Aims. Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Methods. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. Results. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. Conclusion. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression. Cite this article: Bone Joint Res 2023;12(8):476–485

Objectives. Osteoporosis is a systemic bone metabolic disease, which often occurs among the elderly. Angelica polysaccharide (AP) is the main component of angelica sinensis, and is widely used for treating various diseases. However, the effects of AP on osteoporosis have not been investigated. This study aimed to uncover the functions of AP in mesenchymal stem cell (MSC) proliferation and osteoblast differentiation. Methods. MSCs were treated with different concentrations of AP, and then cell viability, Cyclin D1 protein level, and the osteogenic markers of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2) were examined by Cell Counting Kit-8 (CCK-8) and western blot assays, respectively. The effect of AP on the main signalling pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin was determined by western blot. Following this, si-H19#1 and si-H19#2 were transfected into MSCs, and the effects of H19 on cell proliferation and osteoblast differentiation in MSCs were studied. Finally, in vivo experimentation explored bone mineral density, bone mineral content, and the ash weight and dry weight of femoral bone. Results. The results revealed that AP significantly promoted cell viability, upregulated cyclin D1 and increased RUNX2, OCN, ALP, and BMP-2 protein levels in MSCs. Moreover, we found that AP notably activated PI3K/AKT and Wnt/β-catenin signalling pathways in MSCs. Additionally, the relative expression level of H19 was upregulated by AP in a dose-dependent manner. The promoting effects of AP on cell proliferation and osteoblast differentiation were reversed by H19 knockdown. Moreover, in vivo experimentation further confirmed the promoting effect of AP on bone formation. Conclusion. These data indicate that AP could promote MSC proliferation and osteoblast differentiation by regulating H19. Cite this article: X. Xie, M. Liu, Q. Meng. Angelica polysaccharide promotes proliferation and osteoblast differentiation of mesenchymal stem cells by regulation of long non-coding RNA H19: An animal study. Bone Joint Res 2019;8:323–332. DOI: 10.1302/2046-3758.87.BJR-2018-0223.R2

Aim. As the number of performed total hip arthroplasties (THA) and total knee arthroplasties (TKA) has increased over the years, revision surgeries are expected to increase as well. Revision surgeries are associated with a longer operating room time, prolonged length of stay (LOS), and more frequent complications. Postoperative hematomas are a major reason for wound healing disturbances and periprosthetic joint infections (PJI). We aimed to systematically assess the use and safety of a microporous polysaccharide hemosphere (MPH) in revision THA and TKA. We focused on the risk reduction of further revision surgeries in case of wound healing disorders and hematoma, transfusion of packed red blood cells (PRBC), loss of hemoglobin (hb) and mean LOS following the use of MPH. Method. Our prospective study includes 89 patients who underwent revision surgery after THA and TKA with application of MPH and were compared to 102 patients who did not receive MPH and underwent revision surgery after THA and TKA. Five grams of MPH. 1. were applied periarticular before fascia closure and to the subcutaneous soft tissue. The follow-up was conducted in daily clinical visits during the inpatient stay and three months postoperatively in our outpatient clinic. Repeated revision surgery was performed in case of prolonged secretion (>10 days) or clinical suspicion of infection. After matching the cohorts the outcomes were statistically analyzed using paired methods. Results. A significantly lower odds ratio for repeat revisions was found for the MPH cohort (OR=0.312; 95%-CI 0.090, 0.893; p=0.027). Differences between pre- and postoperative hb levels, LOS and transfusions of PRBC did not reach significance. No intra- or postoperative complications to MPH occurred. Moreover, no infection relapse occurred after applying MPH. Conclusions. Routine use of MPH in revision arthroplasty management after TKA and THA appears to be safe and an effective way to support hemostasis, with no observed adverse events related to MPH use. There were noticeably less hematomas and revision surgeries in the MPH group. 1. Arista BD, Franklin Lakes, NJ, USA

Polysaccharide (alginate and chitosan) capsules coated with a unique self-assembled semi-crystalline shell of calcium phosphate provide an enclosed biological system for the spatial and temporal delivery of human cells and bioactive factors. The aim of this study was to demonstrate plasmid DNA entrapment, delivery and transfection of adjacent cells inside capsules, embedded capsules and plated. Bacterial plasmid DNA and/or bone cells (SaOS) was added to solution of sodium alginate solution supplemented with phosphate ions and mixed thoroughly. Alginate droplets were fed through a syringe into a solution of chitosan supplemented with calcium ions. Guest capsules were inserted into soft, pliable host capsules soon after immersion in chitosan solution. Capsules were then immersed in 2mL DMEM 10% FCS in 6-well plastic plates for up to 7 days to enable transfection to occur. Encapsulated bone cells were stained with standard X-Gal to show transfected cells expressing beta-galactosidase. DNA delivery and transfection was demonstrated within capsules containing SaOS cells and plasmid, an admixture of SaOS bone cells and plasmid (51%) and from capsules containing DNA alone suspended in media over plated SaOS one cells. We also demonstrate capsule transfection of encapsulated cells in vivo. Transfection efficiency is highest when plasmid is entrapped and released from embedded capsules followed by plasmid/ SaOS admixture within capsules and lowest efficiency was observed with plated SaOS cells (with a transfection efficiency of 5%). The ability to regulate shell decomposition by manipulating the degree of mineralization and the strength of gelling, and release of capsule contents provides a mechanism for programmed release of gene modulated cells into the biological environment. The beta-galactosidase plasmid was found to be strongly associated with the chitosan/ calcium phosphate shell as shown by ethidium-homodimer-1 staining of encapsulated DNA and this may assist the transfer from gel to cell. Programmed non-viral delivery of genes using biomaterial constructs is an important approach to gene therapy and orchestrated tissue regeneration. These unique biomineralised polysaccharide capsules provide a facile technique, and an enclosed biomimetic micro-environments with specifiable degradation characteristics, for the safe encapsulation and delivery of functional quantities of plasmid DNA with the implicit therapeutic implications therein

Introduction: Staphylococci are a well recognized cause of orthopaedic implant infections, due to their ability to produce biofilms, that limit antibiotic and host defence capabilities. To detect serum IgM levels against staphylococcal slime polysaccharide antigens (SSPA), an original immunoenzymatic assay has been previously developed and tested in patients affected by staphylococcal vascular graft infections [The Lancet • 359:2166–2168, 2002]. This is the first report on the efficacy of this serum ELISA testing of anti-SSPA IgM in Staphylococcal Orthopaedic Implant-Related Infections (SOIRI). Methods: SSPA is extracted and purified using a suitable original patented bacterial strain and method. The immunoenzymatic assay to detect anti-SSPA IgM was performed on sera collected from 53 patients with joint prosthesis (24 hips, 17 knees, 2 shoulders). Each serum sample was tested in triplicate in two different assays and values are expressed as means. Main exclusion criteria were: time from implant < 6 months, rheumatoid arthritis, concurrent known infections. The study was approved by the local ethical committee and all patients gave their informed consent. 28 sera were collected from controls (patients with uninfected joint prosthesis, as judged from clinical, laboratory and radiological data) and 25 sera were obtained from patients with known SOIRI, sustained by S. aureus or coagulase-negative Staphilococci (CNS) (positive joint aspiration and/or intra-operative cultural examination). Results: 25 patients were classified as true negative, 21 true positive, 4 false negative, 3 false positive. Test efficacy calculation provided the following Results: sensitivity: 84 %, specificity: 89.3 %, positive predictive value: 87.5 %, negative predictive value: 86.2 %. Discussion and Conclusion: Anti-SSPA IgM immunoenzymatic assay showed interesting sensitivity and specificity in this preliminary prospective study. The test appear as an innovative, user-friendly, cheap and non-invasive diagnostic tool for orthopaedic implant-related infections. Further studies should be devoted to better define the cut-off value, testing reproducibility and standardization for large scale application

The incidence of PJI in knee replacements is 2.8% and slightly lower with hip replacement surgery. PJI make up 15% (or even more) of knee revisions. To combat PJI, antibiotic laden bone cement has been used for many decades, but antibiotic stewardship dictates more prudent management of antimicrobials. Projected increase in infection rate, due to increased surgery and latent infection to be almost 5-fold up to 2035. Biofilm is a complex structure of bacteria and polysaccharide matrix and, is recognised as a major component in PJI and other orthopaedic infections. Biofilm is responsible for high incidence of resistance to antimicrobials and ineffective host immune response. Method. Stabilized hypochlorous acid has been reported to have a rapid kill rate on all pathogens, including MDR pathogens associated with chronic and acute wound infections. It destroys biofilm on contact, is not cytotoxic, reduces inflammation and stimulates wound healing. 0,038% of Hypochlorous acid was used as prophylaxis against infection and to treat PJI. We report on our experience with hypochlorous acid as a wound irrigation as prophylaxis against infection (more than 600 cases) and for PJI. We also report on a University study where a head to head analysis was done on the anti-biofilm efficacy between hypochlorous acid 0,038% (Trifectiv Surgical Wound Irrigation) and Product X (an industry-standard product for the prevention and treatment of biofilm infection. Hypochlorous acid offers a valuable addition to the armamentarium of wound antiseptics, with added anti-inflammatory value. An in vitro study demonstrated superior efficacy against biofilm when compared to Product X

Introduction. Ink engineering can advance 3D-printability for better therapeutics, with optimized proprieties. Herein, we describe a methodology for yielding 3D-printable nanocomposite inks (NC) using low-viscous matrices, via the interaction between the organic and inorganic phases by chemical coupling. Method. Natural photocurable matrices were synthesized: a protein – bovine serum albumin methacrylate (BSAMA), and a polysaccharide – hyaluronic acid methacrylate (HAMA). Bioglass nanoparticles (BGNP) were synthesized and functionalized via aminosilane chemistry. The functionalization of BSAMA, HAMA, and BGNP were quantified via NMR. To arise extrudable inks, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry was used to link innate carboxylic groups of BSAMA/HAMA and amine-functionalized BGNP. Different crosslinker and BGNP amounts were tested. Visible light photopolymerization is performed, using lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The NC's rheological, mechanical, and biological behavior was evaluated before 3D extrusion printability. Result. All composite formulations effectively immobilized and homogeneously dispersed the BGNP, turning low-viscous materials (< 1 Pa) into shear-thinning formulations with tunable increased elastic/viscous moduli (50-500 Pa). More pronounced increments were found with increasing EDC/NHS and BGNP concentrations. The resulting inks produce robust and stable scaffolds successfully retrieved after post-print photocrosslinking (1-5 kPa). Bioactivity in simulated body fluid and in vitro assays using adipose-derive stem cells revealed a similar calcium/phosphate ratio to that of hydroxyapatite, and increased viability and metabolic activity. BSAMA and HAMA demonstrated distinct natures not only in printability but also in overall cellular performance and mechanical properties, making these ideal for interfacial tissue engineering. Conclusion. This strategy demonstrated being effective and reproducible to advance nanocomposites for 3D printing using different types of biomaterials. Further, we envision using both inks to produce hierarchical constructs via extrusion printing, better mimicking bone-to-cartilage interfaces. Acknowledgements. FCT grants (DOI:10.54499/2022.04605.CEECIND/CP1720/CT0021), (BI/UI89/10303/2022), (PRT/BD/154735/2023); EU's Horizon 2020 research and innovation programs InterLynk (Nº953169) and SUPRALIFE (Nº101079482) projects; CICECO-Aveiro Institute of Materials projects (DOI:10.54499/UIDB/50011/2020), (DOI:10.54499/UIDP/50011/2020), and (DOI:10.54499/LA/P/0006/2020), financed by FCT/MCTES(PIDDAC)

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues. Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002). *. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate. Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads. Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues

Two groups of intervertebral discs, one normal, as obtained from the post-mortem room, the other prolapsed, as removed at operation, have been compared by chemical analysis of their principal constituents. There is a progression of chemical changes associated with the ageing of the normal disc. This shows not only the expected slight increase in collagen as age advances, but also, surprisingly, that the polysaccharide content rises to a maximum in the fourth decade, in the same way as does polysaccharide in costal cartilage. In prolapsed discs the ageing process is superseded by a different and distinctive progression, which advances, not according to age, but according to the duration of the prolapse. There is a critical level to which the polysaccharide content must apparently fall, irrespective of the normal level for the patient's age, before a prolapse occurs. Normal ageing probably consists in the breakdown of a particular polysaccharide/protein linkage, with coincident "maturation" of collagen. In the prolapsing disc multiple, and possibly different, linkages are rapidly broken down. This depolymerisation of a gel structure must be presumed to be the basis of the decreased imbibition capacity of the nucleus pulposus, and to be the source of the hydrostatic abnormalities which result in disc prolapse. In both normal and prolapsing discs the products of mucopolysaccharide breakdown appear to participate in the metabolism of collagen

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections

20 years ago, we designed injectable bioactive suspensions in water of calcium phosphate ceramics for bone and periapical regenerations. Because of leakage of these suspensions, we focused on injectable hydrogels before to set in situ by chemical crosslinking to form 3D scaffolds. We set up a platform to develop a series of innovative hydrogels for bone, cartilages and periodontal tissue regeneration. We based our strategy on polysaccharides macromolecules because they are renewable materials, that originate from biological sources and generally are biocompatible, non-toxic and biodegradable. We developed a family of silated macromolecules able to react forming biocompatible hydrogels. The silated polymers are self-setting hydrogels able to covalently crosslink under pH variation, without addition of toxic crosslinking agent. All these macromolecules could be combined in multicomponent hydrogels, representing a strategy for improving mechanical properties of biomaterials or to tailor particular properties to meet specific needs. For mineral scaffolding, we realized composites of calcium phosphates particles or cements with hydrogel, increasing the ductility and creating macroporous scaffold to propose foam bone cements well adapted to bone biomaterials and Bone tissue engineering. Perspectives are 3D printing and bio printing techniques. We will use our hydrogels platform to prepare tunable (bio)inks in skeletal medicine

All types of regenerative materials, including metal implants, porous scaffolds and cell-laden hydrogels, interact with the living tissue and cells. Such interaction is key to the settlement and regenerative outcomes of the biomaterials. Notably, the immune reactions from the host body crucially mediate the tissue-biomaterials interactions. Macrophages (as well as monocytes and neutrophils), traditionally best known as defenders, accumulate at the tissue-biomaterials interface and secrete abundant cytokines to create a microenvironment that benefits or inhibits regeneration. Because the phenotype of these cells is highly plastic in response to varying stimuli, it may be feasible to manipulate their activity at the interface and harness their power to mediate bone regeneration. Towards this goal, our team have been working on macrophage-driven bone regeneration in two aspects. First, targeting the abundant, glucan/mannan-recognising receptors on macrophages, we have devised a series of glucomannan polymers that can stimulate macrophages to secrete pro-osteogenic cytokines, and applied them as coating polymer of mesenchymal stem cells-laden hydrogels. Second, targeting the toll-like receptors (TLRs) on macrophages, we have screened TLR-activating polysaccharides and picked up zymosan (beta-glucan) to be modified onto titanium and glass implants. We evaluated both the efficacy of integration and safety of immune stimulation in both in vitro and in vivo models. Our future exploration lies in further elaborating the different roles and mechanisms of macrophages of various types and origins in the regenerative process

The recent description of progenitor/stem cells in degenerated intervertebral discs (IVDs) raised the possibility of harnessing their regenerative capacity for endogenous repair. The aim of this work is to develop an intradiscal polysaccharide microbead-based delivery system for the sequential release of chemokines and nucleopulpogenic factors. This delivery system would sequentially contribute to 1) the recruitment of resident progenitors (CXCL12 or CCL5), 2) the differentiation of the mobilized progenitors (TGF-β1 and GDF5), and 3) the subsequent regeneration of NP. To determine the effects of chemokines on in vitro cell recruitment, human mesenchymal stem cells (MSC) were cultured in Transwells for 4h, with or without CXCL12 or CCL5. In parallel, pullulan microbeads (PMBs) (100µm) were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs were loaded with biological factors then release assays were performed at 37°C for 21 days and supernatant concentrations were measured by ELISA. As compared to untreated MSC, MSC migration was improved with a 3.9 (CXCL12) and 7.5 (CCL5) fold increase, respectively. All factors were successfully adsorbed on PMBs and a burst release within the 1. st. day was observed. At day 7, 27.5% and 83% of CXCL12 and CCL5 were released, respectively and at day 21, 20% and 100% of TGF-β1 and GDF5 were released, respectively. Currently, released cytokine bioactivity is being analysed and an ex vivo ovine IVD model is developed to determine the repair potential of this controlled release approach

Cartilage and bone degeneration are major healthcare problems affecting millions of individuals worldwide. Elucidation of the processes modulating the cell-matrix interactions involved in cartilage or bone formation offer tremendous potential in the development of clinically relevant strategies for cartilage and bone regeneration. We have therefore adopted an ex vivo tissue engineering approach to investigate chondrogenesis and osteogenesis using a mix human mesenchymal progenitor populations encapsulated in biomineralised polysac-charide templates with or without the addition of type-I collagen. Alginate/chitosan polysaccharide capsules containing 2.5mg/ml type-I collagen and TGF-beta-3 were encapsulated with human bone marrow cells (HBMC), articular chondrocytes or a co-culture at a ratio of 2:1 respectively and placed in a rotating (Synthecon) biore-actor or held in static 2D culture conditions for 28 days, to determine whether the presence of type-I collagen within the alginate could promote the synthesis of an extracellular matrix. Constructs were stained with alcian blue, sirius red and von Kossa. In bioreactor samples encapsulated with HBMC and type-I collagen, viable cells were present within lacunae, surrounded by a matrix of proteo-glycans and fibrous collagen, which was mineralized. Immunohistochemistry and polarised light microscopy indicated an organised collagenous matrix with extensive expression of type I collagen and bone sialoprotein with small regions of type II collagen. Type X collagen was also expressed indicating the presence of hypertrophic chondrocytes. Within the static HBMC groups, smaller areas of matrix were generated with decreased expression of type-I and type-II collagen. Co-culture bioreactor samples also demonstrated regions of new mineralised bone matrix; however these were less prominent than in the HBMC only groups. No matrix formation was observed in chondrocyte cultures although the cells remained viable as assessed by live/dead staining. Biochemical analysis indicated significantly increased (p< 0.05) DNA in all bioreactor samples in comparison with static constructs and significantly increased protein in HBMC bioreactor constructs in comparison with other cell types. These studies outline a unique tissue engineering approach, utilizing individual and mixed human mesen-chymal progenitor populations coupled with innovative polysaccharide templates containing type I collagen and bioreactor systems to promote chondrogenic and osteo-genic differentiation

Phenotypic drift of stem cells and insufficient production of extracellular matrix (ECM) are frequently observed in tissue-engineered cartilage substitutes, posing major weaknesses of clinically relevant therapies targeting cartilage repair. Microenvironment plays an important role for stem cell maintenance and differentiation and therefore an optimal chondrogenic differentiation protocol is highly desirable. Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates biological processes by several orders of magnitude. MMC was recently shown to significantly increase ECM deposition and to promote chondrogenic differentiation of stem cells. We hypothesise that the addition of sulphated high-molecular weight polysaccharides (carrageenan) to the media positively affects stem cell maintenance and chondrogenic differentiation. Herein, we venture to assess the impact of MMC on the maintenance of stem cell phenotype and multipotency, and ECM deposition in xeno-free human bone marrow mesenchymal stem cell (BMSCs) cultures. We investigate different xeno- and serum-free stem cell media with MMC for expansion of BMSCs, assessing multipotency maintenance (FACS analysis), cell viability, metabolic activity, proliferative capacity and matrix deposition (SDS-PAGE, ICC) at day 4 and day 10. Experiments will be conducted at 2 different passages (p3, p7). Medium without MMC will be used as control. Based on these results, cells expanded with the best protocol will be subsequently investigated for chondrogenic differentiation comparing different xeno-/serum-free and serum containing differentiation media. Chondrogenic differentiation will be assessed via Alcian blue and Safranin O stainings, gene expression for chondrogenic marker genes and quantification of GAG content. Finally, these findings will pave the way for developing more effective strategies for cartilage tissue engineering

