Background: Increased nerve growth into degenerated intervertebral discs is associated with discogenic low back pain [. 1. ]. Many of these growing nerves are in neo-vascularised areas of the tissue [. 1. ,. 2. ] and endothelial cells that penetrate the disc express neurotrophic factors [. 3. ]. Thus, disc neovascularisation and disc innervation may be closely linked. Whilst disc aggrecan has been found to inhibit sensory nerve growth in vitro [. 4. ], the effects of disc aggrecan on endothelial cells are unknown. Methods/Results: Adapting in vitro assays used previously [. 4. ], with HMEC-1 and EAhy-926 cell lines as models of endothelial cell growth, we found that disc aggrecan inhibited endothelial cell migration in a dose-dependent manner. Endothelial cells traversed over collagen substrates until they encountered disc aggrecan substrates (1mg/ml human aggrecan), where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the collagen:aggrecan border (Figure 1). After reaching the aggrecan border, some endothelial cells also migrated away from the disc aggrecan. At lower concentrations of disc aggrecan (0.01mg/ml), no such inhibition of endothelial cell growth was seen. Conclusions: Loss of aggrecan, increased innervation and neovascularisation are all marked features of disc degeneration [. 1. ,. 2. ,. 5. ]. This study provides evidence that disc aggrecan inhibits endothelial migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predisposes the tissue to vascular invasion

Purpose: Glucocorticoids (GCs) are widely prescribed drugs in a large variety of diseases. Their use are strongly influenced by their associated negative side effects. Bone-related effects are mainly osteoporosis and osteonecrosis (ON). Despite the strong link between GCs and ON, the pathogenic mechanisms by which GCs cause ON are still unclear. Cumulative evidence shows that dysfunction or activation of endothelial cells (ECs) play an important role in ON. Method: In this study, we investigated the influence of dexamethasone (Dex) on the Tumor Necrosis Factor-alpha [TNF-alpha] or Lipopolysaccharide [LPS] or Thrombin [IIa] -stimulated Human Umbilical Vein Endothelial Cells (HUVEC). We examined the molecular expression of 9 candidate genes (E-selectin [E-Sel], Intracellular adhesion molecule-1 [ICAM-1], Plasminogen activator inhibitor-1 [PAI-1], Tissue Factor [TF], Tissue plasminogen activator [t-PA], Urokinase plasminogen activator [u-PA], Vascular adhesion molecule-1 [VCAM-1], Von Willebrand Factor [vWF] and Thrombomodulin [THBD]) by real-time PCR. Live cell number of HUVEC under exposure to Dex was also assayed by viability test. All experiments were performed in triplicates and Standard error of the mean (SEM) was obtained. Results: We showed that Dex alone significantly induced the expression of E-Sel, ICAM-1, TF, VCAM-1 and VWF while downregulating THBD and U-PA expression. Our results also showed a significant priming effect of Dex on E-Sel and TF inflammatory-mediated induction by TNF-alpha and LPS respectively. Comparable results were obtained from Northern Blot analysis; results from FACS analysis and Functional assays will be presented at the meeting. Conclusion: Our observations suggest a procoagulant activity of Dex on HUVEC. We also observed a priming activity of Dex on E-Sel and TF inflammatory-mediated induction. These results suggest a potential endothelial cell activation mechanism and subsequent microvascular thrombosis in glucocorticoid-induced ON

Aims. Alcoholism is a well-known detrimental factor in fracture healing. However, the underlying mechanism of alcohol-inhibited fracture healing remains poorly understood. Methods. MicroRNA (miR) sequencing was performed on bone mesenchymal stem cells (BMSCs). The effects of alcohol and miR-19a-3p on vascularization and osteogenic differentiation were analyzed in vitro using BMSCs and human umbilical vein endothelial cells (HUVECs). An in vivo alcohol-fed mouse model of femur fracture healing was also established, and radiological and histomorphometric analyses were used to evaluate the role of miR-19a-3p. The binding of miR-19a-3p to forkhead box F2 (FOXF2) was analyzed using a luciferase reporter assay. Results. miR-19a-3p was identified as one of the key regulators in the osteogenic differentiation of BMSCs, and was found to be downregulated in the alcohol-fed mouse model of fracture healing. In vitro, miR-19a-3p expression was downregulated after ethanol administration in both BMSCs and HUVECs. Vascularization and osteogenic differentiation were independently suppressed by ethanol and reversed by miR-19a-3p. In addition, the luciferase reporter assay showed that FOXF2 is the direct binding target of miR-19a-3p. In vivo, miR-19a-3p agomir stimulated callus transformation and improved the alcohol-impaired fracture healing. Conclusion. This study is the first to demonstrate that the miR-19a-3p/FOXF2 axis has a pivotal role in alcohol-impaired fracture healing, and may be a potential therapeutic target. Cite this article: Bone Joint Res 2022;11(6):386–397

