Several synthetic polymers have been widely investigated for their use in bone tissue engineering applications, but the ideal material is yet to be engineered. Triazine-trione (TATO) based materials and their derivatives are novel in the field of biomedical engineering but have started to draw interest. Different designs of the TATO monomers and introduction of different chemical linkages and end-groups widens the scope of the materials due to a range of mechanical properties. The aim of our work is to investigate novel TATO based materials, with or without hydroxyapatite filler, for their potential in bone tissue engineering constructs. Initially the biocompatibility of the materials was tested, indirectly and directly, according to ISO standards. Following this the osteoconductive properties were investigated with primary osteoblasts and an osteoblastic cell line. Bone marrow derived mesenchymal stem cells were used to evaluate the osteogenic differentiation and consequently the materials potential in bone tissue engineering applications

The current procedures being applied in the clinical setting to address osteoporosis-related delayed union and nonunion bone fractures have been found to present mostly suboptimal outcomes. As a result, bone tissue engineering (BTE) solutions involving the development of implantable biomimetic scaffolds to replace damaged bone and support its regeneration are gaining interest. The piezoelectric properties of the bone tissue, which stem primarily from the significant presence of piezoelectric type I collagen fibrils in the tissue's extracellular matrix (ECM), play a key role in preserving the bone's homeostasis and provide integral assistance to the regeneration process. However, despite their significant potential, these properties of bone tend to be overlooked in most BTE-related studies. In order to bridge this gap in the literature, novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers were developed to replicate the bone's fibrous ECM composition and electrical features. Different HAp nanoparticle concentrations (1–10%, wt%) were tested to assess their effect on the physicochemical and biological properties of the resulting fibers. The fabricated scaffolds displayed biomimetic collagen fibril-like diameters, while also presenting mechanical features akin to type I collagen. The increase in HAp presence was found to enhance both surface and piezoelectric properties of the fibers, with an improvement in scaffold wettability and increase in β-phase nucleation (translating to increased piezoelectricity) being observed. The HAp-containing scaffolds also exhibited an augmented bioactivity, with a more comprehensive surface mineralization of the fibers being obtained for the scaffolds with the highest HAp concentrations. Improved osteogenic differentiation of seeded human mesenchymal stem/stromal cells was achieved with the addition of HAp, as confirmed by an increased ALP activity, calcium deposition and upregulated expression of key osteogenic markers. Overall, our findings highlight, for the first time, the potential of combining PVDF-TrFE and HAp to develop electroactive and osteoinductive nanofibers for BTE. Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), the PhD scholarship (2022.10572.BD) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020)

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications

Scaffold-based bone tissue engineering holds great promise for the future of osseous defects therapies. Prepare the suitable scaffold properties are physiochemical modifications in terms of porosity, mechanical strength, cell adhesion, biocompatibility, cell proliferation, mineralization and osteogenic differentiation are required. We produce various bone tissue scaffolds with different techniques such as lyophilization, 3D printing and electrospinning. We wish to overview all the different novel scaffold methods and materials. To improve scaffolds poor mechanical properties, while preserving the porous structure, it is possible to coat the scaffold with synthetic or natural polymers. An increasing interest in developing materials in bone tissue engineering is directed to the organic/inorganic composites that mimic natural bone. Specifically, bone tissue is a composite of an organic and inorganic matrix. Using PLLA, loofah, chitin and cellulose biomaterials we produced bone tissue scaffold with lyophilization technique. Also, using fish scale powder and wet electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) a sponge structure had created. Using MRI image data and 3D printer method, a bone tissue scaffold is created by PLA filament. Their mechanical properties had analysed with compression tests and their biocompatibilities had investigated. In order to provide novel strategies for future treatment of bone tumours, the properties of the scaffold, including its in vitro extended-release properties, the inhibition effects of chemotherapeutic agent on the bone tumours and its bone repair capacities were investigated in vitro by using MG63 cells. To develop chemotherapeutic agent-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles in a porous nano-hydroxyapatite scaffold we aimed to use double emulsion method