Joint pain, as a consequence of cartilage degeneration or trauma results in severe pain or disability for millions of individuals worldwide. However, the potential for cartilage to regenerate is limited and there is an absence of clinically viable cartilage formation regimes. Cartilage is composed of only one cell type, is avascular and has a relatively simple composition and structure, thus cartilage tissue engineering has tremendous potential. Therefore, to address this clinical need, we have adopted a tissue engineering approach to the generation of cartilage ex vivo from mesenchymal cell populations encapsulated in polysaccharide templates form alginate and chitosan that favours chondrogenesis, and cultured within perfused or rotating bioreactor systems. To drive the chondrogenic phenotype, alginate beads were encapsulated with isolated human bone marrow cells, human articular chondrocytes or a combination of both in a 2:1 ratio, with the addition of TGF-â3, and placed in either a Synthecon rotating-wall bioreactor, perfused at a flow rate of 1ml/hour, or held in static conditions for 28 days. Alcian Blue and Sirius Red staining indicated ordered, structured and even cell distribution within capsules from the rotating bioreactor system in comparison with perfused and static conditions. Furthermore, alginate beads encapsulated with mixed cell populations that were cultured under static and rotating-wall conditions revealed positive staining for both collagen and proteoglycan, and with areas that closely resembled the formation of osteoid. Cell viability, assessed using the fluorescent dye Cell Tracker Green, indicated a higher proportion of metabolically active cells in capsules from the rotating-wall bioreactor than perfused or static under the conditions examined. Immunohistochemistry indicated the expression of type II collagen, SOX9 and C-MYC in samples from all conditions after 28 days. C-MYC is implicated in cell proliferation and differentiation and type II collagen and SOX9 are cartilage-specific markers. Biochemical analysis revealed significantly increased (p < 0.05) protein in samples encapsulated with mixed cell populations compared with alginate samples that were encapsulated with either bone marrow or chondrocytes. There was also a significant increase in protein in all samples that were cultured in the rotating-wall bioreactor in comparison with perfused or static conditions after 28 days. A significant increase in DNA was observed in the rotating-wall than perfused or static for the bone marrow cultures. Interestingly in chondrocyte cultures perfused conditions were found to result in significantly higher DNA than rotating-wall and static, and static conditions resulted in significantly higher DNA for alginate encapsulated with mixed cell populations. The current studies outline a tissue engineering approach utilising progenitor populations, bioreactors and appropriate stimuli to promote the formation of cartilage within a unique innovative polysaccharide capsule structure, and indicate the potential of rotating-wall systems to promote cartilage formation. Understanding the conditions required for the generation of functional cartilage constructs using such bioreactor systems carries significant clinical potential

Hydrogels as scaffolds provide a suitable environment for the cells (biocompatibility, biodegradability). Their biomechanical properties are very important to provide not only direct support to the surrounding tissue but also provide a local microenvironment. There is an interest in composite hydrogels with hydroxylapatite or bioactive glass (BAG) for tuning of their bioactivity and biomechanical properties [1]. Hydrogels were prepared from a polysaccharide gellan gum (GG), dissolved in ultrapure water at 90°C under constant stirring to a final concentration of 2 wt.% GG. Sodium-free BAG (70 wt.% SiO. 2. , 30 wt.% CaO) was synthesized using a sol-gel technique with particles of ∼100 nm, clustered to ∼10 µm large agglomerates [1]. The hydrogel composites were prepared by admixing up to 2–8 wt.% of BAG powder into a solution of GG during sonication, and pouring the hot BAG-GG suspension with following cooling to room temperature. Mechanical properties were evaluated using different protocols in creep (0.1 to 1.2 N), strain sweep (1 to 20 µm) and frequency scan (100 to 0.1 Hz) modes, with specimens immersed in water at 25°C. Maximum load (or deformation) before breaking of scaffold materials is a very important material property but is rarely measured. Here creep experiments at different applied stresses were carried out first. These loads exert more proper stress on the scaffold material that results in deformation, which is not the same as during deformation in relaxation or stress-strain tests [2]. The second set of experiments was made at physiologically relevant conditions (1 Hz frequency and small amplitude-controlled deformation) [3]. Amount of 2% BAG was found to be sufficient to get nearly linear deformation in the whole measured strains region, but at higher concentration stress deviated from linearity at strains exceeding ∼0.5% at 1 Hz. Storage modulus (E') did not significantly change and the loss tangent was found nearly constant (∼0.1) for the whole frequency range, indicating a strong network structure of BAG-doped hydrogel. Additions of 2% BAG give a ten-fold increase in both storage and loss moduli, whereas further increase of BAG content does not show further stiffening. The application of tailored protocols [3] allowed analysis of dynamic, creep and relaxation tests in the same device with same specimens, which might be not possible for other techniques. Creep data would provide valuable information in addition to dynamic modes to predict long-term behaviour of the composite hydrogels. Properly tailored protocols could mimic, for example, articular cartilage or other tissue working conditions and allow evaluation of the side effects like swelling at early stage, which measurements are usually rather cumbersome