Background: Circulating endothelial precursor cells (CEPS) are thought to play a role in postnatal angiogenesis. We investigated the angiogenic stress of musculoskeletal trauma on CEP kinetics in trauma patients and their bone marrow progenitor populations in a murine model. Methods: Peripheral blood mononuclear cells (PB-MNCs) were isolated from patients (n=12) on consecutive days following closed lower-limb diaphyseal fractures. CEP levels, defined by the surface expression patterns of VEGFR2, CD34 and AC133 were determined and cytokine analysis of collected serum was performed. Bone marrow precursors defined by Ly-6A/E and c-Kit expression were harvested following traumatic insult from the murine model and quantified on flow cytometry. Human and murine progenitor populations were cultured on fibronectin and examined for markers of endothelial cell linage (Ulexeuropaeus- agglutinin- 1 binding and acetylated-LDL uptake) and cell morphology. Statistical analysis was performed using variance analysis. Results: A consistent increase in human CEPs levels was noted within 72 hours of the initial insult, the percentage increase over day 1 reaching 300%. Conclusion: We propose that musculoskeletal trauma through the release of chemokines such as VEGF, promotes rapid mobilisation of CEP from born marrow, which have the potential to contribute to reparative neovascularisation. Strategies to enhance CEPs kinetics may accelerate this process and offer a therapeutic role in aberrant fracture healing

Aims: Circulating endothelial precursor cells (CEPs) are thought to play a role in angiogenesis. We investigated the angiogenic stress of musculoskeletal trauma on CEP kinetics in trauma patients and their bone marrow progenitor populations in a murine model. Methods: Peripheral blood mononuclear cells (PB-MNCs) were isolated from patients (n=12) on consecutive days following closed lower-limb diaphyseal fractures. CEP levels, deþned by the surface expression patterns of VEGFR2, CD34 and AC133 were determined and cytokine analysis of collected serum was performed. Bonemarrow precursors deþned byLy-6A/E and c-Kit expression were harvested following the traumatic insult from the murine model and quantiþed on ßow cytometry. Human and murine progenitor populations were cultured on þbronectin and examined for markers of endothelial cell lineage (Ulexeuropaeus- agglutinin-1 binding and acetylated-LDL uptake) and cell morphology. Statistical analysis was performed using variance analysis. Results: A consistent increase in human CEPs levels was noted within 72 hours of the initial insult, the percentage increase over day 1 reaching 300% (p=0.008) and returning to normal levels by day 10. Murine bone marrow precursors were mobilisd within 24 hrs peaking at 48hrs (900% p=0.035). On culture, morphologically characteristic endotheliallike cells binding UEA-1 and incorporating LDL were identiþed. Serum VEGF levels increased signiþcantly within 24 hrs of the insult, (p=0.018) preceeding the peak in CEP mobilisation. Conclusion: We propose that musculoskeletal trauma through the release of chemokines such as VEGF, promotes rapid mobilisation of CEPs from born marrow, which have the potential to contribute to reparative neovascularisation. Strategies to enhance CEPs kinetics may accelerate this process and offer a therapeutic role in aberrant fracture healing

Introduction. Nonunion is a common and costly fracture outcome. Intricate reciprocity between angiogenesis and osteogenesis means vascular cell-based therapy offers a novel approach to stimulating bone regeneration. Hypothesis. The current study compared early and late outgrowth endothelial progenitor cell subtypes (EPCs vs OECs) for fracture healing potential in vitro and in vivo. Methods. Primary cell cultures were isolated and characterized by endothelial assays, immunosorbent assays, and multi-color flow cytometry. Co-cultures of EPC subtypes with/without primary osteoblasts (pObs) were analyzed for tube length and connectivity. In vivo, EPCs or OECs (1×10. 6. ) seeded on a gelfoam scaffold were implanted in a rat model of nonunion. Radiography was used to monitor callus formation. Results. OECs expressed more BMP-2 and less VEGF than EPCs (p<0.05). Analysis of surface markers showed decreased CD34+/CD133+/Flk-1+, CD133+ and CD45+ populations in OECs while CD34+/CD31+/Flk-1+ cells increased. pObs significantly inhibited the strong tubulogenesis of OECs while enhancing connectivity and sprout length of EPCs. In vivo, 0/6 scaffold-control and 1/5 OEC rats achieved union at 10 weeks. In comparison, all EPC rats achieved full or partial union. Discussion and Conclusion. Despite favorable tubulogenic and osteoconductive profiles of OECs, EPCs display enhanced fracture healing in vivo. Differences in CXCR4 expression and cell-mediated effects may contribute to this result

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging. Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1. Cre. -mGmT and Dmp1. Cre. -COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1. Cre. -Mfn2. f/f. mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs. Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1. Cre. -mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1. Cre. -COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain. Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging

Relevant in vitro models emulating tendinopathies are highly needed to study these diseases and develop better treatments. We have recently proposed a new strategy that allows the automated 3D writing of microphysiological systems (MPS) embedded into its own biomimetic fibrillar support platform based on the self-assembling of cellulose nanocrystals (CNCs). Here, we explored this CNC platform for writing humanized in vitro tendon models using tendon decellularized extracellular matrix (dECM)-based bioinks to closely recapitulate the biophysical and biochemical cues of tendon cell niche and self-induce the tenogenic differentiation of stem cells. The proposed concept was further explored to study the crosstalk between the tendon core and vascular compartment. Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages. Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy. Acknowledgments: EU H2020 for ERC-2017-CoG-772817; ERC-PoC-BioCHIPs-101069302; FCT/MCTES for 2022.05526.PTDC, 2020.03410.CEECIND, and PD/BD/129403/2017

An increased number of neutrophils (NEUs) has long been associated with infections in the knee joints; their contribution to knee osteoarthritis (KOA) pathophysiology remains largely unexplored. This study aimed to compare the phenotypic and functional characteristics of synovial fluid (SF)-derived NEUs in KOA and knee infection (INF). Flow cytometric analysis, protein level measurements (ELISA), NEU oxidative burst assays, detection of NEU phagocytosis (pHrodo. TM. Green Zymosan Biparticles. TM. Conjugate for Phagocytosis), morphological analysis of the SF-derived/synovial tissue NEUs, and cultivation of human umbilical vein endothelial cells (HUVECs) using SF supernatant were used to characterise NEUs functionally/morphologically. Results: Compared with INF NEUs, KOA NEUs were characterised by a lower expression of CD11b, CD54 and CD64, a higher expression of CD62L, TLR2 and TLR4, and lower production of inflammatory mediators and proteases, except CCL2. Functionally, KOA NEUs displayed an increased production of radical oxygen species and phagocytic activity compared with INF NEUs. Morphologically, KOA and INF cells displayed different cell sizes and morphology, histological characteristics of the surrounding synovial tissues and influence on endothelial cells. KOA NEUs were further subdivided into two groups: SF containing <10% and SF with 10%–60% of NEUs. Analyses of two KOA NEU subgroups revealed that NEUs with SF <10% were characterised by 1) higher CD54, CD64, TLR2 and TLR4 expression on their surface; 2) higher concentrations of TNF-α, sTREM-1, VILIP-1, IL-1RA and MMP-9 in SFs. Our findings reveal a key role for NEUs in the pathophysiology of KOA, indicating that these cells are morphologically and functionally different from INF NEUs. Further studies should explore the mechanisms that contribute to the increased number of NEUs and their crosstalk with other immune cells in KOA. This study was supported by the Ministry of Health of the Czech Republic (NU20-06-00269; NU21-06-00370)

Aims. CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. Methods. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. Results. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Conclusion. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state. Cite this article: Bone Joint Res 2023;12(3):189–198

