated a pro-inflammatory response in fibroblasts and macrophages, contrasting with an anti-inflammatory response observed in mesenchymal progenitor cells. In mice, repeated intra-articular CXCL16 injections induced histological synovitis and sex-dependent activation of inflammatory and fibrotic transcriptional programs in synovium. Repeated CXCL16 joint injections also induced knee hyperalgesia, which was mitigated by co-administration of the CXCR6 antagonist, ML339. A single intra-articular injection of CXCL16 induced acute knee hyperalgesia as early as 30 minutes post-injection, which was completely abrogated by ML339 co-treatment, suggesting direct CXCL16 binding to nociceptor-expressed CXCR6. In a murine PTOA model, systemic CXCR6 antagonism with ML339 alleviated knee hyperalgesia and altered circulating immune cell profiles. Direct stimulation of mouse dorsal root ganglion-derived nociceptive neurons with CXCL16 induced rapid calcium signaling, which was abolished by co-treatment with ML339. These findings establish CXCL16 as a regulator of joint inflammation and identifies the CXCL16-CXCR6 binding mechanism as key in mediating pain-related behaviors and nociceptor activation, offering a therapeutic target for PTOA-related inflammation and pain management. One Sentence SummaryCXCL16 regulates synovial inflammation and mediates joint nociception via CXCR6, highlighting its potential as a therapeutic target for post-traumatic osteoarthritis.

physiology↗

bioRxiv2025

A standardized, open-source, portable model for noninvasive joint injury in mice

Preclinical models of osteoarthritis (OA) are crucial for the study of disease mechanisms and for the development of critically-needed disease-modifying therapeutics. While surgical OA models, such as the destabilization of the medial meniscus (DMM), have been the gold standard in the field for decades, noninvasive joint loading-based models have increased in popularity and utility. To facilitate standardization of the noninvasive anterior cruciate ligament rupture (ACLR) model in mice, we present the Mobile Joint-Injury Operator (MoJO) - an open-source protocol with accompanying fixtures and data, designed for a low-cost, commercially-available, portable uniaxial testing system with a small footprint. We provide 3d-printable fixture designs and a rapid, highly-repeatable ACLR-mediated joint injury protocol that results in the expected post-traumatic osteoarthritis phenotype in male and female C56Bl/6 mice. We then describe the expected mechanical data from the injury procedure and offer various troubleshooting strategies. Finally, we summarize the resultant PTOA phenotype by knee hyperalgesia testing, {micro}CT imaging, flow cytometry, and histological assessment. Increased standardization of this model is a critical aspect of the overall refinement of animal models of OA.

physiology↗

bioRxiv2023

Intra-Articular Sprouting Of Nociceptors Accompanies Progressive Osteoarthritis: Comparative Evidence In Four Murine Models

ObjectiveKnee joints are densely innervated by nociceptors. Sprouting of nociceptors has been reported in late-stage osteoarthritis (OA), both in human knees and in rodent models. Here, we sought to describe progressive nociceptor remodeling in four mouse models of knee OA, capturing early and late-stage disease. MethodsSham surgery, destabilization of the medial meniscus (DMM), partial meniscectomy (PMX), or non-invasive anterior cruciate ligament rupture (ACLR) was performed in the right knee of 10-12-week old male C57BL/6 NaV1.8-tdTomato mice. Mice were euthanized (1) 4, 8 or 16 weeks after DMM or sham surgery; (2) 4 or 12 weeks after PMX or sham; (3) 1 or 4 weeks after ACLR injury or sham. Additionally, a cohort of naive male wildtype mice was evaluated at 6 and 24 months. Twenty-m thick mid-joint cryosections were assessed qualitatively and quantitatively for NaV1.8+ and PGP9.5+ innervation. Cartilage damage (using a modified OARSI score), synovitis, and osteophytes were assessed blindly. ResultsProgressive OA developed in the medial compartment after DMM, PMX, and ACLR. Synovitis and associated neo-innervation by nociceptors peaked in early-stage OA. In the subchondral bone, channels containing sprouting nociceptors appeared early, and progressed with worsening joint damage. Two-year old mice developed primary OA in both the medial and the lateral compartment, accompanied with neuroplasticity in the synovium and the subchondral bone. All 4 models had an increased nerve signal in osteophytes. ConclusionAnatomical neuroplasticity of nociceptors was observed in association with joint damage in 4 distinct mouse models, suggesting that it is intrinsic to OA pathology.

neuroscience↗

bioRxiv2022

Sexual dimorphism of the synovial transcriptome underpins greater PTOA disease severity in male mice following joint injury

ObjectiveTo elucidate sex differences in synovitis, mechanical sensitization, structural damage, bone remodeling, and the synovial transcriptome in the anterior cruciate ligament rupture (ACLR) mouse model of post-traumatic osteoarthritis (PTOA). MethodsMale and female 12-week-old C57Bl/6 mice were randomized to noninvasive ACLR or sham (n=9/sex/group/timepoint). Knee hyperalgesia, mechanical allodynia, and intra-articular MMP activity (via intravital imaging) were measured longitudinally. Trabecular and subchondral bone remodeling and osteophyte formation were assessed by CT. Histological scoring of PTOA and synovitis and anti-MMP13 immunostaining was performed. NaV1.8-Cre;tdTomato mice were used to document localization and sprouting of nociceptors. Bulk RNAseq of synovium in sham, 7d, and 28d post-ACLR, and contralateral joints (n=6) assessed injury-induced and sex-dependent synovial gene expression. ResultsMale mice exhibited worse joint damage at 7d and 28d and worse synovitis at 28d, accompanied by greater MMP activity, knee hyperalgesia, and mechanical allodynia. Females had catabolic responses in trabecular and subchondral bone after injury, whereas males exhibited greater osteophyte formation and sclerotic remodeling of trabecular and subchondral bone. NaV1.8+ nociceptor sprouting in subchondral bone and medial synovium was induced by injury and comparable between sexes. RNAseq of synovium demonstrated that both sexes had similar injury-induced gene expression at 7d, but only female mice exhibited synovial inflammatory resolution by 28d, whereas males had persistent pro-inflammatory, pro-fibrotic, pro-neurogenic, and pro-angiogenic gene expression. ConclusionWorse overall joint pathology and pain behavior in male mice was associated with persistent activation of synovial inflammatory, fibrotic, and neuroangiogenic processes, implicating persistent synovitis in driving sex differences in murine PTOA.

physiology↗

bioRxiv2022

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis

ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis. MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes. ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury.

cell biology↗