Bone defects can result from different incidents such as acute trauma, infection or tumor resection. While in most instances bone healing can be achieved given the tissue's innate ability of self-repair, for critical-sized defects spontaneous regeneration is less likely to occur, therefore requiring surgical intervention. Current clinical procedures have failed to adequately address this issue. For this reason, bone tissue engineering (BTE) strategies involving the use of synthetic grafts for replacing damaged bone and promoting the tissue's regeneration are being investigated. The electrical stimulation (ES) of bone defects using direct current has yielded very promising results, with neo tissue formation being achieved in the target sites in vivo. Electroactive implantable scaffolds comprised by conductive biomaterials could be used to assist this kind of therapy by either directing the ES specifically to the damaged site or promoting the integration of electrodes within the bone tissue as a coating. In this study, we developed novel conductive heat-treated polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning capable of mimicking key native features of the bone tissue's extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The developed scaffolds were doped with sulfuric acid and mineralized in Simulated Body Fluid to mimic the inorganic phase of bone ECM. As expected, the doped PAN/PEDOT:PSS nanofibers exhibited electroconductive properties and were able to preserve their fibrous structure. The addition of PEDOT:PSS was found to improve the bioactivity of the scaffolds, with a more significant in vitro mineralization being obtained. By seeding the scaffolds with MG-63 osteoblasts and human mesenchymal stem/stromal cells, an increased cell proliferation was observed for the mineralized PAN/PEDOT:PSS nanofibers, which also registered an increased expression of key osteogenic markers (e.g Osteopontin). Our findings appear to corroborate the promising potential of the generated nanofibers for future ES-based BTE applications. Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), BioMaterARISES (EXPL/CTM-CTM/0995/2021) and OptiBioScaffold (PTDC/EME-SIS/32554/2017, POCI-01- 0145-FEDER- 32554), the PhD scholarship (2022.10572.BD) and through institutional funding to iBB (UIDB/04565/2020 and UIDP/04565/2020), Associate Laboratory i4HB (LA/P/0140/2020) and IT (UIDB/50008/2020)

Design of bone tissue engineering scaffolds imposes a number of requirements for their physical properties, in particular porosity and mechanical behaviour. Alginates are known as a potential material for such purposes, usually deploying calcium as a cross-linker. Calcium over-expression was reported having proinflammatory effect, which is not always desirable. Contrary to this, barium has better immunomodulatory outcome but data for barium as a cross-linker are scarce. In this work the objective was to produce Ba-linked alginates and compare their viscoelastic properties with Ca-linked controls in vitro. Sodium alginate aqueous solution (1 wt%) with 0.03 wt.% CaCl. 2. is gelled in dialysis tubing immersed in 27 mM CaCl. 2. (controls) or BaCl. 2. , for 48 h, followed by freeze-drying and rehydration (with 0.3 wt.% CaCl. 2. and 0.8 wt.% NaCl). Hydrogel discs (diameter 8-10 mm, thickness 4-6 mm) were assessed in dry and wet (DMEM immersed) states by dynamic mechanical analysis (DMA) under compressive creep conditions with increased loads, frequency scans and strain-controlled sweeps in physiological range (0.1-20 Hz) at 25°C and 37°C. Resulting data were analysed by conventional methods and by a model-free BEST (Biomaterials Enhanced Simulation Testing) to extract invariant values and material functions. Significant differences were observed in properties of Ba-linked hydrogel scaffolds vs. Ca-linked controls. Specifically, for the similar porosity Ba-samples exhibited lower creep compliance, higher dynamical stiffness and lower loss factor in the whole studied range. Invariant modulus exhibited a non-linear decay vs. applied stress. These differences were observed in both dry and wet states and temperatures. Use of barium as a cross-linker for alginates allows further modification of biomechanical properties of the scaffolds for better compliancy to the tissues in the application. Barium release might have an immunomodulating effect but also promote ion exchange for osteogenesis due to additional Ca/Ba concentration gradient

