Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility

The fixation of titanium or titanium alloy implants is related to their surface composition and topography. Osteoconductive calcium phosphate coatings promote bone healing and apposition, leading to the rapid biological fixation of implants. It’s no doubt that the addition of certain biologically active protein with biomaterial will improve the bioactivity of the material. Previously, we examined the biocompatibility of basic fibroblast growth factor (bFGF) incorporation with titanium implants. Now we investigate the effect of fibronectin (FN) incorporation with thin calcium phosphate film deposited on titanium by electron-beam evaporation since fibronectin is actively involved in cell adhesion, spreading, would healing, cytoskeletal reorganization, and bone tissue formation. A FN-apatite composite layer was formed on the surface of titanium by biomimetic process. The coating process was carried out by immersing thin calcium phosphate film coated Ti in Dulbecco’s Phosphate buffered saline containing FN (20 ug/ml). The surfaces of samples were examined with FESEM, Fourier transform infrared spectroscopy and X-ray diffraction. The quantity of FN taken up and the kinetics of protein release were monitored by BCA method and Elisa. The fibronectin was immobilized in the newly formed apatite layer. The adhesion of osteoblast cells to the FN-apatite composite layer was to show the biocompatibility of implants, and FN-apatite composite layer could enhance osseintegration of implants in vivo. This research was supported by a grant (code #: 08K1501-01220) from Center for Nanostructured Materials Technology under 21st Century Frontier R& D Program of the Ministry of Education, Science and Technology, Korea

In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by Real Time-PCR and normalized with the expression of S29. Percentage of phosphorylated Smad2, Smad1 and JNK were determined through western blot. TGF-β1 content was quantified by sandwich ELISA; MMP-13 and GAG by western blot and DMMB assay, respectively. At least three biological replicates were used. ANOVA test was used for parametric analysis; Kruskal-Wallis and Mann-Whitney post hoc test for non-parametric. Preliminary data show that compression increased collagen II expression in control, but not in CHIR99021 and FB-fs pre-treated group (Fig. 1A-B). This was associated with downregulation of β1-integrin expression, which is the main collagen receptor and further regulates collagen II expression, suggesting inhibition of Erk1/2 pathway. A trend of increase expression of collagen type X after mechanical loading was observed in CHIR and FB-fs group. ALK-1 and ALK-5 showed a trend toward stronger upregulation in CHIR99021 group after compression, suggesting the activation of both Smad1/5/8 and Smad 2/3 pathways. To further investigate pathways leading to these different mechano-responses, the phosphorylation levels of Smad1 and Smad2, Erk1/2 and JNK proteins are currently being studied. Preliminary results show that Smad2, Smad1 and JNK protein levels increased in all groups after mechanical loading, independently of an increase in TGF-β1 expression or content. Compression further increased phosphorylation of Smad2, but not of Smad1, in all groups

Objective. High molecular weight hyaluronan (HA) is widely used in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA) by intra-articular injection. However, comparative studies of HA actions on catalytically activated cartilages in different pathologic conditions have rarely been investigated. Fibronectin fragments increased in OA and RA joints are known to cause cartilage damage through their catabolic activities. This study aimed to compare the inhibitory effects of HA on nitric oxide (NO) production by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) between normal and diseased cartilages. Methods. Articular cartilage explants from normal, OA, or RA joints or isolated chondrocytes in monolayer were incubated with HBFN-f in the presence or absence of HA. Secreted NO levels in conditioned media were determined. Induction of inducible nitric oxide synthase (iNOS) and activation of nuclear factor-?B (NF-?B) were assessed with immunoblotting. Cultures were pre-treated with the specific inhibitor to evaluate the role of NF-?B in HBFN-f action. Immunofluorescence histochemistry was performed using fluoresceinated anti-CD44 antibody. Results. When articular cartilage explants from normal, OA, or RA joints were incubated with HBFN-f, the RA and OA cartilages produced higher levels of NO compared with normal cartilage. Pre-treatment with 2700 kDa HA resulted in significant suppression of HBFN-f-stimulated NO production in OA and RA cartilages. While CD44 was up-regulated in OA and RA cartilages, anti-CD44 antibody reversed HA inhibition of HBFN-f action in those cartilages. While NF-?B activation contributed to HBFN-f-stimulated NO production, HA inhibited HBFN-f-activated phosphorylation and nuclear translocation of NF-?B. Conclusion. The present results clearly demonstrated that HA blocked HBFN-f actions in OA and RA cartilages through interaction with CD44. Down-regulation of NF-?B could involve HA inhibition of HBFN-f action. Intra-articular administration of HA, which targets CD44 highly expressed on OA and RA chondrocytes, could suppress catabolic actions by fibronectin fragments like HBFN-f in diseased cartilage

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development. Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining. Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production. Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA

Material-based strategies seek to engineer synthetic microenvironments that mimic the characteristics of physiological extracellular matrices for applications in regenerative therapies, including bone repair and regeneration. In our group, we identified a specific chemistry, poly(ethyl acrylate) (PEA), able to induce the organization of fibronectin (FN), upon adsorption of the protein, into fibrillar networks similar to the physiological ones, leading to enhanced cellular response, in terms of cell adhesion and differentiation. In this work, we exploit these FN networks to capture and present growth factors (GF) in combination with the integrin binding domain of FN during bone tissue healing. Fibrillar conformation of FN adsorbed on PEA favors the simultaneous availability of the GF binding domain (FNIII12–14) next to the integrin binding region (FNIII9–10), compared to poly(methyl acrylate) (PMA), a material with similar chemistry, where FN adopts a globular conformation. The combined exposure of specific adhesive sequences recognized by integrins and GF binding domains was found to improve the osteogenic differentiation of mesenchymal stem cells. A higher expression of bone proteins was found when BMP2 is bound or sequestered on the material surface versus its administration in the culture media in vitro. The potential of this system as recruiter of GFs was also investigated in a critical-size bone segmental defect in mouse. The synergistic integrin-GF signalling, induced by fibrillar FN, promoted bone formation in vivo with lower BMP2 doses than current technologies. Furthermore, we optimized the system for its potential use in translational research, seeking to address the clinical need of using biocompatible and biodegradable material implants. Polycaprolactone scaffolds were synthesized and coated with a thin layer of plasma- polymerized PEA that recruits and efficiently presents GF during healing of critical size defects. The material-driven FN fibrillogenesis provides a new strategy to efficiently reduce the GF doses administrated in bone regenerative therapies

The presence of the connective tissue components fibronectin and the different types of collagen was demonstrated by histological and immunohistological methods in the granulation and scar tissue of a healing injury in rat muscle. The effects of physical activity on granulation tissue production, scar formation and muscle regeneration at various stages of healing were studied. It was shown that immobilisation after injury accelerates granulation tissue production, but if continued too long, leads to contraction of the scar and to poor structural organisation of the components of regenerating muscle and scar tissue. However, a certain period of immobilisation, about five days for rat muscle, is required to allow newly-formed granulation tissue to cover the injured area and to have sufficient tensile strength to withstand subsequent mobilisation. This mobilisation, at the correct interval, seems essential for the quicker resorption of scar tissue and the better structural organisation of the muscle

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls. . Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

Background: Osseointegrated amputation prostheses avoid soft tissue complications associated with traditional socket prostheses. Forces are transmitted directly to the skeleton resulting in improved function. However, approximately 50% of transcutaneous implants become infected due to the lack of a successful skin-implant seal. Intraosseous Transcutaneous Amputation Prostheses (ITAP) are designed to integrate with the skin preventing epithelial downgrowth and infection. Fibronectin adsorption enhances fibroblast adhesion in vitro; however, in vivo, fibronectin becomes desorbed from the implant surface. Covalent attachment of fibronectin by silanisation has been shown to be durable in vitro. The silanisation process for fibronectin includes a stage of passivation with sulphuric acid which alters surface characteristics. Aims: The aim of this study was to determine if in vitro fibroblast adhesion to silanised fibronectin (SiFn) titanium alloy could be improved by omitting or reducing the length of time of passivation. The study also assessed the effects of SiFn on dermal attachment in vivo comparing the results with adsorbed fibronectin substrates and with uncoated controls. Methods: Scanning electron microscopy, Ra profilometry and contact angle measurement (n=6) were used for topographical characterization of surfaces. Anti-vinculin antibodies were used to immunolocalize fibroblast adhesion sites after 24 hours. The morphology of fibroblasts on each surface was evaluated using scanning electron microscopy. Subcutaneous plates were implanted onto the tibiae of an ovine model (n=3) in order to evaluate the performance of the modified SiFn surface in vivo. Hydroxyapatite (HA) and adsorption of fibronectin to HA (HAFn) were also tested because HA coatings are currently applied to the dermal section of ITAP in clinical trials. After four weeks, a histological assessment of the percentage of soft-tissue attachment and cell alignment relative to the implant was performed. Results: Passivation produced rougher, more hydrophobic surfaces with numerous microcracks and was associated with poorer fibroblast adhesion and spreading than un-passivated controls in vitro. SiFn with passivation resulted in poorer cell adhesion than SiFn without passivation. Reducing the time period for passivation did not reduce the detrimental effects of passivation In vivo, HAFn and SiFn resulted in higher median values for soft-tissue attachment than simple adsorption of fibronectin; however, the differences were not statistically significant. Cell alignment was significantly different for HAFn and SiFn compared with controls (p< 0.05), with cells on the fibro-nectin treated surfaces orientated more perpendicular to the implant surface. Conclusion: Omission of passivation improves fibro-blast adhesion to SiFn surfaces in vitro. Coating with fibronectin either by silanisation onto titanium alloy or by adsorption onto HA surfaces affected the orientation of cells in vivo, implying that tissue attachment was enhanced. A time course may be of value to determine if fibronectin coatings are lost over time in vivo

The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL. We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects. [1]. Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and collagen II, and expressed higher amounts of collagen II, X and tenomodulin as compared to aligned scaffolds. In-vivo implantation in rabbits of triphasic scaffolds accounting for aligned-wavy-aligned zones showed a high cellular infiltration and the formation of an oriented ECM, as compared to traditional aligned scaffolds. [2]

Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA

Backgrounds and aim. Low back pain resulting from Intervertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect, however, their behaviour in the harsh degenerate environment is unknown. Thus, we aimed to investigate and compare their physiological behaviour in in vitro niche that mimics the healthy and degenerated intervertebral disc environment. Methodology. Porcine NC cells were encapsulated in 3D alginate beads to maintain their phenotype then cultured in media to mimic the healthy and degenerate disc environment, together with control NC media for 1 week. Following which viability using PI and Calcein AM, RNA extraction and RT-PCR for NC cell markers, anabolic and catabolic genes analysed. Proteomic analysis was also performed using Digiwest technology. Results. A small increase in cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in NCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Discussion. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The initial work has been then translated to investigate the potential use of iPSCs differentiated into notochordal like cells as potential regenerative cell sources. Conflicts of interest: No conflicts of interest. Sources of funding: This project has received funding from the European Union Horizon 2020 research and innovation programme under grant agreement No 825925

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source

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

Introduction. Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative means of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including; pressure sores and tissue necrosis. ITAP resolves these problems by attaching the exo-prosthesis transcutaneously to the skeleton. The aim of this study is to increase the attachment of dermal fibroblasts to titanium alloy in vitro. Fibronectin (Fn) and laminin 332 (Ln) enhance early cell growth and adhesion. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable when compared with adsorbed dual coating (AdFnLn), and will enhance early fibroblast growth and adhesion compared to single coatings. Methods. The kinetics of dual single and dual protein coating attachment onto titanium alloy was quantified on silanized 10mm diameter discs using radiolabelled Fn (125I-Fn) and Ln (125I-Ln). Sixty discs were polished, sterilized and silanized. Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48 and 72hrs. Data was compared to un-silanized Ti discs with the same coatings. Five thousand human dermal fibroblasts were seeded on discs (n = 6) of Ti polished alone (Pol), Ti with adsorbed fibronectin (AdFn), Ti with adsorbed laminin (AdLn), Ti adsorbed dual coating (AdFnLn), Ti silanized (Si), Ti silanized with fibronectin (SiFn), Ti silanized with laminin (SiLn), Ti silanized with a dual coating (SiFnLn) for 24hrs. In order to measure cell adhesion fibroblasts were fixed, vinculin stained using mouse vinculin antibody and alexa fluor. Axiovision Image Analysis software was used to measure cell area, vinculin focal adhesion markers per cell and per unit cell area. Data was analysed in SPSS and significance was assumed at the 0.05 level. Results. Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings at all time points (all p values < 0.05). Fibroblasts cultured on dual coatings were significantly larger, produced more vinculin markers per cell, and per unit cell area compared with single coatings. Cells on SiFnLn were larger with more numerous vinculin markers per cell, and per unit cell area compared with AdFnLn (p<0.05). Conclusion. This study has demonstrated that covalently bonding both fibronectin and laminin to Ti alloy provides a durable, dual coating that enhances early fibroblast growth and attachment compared with either protein coating alone in vitro. Our study showed that there is non-competitive binding of laminin on Ti surfaces in the presence of fibronectin. Dual coatings may be applied to the skin-penetrating region of transcutaneous devices to improve the skin seal and this may have positive implications for the development of ITAP

Dupuytren’s disease is a chronic inflammatory process which produces contractures of the fingers. The nodules present in Dupuytren’s tissue contain inflammatory cells, mainly lymphocytes and macrophages. These express a common integrin known as VLA4. The corresponding binding ligands to VLA4 are vascular cell adhesion molecule-1 (VCAM-1) present on the endothelial cells and the CS1 sequence of the fibronectin present in the extracellular matrix. Transforming growth factor-beta (TGF-ß) is a peptide hormone which has a crucial role in the process of fibrosis. We studied tissue from 20 patients with Dupuytren’s disease, four samples of normal palmar fascia from patients undergoing carpal tunnel decompression and tissue from ten patients who had received perinodular injections of depomedrone into the palm five days before operation. The distribution of VLA4, VCAM-1, CS1 fibronectin and TGF-ß was shown by immunohistochemistry using an alkaline phosphorylase method for light microscopy. In untreated Dupuytren’s tissue CS1 fibronectin stained positively around the endothelial cells of blood vessels and also around the surrounding myofibroblasts, principally at the periphery of many of the active areas of the Dupuytren’s nodule. VCAM-1 stained very positively for the endothelial cells of blood vessels surrounding and penetrating the areas of high nodular activity. VCAM-1 was more rarely expressed outside the blood vessels. VLA4 was expressed by inflammatory cells principally in and around the blood vessels expressing VCAM-1 and CS1 but also on some cells spreading into the nodule. TGF-ß stained positively around the inflammatory cells principally at the perivascular periphery of nodules. These cells often showed VLA4 expression and co-localised with areas of strong production of CS1 fibronectin. Normal palmar fascia contained only scanty amounts of CS1 fibronectin, almost no VCAM-1 and only an occasional cell staining positively for VLA4 or TGF-ß. In the steroid-treated group, VCAM-1 expression was downregulated in the endothelium of perinodular blood vessels and only occasional inflammatory cell expression remained. Expression of CS1 fibronectin was also much reduced but still occurred in the blood vessels and around the myofibroblast stroma. VLA4-expressing cells were also reduced in numbers. A similar but reduced distribution of production of TGF-ß was also noted. Our findings show that adherence of inflammatory cells to the endothelial wall and the extravasation into the periphery of the nodule may be affected by steroids, which reduce expression of VCAM-1 in vivo. This indicates that therapeutic intervention to prevent the recommencement of the chronic inflammatory process and subsequent fibrosis necessitating further surgery may be possible

Mesenchymal stem cells (MSC) have a well recognised potential for tissue repair. This potential is two pronged: they can differentiate into the functional cell types of the damaged tissues and they can support tissue recovery by secreting trophic factors, depositing an extracellular matrix (ECM) and dampening inflammation. Three-dimensional microscopy recently shown that MSCs in the bone marrow create an intricate proteo-cellular scaffold with the ECM forming an interconnected cellular continuum whose structure is guided by the deposited ECM. This proteo-cellular scaffold controls bone marrow functions from hematopoiesis to osteogenesis. In the current study we aimed to optimise ECM production under in vitro conditions by immortalised MSCs with the view that the generated ECM can be utilised for tissue repair. With immunocytochemistry we determined the deposition of bone marrow-characteristic ECM proteins: collagen I, III, IV, V, VI, laminin and fibronectin. While primary MSCs produced slightly higher amount ECM proteins than immortalised MSCs, the relative abundancy of the ECM proteins was very similar. In order to isolate the ECM, we optimised a decellularisation method based on gentle lysis with sodium-deoxycholate and DNase digestion. Immunostaining for collagen I, III, VI and fibronectin and labelling the nuclei with Hoechst-33342 confirmed removal of all cells while retaining the ECM in its original architecture. Ideally, the decellularised ECM retains associated cytokines and chemokines, such as CXCL12, important for attracting MSCs. To test this, we seeded Molm-13 leukemia cells on decellularised ECM as MSC-produced CXCL12- and other cytokines protect leukemia cells against chemotherapeutics. We found that the decellularisation process however removed these factors and thus for therapeutic purposes, the decellularised ECM would need to be re-loaded with the essential chemo/cytokines. Overall, we developed a system for decellularised ECM production by immortalised MSCs and the results warrant further exploration of this avenue

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Introduction. Following amputation, residual stumps used to attach the external prostheses can be associated with sores, infection and skin necrosis. These problems could be overcome by off loading the soft tissues. Intraosseous transcutaneous amputation prostheses (ITAP) attach external implants directly to residual bone reducing these complications. However, a tight seal at the skin implant interface is crucial in preventing epithelial down-growth and infection. Fibronectin (Fn) and laminin 332 (Ln), enhance early cell growth and adhesion of keratinocytes. Silanization to titanium alloy (Ti) allows these proteins to bond to the metal directly. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable than absorbed proteins and that keratinocyte adhesion will be increased compared with Ti controls and single silanized proteins. Methods. 10 mm diameter Ti alloy discs were polished, sterilized and silanized. The kinetics of silanized single and dual protein coating attachment onto titanium alloy was quantified using radio-labelled Fn(125I-Fn) and Ln(125I-Ln). Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48, 72hrs. Data was compared to un-silanized Ti discs with the same amount of adsorbed proteins. In order to study cell attachment 20 × 103 keratinocytes were seeded on the discs (n = 6): silanized (Si), silanized fibronectin (SiFn), silanized laminin (SiLn), silanized dual coating (SiFnLn) for 1, 4 and 24hrs. Adhesion of cells was assessed using mouse vinculin antibody for 2hrs and alexafluor for 1hr which stains focal adhesions responsible for attaching cells to surfaces. Axiovision Image Analysis software was used to measure cell area, vinculin markers per cell unit and per unit cell area on 15 cells per disc. Data was analysed in SPSS and significance was assumed at the 0.05 level. Results. Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings (all p values < 0.05). When proteins were combined on silanized discs the same amount of each protein was attached as when used as a single coating (i.e. non competitive binding). Keratinocytes cultured on silanized dual coatings were significantly larger, produced more vinculin markers per unit cell and per cell area compared with single coatings at all time points. Conclusion. This study has demonstrated that silanized using dual proteins on Ti alloy enhances early keratinocyte growth and attachment in vitro. It also shows that there is non-competitive binding of laminin to Ti alloys in presence of fibronectin. This may lead to improved epidermal attachment to ITAP creating a tight seal at the implant interface, which will prevent migration of the epithelium and subsequent infection in vivo

The August 2014 Research Roundup. 360 . looks at: Antibiotic loaded ceramic of use in osteomyelitis; fibronectin implicated in cartilage degeneration; Zinc Chloride accelerates fracture healing in rats; advertisements and false claims; Net Promoter Score: substance or rhetoric?; aspirin for venous thromboembolism prophylaxis and dissection, stress and the soul

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM

Background. Osteoarthritis (OA), a common degenerative disorder of synovial joints, is characterized by disruption of the extracellular matrix (ECM) homeostasis with an overall misbalance towards cartilage catabolism. Integrins are alpha/beta heterodimeric transmembrane proteins transmitting chemical and biomechanical signals into the cells. There is a growing consensus that changes of ECM composition by proteolytic degradation of matrix constituents, or alteration of the biomechanical microenvironment of chondrocytes caused by chronic stress or injury significantly increase the risk of OA through the perturbation of integrin signaling. In order to further investigate the role of the b1 integrin subfamily in OA, we have challenged hip cartilage explants dissected for mice lacking beta1 integrins in chondrocytes by cytokines, ECM degradation products or mechanical stimulation. Methods. Femoral articular cartilages were avulsed from hip joints of 6 weeks old wild type (WT) and b1fl/fl-PrxCre mutant (MT) mice. For the chemically-induced OA-like stimulation, femoral caps were cultured for 3 days in serum-free DMEM/F12 with or without the supplementation of interleukin-1a (IL1a), 120kDa cell-binding fibronectin fragments (120FNf), or tumor necrosis factor-alpha (TNFa) + oncostatin M (OM). Sulphated glycosaminoglycan (sGAG) release of the explants were measured in the supernatants by the 1,9-dimethylmethlene blue (DMMB) assay. Proteoglycan loss was monitored by Safranin-O (SO) staining on cryo-sections of the explants. For the cartilage injury model, avulsed femoral caps were either directly snap-frozen or kept in serum-free DMEM/F12 for 4 hours before snap-freezing. Gene expression changes were analyzed by quantitative RT-PCR using a pre-determined set of genes regulated by injury. Results. Articular cartilages of MT mice were found to have consistently higher release of GAGs when exposed to cytokines or 120FNf. IL-1a exerted the highest catabolic stimulation. The ex vivo biochemical analysis was further verified by SO staining demonstrating more pronounced proteoglycan loss on MT sections compared to WT. Assessing the mRNA of articular cartilages subjected to the injury model, revealed expression changes in genes which have been previously implicated in OA: Il1a (interleukin 1, alpha) and Ptgs2 (prostaglandin-endoperoxide synthase 2) were upregulated in MT mice; whereas Il1rl1 (interleukin 1 receptor-like 1) and Nos2 (nitric oxide synthase 2) expression levels were significantly reduced in MT compared to WT. Conclusion. The data imply that b1 integrins play a protective role against cytokine- and fibronectin fragment-induced cartilage degradation. Our findings also suggest that b1 integrins modulate the expression of catabolic factors upon mechanical insults

The steric and electrostatic complementarity of natural proteins and other macromolecules are a result of evolutionary processes. The role of such complementarity is well established in protein-protein interactions, accounting for the known protein complexes. To our knowledge, non-biological systems have not been a part of such evolutionary processes. Therefore, it is desirable to design and develop nonbiological surfaces, such as implant devices (e.g. bone growth for non-cemented fixation), that exhibit such complementarity effects with the natural proteins. Cell attachment and spreading in vitro is generally mediated by adhesive proteins such as fibronectin and vitronectin [. 1. ]. The primary interaction between cells and adhesive proteins occurs through integrin and an RGD amino acid sequence. The adsorption of adhesive proteins plays an important role in cell adhesion and bone formation to an implant surface [. 1. ]. The ability of the implant surface to adsorb these proteins determines its aptitude to support cell adhesion and spreading and its biocompatibility. For example, the enhancement of osteoblast precursor attachment on hydroxyapatite (HA) as compared to titanium and stainless steel was related to increased fibronectin and vitronectin absorption [. 2. ]. The role of surface characteristics, such as topography, has been studied in recent years without the emergence of a comprehensive and consistent model [. 1. ]. For example, while no statistically significant influence of surface roughness on osteoblast proliferation and cell viability was detected in the study of metallic titanium surfaces [. 3. ], the TiO2 film enhances osteoblast adhesion, proliferation and differentiation upon an increase in roughness [. 4. ]. We designed and produced ceramic [. 5. ] and metallic coatings via an ion beam assisted deposition process with spatial dispersion (roughness) comparable to the size of proteins (3–20nm). Our ceramic and cobaltchrome (CoCr) coatings exhibit high hardness and contact angles with serum of 0° and 40° to 50°, respectively. Furthermore, our theoretical calculations and quantum-mechanical modeling clearly indicate that the spatial electric potential variation across our designed ceramic surfaces is comparable to the electrostatic potential variation of proteins such as fibronectin, promoting increased absorption on these surfaces. Therefore, an increase in the concentration of adhesive proteins on the designed surfaces results in the enhancement of the focal adhesion of cells. Our experimental results of the adhesion and proliferation of osteoblast-like stromal cells from mouse bone marrow indicate that our nanostructured coatings are three to five times better than growing on HA and orthopaedic grades of titanium and CoCr. Our results are consistent with the steric and electrostatic complementarity of nanostructured surfaces and adhesive proteins. This paper presents the adhesion and proliferation of osteoblast-like cells on micro-and nanostructured surfaces and provides new models describing the mechanism responsible for the enhancement of cell adhesion on nanostructured ceramic and metallic surfaces compared with orthopaedic materials

The main obstacle for tissue engineering is the difficulty in producing structurally and functionally well-organized tissues from in vitro cultured cells. Thus, on one hand the research is focusing towards bioactive three-dimensional materials (scaffolds) able to stimulate specific cellular processes. In fact the problem exists that cells cultured in scaffolds have great difficulty to adhere and proliferate if they don’t recognize bioactive molecules. In this respect biological polymers are used in the preparation of synthetic matrices to be used as tissue engineering scaffolds. On the other hand biological research is focusing on morphological and functional properties of cells seeded onto bioactive materials to evaluate their viability, adherence and proliferation, fundamental steps for successful tissue engineering. Surgical specimens were treated with type Ia collagenase and cultured in FCS/EGF supplemented DMEM. Cellular characterization was carried out on 3rd passage cells. Fibroblasts were seeded on Matri-cell, a substrate rich in basal lamina constituents, or PVA-gelatin sponges. Pulmonar ovine fibroblasts were also employed to set up the experimental procedures of cell seeding on scaffolds and histological methods. Immunocytochemistry was carried out to evaluate the presence of cytokeratin, fibroblast antigen, S-100 protein, TGF-beta1, fibronectin, type I collagen. Cytochemistry allowed to examine the synthesis of glysosoaminoglycans (Alcian blu method) and glycoproteins (PAS reaction). A fibroblast-like morphology and phenotype were found in the human cells isolated and selected from yellow ligaments. An high expression of fibroblast Ag, fibronectin and type I collagen but low TGF-beta1 and no cytokeratin immunoreaction were observed. A different localization of the detected antigens was found in the isolated fibroblasts depending on the type of substrate: a strong immunoreactive network of collagen I fibres was observed around cells grown on Matri-cell compared to the granular immunoprecipitates observed in the cytoplasm of fibroblasts grown on non coated plastic. Fibronectin was detected mainly at the extracellular level in Matri-cell cultured fibroblasts. Fibroblasts seeded on PVA-gelatin are viable and adherents to the substrates. Alcian blue reaction demonstrated the active production of glysosoaminoglycans both in Matri-cell or PVA-gelatin cultured cells, suggesting that these substrates allow extracellular matrix molecule production

Aberrant infrapatellar fat metabolism is a notable feature provoking inflammation and fibrosis in the progression of osteoarthritis (OA). Irisin, a secretory subunit of fibronectin type III domain containing 5 (FNDC5) regulate adipose morphogenesis, energy expenditure, skeletal muscle, and bone metabolism. This study aims to characterize the biological roles of Irisin signaling in an infrapatellar fat formation and OA development. Injured articular specimens were harvested from 19 patients with end-stage knee OA and 11 patients with the femoral neck fracture. Knee joints in mice that overexpressed Irisin were subjected to intra-articular injection of collagenase to provoke OA. Expressions of Irisin, adipokines, and MMPs probed with RT-quantitative PCR. Infrapatellar adiposity, articular cartilage damage, and synovial integrity verified with histomorphometry and immunohistochemistry. Infrapatellar adipose and synovial tissues instead of articular cartilage exhibited Irisin immunostaining. Human OA specimens showed 40% decline in Irisin expression than the non-OA group. In vitro, the gain of Irisin function enabled synovial fibroblasts but not chondrocytes to display minor responses to the IL-1β provocation of MMP3 and MMP9 expression. Of note, Irisin signaling reduced adipogenic gene expression and adipocyte formation of mesenchymal progenitor cells. In collagenase-mediated OA knee pathogenesis, forced FNDC5 expression in articular compromised the collagenase-induced infrapatellar adipose hypertrophy, synovial hypercellularity, and membrane hyperplasia. These adipose-regulatory actions warded off the affected knees from cartilage destruction and gait aberrance. Likewise, intra-articular injection of Irisin recombinant protein mitigated the development of infrapatellar adiposity and synovitis slowing down the progression of cartilage erosion and walking profile irregularity. Affected joints and adipocytes responded to the Irisin recombinant protein treatment by reducing the expressions of cartilage-deleterious adipokines IL-6, leptin, and adiponectin through regulating PPAR&gamma, function. Irisin dysfunction is relevant to the existence of end-stage knee OA. Irisin signaling protects from excessive adipogenesis of mesenchymal precursor cells and diminished inflammation and cartilage catabolism actions aggravated by adipocytes and synovial cells. This study sheds emerging new light on the Irisin signaling stabilization of infrapatellar adipose homeostasis and the perspective of the therapeutic potential of Irisin recombinant protein for deescalating knee OA development

Osteoinductive bone substitutes are in their developmental infancy and a paucity of effective grafts options persists despite clinical demand. Bone mineral substitutes such as hydroxyapatite cause minimal biological activity when compared to osteoinductive systems present biological growth factors in order to drive bone regeneration. We have previously demonstrated the in-vitro efficacy of a bioengineered system at presenting growth factors at ultra low-doses. This study aimed to translate this growth factor delivery system towards a clinically applicable implant. Osteoinductive surfaces were engineered using plasma polymerisation of poly(ethyl acrylate) onto base materials followed by adsorption of fibronectin protein and subsequently growth factor (BMP-2). Biological activity following ethylene oxide (EO) sterilisation was evaluated using ELISAs targeted against BMP-2, cell differentiation studies and atomic force microscopy. Scaffolds were 3D printed using polycaprolactone/hydroxyapatite composites and mechanically tested using a linear compression models to calculate stress/strain. In-vivo analysis was performed using a critical defect model in 23 mice over an 8 week period. Bone formation was assessed using microCT and histological analysis. Finally, a computer modelling process was developed to convert patient CT images into surface models, then formatted into 3D-printable scaffolds to fill critical defects. Following EO sterilisation, there was no change in scaffold surface and persistent availability of growth factors. Scaffolds showed adequate porosity for cell migration with mechanical stiffness similar to cancellous bone. Finally, the in vivo murine model demonstrated rapid bone formation with evidence of trabecular remodelling in samples presenting growth factors compared to controls

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Existing regenerative approaches rely on high doses of growth factors, such as BMP-2 in bone regeneration, which can cause serious side effects. We describe an ultra-low-dose growth factor technology yielding high bioactivity based on a simple polymer, poly (ethyl acrylate) (PEA), and report its translation to a clinical veterinary setting. This polymer-based technology triggers spontaneous fibronectin organization and stimulates growth factor signaling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, we use plasma-polymerized PEA on 2D and 3D substrates to enhance cell signaling in vitro, showing the complete healing of a critical-size bone injury in mice in vivo. We demonstrate its safety and efficacy in a Münsterländer dog with a non-healing humerus fracture, establishing the clinical translation of advanced ultra-low-dose growth factor treatment

INTRODUCTION: We have previously demonstrated the efficacy of a modified Ti-surface tethered with antibiotics in preventing bacterial colonization. It is not known if coverage of this surface with serum or other physiological material may hinder the bactericidal properties of such a surface.. The in vitro activity and efficacy of such a surface against S. aureus and S. epidermidis was tested following coverage of the surface with serum. METHODS: Vancomycin was coupled to Ti6Al4V pins by aminopropylation, linker addition, and vancomycin coupling (VancTi). Bactericidal activity was tested in solutions of bacteria (Ci=1×104cfu/ml) incubated with pins±pre-incubation with fetal bovine serum (FBS). Anti fibronectin and anti-vancomycin antibodies were used to detect surface coating or modification. Bacteria were detected by fluorescent labeling (Syto9) or by direct counting after solubilization. RESULTS: By immunofluorescence, pins were extensively covered with serum fibronectin which did not interfere with the diffuse, intense vancomycin staining. When incubated with S. epidermidis or S. aureus, VancTi showed little colonization compared to control pins (> 95% reduction in cfu). DISCUSSION: In a physiological environment, implants will be coated with serum proteins. Activity of the VancTi was unaffected by this coating and maintained potent inhibition of bacterial colonization. We have described a surface modification that allows Ti implants to resist colonization and subsequent periprosthetic infection. Such surfaces hold great promise for the prevention and treatment of periprosthetic infections

The study describes the changes of condrocytes and extracellular matrix occurring in Hip OA. 16 femoral heads were included in the study. Cartilage explants were removed from 3 anatomical sites over the surface of 14 OA and 2 non-OA patients. Cartilage sections were evaluated with histological (EE, Alcian Blu and Mallory-Azan stainings) and immuno-histochemichal (antibodies directed against fibronectin, tenascin, laminin, type I and type IV collagen, metallo-proteinase-1,-2,-7 and -7) analysis. Histological analysis of cartilage of central and per-hipheral biopsies from patients with severe OA showed significant reduced number of chondrocytes in both superficial and middle zones. In the lower cartilage layer with severe structural lesions a cospicous number of cartilagineous repair-islands were noticed. Immunohistochemical analysis showed high levels of tenascin in all cartilage layers of byopses showing structural damages. Frequently we observed an altered distribution of fibronectin. Metalloproteinase-2 (constitutive) is present in all stages during coxarthritis. Metalloproteinase-9 (not constitutive) is expressed at the final stages suggesting an important late role. Obtained results show that metalloproteinases have a peculiar behaviour during coxarthritis vs. other pathologies. Costitutive metal-loproteinases have a fundamental role in extracellular matrix remodelling, MMP-2 especially

Osteoarthritis (OA) is the most common form of joint disease leading to disability and dependence. In severe cases of knee OA, the joint is deemed irrecoverable and total knee replacements are indicated. Tissue engineering is a possible solution for this pathology and previous work from our laboratory has demonstrated that it is possible to isolate and expand chondroprogenitor cells in vitro from healthy knee-joint articular cartilage. Work presented here describes the detection and isolation of chondroprogenitor cells derived from osteoarthritic cartilage following total knee replacement in patients with severe OA, suggesting a pool of viable cells from this degenerate region which has been previously deemed non-recoverable. Human articular cartilage was excised from tibial plateaux (TP's) obtained from total knee replacements following the diagnoses of severe OA. Cells were isolated by a sequential pronase and collagenase digestion and subject to a fibronectin adhesion assay. Cells were expanded in monolayer in supplemented growth medium. Clonal 3D pellet cultures were established in chondrogenic and osteogenic differentiation media. Adipogenic cultures were also established in monolayer cultures. Histological procedures, immunohistochemistry and molecular biology were undertaken in order to determine the extent of differentiation. In addition, osteochondral plugs were excised from the TP's and wax embedded for further histological and immunohistochemical analysis. Clonal cell lines obtained from osteoarthritic knee-joint cartilage using the fibronectin adhesion assay were isolated and successfully cultured to a maximum of 60 population doublings whilst still demonstrating a chondrogenic capacity. Three-D pellet cultures after 21 days of chondrogenic induction produced smooth and iridescent pellets which stained positively for toluidine blue and safranin O. Positive labelling for collagen type II and aggrecan were also observed. Following osteogenic induction; evidence of mineralisation was indicated by the von Kossa stain. Adipogenic induction revealed a positive result. Osteochondral plugs demonstrated sporadic positive labelling in the surface region for putative stem cell marker Stro-1. Chondroprogenitor cells isolated from osteoarthritic display a strong chondrogenic phenotype, and have the ability to be induced into different lineages. These findings suggest the presence of a pool of viable chondroprogenitors from osteoarthritic tissue which was otherwise deemed irrecoverable

Summary. Attachment, proliferation and osteogenic maturation of hMSCs are enhanced on a sub-micron grooved Ti6Al4V alloy, while osteoblasts are less sensitive. These effects are attributed to their different maturation stage and may be mediated through differential activation of the RhoA/ROCK pathway. Introduction. Ti6Al4V alloy is the most widely used titanium-based biomaterial for manufacturing bone-anchoring devices. We report on the interactions of human bone-forming cells, mesenchymal stem cells from bone marrow (hMSCs) and primary osteoblasts (hOBs), with an anisotropic Ti6Al4V alloy that displays submicron grooves. Materials. Submicrometric Ti6Al4V surfaces (GV) with parallel grooves and mean average roughness values around 200 nm were generated by mechanical abrasion. A polished Ti6Al4V surface (PL) was used for comparative purposes. hMSCs were phenotypically characterised as progenitors and hOBs as committed osteogenic cells at a late stage of maturation. Cell attachment, proliferation, cytoskeleton organisation, adhesion sites and fibronectin matrix distribution, RhoA and ROCK distributions and activities, and osteogenic maturation, were analysed in cells cultured on the investigated alloys by means of scanning electron and confocal microscopy and biochemical assays. Results. hMSCs adhered and proliferated at a higher rate on GV alloy than on PL, while no differences were found in hOBs behavior. Patterned Ti6Al4V alloy promoted the orientation of hOBs and hMSCs in the direction of the anisotropy. Filopodia were involved in the alignment of both cell types along the grinding direction while cells exhibited a lamellipodia-dominated state on PL samples. In both cell types, focal adhesions, actin and tubulin networks and fibronectin extracellular matrix aligned with the direction of the grooves. RhoA/ROCK signaling contributed to hMSCs alignment on the patterned alloy, while osteoblastic orientation does not rely on the activation of this pathway. RhoA activity increased in both cell types cultured on GV alloy and interference on RhoA functioning by treatment with C3 transferase led to loss of organisation of actin and tubulin cytoskeletons. ROCK activity of hMSCs was up-regulated on GV samples, but not affected in hOBs. Treatment with hydroxyfasudil, an inhibitor of ROCK activity, disrupted the alignment of adhesion sites in hMSCs but not in hOBs. RhoA/ROCK signaling is not involved in the orientation of microtubule bundles of hOBs neither hMSCs on any surface, suggesting that such process is controlled by the RhoA effector mDia. When cultured on media that support osteogenic maturation, hMSCs and hOBs behaved differently on the anisotropic surfaces. OPN secretion increased in hMSCs cultured on GV alloy while it remained unaffected in hOBs. Cell mineralisation proceeded to a same extent in hMSCs cultured on the two metallic surfaces but decreased in hOBs cultured on the patterned samples. Discussion/Conclusion. Collected results indicate that hOBs are less sensitive to the patterned topography of Ti6Al4V alloy than hMSCs. These effects could be attributed to their different stages of maturation and can be mediated, at least in part, through ROCK since its activity increased on hMSCs cultured on the patterned alloy, while hOBs failed to up-regulate it. Collectively, these findings indicate that topography of Ti6Al4V can be manipulated to control cell functions via contact guidance responses. Altogether with the available data in the literature, results herein may also provide guidance to the implant technology for the design of Ti-based alloys with bioinspired surfaces topographies able to enhance specific bone cell activities

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. In vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with MMC at 2 % oxygen tension showed increased synthesis and deposition of collagen type I. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2 % oxygen tension. In addition, western blot analysis revealed increased expression of tendon-specific protein Scleraxis, while a detailed gene analysis illustrated upregulation of tendon-specific genes and downregulation of trans-differentiation genes again when cells cultured with MMC under hypoxic conditions. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in different ways. At present, to devitalize tumor-bearing osteochondral segments, mainly extracorporal irradiation or autoclaving is used. Both methods have substantial disadvantages, e.g. loss of biomechanical and biological integrity of the bone. In particular integration at the autograft-host junction after reimplantation is often impaired due to alterations of the osteoinductivity following irradiation or autoclaving. As an alternative approach, high hydrostatic pressure (HHP) treatment of bone is a new technology, now being used in preclinical testing to inactivate tumor cells without alteration of biomechanical properties of bone, cartilage and tendons. The aim of this study was to investigate the influence of HHP on fibronectin (FN), vitronectin (VN), and type I collagen (col. I) as major extracellular matrix proteins of bone tissue, accountable among others for the osteoinductive properties of bone. Fibronectin, vitronectin and type I collagen were subjected to HHP (300 and 600 MPa) prior to the coating of cell culture plates with these pre-treated proteins. Following the biological properties were measured by means of cell proliferation, adherence, and spreading of the human osteosarcoma cell line (Saos-2) and primary human osteoblast-like cells. Up to 600 MPa all tested matrix proteins did not show any changes, regarding the biological properties adherence, spreading and proliferation. We anticipate that, in orthopedic surgery, HHP can serve as a novel, promising methodical approach, by damaging normal and tumor cells without alteration of osteoinductive properties, thus facilitating osteointegration of the devitalized bone segment in cancer patients after reimplantation

Aim of study: The development of tissue engineering techniques evidenced that the healing of injured ligaments require the interactions of different cell types, local cellular environment and the use of devices. In order to gain new information on the complex interactions between mesenchymal stem cells (MSCs) and biodegradable scaffold, we analysed in vitro the proliferation, vitality and phenotype of MSCs grown onto a multilayered-woven-cylindric-array of Hyaff-11A8 fiber configured as ligament scaffold. Methods: Sheep MSCs were isolated from bone marrow aspirates and grown at two different density (7,5x106/cm and 15x106/cm) in the scaffold. At different time points (2, 4, 6 days) cellular proliferation was analysed by MTT test and cellular viability by calcein-AM immunofluorescence dye and confocal microscopy analysis. Moreover, hyaluronic acid receptor (CD44) and typical matrix ligament proteins (collagen type I, III, laminin, fibronectin, actin) were evaluated by immunohistochemistry. Results: MSCs growth was cell density-dependent and cells were uniformly distributed inside and along the scaffold. Confocal analysis showed that MSCs completely wrap the fibers at both cell concentrations analysed and were all viable both outside and inside the scaffolds only using the lower cell concentration. Moreover, MSCs expressed CD44, collagen type I, III, laminin, fibronectin and actin. Conclusion: These data demonstrate that MSCs well survive in a hyaluronic acid-configured ligament scaffold expressing a protein important for scaffold interaction, like CD44, and proteins responsible of the functional characteristic of the ligaments

Introduction. Despite the high regenerative capacity of bone, large bone defects often require treatment involving bone grafts. Conventional autografting and allografting treatments have disadvantages, such as donor site morbidity, immunogenicity and lack of donor material. Bone tissue engineering offers the potential to achieve major advances in the development of alternative bone grafts by exploiting the bone-forming capacity of osteoblastic cells. However, viable cell culture models are essential to investigate osteoblast behavior. Three-dimensional (3D) cell culture systems have become increasingly popular because biological relevance of 3D cultures may exceed that of cell monolayers (2D) grown in standard tissue culture. However, only few direct comparisons between 2D and 3D models have been published. Therefore, we performed a pilot study comparing 2D and 3D culture models of primary human osteoblasts with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation. Patients and Methods. Primary human osteoblasts were extracted from femoral neck spongy bone obtained during surgery procedures. Primary human osteoblasts of three donor patients were cultured in monolayers and in three different 3D culture models: 1) scaffold-free cultures, also referred to as histoids, which form autonomously after multilayer release of an osteoblast culture; 2) short-term (10-day) collagen scaffolds seeded with primary human osteoblasts (HOB); 3) long-term (29-day) collagen scaffolds seeded with HOB. Expression levels of transcription factors RUNX2 and osterix, both involved in osteoblast differentiation, were investigated using quantitative PCR and immunohistochemical staining. Furthermore, markers of osteogenic differentiation were evaluated, such as alkaline phosphatase activity, osteocalcin expression, and mineral deposition, as well as the expression of collagen type I and fibronectin extracellular matrix proteins. Results. Cells of the same origin, which were cultivated in different culture models, showed varying expression levels with regard to transcription factors RUNX2 and osterix as well as osteogenic markers. Increased levels of transcription factor RUNX2 and the extracellular matrix protein fibronectin were observed in all 3D cell culture models compared to monolayers. Furthermore, long-term cultivated histoids showed increased levels of osteogenic late-stage marker osteocalcin and transcription factor osterix. Additionally, long-term collagen scaffolds seeded with HOB showed elevated levels of osteocalcin compared to monolayers and short-term scaffolds. Moreover, alkaline phosphatase activity and mineralization capacity were increased in histoids. Conclusion. Considering the complex biochemical interactions of cells with surrounding cells and the extracellular matrix in vivo, important biological properties are disregarded when cells are only studied in 2D study models. Hence, we compared different 3D HOB cell culture models to 2D HOB monolayers with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation in vitro. Our pilot study indicated that three-dimensional study models may promote osteogenic differentiation in vitro. Additionally, a beneficial effect of longer culture duration on osteogenic differentiation was observed. Hence, our findings emphasise the importance of dimension and culture duration when studying osteoblast function. Subsequent studies with higher sample sized may lead to the development of viable primary human osteoblast cell culture models for bone tissue engineering. Summary. Three-dimensional cell culture models of primary human osteoblasts (HOB), including collagen scaffolds and scaffold-free cultures, were compared to HOB monolayers with regard to osteogenic differentiation. Our study indicated that three-dimensional study models may promote osteogenic differentiation of HOB in vitro

Purpose: Bone allografting appears to be optimised by in situ stromal cells which have potential to evolve into a bone line. The purpose of this study was to test the bio-compatibility of stromal cells and an allogenic human bone support treated with stromal cells as well as their evolutive potential. Material and methods: The bone support was a human femoral head allograft harvested during total hip arthroplasty. After validation of the safety of the femoral heads by the bone bank, they were treated using the Osteopure(r) method. Human stromal cells were harvested during cardiac surgery from the sternotomy. The in vitro study was conducted in a sterile atmosphere in an incubator. Different adhesion molecules were used: collagen, gelatin, fibronectin, human serum AB, in addition to an adhesion molecule-free medium. Microscopic qualitative evaluation determined the adhesion of stromal cells and the absence of difference between the morphology of cultured stromal cells and stromal cells found in the bone marrow. Cell counts were made on days 24; 32; 48, and 64. The functional properties of the new cultured stromal cells was evaluated by seeding CD34+ cells on day0 and counting the number of CFC produced on day45 (LTCIC1). This LTCIC1 line was cultured in the different media and re-evaluated at day45 (LTCIC2). Results: The first microscopic observations showed that the stromal cells oriented naturally in the bone architecture with no particular rejection and that they maintained their adhesion properties with each other and with the bone support. Cell counts showed increased proliferation for the stromal cells cultured on the bone support compared with cultures without bone support. Stromal cultures were favoured by the presence of bone and culture media containing collagen, gelatin, and fibronectin. But the LTCIC2 cultures demonstrated better performance with bone and gelatin. Discussion: Proliferation of stromal cells cultured in contact with an allograft demonstrated the biocompatibility of stromal cells/treated allografts. After twelve weeks incubation, the first cell counts tended to show that stromal cells cultured in vitro on human bone substitute preserve their functional potential and allow the proliferation of certain cells participating in osteogenesis. Further research to identify the capacity of these cells to induce an osteoblastic line must be conducted to allow in situ graft osteogenesis

Aims

Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA.

Introduction: The highest goal after meniscus damage is the preservation of the meniscus, which is often not possible due to the bad healing of meniscus lesions in the avascular zone. Therefore, the goal of our investigations was the analysis of expression of different angiogenic factors, growth hormones and cytokines in human meniscus cells (fibrochondrocytes). The mutual influence of the fibrochondrocytes by endothelial cell cocultures was analyzed, in order to examine the molecular bases of the healing of meniscus tears in vascularized zones more exactly. For this purpose, commercially available HUVEC [human umbilical vein endothelial cells] were used as well established and stable endothelial cell model. Material and Methods: Meniscal fibrochondrocytes were expanded in DMEM medium enriched with antibiotics and 10 % FCS. Cocultures of mensical cells and HUVEC were incubated in transwells over four and twelve days, separated by a semipermeable membrane. The expression of Angiopoietin-1, Angiopoietin-2, End-ostatin, VEGF, SMAD-4, Thrombospondin-1, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, IL-1β, iNOS, MMP-1, MMP-3, MMP-13, collagen-I, -II, -III, -VI, X, and -XVIII were examined by RT-PCR and immunhistochemistry in fibrochondrocytes in the comparison to cultures without endothelial coculture. A proliferation assay was used to investigate the mitotic activity in the coculture compared to the control culture after 4 and 12 days. Results: In presence of HUVEC, meniscal fibrochon-drocytes expressed the following factors at rates comparable to cells w/o HUVECS: Angiopoietin-1, Angiopoietin-2, VEGF, SMAD-4, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, iNOS, MMP-1, MMP-3, MMP-13, Thrombostatin-1, collagen-I, -II, -III, -VI, X, and -XVIII. In contrast, expression of end-ostatin (5.1-fold ± 1.2, p< 0.01) and IL-1β (10.3-fold ± 2.3, p< 0.003) were expressed significantly higher in the coculture when compared to the individual cell cultures. The proliferation rate of HUVEC was significantly decreased in coculture when compared to controls: 22 % after 7 days and 35 % after 14 days (p< 0.001). Discussion/ Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint. We could show that coculture of meniscus cells with endothelial cells revealed an increased expression of the anti-angiogenetic factor endostatin and the pro-inflammatory IL-1β. This suggests that meniscus cells are trying to inhibit proliferation of endothelial cells in their neigbourhood, which implicates huge problems in the research field of neoangiogenisis and tissue engineering in meniscus tissue for new healing methods after meniscus trauma

Cellular therapies play an important role in tendon tissue engineering and regenerative medicine with tenocytes being described as the most prominent cell population for these applications if available in large numbers. However, this is difficult to achieve, because in vitro expansion of tenocytes leads to phenotypic drift and loss of function. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media to mimic the dense extracellular matrix and accelerate the production of ECM-rich substitutes. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing deposition of tissue-specific extracellular matrix. SDS-PAGE and immunocytochemistry analysis, demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased collagen type I synthesis and deposition after 7 days. Moreover, immunocytochemistry for collagen type III, type V, VI, elastin and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, it was shown that low oxygen tension and MMC did not affect the spindle-shape morphology, metabolic activity, proliferation and viability of human tenocytes Collectively, these results suggest that the synergistic effect of optimal macromolecular crowding concentration and low oxygen tension (2%) can accelerate the formation of ECM-rich substitutes, which may stimulate tenogenic phenotype maintenance. Further gene and protein analysis for tendon specific markers should be performed to validate our promising results

Aims

This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).

Background. Intervertebral disc degeneration is implicated as a major cause of chronic lower back pain. Current therapies for lower back pain are aimed purely at relieving the symptoms rather than targeting the underlying aberrant cell biology. As such focus has shifted to development of cell based alternatives. Notochordal cells are progenitors to the adult nucleus pulposus that display therapeutic potential. However, notochordal cell phenotype and suitable culture conditions for research or therapeutic application are poorly described. This study aims to develop a suitable culture system to allow comprehensive study of the notochordal phenotype. Methods & Results. Porcine notochordal cells were isolated from 6 week post natal discs using dissection and enzymatic digestion and cultured in vitro under different conditions: (1)DMEM vs αMEM (2)laminin-521, fibronectin, gelatin and untreated tissue culture plastic (3)2% 02 vs normoxia (4)αMEM (300 mOsm/L) vs αMEM (400 mOsm/L). Notochordal cells were cultured in alginate beads as a control. Adherence, cell viability, morphology and expression of known notochordal markers (CD24, KRT8, KRT18, KRT19 and T) were assessed throughout the culture period. Use of αMEM media and laminin-521 coated surfaces displayed the greatest cell adherence, viability and retention of notochordal cell morphology and gene expression, which was further enhanced through culture in hypoxia and hyperosmolar media mimicking the intervertebral disc niche. Conclusions. Assessment of the therapeutic potential of notochordal cells is potentially valuable to development of a cell based therapy for chronic lower back pain. Our model has provided a system in which notochordal cells can be studied extensively. Conflicts of Interest: None. Funding obtained from the Henry Smith Charity, London

Introduction: Cell adhesion to titanium alloy implants is important in osseointegration [1,2] and attachment of the soft tissues to skin penetrating implants e.g. external fixator pins and Intraosseous Transcutaneous Amputation Prostheses [3,4]. Cell adhesion can be assessed using cell area data and immunolocalisation of focal contact proteins e.g. vinculin; however no method of assessing biophysical attachment is performed routinely. Cell adhesion can be enhanced with adhesion proteins including fibronectin (Fn)[5]. We have previously shown that covalently binding Fn to titanium also increases cell adhesion, and produces a more robust protein coating [6]. However the strength of adhesion of cells to this coating has not been measured. Our hypothesis was that biophysical cell adhesion measured using novel radial flow apparatus would correlate with cell area and focal contact data and that covalently bound fibronectin substrates would increase cell adhesion compared with adsorbed and uncoated controls. Method: Dermal fibroblasts were cultured for 1, 4, and 24 hours on 30mm and 10mm diameter polished titanium alloy discs (n = 6). Cells on 30mm discs were calcein stained and subjected to shear stress in a submerged, media filled, custom-made radial flow apparatus at 37¬C at 1.66ml/s for 15s. Cells were fixed in 10% formal saline and photographs were taken using a tangential light source. Fluorescent microscopy was performed at 2mm intervals along two perpendicular diameters. Using image analysis, the central cell free zone was measured and radial distance and shear stress calculated. Cells on 10mm discs were fixed, permeablised and vinculin stained (mouse vinculin antibody (1:200) 2hrs; FITC mouse antibody (1:100)1hr). Images were analyzed with a Zeiss microscope linked to image analysis software and the number of focal contacts were counted per cell area. The medians of the radial flow data were compared with data for cell area and focal contact production at the same time points using Spearman¡s regression correlation. This method was subsequently used to compare cell adhesion at one hour with adsorbed and covalently bound Fn substrates (10¥ìg/disc). Results/Discussion: The shear strength of cells increased between 4 and 24hrs (p=0.002) on polished untreated control substrates. Attachment values (dynes/cm2) were 84.90 (73.98–97.19), 96.30 (91.66–100.89), and 136.69 (134.68–140.30) for 1, 4 and 24 hours respectively. At 1hr, covalently bound Fn (509.90 dynes/cm2 (490.55–528.49) significantly increased cell adhesion compared with adsorbed Fn(434.45 dynes/cm2(385.25–465.62)) and control substrates(p=0.002). There was significant correlation between shear stress and focal contacts/cell (1.00(p< 0.01)) and focal contacts/cell area (0.900(p=0.037)), but not cell area (0.600(p=0.285)). Conclusion: Radial flow measurement is a useful direct method to quantify cell adhesion to orthopaedic implants and correlates well with other methods of measurement. Covalently bound Fn significantly increases biophysical cell attachment compared with adsorbed and uncoated controls

Introduction: The knee meniscus is prone to injury and has limited intrinsic healing potential despite surgical repair. Methods to enhance fibrochondrocyte function and augment meniscal repair would be invaluable in the treatment of meniscal injuries. Ultraviolet Ozone (UVO) modified surfaces have been characterised chemically and topographically. These surfaces have been shown to promote the function of certain cell types. This study investigated the attachment, proliferation and extracellular matrix production of fibrochondrocytes cultured on UVO modified polystyrene surfaces. Interest was paid to the integrins, a group of transmembrane extracellular matrix attachment glycoproteins. In particular the subunits alpha2 and alpha5, as they specifically bind to the ligands Collagen Type I and Fibronectin, major components in the human meniscus. Methods: Tissue samples from adult human medial meniscal tears were obtained at knee arthroscopy. Fibrochondrocytes were isolated by standard cell culture techniques and cultured to 100% confluence before seeding onto UVO modified polystyrene surfaces. The untreated polystyrene surfaces of culture dishes were oxidatively modified with an ultraviolet ozone treater. The response of fibrochondrocytes to various surface oxygen concentrations was investigated. Untreated, hydrophobic surfaces acted as controls. Images of cells in culture were obtained with a Leica digital camera mounted on a microscope. Cells were counted at 24, 48, 72 and 96 hours. After 48 and 96 hours of culture standard wet transfer Western Blots were undertaken using antibodies to the alpha2 and aalpha5 integrin subunits (Santa Cruz Biotechnology). To evaluate potential extracellular matrix production total protein assays were undertaken at 1, 2, 3, 4, 6, 8 and 10 days of culture (Bio-Rad Laboratories). Results: Fibrochondrocytes attached preferentially to the UVO treated surfaces. They proliferated steadily until they reached confluence at 96 hours. Western Blot analysis showed the integrin subunit a5 to be present in the cell lysate after both 48 and 96 hours of culture. The a2 subunit was not detected at these times. There was no increase in total protein concentration on surfaces after fibrochondrocytes had reached confluence. Discussion: UVO modified surfaces promote the attachment and proliferation of human fibrochondrocytes. The alpha2 subunit was not detected in the cell lysate of these surfaces after culture for 96 hours. Whether this is due to defective or absent Collagen Type I at this stage of culture remains to be answered. The presence of the alpha5 subunit suggests that Fibronectin may be involved in the process of fibrochondrocyte attachment to UVO modified polystyrene surfaces. It is suggested that there is little or no extracellular matrix production after 4 days as there is no increase in total surface protein concentration after confluence is reached. The increase in total surface protein concentration up to this point most likely reflects cell proliferation. Conclusion: Ultraviolet Ozone modified surfaces enhance certain aspects of fibrochondrocyte function and therefore have a potential role in the development of novel therapies for meniscal repair

Introduction: MEPE was identified in patients with tumors and oncogenic hypophosphatemic osteomalacia (OHO), and therefore thought to inhibit osteoblast differentiation and proliferation. However when looking at the structure of MEPE in detail a number of important domains are observed, including a glycosamino-glycan-attachment site, and a RGD cell-attachment motif. The RGD motif is by far the best characterized peptide sequence for stimulating cell adhesion on synthetic surfaces. Glycosaminoglycan attached to MEPE also has the potential to interact with numerous growth factors, proteases and cell surface receptors. MEPE shares molecular similarities with several dentin-bone phosphoglycoproteins which exhibit an ASARM motif shown to potently inhibit calcium crystallization and crystal growth in the salivary duct system. More recently the ASARM peptide sequence has been shown to be a inhibitor of osteoblast mineralization. Method: To test the hypothesis that MEPE has multiple functional sites, PCR Primers were designed to provide a truncated MEPE protein, which contained pro-osteogenic motifs and had the anti-osteogenic ASARM motif removed. PCR products were cloned using the pBAD TOPO® TA Expression Kit. MEPE was than expressed in E. coli and purified by HIS column chromatography. Expression of truncated MEPE was confirmed by coomassie staining, Western blot with an antibody to an epitope tag and sequence analysis. Truncated MEPE was passively absorbed overnight at 4 oC in a 96 well plate (0.3–50 micrograms) and Fibronectin was laid down (30 micrograms) as a positive control. Primary rat osteoblasts in serum free medium were seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 24 hours. MTT assay was used to estimate cell number, the coloured product absorbance was then determined at 490nm and adhesion was expressed relative to fibronectin. In addition we laid down truncated MEPE into three 8 well chamber slides as above. This was left overnight at 4 oC. Primary rat osteoblasts were then seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 4 hours in serum free medium. Cells were viewed and images captured with a phase contrast microscope. Results: We have successfully expressed MEPE in E. Coli and devised a purification strategy for obtaining protein. This has been confirmed by coomassie, silver stain and Western blot analysis. The MTT assay showed a significant increase in cell adhesion and proliferation within wells coated with 50 micrograms (70% +/− 0.67(relative to fibronectin)), 30 micrograms (63% +/− 0.81), 3 micrograms (54% +/− 2.4) of MEPE when compared with TCP (32% +/− 0.56). Furthermore we have shown increased osteoblast spreading with increasing dose when compared to tissue culture plastic alone. Conclusion: The data shows a dose dependent response of osteoblast to increasing concentrations of the novel MEPE protein. This provides evidence that MEPE without the ASARM domain increases osteoblast adhesion, cell anchorage and spreading. Further studies are currently been undertaken to establish its long term effects on osteoblast function and suitability for incorporation into orthopaedic biomaterials

Background. Auxetic materials have a negative poisons ratio, and a number of native biological tissues are proposed to possess auxetic properties. One such tissue is annulus fibrosus (AF), the fibrous outer layers of the intervertebral disc (IVD). However, few studies to date have investigated the potential of these materials as tissue engineering scaffolds. Here we describe the potential of manually converted polyurethane (PU) foams as three dimensional cellular scaffolds for AF repair. Methods. Rat MSCs were seeded onto fibronectin coated auxetic foams at a cell density of 6.4 × 10. 3. cells/mm. 3. , and cultured for up to 3 weeks. Cell viability was assessed throughout culture and following culture scanning electron microscopy (SEM) was used to assess morphological characteristics. Histological assessment was performed to assess production of matrix proteins. Results. Cells adhered to the surface auxetic foams and remained viable for the 3 weeks investigated. Histology and SEM demonstrated cells within the full thickness of the auxetic foams, where extracellular matrix was starting to be produced following 3 weeks, including collagens suggesting differentiation of the MSCs. Conclusion. Auxetic PU foams have a significant potential for use in tissue engineering applications, potentially mimicking the multiaxial strains of annulus fibrous tissue. MSCs were shown to adhere, survive and produce matrix within the foams after 3 weeks, future work will focus on longer term studies and in depth analysis of the phenotype of the cells. No conflicts of interest. Funding provided by a grant from Sheffield Children's Hospital NHS trust

Polyether ether ketone (PEEK) has been increasingly employed as biomaterials for trauma, orthopeadic, and spinal implants. However, concern has been raised about the inertness of PEEK which limits bone integration. In this study, we have coated PEEK with a functional material seeking to promote osteogenic differentiation of human mesenchymal stem cells (hMSC). We have used spray drying to coat poly(ethyl acrylate) (PEA) as a coating on PEEK. This technique is simple, allows a range of controlled coating thicknesses (from hundred nm to a few um), cost effective and easily translatable to scaffolds or implant surfaces for existing or new orthopaedic applications. PEA induces the organisation of fibronectin (FN) into nanonetworks upon simple adsorption from protein solutions. These FN nanonetworks on PEA represent a microenvironment for efficient growth factor binding and presentation in very low but effective doses. In this study we show cell adhesion and stem cell differentiation towards an osteogenic lineages when bone morphogenetic protein 2 (BMP2) was adsorbed on these engineered PEEK/PEA/FN microenvironments in very low doses. Overall, the developed functional coatings on PEEK has the potential to allow the translation of this material into orthopaedic applications

Aims

Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

Background. The cartilaginous growth plate (GP) is a zonal structure, in which chondrocytes are organized into columns and drive the longitudinal elongation of the endochondral bones. In the proliferative zone (PZ), cells exhibit high mitotic activity, are flattened and oriented along the mediolateral (ML) axis of the GP. Mitotic figures in the elongated chondrocytes lie perpendicular to the proximo-distal (PD) direction of growth, while cell divisions occur parallel to the columns followed by a gliding movement of the daughter cells. The mechanisms responsible for the geometrical anisotropy and columnar arrangement of PZ chondrocytes are poorly understood. Here, we assessed the function of the adhesive receptor β1 integrins on spindle and division geometry in chondrocytes using mouse genetics. Methods. GP slices were prepared from wild type (wt) and β1fl/fl-Col2a1cre mice. Primary rib chondrocytes were cultured on substrate-coated glass slides and fluorescently stained with anti-alpha-tubulin and anti-pericentrin antibodies, with phalloidin and DAPI. Confocal stacks were built from images acquired by confocal microscopy. Cell and spindle orientation relative to the PD axis (in vivo) or to the substrate plane (SP) in vitro were determined by geometric functions. Shape indexes (SI) were calculated as the ratio of the length and height of the cell. Results. During GP morphogenesis, aggregating mesenchymal cells (E11) were polygonal (SI=1.43) and nonoriented. Upon chondrogenic differentiation at E12, wt GP chondrocytes showed moderate flattening (SI=1.93) and tend to align perpendicular to the PD axis. At E13, PZ chondrocytes further flattened (SI=3.41) and largely organized into lines along the ML axis. At E14, the first vertical stacks formed, which were gradually elongated along the PD axis at later stages and composed of extremely flat (SI=4.91), ML-oriented chondrocytes. Early spindles were randomly oriented at all stages, whereas late spindles were aligned along the long axis of the cell. In contrast, β1 integrin null PZ chondrocytes were rounded (SI=1.37), displayed random orientation of both early and late spindles, and failed to organize into columns. On collagen II, both wt and β1-null primary chondrocytes remained rounded and displayed random spindle orientation relative to the SP. On fibronectin (FN), wt chondrocytes were flattened (SI=3.26) and their spindle was aligned parallel to SP. Mutant cells lacking the major FN receptor α5β1 integrin did not spread, were rounded (SI=1.73) and aligned their spindle randomly. On vitronectin, both cell types spread and flattened well (SI=4.11 and SI=4.20) accompanied by parallel spindle orientation to the SP. Conclusion. We propose that β1 integrins provide adhesive cues for chondrocyte geometry and orientation, which in turn orients the mitotic spindle and determines the division axis; and for chondrocyte intercalation to shape the three-dimensional structure of the GP