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

Angiogenesis is the formation of new blood vessels occurring in an adult through migration and proliferation of endothelial cells, and tubular structures formation. Angiogenesis is modulated by growth factors, cytokines, adhesion molecules, integrins, and enzymes. Angiogenesis plays a role in many physiological processes (i.e. remodeling of ischemic muscle, woumd healing, fracture repair) as well as in pathological process such as rheumathoid arthritis and metastases. In bone, vasculature is essential for cartilage resorption and angiogenesis temporally precedes osteogenesis: the origin of bone is the artery carrying calcium and phosphate ions. Osteogenesis takes place near newly formed vessels, that mediate delivery of osteoprogenitor cells, secrete mitogens for osteoblasts, and transport nutrients and oxygen. Inadequate bone vascularity is associated with decreased bone formation and bone mass. In animals, inhibition of angiogenesis during fracture repair results in the formation of fibrous tissue. A poor blood supply is therefore considered as a risk factor for an impaired bone healing. Angiogenesis is vital in tissue engineering, especially when matrices are colonized by cells with an aerobic metabolism. The scaffold must not only support the growth of the cells making up the organ which should be replaced in vivo (i.e. osteoblasts); it must also support the growth of endothelial cells and develop an effectively functioning vasculature to supply the cells with oxygen. Osteogenesis of tissue engineered materials could be limited by a lack of vascularization, and the bioengineered graft may be potentially resorbed in the same way as a conventional bone graft. In rats, angiogenesis in coralline materials implanted in ectopic muscular sites, was higher when the biomaterial was combined with a vascular pedicle or was coated with bone marrow stromal cells. A combination of both enhanced vascularization and osteogenesis to a greater extent. Endothelial cells release growth factors and cytokines promoting bone deposition: PDGF-AB, TGF-beta 1 and 2, FGF-2, EGF, BMP. However, under inflammatory stimula, endothelial cells release bone resorbing cytokines: IL-6, M-CSF, GM-CSF. Bone marrow stromal cells release angiogenetic proteins such as VEGF, FGF-2, PDGF, TGF, and, after induction with BMP, PlGF. A conversation between bone marrow stromal cells and endothelial cells may therefore be hypothesized. Cultures of bone marrow stromal cells with endothelial cell conditioned medium showed significantly higher phosphatase alkaline activity and osteocalcin production. It was also be hypothesized that stromal cells may acquire immunophenotypic characteristics consistent with endothelial cells. Therefore scaffold requirements are also the ability to favour angiogenesis; endothelial cells growing on the artificial scaffold should mantain a normal phenotype and should not exhibit a pro-inflammatory and bone.resorbing phenotype. Endothelial cell cultures are useful supplementary in vitro tests for the evaluation of scaffolds for bone tissue engineering. Endothelial cell cultures are derived both from animals (usually ox, calf or pig vessels) and from human tissues, mainly the human umbilical vein and the vessels of microcirculation (derma or subcutaneous fat). Endothelial cells in non-human species show different reactions: they have usually a faster replication rate and grow better on the artificial substrata. Endothelial cells from different organs are intrinsically different and exhibit different responses to stimula. if the use of endothelial cells from bone microcirculation should be desirable, they require transfection with viral vectors to be immortalized. To study the response of endothelial cells cultured in vitro on artificial scaffolds, their adhesion, growth, viability and production of metabolites should be evaluated. Adhesion and growth on the materials may be evaluated indirectly by the uptake of Alamar Blue, which measures the amount of oxido-reduction reactions in the cell. A direct evaluation may be obtained by fluorescence microscopy using specific staining for the different cell structures. By studying the expression of adhesins and integrins, the interference of the scaffold with the cell/cell and cell/substrate adhesion should be verified. The release of substances in conditioned medium, as well as the evaluation of specific mRNAs in cells, should be assayed. Among the metabolites released by endothelial cells, the substances promoting bone deposition or favouring resorption, should be investigated. In particular, the release of growth factors may be explored, as they favour cell proliferation and the incorporation of the engineered scaffold within tissues. For the enhancement of bone formation, growth factors may be delivered in different ways: through incorporation on the scaffold, through transfection of bone marrow stromal cells, through platelet gel. Angiogenic growth factors are stored in platelet alpha granules and released during activation. A significant increase in the proliferation of bovine bone endothelial cells was demonstrated after 72 hour incubation with platelet gel in comparison with serum free conditions; the proliferation was similar to the growth induced by the fetal calf serum supplementation (platelet gel: 82.2B18.1x103 cells; serum free: 19.5B11.1x103 cells; fetal calf serum: 72.4B12.4x103 cells). However, the platelet gel inhibited the formation of tubular structures on Matrigel. In conclusion, the development of newly formed vessels on the bone cell engineered scaffold improves the incorporation in the host tissues and the success of the device. The use of exogenous growth factors or of platelet gel favours angiogenesis, besides osteoblast differentiation. The in vitro evaluation of the scaffold should be supplemented by tests on the adhesion, growth and functionality of endothelial cells