Worldwide 500,000 cases of maxillofacial cancer are diagnosed each year. After surgery, the reconstruction of large bone defect is often required. The induced membrane approach (Masquelet, 2000) is one of the strategies, but exhibits limitations in an oncological context (use of autografts with or without autologous cells and Bone Morphogenetic Proteins). The objectives of this work are to develop an injectable osteoinductive and osteoconductive composite matrix composed of doped strontium (Sr) hydroxyapatite (HA) particles dispersed within a polysaccharide scaffold, to evaluate in vitro their ability to stimulate osteoblastic differentiation of human mesenchymal stem cells (hMSC) and to stimulate in vivo bone tissue regeneration. HA particles were synthesized with different ratios of Sr. X-ray diffraction (XRD), Inductively Coupled Plasma (ICP), and particle size analysis (Nanosizer™) were used to characterize these particles. HA and Sr-doped HA were dispersed at different ratios within a pullulan-dextran based matrices (Autissier, 2010), Electronic scanning microscopy Back Scattering Electron microscopy (ESEM-BSE) and ICP were used to characterize the composite scaffolds. In vitro assays were performed using hMSC (cell viability using Live/Dead assay, expression of osteoblastic markers by quantitative Polymerase Chain Reaction). Matrices containing these different particles were implanted subcutaneously in mice and analyzed by Micro-Computed Tomography (micro-CT) and histologically (Masson's trichrome staining) after 2 and 4 weeks of implantation. XRD analysis was compatible with a carbonated hydroxyapatite and patterns of Sr-doped HA are consistent of Sr substitution on HA particles. Morphological evaluation (TEM and Nanosizer™) showed that HA and Sr-doped HA particles form agglomerates (150 nm to 4 µm). Matrices composed with different ratios of HA or Sr-doped-HA, exhibit a homogenous distribution of the particles (ESEM-BSE), whatever the conditions of substitution. In vitro studies revealed that Sr-doped HA particles within the matrix stimulates the expression of osteoblastic markers, compared to non-doped HA matrices. Subcutaneous implantation of the matrices demonstrated the formation of a mineralized tissue. Quantitative analyses show that the mineralization of the implants is dependent of the amount of HA particles dispersed, with an optimal ratio of 5% of particles. Histological analysis revealed osteoid tissue in contact to the matrix. In conclusion, the ability of this injectable composite scaffold to promote ectopically tissue mineralization is promising for bone tissue engineering. Osseous implantation in a femoral bone defect in rats is now in progress. 5% of doped HA particles were implanted within the induced membranes in a context of radiotherapy procedure. Micro-CT analyses are ongoing. This new matrix could represent an alternative to the autografts for the regeneration of large bone defects in an oncological context

Aims

This study aimed to explore the diagnostic value of synovial fluid neutrophil extracellular traps (SF-NETs) in periprosthetic joint infection (PJI) diagnosis, and compare it with that of microbial culture, serum ESR and CRP, synovial white blood cell (WBC) count, and polymorphonuclear neutrophil percentage (PMN%).

Aims

Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella.