Ankle fractures are among the most common fragility fractures in older adults. Osteoporosis, age-related impairment of bone regeneration, and multimorbidity increase the risk of delayed union, nonunion, and postoperative complications. Besides anatomical reduction and stable fixation, adjunctive therapies aimed at enhancing biological fracture healing have gained growing interest. This review summarizes the pathophysiological aspects of fracture healing in elderly patients and evaluates current biological, pharmacological, and physical strategies to promote healing after geriatric ankle fractures. Osteoporosis is the major risk factor for impaired fracture healing, affecting both bone regeneration and implant stability. Comprehensive osteoporosis management forms the basis of adjunctive treatment. Antiresorptive therapies mainly reduce the risk of subsequent fragility fractures without significantly impairing fracture consolidation. Among osteoanabolic agents, teriparatide has shown promising effects on callus formation and fracture union. Growth factors such as bone morphogenetic proteins (BMPs) and platelet-derived growth factor (PDGF) possess osteoinductive properties but are limited to selected indications. Although platelet-rich plasma (PRP) is biologically attractive, current evidence does not support its routine use in geriatric ankle fractures. Cell-based therapies and biophysical stimulation techniques, including low-intensity pulsed ultrasound, may benefit selected high-risk patients but require further clinical validation. Early functional mobilization remains a key component of successful treatment. Enhancing fracture healing in geriatric ankle fractures requires a multimodal approach combining mechanical stability with biological augmentation.

Sprunggelenkfrakturen zählen zu den häufigsten Fragilitätsfrakturen im höheren Lebensalter. Osteoporose, altersbedingte Einschränkungen der Knochenregeneration und Multimorbidität erhöhen das Risiko verzögerter Frakturheilungen und postoperativer Komplikationen. Neben anatomischer Reposition und stabiler Osteosynthese gewinnen additive Therapien zur biologischen Unterstützung der Frakturheilung zunehmend an Bedeutung. Ziel dieses Übersichtsartikels ist die Bewertung aktueller ergänzender biologischer, pharmakologischer und physikalischer Therapiekonzepte bei geriatrischen Patienten mit Sprunggelenkfrakturen. Die Osteoporose stellt den wichtigsten Risikofaktor für eine gestörte Frakturheilung dar und beeinflusst sowohl die Knochenregeneration als auch die Implantatstabilität. Eine strukturierte Osteoporosediagnostik und -therapie bilden die Grundlage jeder additiven Behandlung. Antiresorptive Medikamente reduzieren vor allem das Risiko weiterer Fragilitätsfrakturen, ohne die Frakturheilung wesentlich zu beeinträchtigen. Unter den osteoanabolen Therapien zeigt insbesondere Teriparatid positive Effekte auf Kallusbildung und Frakturkonsolidierung. Wachstumsfaktoren wie „bone morphogenetic proteins“ (BMP) und „platelet-derived growth factor“ (PDGF) besitzen osteoinduktive Eigenschaften, bleiben jedoch ausgewählten Indikationen vorbehalten. Für „platelet-rich plasma“ (PRP) fehlen bislang ausreichende klinische Evidenzen für eine routinemäßige Anwendung. Zellbasierte Verfahren sowie physikalische Therapien wie niedrigintensiver gepulster Ultraschall zeigen potenziellen Nutzen in Risikokonstellationen, bedürfen jedoch weiterer Forschung. Die Förderung der Frakturheilung bei geriatrischen Sprunggelenkfrakturen erfordert einen multimodalen Ansatz, der mechanische Stabilität mit biologischer Augmentation kombiniert.

Background/objective Increased lysyl oxidase (LOX) activity favors pathologic cartilage and vessel calcification. LOX promotes disease through enhanced collagen cross-linking, inflammation, reactive oxygen species (ROS) production, cell trans-differentiation, and fibrosis. This study investigates the therapeutic potential of cystathionine gamma lyase (CSE)-generated hydrogen sulfide (H2S) to inhibit tendon calcification by targeting LOX in human samples and murine models of calcific tendinopathy (CT). Methods Human shoulder supraspinatus tendons with varying degrees of CT were analyzed using Alizarin Red staining and LOX and CSE immunohistochemistry to evaluate the correlation between CSE and LOX/calcification. Mechanistic studies were performed using wild-type (WT) and CSE knockout murine tenocytes cultured in calcification-inducing medium with or without H2S donors or the LOX inhibitor β-aminopropionitrile (BAPN). Achilles tendon CT was induced in WT and CSE knockout mice via surgical intervention or aging. Tendon calcification, LOX expression, biomechanical integrity, and transcriptomic changes were assessed. Persulfidation of total proteins and recombinant human LOX (rhLOX) was measured using the dimedone-switch method. Results An inverse correlation between CSE levels and LOX/calcification was observed in human tendons and in the surgery-induced CT murine model. In murine tenocytes and in the aging murine model, CSE deficiency led to increased LOX expression, enhanced calcification, and reduced tendon biomechanical integrity.Transcriptomic analysis confirmed the negative association between CSE and LOX in murine CT. Mechanistically, H2S increased total cellular protein persulfidation, including rhLOX, resulting in inhibition of its enzymatic activity. Conclusion Dysregulated LOX activity is a key driver of calcific tendinopathy. CSE-generated H2S effectively suppresses LOX activity, highlighting its potential as a therapeutic strategy for CT and other calcification-related disorders. The translational potential of this article This study identifies LOX as a therapeutic target in CT and supports H2S as a promising treatment strategy for this condition.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that plays a critical role in cellular communication, mechanotransduction, and tissue repair. It is ubiquitously expressed in musculoskeletal tissues, including cartilage, tendons, and skeletal muscles, where it regulates cell-matrix interactions, modulates signaling pathways, and influences the biomechanical properties of these tissues. Given its functional significance, particularly its regulation of the collagen-crosslinking-enzyme lysyl oxidase, we investigated the structural and functional properties of tendons lacking syndecan-4. In this study, tendons from syndecan-4-deficient mice (C57Bl/6J, 12 weeks, male) were subjected to biomechanical testing, including dynamic loading and load-to-failure assays, to evaluate their mechanical stability. Histological analyses of paraffin-embedded sections were performed to examine tissue composition. Collagen organization and intrinsic disorder were assessed using Azan staining and polarization microscopy. Fibril diameter was quantified using transverse electron microscopy. Our findings revealed that tendons deficient for syndecan-4 exhibit significantly increased stiffness compared to wild-type controls, as demonstrated in both dynamic testing and load-to-failure assays. Azan staining highlighted reduced collagen fibril packing in syndecan-4-deficient tendons, which was confirmed by grayscale image analysis of collagen intensity. Polarization microscopy demonstrated increased intrinsic disorder in these tendons, accompanied by a reduced mean fibril diameter. Electron micrographs further revealed disrupted fibril maturation and organization, with fewer large-diameter fibrils observed in the syndecan-4-deficient samples. The absence of syndecan-4 profoundly alters the topology and function of murine Achilles' tendons, leading to increased stiffness despite histological evidence of impaired collagen organization and maturation. These results suggest that the increased stiffness may be attributed to heterotopic mineralization as compensatory mechanisms arising from collagen fibril disarray. Further investigations are warranted to elucidate the underlying mechanisms linking syndecan-4 deficiency to the observed biomechanical and structural abnormalities

OBJECTIVES:In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) acquire an aggressive, tumour-like phenotype characterised by increased adhesion to extracellular matrix, contributing to joint degradation. The collagen-binding integrin alpha11beta1 is involved in similar processes in cancer-associated fibroblasts, but its role in RA and arthritic mice remains unclear. METHODS:Integrin α11 expression was analysed in synovial tissue and FLS from RA and osteoarthritis patients and human tumour necrosis factor transgenic (hTNFtg) and wild-type mice supported by Accelerating Medicines Partnership Rheumatoid Arthritis and Pathobiology of Early Arthritis Cohort data. A novel 3-dimensional (3D) organoid coculture model and electron microscopy were used to analyse FLS invasion into cartilage explants, Itga11-/- were crossed with hTNFtg mice, and disease severity was evaluated using microcomputed tomography (µCT) and histology. Functional assays using FLS included cell morphology, adhesion, degradation, and matrix metalloproteinase expression and were complemented by osteoclast and coculture studies. RESULTS:In the context of RA, strong α11 expression was detected in the synovium, particularly in sublining clusters of FLS within fibroid-type synovial tissue in vivo and at focal adhesions of arthritic FLS and at invasion sites within the 3D coculture model in vitro. Clinical scores, µCT imaging, and histomorphological analyses revealed significantly reduced cartilage degradation, bone erosions, and FLS attachment to cartilage in Itga11-/-hTNFtg compared to hTNFtg mice. In vitro studies revealed that α11 deficiency led to a decreased receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio along with reduced TNFα-induced proteolytic degradation activity, and signalling pathway activation. CONCLUSIONS:Integrin α11 levels are increased in RA, and its deficiency notably diminishes joint destruction in hTNFtg mice, emphasising its potential as promising therapeutic target.

Alpha-melanocyte-stimulating-hormone (α-MSH) has been identified as a new anti-inflammatory treatment compound in rheumatoid arthritis (RA) and other inflammatory diseases. However, its direct effect on bone cell differentiation or on bone regeneration, which is an inflammatory process, too, has not been investigated, yet. Bone tissue is significantly affected in inflammatory joint diseases. Additionally, inflammatory signaling is essential -in bone regeneration during fracture healing. Therefore, we evaluated the impact of α-MSH-treatment on bone forming cells in an inflammatory setting in vitro and as a treatment approach in a murine fracture healing model in vivo. The influence of α-MSH treatment and melanocortin-receptor expression patterns was investigated in vitro in the presence of either IL-1β or/and TNF-α as an inflammatory stimulus. Osteoblast cell function was evaluated by analyzing proliferation and mineralisation capacities. Using quantitative real time PCR, we analyzed mRNA expression of receptors. To explore the impact of α-MSH on bone regeneration in vivo, treatment with α-MSH or NaCl (control) was performed in a murine fracture-healing model using a closed femur fracture stabilized with an intramedullary implant (female, n = 6–8 mice per group). α-MSH-treatment did not impair either proliferation nor mineralisation of osteoblastic cells under native or inflammatory conditions (no significant differences found). All four melanocortin receptor-molecules were expressed in murine osteoblastic cells but in very limited amounts and this did not change upon treatment with inflammatory cytokines or α-MSH or both at the same time. Callus formation in fractured femurs of α-MSH-treated mice was slightly delayed at day 14 post fracture with regard to less cartilage formation (NaCl: 19.9

This study evaluates four compression testing methods to determine the most reliable and reproducible technique for assessing the compression strength of murine lumbar vertebral bodies. Twenty female C57BL/6 mice (12 weeks old) were randomized into four groups: Group 1, compression of the complete lumbar vertebral body (LVB) with dorsal spinal processes; Group 2, compression at the vertebral body surface; Group 3, compression at the vertebral body surface after vertebral arch resection; Group 4, resection of the vertebral arch with straightening of the intervertebral joint surface. A mono-axial static testing machine applied compression, measuring load to failure, stiffness, yield load, and elasticity modulus. Method 1 resulted in significantly higher load-to-failure and yield-to-failure (25.9 N compared to 18.2 N, and twice 12 N for Methods 2–4), with the least variation in relative values. Method 3 had increased stiffness and a significantly higher Young’s modulus (232 N/mm, in contrast to 101, 130, and 145 N/mm for Methods 1, 2, and 4, respectively) but yielded inconsistent results. Method 4 showed the greatest variability across specimens. Method 2 yields suitable data quality as well, albeit with a slightly higher variation, and is the recommended procedure if the spinal processes have to be excluded from the measurement. Based on these findings, Method 1 produced the most consistent and reproducible data and is recommended for future studies evaluating vertebral biomechanics in mice.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL Copy DOI

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Numerous genetically modified mouse models are available to evaluate gene functions related to bone metabolism regulation. This study aims to evaluate four prevalent biomechanical testing methods for assessing the structure and stability of murine lumbar vertebral bodies, identifying the most reliable, valid, and reproducible technique. The fourth lumbar vertebrae of 20 female C57BL/6 mice (wild type, 12 weeks) were tested, randomized into four testing groups: Method 1 - compression of the complete lumbar vertebral body (LVB) including the dorsal spinal processi; Method 2 - compression at the vertebra body surface; Method 3 - isolated resection of the vertebral arch; Method 4 - resection of the vertebral arch and straightening of the intervertebral joint surface. Compression was applied using a mono-axial static testing machine. Maximum load, stiffness, yield load, and the elasticity modulus were evaluated. Load-to-failure and yield-to-failure were significantly higher in Method 1. Method 3 showed increased stiffness and significantly increased Young's modulus. The least variation in relative load-to-failure and yield-to-failure was observed with Method 1. Method 4 exhibited the greatest overall variation in specimen values. Method 3 yielded divergent results and is not recommended. Method 1 led to the most consistent and reproducible data and is recommended.

Although the rate of infection after the reconstruction of a ruptured anterior cruciate ligament (ACL) is low, prophylactic incubation of the graft with vancomycin (Vanco-wrap or vancomycin soaking) is routinely performed. A cytotoxic effect of vancomycin is reported for several cell types, and the prophylactic treatment might prevent infection but harm the tissue and cells. Aim: A comprehensive study was performed to investigate the effect of vancomycin on tendon tissue and isolated tenocytes using cell viability, molecular and mechanical analysis. Material and methods: Rat tendons or isolated tenocytes were incubated in increasing concentrations of vancomycin (0–10 mg/mL) for different times, and cell viability, gene expression, histology and Young’s modulus were analyzed. Results: The clinically used concentration of vancomycin (5 mg/mL for 20 min) had no negative effect on cell viability in the tendons or the isolated tenocytes, while incubation with the toxic control significantly reduced cell viability. Increasing the concentration and prolonging the incubation time had no negative effect on the cells. The expression of Col1a1, Col3a1 and the tenocyte markers mohawk, scleraxis and tenomodulin was not affected by the various vancomycin concentrations. The structural integrity as measured through histological and mechanical testing was not compromised. Conclusion: The results proved the safe application of the Vanco-wrap on tendon tissue. Level of evidence: IV.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

Background Pathological calcification ((PC), i.e. the deposition of calcium-containing crystals) in tendons is a hallmark of calcific tendinopathy (CT), a disease associated with pain, tendon rupture, and disability. Currently, only symptomatic treatments exist for CT and none of them target specifically calcification. Intriguingly, the gasotransmitter H2S has recently emerged as a potential anti-calcifying molecule [1]. Objectives To establish in CT the potential anti-calcifying effect of hydrogen sulfide (H2S) and of the H2S-producing enzyme cystathionine gamma lyase (CSE), and the underlying mechanisms involved. Methods Wild type (WT) and CSE knock-out (KO) tenocytes were isolated from adult mice Achilles’ tendons. Tenocyte were cultured in αMEM + 10% FBS + 50μg/ml ascorbic acid (Control medium) in presence of 10% calciprotein particles (calcification medium, CM). Spontaneous Achilles’ tendon CT in old mice (35 weeks old) and surgery-induced Achilles’ tendon CT in8-12 weeks old mice were quantified by micro computed tomography. Dynamic Young’s modulus was measured in 35 weeks old mice as previously described [2]. Immunohistochemistry was performed on human and murine tendon sections with anti-CSE and anti-LOX and anti-LOXL2rabbit polyclonal antibodies. LOX expression was analyzed by qPCR and by Western-blot. LOX(L) activity was quantified in cells supernatants and lysates. Results In vitro, tenocyte calcification (in CM) was inhibited by exogenous H2S-donors, while it was exacerbated in CSE KO tenocytes, producing as expected less H2S than WT. Reduced calcification in tenocytes exposed to H2S was accompanied by decreased expression of genes coding for BMP2, BMP4 and decreased activation of the BMP signaling pathway (pSMAD1/5/8). Accordingly, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes. The protective role of CSE-H2S was confirmed in vivo. Indeed, in aged mice, micro computed tomography revealed exacerbated Achilles’ tendon calcification in CSE KO mice compared to WT. Interestingly, CSE deficiency led to reduced biomechanical strength, as the dynamic Young’s modulus was significantly decreased upon increased tendon displacement. Furthermore, using a tenotomy model of CT, we found an inverse correlation between CSE expression and calcification in tendons. This was confirmed in human tendons from CT patients, which exhibited decreased CSE expression where calcification was present. In parallel experiments, we found that calcification in tenocytes was significantly reduced by addition of BAPN, a pan-inhibitor of lysyl oxidases (LOX(L)) enzymes. LOX(L) family includes 5 enzymes LOX and LOX1-4, catalysing elastin and collagen cross-links. We next investigated if the anti-calcfiying effect of H2S in CT could be mediated by inhibition of LOX(L). Indeed, in CM-stimulated tenocytes we found that H2S impacts both LOX/LOX2 expression (as CSE deficiency increased significantly LOX/LOX2 gene expression) and LOX(L) activity (as CSE deficiency increased LOX(L) activity and conversely H2S dose-dependently inhibited LOX(L) activity in WT tenocytes. Finally, in vivo we discovered that, CSE was inversely correlated with calcification and LOX and LOXL2 expression in mouse and human tendons. Conclusion Altogether, our results suggest that increasing H2S levels in tenocytes could represent a future strategy to prevent or decrease calcification in CT, likely via down-modulation of LOX(L) expression and activity. References [1]Castelblanco, M., et al., The role of the gasotransmitter hydrogen sulfide in pathological calcification. Br J Pharmacol, 2020. 177(4): p. 778-792. [2]Kronenberg, D., et al., Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin alpha2beta1-Deficient Mice. Int J Mol Sci, 2020. 21(8). Acknowledgements: NIL. Disclosure of Interests None Declared.

BMP-1 is the major procollagen-C-peptidase activating, besides fibrillar collagen types I-III, several enzymes and growth factors involved in the generation of extracellular matrix. This study investigated the effect of adding and inhibiting BMP-1 directly post fracture. Standardised femoral fractures were stabilized by an intramedullary nail in 12 week-old female C57Bl/6J mice. We injected either 20 µL recombinant active BMP-1, activity buffer or the BMP-1 specific inhibitor “sizzled”. After 7, 14 and 28 days, mice were sacrificed. Femurs were dissected and paraffin slides were prepared. Callus composition was divided into soft tissue, mineralized and cartilaginous callus. Murine MC3T3 pre-osteoblastic cells were kept in culture adding BMP-1 and sizzled during osteoblastic differentiation. Putative cytotoxicity was determined using MTT-vitality assay. Cell calcification, collagen deposition, and BMP-2 and myostatin protein quantity were characterized. Adding BMP-1 displayed a weak positive effect on the outcome. After 7 days, more mineralised callus was present, meanwhile the cartilaginous callus was apparently remodelled at higher rate. In the case of BMP-1 inhibition, we observed more cartilaginous callus, which may indicate reduced stability. In cell culture, we could observe a high interference with mineralisation capabilities depending on the stage of osteoblastic development when adding BMP-1 or inhibiting it. Addition and inhibition impaired myostatin (anti-osteogen) and BMP-2 (pro-osteogen) expression. Interfering with BMP-1 homeostasis in this early stage of fracture repair seems to have rather negative effects. Inhibition apparently yields lower callus quality while the addition of BMP-1 does not significantly accelerate the healing outcome. Cell culture experiments show that BMP-1 application after 7 days of healing leads to higher collagen output but has no effect on mineralisation. This may suggest that BMP-1 application at a later time-point may lead to more pronounced beneficial effects on fracture repair.

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised. Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel. Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT. Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner.

Overuse injuries or acute trauma in joints often lead to painful tendinopathy, and pharmacological treatment effects are limited. The site of the disease is hard to reach with drugs, both systemically and through the skin. Therapeutic gases may close this gap, as they permeate easier through tissues than conventional small molecules. We present a patch device releasing the anti-inflammatory gas carbon monoxide (CO) through the skin to the subcutaneous tendons and tissues. CO is chemically generated upon device activation and its design maximizes CO exposure to the underlying skin and protects the patient from all side and degradation products. The patch delivered CO successfully through the intact skin, granting lasting, subcutaneous CO exposure for up to 16 h. Furthermore, the released CO induced the proliferation of fibroblasts and the polarization of monocytes into antiinflammatory M2 macrophages. In conclusion, the CO-releasing device might open an entirely new treatment option against tendinopathies in case of a positive outcome of future in vivo studies.

Introduction: Neoangiogenesis drives the replacement of mineralized cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) while interacting with VEGF and OPG, plays a role in the communication of osteoclasts and osteoblasts and in the development of blood vessels. To understand the function of Sdc1 in enchondral ossification we analysed bone structure and vessel development in bone growth and fracture healing in mice deficient in Sdc1.

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2 -/- ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones. Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.

Osteoporotic fractures are often linked to persisting chronic pain and poor healing outcomes. Substance P (SP), α-calcitonin gene-related peptide (α-CGRP) and sympathetic neurotransmitters are involved in bone remodeling after trauma and nociceptive processes, e.g., fracture-induced hyperalgesia. We aimed to link sensory and sympathetic signaling to fracture healing and fracture-induced hyperalgesia under osteoporotic conditions. Externally stabilized femoral fractures were set 28 days after OVX in wild type (WT), α-CGRP- deficient (α-CGRP −/−), SP-deficient (Tac1−/−) and sympathectomized (SYX) mice. Functional MRI (fMRI) was performed two days before and five and 21 days post fracture, followed by µCT and biomechanical tests. Sympathectomy affected structural bone properties in the fracture callus whereas loss of sensory neurotransmitters affected trabecular structures in contralateral, non-fractured bones. Biomechanical properties were mostly similar in all groups. Both nociceptive and resting-state (RS) fMRI revealed significant baseline differences in functional connectivity (FC) between WT and neurotransmitter-deficient mice. The fracture-induced hyperalgesia modulated central nociception and had robust impact on RS FC in all groups. The changes demonstrated in RS FC in fMRI might potentially be used as a bone traumata-induced biomarker regarding fracture healing under pathophysiological musculoskeletal conditions. The findings are of clinical importance and relevance as they advance our understanding of pain during osteoporotic fracture healing and provide a potential imaging biomarker for fracture-related hyperalgesia and its temporal development. Overall, this may help to reduce the development of chronic pain after fracture thereby improving the treatment of osteoporotic fractures.

Background In rheumatoid arthritis (RA), fibroblast like synoviocytes (FLS) undergo a “tumor-like” transformation, wherein they develop an aggressive phenotype that is characterized by increased adhesion to components of cartilage extracellular matrix (ECM) and that contributes extensively to joint destruction. The collagen binding integrin α11β1 was previously shown to be involved in similar processes in cancer-associated fibroblasts mediating tumorigenicity and metastasis in certain tumors. Therefore, this study aimed to study the role of integrin α11β1 in RA and to characterize the effects of α11β1 deficiency on the disease course and severity in arthritic hTNFtg mice. Methods The expression levels of integrin α11β1 were analyzed by immunohistochemistry, immunofluorescence, and western blot analysis in synovial samples and FLS of patients with RA and osteoarthritis (OA) as well as in samples from wild type (wt) and arthritic hTNFtg mice. Furthermore, the subcellular expression of integrin α11β1 was investigated in co-culture experiments with cartilage explants and analyzed by transmission electron microscopy. To investigate the effects of integrin α11β1 deficiency, itga11 -/- mice were interbred with hTNFtg mice and disease severity was assessed by clinical scoring of grip strength and paw swelling over the disease course. Hind paws of 12-weeks-old mice of all genotypes were analyzed by µCT imaging followed by stainings of paraffin-embedded tissue sections with Toluidine-blue and tartrate-resistant acid phosphatase (TRAP) to evaluate established parameters of joint destruction such as inflammation area, cartilage destaining, FLS attachment to the cartilage surface, and bone damage. Results Expression levels of integrin α11β1 were clearly elevated in synovial tissues and FLS from RA patients and hTNFtg mice, compared to the controls derived from OA patients and wt mice. Interestingly, this expression was shown to be particularly localized in focal adhesions of the FLS. As revealed by transmission electron microscopy, integrin α11β1 expression was particularly evident in areas of direct cellular contact with the ECM of cartilage. Evaluations of clinical scorings and histomorphological analyses demonstrated that itga11 -/- hTNFtg displayed alleviated clinical symptoms, higher bone volume, less cartilage destruction and reduced FLS attachment to the cartilage in comparison to hTNFtg mice. Conclusions The collagen-binding integrin α11β1 is upregulated in the context of RA and its deficiency in mice with an inflammatory hTNFtg background leads to a significant reduction in the arthritic phenotype which makes integrin α11β1 an interesting target for therapeutical intervention.