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 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

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

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

Objectives

Intra-articular injections of local anaesthetics (LA), glucocorticoids (GC), or hyaluronic acid (HA) are used to treat osteoarthritis (OA). Contrast agents (CA) are needed to prove successful intra-articular injection or aspiration, or to visualize articular structures dynamically during fluoroscopy. Tranexamic acid (TA) is used to control haemostasis and prevent excessive intra-articular bleeding. Despite their common usage, little is known about the cytotoxicity of common drugs injected into joints. Thus, the aim of our study was to investigate the effects of LA, GC, HA, CA, and TA on the viability of primary human chondrocytes and tenocytes in vitro.

Objectives

The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA.

Ciprofloxacin hydrochloride-loaded microspheres were prepared by a spray-drying method using pectin and chitosan. The effects of different polymers and drug ratios were investigated.

The most appropriate carriers were selected by in vitro testing. A rat methicillin-resistant Staphylococcus aureus osteomyelitis model was used to evaluate the effects of the loaded microspheres.

The drug was released rapidly from the pectin carrier but this was more sustained in the chitosan formulation.

Chitosan microspheres loaded with ciprofloxacin hydrochloride were more effective for the treatment of osteomyelitis than equivalent intramuscular antibiotics.

We evaluated the possible induction of a systemic immune response to increase anti-tumour activity by the re-implantation of destructive tumour tissue treated by liquid nitrogen in a murine osteosarcoma (LM8) model. The tumours were randomised to treatment by excision alone or by cryotreatment after excision. Tissue from the tumour was frozen in liquid nitrogen, thawed in distilled water and then re-implanted in the same animal. In addition, some mice received an immunological response modifier of OK-432 after treatment. We measured the levels of interferon-gamma and interleukin-12 cytokines and the cytotoxicity activity of splenocytes against murine LM8 osteosarcoma cells. The number of lung and the size of abdominal metastases were also measured.

Re-implantation of tumour tissue after cryotreatment activated immune responses and inhibited metastatic tumour growth. OK-432 synergistically enhanced the anti-tumour effect. Our results suggest that the treatment of malignant bone tumours by reconstruction using autografts containing tumours which have been treated by liquid nitrogen may be of clinical value.