Millions of patients each year suffer from challenging non-healing bone defects secondary to trauma or disease (e.g. cancer, osteoporosis or osteomyelitis). Tissue engineering approach to non-healing bone defects has been investigated over the past few decades in a search for a novel solution for critical size bone defects. The success of the tissue engineering approach relies on three main pillars, the right type of cells; and appropriate scaffold; and a biologically relevant biochemical/ biophysical stimuli. When it comes to cells the mesodermal origin of mesenchymal stem cells and its well demonstrated multipotentiality makes it an ideal option to be used in musculoskeletal regeneration. For the presented set of experimental assays, fully characterised (passage 3 to 5)ovine adipose-derived mesenchymal stems cells (Ad-MSC) were cultured either in growth medium (GM) consisting of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% penicillin-streptomycin as a control or in osteogenic differentiation medium (DM), consisting of GM further supplemented with L- ascorbic acid (50 μg/ml), β-glycerophosphate (10 mM) and dexamethasone (100nM). Osteogenic differentiation was assessed biochemically by quantifying alkaline phosphatase (ALP) enzyme activity and alizarin red staining after 3, 7, 14 and 21 days in culture (where 1×105 cells/well were seeded in 24 well-plate, n=6/media type/ time point). Temporal patterns in osteogenic gene expression were quantified using real-time PCR for Runx-2, osteocalcin (OC), osteonectin (ON) and type 1 collagen (Col 1) at days 7, 15 and 21 (where 1×105 cells were seeded in T25 cell culture flasks for RNA extraction, n= 4 / gene/ media type/time point). The morphology of osteogenic cells was additionally evaluated by scanning electron microscopy (SEM) of cells seeded at low-density (1×102 cells) on glass coverslips for 2 weeks in GM or DM. The level of ALP activity of cells grown in osteogenic DM was significantly higher than the control growing in the standard growth medium (p ≤ 0.05) at days 3, 7 and 14. At 21 days there was a sharp drop in ALP values in the differentiating cells. Mineralisation, as evidenced by alizarin red staining, increased significantly by day 14 and then peaked at day 21. Quantitative real-time PCR confirmed early increases in Runx-2, Col 1 and osteonectin, peaking in the second week of culture, while osteocalcin peaked at 21 days of culture. Taken as a whole, these data indicate that ovine-MSCs exhibit a tightly defined pathway of initial proliferation and matrix maturation (up to 14 days), followed by terminal differentiation and mineralisation (days 14 to 21). SEM analysis confirmed the flattened, roughened appearance of these cells and abandoned extracellular matrix which resembled mature osteoblasts. Given the ready availability of adipose tissues, the use of Ad-MSCs as progenitors for bone tissue engineering applications is both feasible and reasonable. The data from this study indicate that Ad-MSCs follow a predictable pathway of differentiation that can be tracked using validated molecular and biochemical assays. Additional work is needed to confirm that these cells are osteogenic in vivo, and to identifying the best combination of scaffold materials and cell culture techniques (e.g. static versus dynamic) to accelerate or stimulate osteogenic differentiation for bone tissue engineering applications

Summary Statement. 3D porous and nano-structured polyetheretherketone (PEEK) surface embedded with biofunctional groups can not only induce the up-regulation of osteogenic genes and proteins in-vitro, but also help promote new bone formation in-vivo. Introduction. Porous biomaterials with three-dimensional (3D) surface structure can enhance biological functionalities especially in bone tissue engineering. Many techniques have hitherto been utilised to fabricate porous structures on metal surfaces, including machining, shotblasting, anodic oxidation, alkali treatment and acid-etching. However, it has been difficult to accomplish this on polyetheretherketone (PEEK) due to its inherent chemical inertness. In this study, we have applied a method comprising of sulfonation and water immersion to establish a 3D porous and nanostructured network on the PEEK surface. This newly established 3D network embedded with bio-functional groups can help promote new bone formation in-vivo. Methods. Two kinds of sulfonation-treated PEEK (SPEEK) samples, SPEEK-W (water immersion and rinsing after sulfonation) and SPEEK-WA (SPEEK-W with further acetone rinsing) were prepared. The surface characteristics, in vitro cellular behavior, in-vivo osseointegration, and apatite-forming ability are systematically investigated by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, cell adhesion and cell proliferation assay, real-time PCR analysis, micro-CT evaluation, and immersion tests. Results and discussion. SPEEK-WA sample induced superior pre-osteoblast functions including initial cell adhesion, proliferation, and osteogenic differentiation in vitro. In general, the pre-osteoblasts cultured on SPEEK-WA exhibited the highest gene expressions including Runx2, ALP, and Col1a1 expressions followed by SPEEK-W as compared with the PEEK control (p < 0.05). The ALP and Col1a1 expressions on SPEEK-WA were dramatically higher than those on SPEEK-W and PEEK control on days 7 and 14. In addition, significant higher apatite-forming ability and substantially enhanced osseointegration were found as well. In particular, bone ingrowth could be observed from SPEEK-WA. Although SPEEK-W sample presented a similar surface morphology and chemical composition as SPEEK-WA, its cytocompatibility was inferior due to residual sulfuric acid. Our results indicated the cell functions, bone growth, and apatite formation on the SPEEK surfaces were controlled by multiple factors, which included 3D porous and nano-structure, SO. 3. H groups and local pH environment. Conclusion. Surface functionalization and 3D porous networking may broaden the use of PEEK in bone tissue engineering

Bone fractures are highly observed clinical situation in orthopaedic treatments. In some cases, there might be non-union problems. Therefore, recent studies have focused on tissue engineering applications as alternative methods to replace surgical procedures. Various biopolymer based scaffolds are produced using different fabrication techniques for bone tissue engineering applications. In this study, hydroxyapatite (HAp) and loofah containing carboxymethyl chitosan (CMC) scaffolds were prepared. In this regard, first 4 ml of CMC solution, 0.02 g of hydroxyapatite (HAP) and 0.06 g of poly (ethylene glycol) diglycidyl ether (PEGDE) were mixed in an ultrasonic bath until the HAp powders were suspended. Next, 0.04 g of loofah was added to the suspension and with the help of PEGDE as the cross-linking agent, then, the mixture was allowed to cross-link at 40. o. C overnight. Finally, the three-dimensional, porous and sponge-like scaffolds were obtained after lyophilization (TELSTAR - LyoQuest −85) at 0.1 mbar and −25°C for 2 days. Morphologies, chemical structures and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier Transform infrared spectroscopy (FT-IR) and thermogravimetric differential thermal analysis (TGA/DTA), respectively. In addition, swelling behavior and mechanical properties of the scaffolds under compression loading were determined. In order to investigate biocompatibility of the scaffolds, WST-1 colorimetric assay at days 0, 1, 3, 5 and 7 was conducted by using human dermal fibroblast. Also, histological and morphological analysis were performed for cell attachment at day 7. In conclusion, the produced scaffolds showed no cytotoxic effect. Therefore, they can be considered as a candidate scaffold for bone tissue regeneration. Further studies will be performed by using bone marrow and periosteum derived mesenchymal stem cells with these scaffolds

The development of functional biomaterials scaffolds for bone tissue engineering applications includes the control of specific biological and mechanical parameters that are involved in the growth of bone tissue in a way that mimics the physiological process of healing bone defects. Here, we report on the development of composite scaffolds made from biodegradable natural and synthetic biomaterials with characteristic architectural features, functionalized with the osteoinductive growth factor bone morphogenetic protein BMP-2, and evaluating their osteogenic response in static and dynamic cell culture systems. The results show that scaffold designing with advanced technologies combined with appropriate biochemical and mechanical stimulating factors, results to an enhanced proliferative and osteogenic/chondrogenic differentiation response of cells cultured on the developed scaffolds, and thus controlling the new tissue formation and reconstruction

There has been a significant increase in the demand of polymeric scaffolds with promising affects in bone regeneration. However, inflammation is still a problem in transplantations to overcome with local antibiotic therapy. In this study, it is aimed to develop a functional POSS nanocage reinforced chitosan scaffold (CS/POSS) coated with drug loaded chitosan composite nanospheres to provide a controlled antibianyiotic delivery at the defect site. Gentamicin and vancomycin were selected as model antibiotic drugs. Drug loaded nanospheres were fabricated with electrospray method and characterized in terms of morphology, hydrodynamic size, surface charge, FT-IR, in vitro drug release, antimicrobial activity and cytotoxicity. CS/POSS scaffolds were fabricated via lyophilisation and characterized with mechanic, swelling test, SEM and micro CT analyses. Positively charged nanospheres with uniform morphology were obtained. High drug encapsulation efficiency (80–95%) and sustained release profile up to 25 days were achieved with a cumulative release of 80–90%. In addition, the release media of the nanospheres (in 6 hours, 24 hours and 25 days of incubation period) showed a strong antimicrobial activity against S.aureus and E.coli, and did not show any cytotoxic effect to 3T3 and SaOS-2 cell lines. CS/POSS scaffolds were obtained with high porosity (89%) and 223.3±55.2μm average pore size. POSS reinforcement increased the compression modulus from 755.7 to 846.1Pa for 10 % POSS addition. In vitro studies of nanosphere coated bilayer scaffolds have showed high cell viability. Besides ALP activity results showed that POSS incorporation significantly increased the ALP activity of Saos-2 cells cultured on the scaffold. In conclusion, these composites can be considered as a potential candidate in view of its enhanced physico-chemical properties as well as biological activities for infection preventive bone tissue engineering applications

First works focuses on the characterization (physical and biological) of this biomaterial. Current work had studied osteoinductive and osteoconductive capacity of these hydrogels. In vivoresults highlight a significant bone reconstruction two months after implantations on bone lesions in mice. Bone is a dynamic and vascularized tissue that has the ability of naturally healing upon damage. Nevertheless, in the case of critical size defects this potential is impaired. Present approaches mainly consider autografts and allografts, which presents several limitations. Bone Tissue Engineering (BTE) is based on the use of 3D matrices to guide both cellular growth, differentiation to promote bone regeneration. Hence, matrices can contain biological materials such as cells and growth factors. Our project aims to design a hydrogel for BTE, particularly for bone lesion filling. We previously showed that a porous 3D hydrogel, Glycosyl-Nucleoside-Fluorinated (GNF) is: 1) non-cytotoxic to clustered human Adipose Mesenchymal Stem Cells (hASCs), 2) bioinjectable and 3) biodegradable. Therefore, this novel class of hydrogels show promise for the development of therapeutic solutions for BTE [1]. The hypothesis of this research was that improving the capacity to promote the adhesion of cells by adding collagen gel matrices and bone morphogenic protein 2 (BMP-2) to improve the bone regenerative potential of this gel. Collagen is a protein matrix well known for its cytocompatibility [2]. BMP-2, have been shown ability to induce bone formation in combination with an adequate matrix [3]. Thereby, the overall aim of this work was to design, develop and validate a new composite hydrogel for BTE. GNF was prepared as previously described in detail[1], at a concentration of 3% (w/v). Type I-collagen gel was prepared from rat-tail tendons at a concentration of 4 g/L [2]. hASCs were isolated from human adipose tissue in our laboratory. To establish a suitable microenvironment for cell proliferation and differentiation cells were seeded in collagen and then GNF gel was added and the resulting mixture was blended, BMP-2 (InductOs ® Kit) is added to this preparation (5µm BMP-2/ml). Fluorometry was used to follow BMP2 release in vitro andin vivo(NOG mices;n=6), orthotopic calvariumbone critical defect (3.3 mm) has been selected to challenge the bone repair. Adding collagen hydrogel improve cell adhesion, survivals and proliferation rather than simple GNF hydrogel. This novel gel composite has the ability to sustain hASCs adhesion and differentiation towards the osteoblastic lineage (positive ALP cells). Fluorometry showed the ability of our hydrogel to prolong the residence of BMP-2 (in vitro and in vivo) compared to collagen hydrogel sponges. Implantation of hydrogel containing hASC and BMP-2 has shown encouraging results in bone reconstruction: 2 months after implantation of biomaterials a significant bone reconstruction can be observed using X-Ray imaging. Adding collagen to GNF allowed to obtain gels showing satisfactory cell-behaviour. In parallel, the presence of GNF hydrogel helps to improve mechanical properties of the biomaterial (hydrogel stability and controlled release of BMP-2). The first in vivostudies have shown encouraging bone regeneration capacity of these hydrogels. The implantation performed on a larger number of animals and quantitative microCT analysis will enable us to judge the effectiveness of this hydrogel as a new injectable biomaterial for BTE. This work was partially supported by NSERC-Canada, FRQ-NT-Quebec, FRQ-S- Quebec, and CFI-Canada. Mathieu Maisani was awarded of a NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca)

Summary Statement. Mesenchymal stem cells from minced umbilical cord fragments may represent a valuable cell population for cartilage and bone tissue engineering. Introduction. A promising approach for cartilage and bone repair is the use of umbilical cord mesenchymal stem cell (UC-MSC)-based tissue engineering. Through a simple and efficient protocol based on mincing the umbilical cord, a consistent number of multipotent UC-MSCs can be obtained. The aim of this in-vitro study is to investigate the pluripotency of UC-MSCs and, in particular, the chondrogenic and osteogenic potential of UC-MSCs grown in tridimensional scaffold, in order to identify a potential clinical relevance for patients who might benefit from MSCs-therapy. Materials/Methods. Fresh UC samples from women with healthy pregnancies were retrieved at the end of caesarean deliveries. The UC samples were manually minced without any enzymatic digestion into very small fragments (less than 4 mm length) and cultivated in an MSC expansion medium. At day 14, the UC tissue was removed and adherent cells were allowed to expand for 2 additional weeks. At day 28, the adherent cells were collected and replated until confluence was reached (Passage 1 or P1). Immunophenotypic characterization, Fluorescence In Situ Hybridization (FISH), telomere length analysis, Immunosuppression of T lymphocyte cultures, and multilineage differentiation was performed in UC-MSCs at P1 or P2. For chondrogenic differentiation on scaffold, UC-MSCs at P2 were loaded either on a hyaluronic-acid (HA)-felt(Hyaff-11) or on a collagen-I/III membrane(Chondro-gide), with a coating of fibrin glue(Tisseel), and grown in chondrogenic medium both in normoxic and hypoxic(10%O. 2. ) conditions. After 1 month, sections were stained with haematoxylin/eosin and Safranin-O. Expression of chondrocyte markers(sox-9, type II collagen) and hypoxic markers(HIFs) was assessed using immunofluorescence(IF). For osteogenic differentiation on scaffold, UC-MSCs at P2 were loaded into a bone-graft-substitute(Orthoss) and grown in osteogenic medium. After 10, 20,30 days, sections were stained with haematoxylin/eosin and expression of osteocalcin and RunX2 was assessed using immunofluorescence. Results. At P1, we obtained a mean value of 22.8 × 10. 6. cells(SD 1.7) from each UC, corresponding to 0.66 (SD 0.14) × 10. 6. cells/gram of UC. Cells were positive for CD73, CD90, CD105, CD44, CD29 and HLA-I, and negative for CD34 and HLA-class II, with a subpopulation that was negative for both HLA-I and HLA-II. Results from FISH demonstrated that 95–100% of UC-MSCs were of fetal origin. Telomere length of UC-MSCs was similar to that of Bone Marrow (BM) MSC from young donors (aged 20–30 years). At 5 days, the supernatant of UC-MSC cultures had immunosuppressive activity upon T-Lymphocyte cultures. The mixed UC-MSC population was able to differentiate towards osteogenic (monolayer), adipogenic, miogenic and chondrogenic (pellet culture). Chondrogenic differentiation on scaffolds was also confirmed; hypoxic condition improved the expression of chondrogenic markers. Osteogenic differentiation on scaffold was also confirmed after 20 days of culture. Discussion/Conclusions. These results suggest that the straightforward procedure of collecting UC-MCS at P1 from minced umbilical cord fragments can achieve a valuable cell population that seems to have a potential for orthopaedics tissue engineering such as the on-demand cell delivery using chondrogenic or osteogenic scaffold. The concept of this study may have a clinical relevance as a future hypothetical option for single-stage cartilage repair and bone regeneration

3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations. Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×10. 4. , 5×10. 4. , 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&E) to identify any visible necrotic core. Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×10. 4. cells proliferated faster than 5×10. 4. and 1×10. 5. cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core. This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs

Bioactive glasses were first discovered in the late 1960s by Larry Hench. In the 1980s and 1990s bioactive glasses experienced a surge of research interest, an interest which has since declined. This talk will examine the current status of bioactive glasses and discuss future roles and applications for bioactive glasses in regenerative medicine, specifically those related to orthopaedic tissue engineering. Bioactive materials are often considered as those that have the ability to bond to mineralised bone tissue in the physiological environment, however, this talk, as well as examining this aspect, will consider the broader sense of bioactive as ‘having or eliciting a biological effect’. It will examine the role of bioactive glasses as active drug carriers and the influence which enhanced nanotechnology will have on the application of bioactive glasses in vivo.

It is well known that environmental cues such as mechanical loading and/or cell culture medium composition affect tissue-engineered constructs resembling natural bone. These studies are mostly based on an initial setting of the influential parameter that will not be further changed throughout the study. Through the growth of the cells and the deposition of the extracellular matrix (ECM) the initial environmental conditions of the cells will change, and with that also the loads on the cells will change. This study investigates how changes of mechanical load or media composition during culture influences the differentiation and ECM production of mesenchymal stromal cells seeded on porous 3D silk fibroin scaffolds. ECM formation, ECM mineralization and cell differentiation in 3D tissue-engineered bone were analyzed using microscopic tools. Our results suggest that mechanical stimuli are necessary to differentiate human mesenchymal stromal cells of both bone marrow and adipose tissue origin into ECM producing osteoblasts which ultimately become ECM-embedded osteocytes. However, the influence of this stimulus seems to fade quickly after the onset of the culture. Constructs which were initially cultured under mechanical loading continued to deposit minerals at a similar growth rate once the mechanical stimulation was stopped. On the other hand, cell culture medium supplementation with FBS was identified as an extremely potent biochemical cue that influences the mechanosensitivity of the cells with regards to cell differentiation, ECM secretion and mineral deposition.

Only through a thorough understanding on these influences over time will we be able to predictably control tissue development in vitro.

Conventional 3D printing by itself is incapable of creating pores on a micro scale within deposited filaments throughout 3D scaffolds. These pores and hence larger surface areas are needed for cells to be adhered, proliferated, and differentiated. The aim of this work was to fabricate 3D polycaprolactone (PCL) scaffolds with internal multiscale porosity by using two different 3D printing techniques (ink/pellet of polymer-salt composite in low/high temperature printing) combined with salt leaching to improve cell adhesion, and cell proliferation besides to change degradation rate of PCL scaffolds:

1. Non-solvent phase separation integrated 3D printing of polymer-salt inks with various salt content (i.e., low temperature ink-based printing, LT).

2. FDM printing of composite polymer-salt pellets which will be obtained by casting and evaporating of prepared ink (i.e., high temperature composite-pellet-based printing, HT).

Further, the two approaches were followed by post salt leaching. Stem cells were able to attach on the surface and grow up to 14 days based on increasing cellular activities.

Biphasic calcium phosphates (BCP) are the most frequently used materials because of their mineral analogy with bio-mineral part of bones. Their chemical synthesis can be modulated by doping, in order to respond to the biological needs. We present here the biological responses induced by copper ions in solution, to characterize its cytotoxicity and antibacterial activity. We also investigate the antibacterial property of Cu-doped BCP (Ca10 Cu0.1 (PO4)6 (OH)1.8 O0.2) on a strain of clinical interest: S. aureus, compared to undoped BCP. The sol-gel route has been used to prepare the BCP ceramics. Human BMC (Bone Marrow Cells) were obtained from metaphysal cancellous bone collected during hip arthroplasty and used for cytotoxicity evaluations. A strain of Staphylococcus aureus isolated from an osteoarticular infection after total knee arthroplasty was used to evaluate antibacterial activities. Results indicate that 3 ppm of copper ions leads to the death of all cultured bacteria in 24 hours and 25 ppm caused the death of all cells in 15 days. Regarding BCP, the undoped bioceramics increased the bacterial growth compared to a control without bioceramic. After 16 hours of contact, the copper ions released by the Cu-doped BCP induced a significant decrease of the bacterial concentration, indeed no viable bacteria were found. These materials seem to be a promising alternative for the preparation of multifunctional bone substitutes.

By combining cells, biological factors, and biomaterials the field of tissue engineering has generated technologies capable of supporting regeneration. However, the regulatory hurdles associated with the use of cell-based therapies often hinder translation. Consequently, to meet the growing demand for regenerative technologies new approaches are needed. Emerging evidence suggests that cell-derived extracellular vesicles (EVs) are critical in cell-cell communication and regulation of bone formation. This talk will explore the role of osteoblast EVs in directing stem-cell differentiation in-vitro. EVs were isolated from cell culture media by ultracentrifugation and profiled for size and composition using a range of techniques. Notably, proteomic analysis revealed the presence of calcium channelling annexins and bridging collagens that may be key to their role in mineralisation. To minimise the concentration of EVs required to induce a pro-osteogenic effect we propose that they may be locally delivered. Opportunities to incorporate these pro-osteogenic EVs into injectable biomaterials will be discussed, in particular the formulation of microcapsules and fluid-gels. In summary, incorporation of EVs in tissue-engineered scaffolds has the potential to deliver all the advantages of a cell-based therapy but without using viable cells. The advantages of this approach may represent a new phase of tissue engineering.

Harnessing the potential of mesenchymal stem cell (MSC) mediated endochondral ossification for the repair of large bone defects represents a promising avenue of investigation as an alternative option to autologous bone transplantation. To date, it has been shown that undifferentiated MSCs are somewhat immune-privileged. In order to induce bone formation from MSCs by endochondral ossification it is usually necessary to first differentiate these cells chondrogenically. However, the status of differentiated cells is less clear than that of undifferentiated MSCs. Furthermore, the fate of implanted bone forming constructs in an allogeneic setting is not known. The potential to use allogeneic MSCs for large bone defect repair would offer opportunities to researchers to develop new therapies using more potent MSC sources and in a more readily available manner with regard to the patient. I will present our research investigating the interactions between chondrogenically primed MSCs and immune cell subsets, namely T cells and dendritic cells. Furthermore, I will discuss the ability of human paediatric MSCs to form bone in the in vivo allogeneic setting.