We developed a 3D vascularized bone remodeling model embedding human osteoblast and osteoclast precursors and endothelial cells in a mineralized matrix. All the cells included in the model exerted their function, resulting in a vascularized system undergoing mineralized matrix remodeling. Bone remodeling is a dynamic process relying on the balance between the activity of osteoblasts and osteoclasts which are responsible for bone formation and resorption, respectively. This process is also characterized by a tight coupling between osteogenesis and angiogenesis, indicating the existence of a complex cross-talk between endothelial cells and bone cells. We have recently developed microscale in vitro hydrogel-based models, namely the 3D MiniTissue models, to obtain bone-mimicking microenvironments including a 3D microvascular network formed by endothelial cell self-assembly [1–2]. Here, we generated a vascularized 3D MiniTissue bone remodeling model through the coculture of primary human cells in a 3D collagen/fibrin (Col/Fib) matrix enriched with CaP nanoparticles (CaPn) to mimic bone mineralized matrix. Human umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (BMSCs), osteoblast (OBs) and osteoclast (OCs) precursors were cocultured in plain and CaPn-enriched Col/Fib according to the following experimental conditions: a) HUVECs-BMSCs; b) OBs-OCs; c) HUVECs-BMSCs-OBs-OCs. Undifferentiated BMSCs were used to support HUVECs in microvascular network formation. BMSCs and peripheral blood mononuclear cells were respectively pre-differentiated into OB and OC precursors through 7 days of culture in osteogenic or osteoclastogenic medium. Needle-shaped CaPn (Ø ∼20 nm, length ∼80 nm) were added to a collagen/fibrinogen solution. Cells were resuspended in a thrombin solution and then mixed with plain or CaPn-enriched collagen/fibrinogen. The cell-laden mix was injected in U-shaped PMMA masks and let to polymerize to generate constructs of 2×2×5 mm. 3. Samples were cultured for 10 days. Microvascular network formation was evaluated by confocal microscopy. OB differentiation was analyzed by quantification of Alkaline Phosphatase (ALP) and cell-mediated mineralization. OC differentiation was assessed by Tartrate-Resistant Acid Phosphatase (TRAP) and cell-mediated phosphate release quantification. HUVECs developed a robust 3D microvascular network and BMSCs differentiated into mural cells supporting vasculogenesis. The presence of CaPn enhanced OB and OC differentiation, as demonstrated by the significantly higher ALP and TRAP levels and by the superior cell-mediated mineralization and phosphate release measured in CaPn-enriched than in plain Col/Fib. The coculture of OBs and OCs with HUVECs and BMSCs further enhanced ALP and TRAP levels, indicating that the presence of HUVECs and BMSCs positively contributed to OB and OC differentiation. Remarkably, higher values of ALP and TRAP activity were measured in the tetraculture in CaPn-enriched Col/Fib compared to plain Col/Fib, indicating that also in the tetraculture the mineralized matrix stimulated OB and OC differentiation. The 3D MiniTissue bone remodeling model developed in this study is a promising platform to investigate bone cell and endothelial cell cross-talk. This system allows to minimize the use of cells and reagents and is characterized by a superior ease of use compared to other microscale systems, such as microfluidic models. Finally, it represents a suitable platform to test drugs for bone diseases and can be easily personalized with patient-derived cells further increasing its relevance as drug screening platform

Introduction and Objective. Klinefelter Syndrome (KS, karyotype 47,XXY) is the most frequent chromosomal aneuploidy in males, as well as the most common cause of infertility in men. Patients suffer from a lack of testosterone, i.e. hypergonadotropic hypogonadism provoking infertility, but KS men also show an increased predisposition to osteoporosis and a higher risk of bone fracture. In a mouse model for human KS, bone analysis of adult mice revealed a decrease in bone mass that could not be rescued by testosterone replacement, suggesting a gene dosage effect originating from the supernumerary X-chromosome on bone metabolism. Usually, X chromosome inactivation (XCI) compensates for the dosage imbalance of X-chromosomal genes between sexes. Some studies suggested that expression of genes that escape silencing of the supernumerary X-chromosome (e.g. androgen receptor) has an impact on sex differences, but may also cause pathological changes in males. As a promising new such candidate for a musculoskeletal escape gene, we identified the integral membrane protein (ITM) 2a, which is encoded on the X-chromosome and related to enchondral ossification. The aim of the project was to characterize systemic bone loss in the course of aging in our KS mouse model, and whether the supernumerary X-chromosome causes differences in expression of genes related to bone development. Materials and Methods. Bone structure of 24 month (=aged) old male wild type (WT) and 41, XXY mice (B6Ei.Lt-Y) were analysed by μCT. Afterwards bones were paraffin embedded and cut. In addition, tissue of brain, liver, kidney, lung and heart were also isolated and embedded for IHC staining. Using an anti-ITM2a antibody, expression and cellular localization of ITM2a was evaluated. IHC was also performed on musculoskeletal tissue of WT embryos (E18.5) and neonatal mice to determine possible age-related differences. Results. In 24 month old mice, the analysis of the lumbar vertebrae revealed a significantly lower BV/TV, trabecular bone volume and trabecular number in the XXY- group compared to WT. Trabecular thickness appeared lower but did not reach significance, with the cortical thickness being significantly higher in the XXY- group. High expression of ITM2a was detected in bone slices of both karyotypes in the chondrocytes inside the growth plate, as well as in megakaryocytes and leucocytes as well as endothelial cells of blood vessels inside the bone marrow. Osteocytes, along with erythrocytes and erythropoetic stem cells were negative for ITM2a. Other organs that showed ITM2a positive staining were kidney (blood vessels), heart (muscle) and brain (different structures). Liver and lung tissue were negative for ITM2a. No obvious difference in the intensity of the ITM2a-expression was observed between the WT and the XXY-karyotype. Analyses of embryotic bone tissue (WT) showed high expression of ITM2a in proliferating, hypertrophic and resting chondrocytes in the growth plates of tibia and femur. In comparison, the neonatal animals (WT) did not show any protein-expression in chondrocytes. Furthermore, within the metaphysis of both, embryotic and neonatal bones, endothelial cells and osteoblasts were ITM2a-positive. Further analyses of bones and tissues from young mice (4–6 month) are ongoing. Conclusions. Bone analyses revealed a significant reduction in trabecular bone mass along with fewer and thinner trabeculae in XXY mice compared to the WT, especially in the spine. ITM2a expression was visible in different cell types inside the bone, and in addition, different expression patterns at different stages of development (embryonic/neonatal) were observed. However, we have not found a significant difference in the quantity of ITM2a between tissues of XXY-karyotypes and WT. Further analyses of X-chromosomal encoded and therefore dysregulated modulators in XXY-karyotype mice and patients may reveal new sex chromosomal effector proteins in bone metabolism

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering

Bottom-up tissue engineering (TE) strategies employing microscale living materials as building blocks provide a promising avenue for generating intricate 3D constructs resembling native tissues. These microtissue units exhibit high cell densities and a diverse extracellular matrix (ECM) composition, enhancing their biological relevance. By thoughtfully integrating different cell types, the establishment of vital cell-cell and cell-matrix interactions can be promoted, enabling the recreation of biomimetic micro-niches and the replication of complex morphogenetic processes. Notably, by co-assembling blood vessel-forming endothelial cells with supportive stromal cells, microtissues with stable capillary beds, referred to as vascular units (VUs), can be generated. Through a modular TE approach, these VUs can be further combined with other microtissues and biomaterials to construct large-scale vascularized tissues from the bottom up. Integration of VUs with technologies such as 3D bioprinting and microfluidics allows for the creation of structurally intricate and perfusable constructs. In this presentation, we will showcase examples of VUs and explore their applications in regenerative medicine and tissue modeling. Acknowledgements: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology)

Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration. We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP. Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO. We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications