Cartilage lacks the ability to self-repair when damaged, which can lead to the development of degenerative joint disease. Despite intensive research in the field of cartilage tissue engineering, there is still no regenerative treatment that consistently promotes the development of hyaline cartilage. Extracellular matrix (ECM) derived hydrogels have shown to support cell adhesion, growth and differentiation [1,2]. In this study, porcine articular cartilage was decellularized, solubilised and subsequently modified into a photo-crosslinkable methacrylated cartilage ECM hydrogel. Bone marrow derived mesenchymal stem/stromal cells (MSCs) were encapsulated into both methacrylated ECM hydrogels (ECM-MA) and gelatin methacryloyl (GelMA) as control hydrogel, and their chondrogenic potential was assessed using biochemical assays and histological analysis. We found that successful decellularization of the cartilage tissue could be achieved while preserving key ECM components, including collagen and glycosaminoglycans. A live-dead assay demonstrated good viability of MSCs withing both GelMA and ECM-MA hydrogels on day 7. Large increases in sGAG accumulation was observed after 21 days of culture in chondrogenic media in both groups. Histological analysis revealed the presence of a more fibrocartilage tissue in the GelMA group, while cells embedded within the ECM-MA showed a round and chondrocytic-like morphology. Both groups stained positively for proteoglycans and collagen, with limited evidence of calcium deposition following Alizarin Red staining. These results show that ECM-MA hydrogels support a hyaline cartilage phenotype and robust cartilaginous matrix production. Future studies will focus on the printability of ECM-MA hydrogels to enable their use as bioinks for the biofabrication of functional tissues

Introduction and Objective. Regeneration of cartilage injuries is greatly limited. Therefore, cartilage injuries are often the starting point for later osteoarthritis. In the past, various bioactive glass (BG) scaffolds have been developed to promote bone healing. Due to the fact that they induce the deposition of hydroxyapatite (HA) -the main component of bone matrix, these BG types are not suitable for chondrogenesis. Hence, a novel BG (Car12N) lacking HA formation, was established. Since BG are generally brittle the combination with polymers is helpful to achieve suitable biomechanic stability. The aim of this interdisciplinary project was to investigate the effects of biodegradable polymer Poly(D,L-lactide-co-glycolide) (PLLA) infiltration into a Car12N scaffold for cartilage tissue engineering. Materials and Methods. BG scaffolds were infiltrated with PLLA using phase separation within a solvent. Pure BG Car12N scaffolds served as control. To assess whether the polymer was homogeneously distributed the polymer to glass ratio and pore contents in the upper, middle and lower third of the scaffolds were examined by light microscopy. For a more precise characterization of the scaffold topology, the glass strut length, the glass strut diameter and the pore circumference were also measured. Leaching tests in 0.1M HCl solution over 8 days were used to allow a gel layer formation on the scaffolds surface. Non-leached and leached scaffolds were subjected to strength testing. Cytotoxicity of the scaffolds with and without polymer was tested according to standards. Scaffolds were colonized with 27.777.8 per cm. 3. primary porcine articular chondrocytes (pACs) or primary human mesenchymal stromal cells (hMSCs), respectively. After cultivation for up to 35 days, the vitality, quantitative DNA and sulfated glycosaminoglycan (sGAG) contents per scaffold were determined. Results. The polymer distribution was not homogeneous in the scaffolds. There were significant differences in glass strut length and pore size. Leaching increased the biomechanical strength. All scaffolds were not cytotoxic. pACs and hMSCs were able to adhere to the scaffold with and without polymer and remained viable during the whole culturing period of 35 d. The DNA content was higher in the pAC colonized scaffolds with polymer than without polymer. The sGAG content was higher in hMSCs seeded scaffolds with polymer than in pACs seeded ones with polymer. Conclusions. Polymer infiltration leads to an increase in mechanical stability of Car12N scaffolds and chondrogenic cells are able to colonize these composites suggesting them as a promising

Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function to damaged articular cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells, but more importantly, the generation of engineered tissue of sufficient quality of clinically relevant size. We describe a novel approach to engineer cartilage grafts using pre-differentiated micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds. Expanded (P2) human nasal chondrocytes (HNCs) or bone marrow-derived mesenchymal stem cells (MSCs) from 3 donors (age 47–62 years) were micro-mass cell pellet cultivated at 5 × 105 cells/pellet for 4 days. Subsequently, pellets were integrated into degradable 3D Printed polymer (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a total of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression. After 4 days, micro-mass cultured pellets could be successfully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seeded constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis observed within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively). We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency (∼100%), as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Furthermore, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage

Cell-based tissue engineering is a promising approach for treating cartilage lesions but the optimal cell-scaffold combination for hyaline cartilage regeneration has yet to be identified. Novel hydrogels allow including tailored tissue type specific modifications with physiologically relevant peptides, by this selectively influencing the cell response. Aim of this study was to modify a poly(ethylene glycol) (PEG)/heparin hydrogel by functionalization with cell instructive peptides introducing matrix-metalloprotease (MMP)-degradability, the cell adhesion motif RGD, or collagen binding motifs (CKLER, CWYRGRL) to improve cartilage matrix deposition in tissue engineering constructs. The hydrogels were formed by mixing thiol-endfunctionalized (MMP-insensitive) starPEG or starPEG-MMP-conjugates carrying MMP-sensitive peptides at every arm and maleimide-functionalized heparin [1] in the presence or absence of cell instructive peptides. Human mesenchymal stromal cells (MSC) or porcine chondrocytes were grown in the hydrogels for up to 4 weeks in vitro under chondrogenic conditions, and in vivo in subcutaneous pockets of immunodeficient mice. MMP-sensitive and –insensitive starPEG/heparin hydrogels supported chondrogenic differentiation of MSC according to induction of COL2A1, BGN and ACAN mRNA expression. Enhanced MMP-sensitivity and therefore degradability increased cell viability and proliferation. RGD-modification of the hydrogels induced cell-spreading and an intensively interconnected cell network. Other than hypothesized, CKLER and CWYRGRL were unable to raise collagen deposition in constructs in vitro. Matrix deposition in chondrocyte-containing peptide-functionalized hydrogels was high and the instructive effect of the hydrogels on chondrocytes appeared stronger in vivo where the merely pericellular cartilaginous matrix deposition was overcome in RGD-functionalized starPEG/heparin hydrogels. Peptide-functionalized starPEG/heparin hydrogel altered cell morphology, proliferation and differentiation with MSC being similar sensitive to cell-matrix interaction peptides like articular chondrocytes. We also demonstrated that in vivoperformance of cell instructive hydrogels can exceed results gained by in vitromodels. Altogether, the manipulation of hydrogel constructs with signaling cues is considered promising for functional cartilage tissue engineering

In the field of tissue engineering (TE), mainly two approaches have been widely studied and utilised throughout the last two decades. Ovsianikov et al. proposed a third strategy for tissue engineering to combine the advantages of the scaffold-based and scaffold-free approach [1].

We utilise the third strategy for TE by fabrication of cell spheroids that are reinforced by microscaffolds, called tissue units (TUs). Aim of the presented study is to differentiate TUs towards a chondrogenic phenotype to show the self-assembly of a millimetre sized cartilage-like tissue in a bottom-up TE approach in vitro.

Two-Photon polymerization (2PP) was utilised to fabricate highly porous microscaffolds with a diameter of 300 µm. The biocompatible and biodegradable, resin Degrad INX (supplied from Xpect INX, Ghent, Belgium) was used for 3D-printing. Each microscaffold was seeded with 4000 human adipose derived stem cells (hASCs) in low-adhesive 96-well plates to allow spheroid formation. TUs were differentiated towards the chondrogenic lineage by application of chondrogenic media, subsequently merged in a cylindrical agarose mold, to fuse into a connected tissue with a diameter of ~1.8 mm and a height of 8 mm.

The characterization of TUs differentiated towards the chondrogenic phenotype included gene expression and protein analysis. Furthermore, immunohistochemically staining for Collagen II and Alcian blue staining were performed to investigate the matrix deposition and fusion of the self-assembled tissue.

Our results suggest that the utilised method could be a promising approach for a variety of tissue engineering approaches, due to the good applicability to a defect side combined with the self-assembly properties of the TUs. Furthermore, the differentiation potential of hASCs is not limited to chondrogenic lineages only, which could pave the way to further TE applications in the future.

Acknowledgements:

This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)

Articular cartilage has limited regenerative potential. Regeneration via autografts or cell therapy is clinically efficacious but the extent of regenerative success depends upon use of an appropriate cell source. The aim of this study was to compare the proliferative and chondrogenic potentials of three human cell types (human bone marrow stromal cells - HBMSCs, neonatal and adult chondrocytes) commonly used in cartilage tissue engineering. HBMSCs, neonatal and adult chondrocytes (passage 2) were cultured in basal and chondrogenic media. At 2, 4 and 6 days, the cells were analysed for morphology and doubling time. Alkaline phosphatase specific activity (ALPSA) was quantified for each group at 2, 4 and 6 weeks. Chondrogenic potential of each cell type was assessed via a pellet culture model. Cryosections were stained with Alcian blue/Sirius Red. HBMSCs showed either elongated or polymorphic phenotypes, with a doubling time of 40 h. Neonatal chondrocytes showed a uniform spindle shape and had the shortest doubling time (16 h). Adult chondrocytes, were also spindle shaped, though slightly larger than the neonatal cells, with a longer doubling time of 22 h. Expression of ALPSA in basal media was of the order HBMSCs > adult chondrocytes > , neonatal chondrocytes. In chondrogenic culture, this order changed to adult chondrocytes > HBMSCs > neonatal chondrocytes. In 3D pellet cultures, all three cell types stained positive for Alcian Blue and showed the presence of chondrocyte-like cells enclosed in lacunae. This comparative study suggests that neonatal chondrocytes are the most proliferative with lowest ALP expression. However, in terms of clinical applications, HBMSCs may be better for cartilage regeneration given their lower ALP expression under chondrogenic conditions when compared with adult chondrocytes under the same conditions. The study has provided information to inform clinical cell therapy for cartilage regeneration

Articular cartilage has a limited regenerative capacity. Tissue engineering strategies adopting seeding and differentiation of individual chondrocytes on porous 3D scaffolds of clinically relevant size remains a considerable challenge. A well documented method to produce small samples of differentiated cartilage tissue in vitro is via micro-mass (pellet) culture, whereby, high concentrations of chondrocytes coalesce to form. a spherical tissue pellet. However, pellet culture techniques are not applied clinically as it is only possible to produce small amounts of tissue (1–2mm). The aims of this study were to develop a method for mass-production of pellets, and investigate whether an alternative “pellet seeding” approach using smart 3D scaffold design would allow large numbers of spherical pellets to be fixed in place. Chondrocytes were isolated from bovine articular cartilage via enzymatic digestion. Freshly isolated and expanded (passage 2) chondrocytes were placed in 96-well plates with round- or v-shaped wells at a range of densities from 0.1, 0.25 and 0.5 million cells per pellet, and centrifuged at 500g for 2 min. In order to assess pellet forming conditions, cells were treated with or without 300 mg/mL fibronectin (FN, Sigma) to improve cell-cell adhesion. Wells were also coated with or without silicone (Sigmacote) to prevent cell adhesion to wells. Pellets were cultured in vitro for up to 14 days and were assessed at various timepoints for size, shape, cell number (DNA assay) and cell differentiation capacity (histology). A robotic Bioplotter device was used to produce porous, biodegradable scaffolds by plotting −250μm polymer (PEGT/PBT) fibres in a layer-by-layer process. Scaffolds with specific 3D pore architecture were produced to allow spherical pellets to be press-fit in each pore thereby fixing them in place throughout the scaffold. Primary and expanded chondrocytes plated at a density of 0.25 million cell/pellet in v-shaped 96-well plates without both FN and silicone treatment produced pellets with consistently better spherical shape and total cell number (as determined via DNA). Under these conditions, cell (re)differentiation and cartilage extracellular matrix formation was observed via positive staining for safranin-O. Mass production of pellets was achieved by culturing multiple 96-well plates in parallel. FN treatment promoted cell-cell adhesion, but also cell adhesion to well plates, irrespective of silicone treatment, resulting in irregular shaped pellets, as did the use of round-bottom shaped wells. Smart scaffold design and layer-by-layer fabrication process allowed direct control over the fibre spacing and pore size (1.0–1.25mm). Multiple layers of spherical pellets (1.25–1.5mm) were press-fit in place, thereby limiting the need for direct cell adhesion to the scaffold. Continued culture of constructs containing pellets resulted in consistent tissue formation throughout the scaffold. In this study, we describe an alternative approach to the design and seeding of scaffolds for cartilage tissue engineering. Current limitations involved with adherence and de-differentiation of single cell populations were avoided by taking advantage of smart 3D scaffold design and pellet-seeding and culture techniques. Further optimisation and automation of the process is necessary, however, such strategies could be beneficial for future scaffold-based cell therapies for repairing articular cartilage defects

Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies.

Acknowledgments: The National Council for Scientific and Technological Development (CNPq, Brazil – Grants # 314 724/2021-4, 307 829/2018-9, 430 860/2018-8, 142 050/2018-0 and 465 656/2014-5), the Coordination for the Improvement of Higher Educational Personnel (CAPES, Brazil – PrInt 88 887.364849/2019-00 and PrInt 88 887.310405/2018-00), the Fund for Support to Teaching, Research and Extension from the University of Campinas (FAEPEX/UNICAMP, Brazil – Grants # 2921/18, 2324/21), and the European Union's Horizon 2020 JointPromise project – Precision manufacturing of microengineered complex joint implants, under grant agreement 874 837 are acknowledged for the financial support of this study.

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration.

Autologous chondrocyte transplantation is a widely used technique for the treatment of cartilage lesions. This therapeutic strategy has been recently improved by the use of biocompatible scaffolds which allow a better fixation of the cells inside the defect together with the maintenance of their original phenotype. We have recently reported that human chondrocytes can efficiently grow on a hyaluronan acid derivative biomaterial (Hyaff-11, Fidia Advanced Biopolymers, Abano Terme, Italy) and are able to express and produce collagen type II and proteoglycans, molecules expressed by differentiated cells (Grigolo et al. Biomaterials 2002). However, from the histological evaluations of the grafted tissues there is not always evidence of hyaline cartilage neo-formation even in presence of good clinical symptoms. Only few studies deals with cellular, and biochemical processes that occur during the remodeling of the graft tissue after transplantation in humans. Biopsy samples harvested from the graft have been examined using a panel of specific antibodies. It was found that cell transplantation is followed not only by a process of cartilage repair but in some cases also by a regeneration achieved through the turnover of the initial fibrocartilagineous tissue via enzymatic degradation and synthesis of newly formed collagen type II. Therefore, we examined the expression of genes encoding extracellular matrix proteins and regulatory factors essential for cell differentiation in human cartilage biopsies of patients who underwent autologous chondrocyte transplantation.

Human cartilage biopsies of patients treated by autologous chondrocyte transplantation and from a multi-organ donor were used. A Real-Time RT-PCR analysis was performed in isolated chondrocytes to evaluate the expression of collagen type I, II, X, aggrecan, cathepsin B, early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for ECM proteins and regulatory proteins was carried out on paraffin embedded sections.

Real-time RT-PCR analysis showed that collagen type I mRNA was expressed in all the samples evaluated while collagen type II was present even if at lower levels compared to control. Collagen type X messenger was undetectable. Aggrecan mRNA was present in all the samples at lower levels compared to donor. Cathepsin B messenger was higher in the samples compared to control. Egr-1 and Sox-9 mRNAs were expressed at lower levels compared to donor. The immunohistochemical analysis showed a slight positivity for collagen type I in all the sections. Collagen type II was found in all the samples evaluated with a positivity confined inside the cells, while the control displayed a positivity which was diffuse in the ECM. Cathepsin B was slightly positive in all the samples while the control was negative. Egr-1 protein was particularly evident in the areas negative for collagen type II. Sox-9 was positive in all the samples, with evident localization in the superficial layer.

Our results provide evidence that the remodelling of the graft tissue after autologous chondrocyte transplantation is regulated by a sophisticated gene expression machinery control addressed to new cartilage formation.

Objectives

We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling.

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated. Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis. These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE. Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis)

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1]. In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2]. The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies. Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020)

Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study. Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I, collagen II, and aggrecan at day 7 and 14, was studied. All experiment were done in triplicated samples. Generally, method 1 SF extraction showed higher cell viability in all strains. Cell viability from Nanglai Saraburi, Laung Saraburi and Nangtui strains were higher than those without SF in every time point while Wanasawan and J108 had higher viability at day 1 and decreased by time. Expression in collagen 1, collagen 2 and aggrecan in method 1 are higher at day 7 and day 14. Collagen 1 expression was highest in Nangnoi Srisaket, followed by Laung Saraburi and Nanglai Saraburi in day 7. Nangnoi Srisaket also had highest expression at day 14, followed by Nanglai Saraburi and Laung Saraburi respectively. Nangseaw had highest collagen 2 expression, follow by Laung Saraburi and Nangnoi Srisaket respectively. Higher aggrecan gene expression of Tubtimsiam, Wanasawan, UB 1 and Nangnoi Srisaket was observed at day 7 and increased expression of all strains at day 14. SF extraction using CaCl2/Ethanol/H2O offered better cell viability and chondrogenic expression. Nangseaw, Laung Saraburi and Nangnoi Srisaket strains expressed more chondrogenic phenotype

Introduction. Articular cartilage injuries have a limited potential to heal and, over time, may lead to osteoarthritis, an inflammatory and degenerative joint disease associated with activity-related pain, swelling, and impaired mobility. Regeneration and restoration of the joint tissue functionality remain unmet challenges. Stem cell-based tissue engineering is a promising paradigm to treat cartilage degeneration. In this context, hydrogels have emerged as promising biomaterials, due to their biocompatibility, ability to mimic the tissue extracellular matrix and excellent permeability. Different stimulation strategies have been investigated to guarantee proper conditions for mesenchymal stem cell differentiation into chondrocytes, including growth factors, cell-cell interactions, and biomaterials. An interesting tool to facilitate chondrogenesis is external ultrasound stimulation. In particular, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to have a role in regulating the differentiation of adipose mesenchymal stromal cells (ASCs). However, chondrogenic differentiation of ASCs has been never associated to a precisely measured ultrasound dose. In this study, we aimed to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D hydrogel. Materials and Methods. Human adipose mesenchymal stromal cells at 2∗10. 6. cells/mL were embedded in a hydrogel ratio 1:2 (VitroGel RGD®) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm. 2. , duty cycle: 20%, pulse repetition frequency: 1 kHz, stimulation time: 5 min) in order to assess its influence on cell differentiation. Hydrogel-loaded ASCs were cultured and differentiated for 2, 7, 10 and 28 days. At each time point cell viability (Live&Dead), metabolic activity (Alamar Blue), cytotoxicity (LDH), gene expression (COL2, aggrecan, SOX9, and COL1), histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated respect to a non-stimulated control. Results. Histological analysis evidenced a uniform distribution of ASCs both at the periphery and at the center of the hydrogel. Live & Dead test evidenced that the encapsulated ASCs were viable, with no signs of cytotoxicity. We found that LIPUS induced chondrogenesis of ASCs embedded in the hydrogel, as demonstrated by increased expression of COL2, aggrecan and SOX9 genes and proteins, and decreased expression of COL1 respect to the non-stimulated control. Conclusions. These results suggest that the LIPUS treatment could be a valuable tool in cartilage tissue engineering, to push the differentiation of ASCs encapsulated in a 3D hydrogel

Mesenchymal Stem Cells (MSCs) are key regulators in senile osteoporosis and in bone formation and regeneration. MSCs are therefore suitable candidates for stem cells mediated gene therapy of bone. Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is a highly osteoinductive cytokine, promoting osteogenic differentiation of MSCs. We hypothesized that genetically engineered MSCs, expressing rhBMP2, can be utilized for targeted cell mediated gene therapy for local and systemic bone disorders and for bone/cartilage tissue engineering. Engineered MSCs expressing rhBMP-2 have both autocrine and paracrine effects enabling the engineered cells to actively participate in bone formation. We conditionally expressed rhBMP2 (tet-controlled gene expression, tet-off system) in mouse and human mesenchymal stem cells. RhBMP2 expressing clones (tet-off and adeno-BMP2 infected MSCs), spontaneously differentiated into osteogenic cells in vitro and in vivo. Engineered MSCs were transplanted locally and tracked in vivo in radial segmental defects (regenerating site) and in ectopic muscular and subcutaneous sites (non-regenerating sites). In vitro and in vivo analysis revealed rhBMP2 expression and function, confirmed by RT-PCR, ELISA, western blot, immunohistochemistry and bioassays. Secretion of rhBMP2 in vitro was controlled by tetracycline and resulted in secretion of 1231 ng/24 hours/106 cells. Quantitative Micro-CT 3-Dimentional reconstruction revealed complete bone regeneration regulated by tetracycline in vivo, indicating the potential of this platform for bone and cartilage tissue engineering. Angiogenesis, a crucial element in tissue engineering, was increased by 10-folds in transplants of rhBMP2 expressing MSCs (tet-off), shown by histomorphometry and MRI analysis (p< 0.05). In order to establish a gene therapy platform for systemic bone disorders, MSCs with tet-controlled rhBMP-2 expression, were injected systemically (iv). These engineered MSCs were genetically modified in order to achieve homing to the bone marrow. Systemic non invasive tracking of engineered MSCs was achieved by recording topographical bioluminescence derived from luciferase expression detected by a coupled charged CCD imaging camera. For clinical situations that require immuno-isolation of transplanted cells, we developed an additional platform utilizing cell encapsulation technique. Immuno-isolated engineered MSCs, with tet-controlled rhBMP-2 expression, encapsulated with sodium alginate induced bone formation by paracrine effect of secreted rhBMP-2. Finally, we have characterized a novel tissue-engineering platform composed of engineered MSCs and biodegradable polymeric scaffolds, creating a 3D bone tissue in rotating Bioreactors. Our results indicate that engineered MSCs and polymeric scaffolds can be utilized for ex vivo bone tissue engineering. We therefore conclude that genetically engineered MSCs expressing rhBMP-2 under tetracycline control are applicable for: a) local and systemic gene therapy to bone, and b) bone tissue engineering. Our studies should lead to the creation of gene therapy platforms for systemic and local bone diseases in humans and bone/cartilage tissue engineering

Introduction. Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids. Materials and Methods. Pellet cultures were prepared either from hMSC single cells (250’000 cells/pellet) or hMSC spheroids (282 cells/spheroid) at the same final cell concentration (250’000 cells/pellet = 887 spheroids/pellet). The effect of polymer concentration on encapsulated hMSC spheroids (887 spheroids/hydrogel) was investigated in THA-Col hydrogels (50μl) at the following concentrations (THA-Col mg/ml): Group (1) 12.5–2.5, (2) 16.7–1.7, (3) 12.5–1.7, (4) 16.7–2.5 mg/ml. All samples were cultured for 21 days in standard chondrogenic differentiation medium containing 10ng/ml TGF-β1. Chondrogenic differentiation and hypertrophy of both pellet cultures and hMSCs spheroids encapsulated in THA-Col were analysed using gene expression analysis (Aggrecan (ACAN), COL1A1, COL2A1, COL10A1), dimethylmethylene-Blue assay to quantify glycosaminoglycans (GAGs) retained in the samples and (immuno-) histological staining (Safranin-O, collagen II, aggrecan) on day 1 and day 21 (n=3 donors). Results. The culture of hMSCs in pellets based on single cells or spheroids resulted in an increase in chondrogenic-associated markers COL2A1 (2’900–3’400-fold) and ACAN (45–47-fold) compared to respective samples on day 1 in both groups. GAGs increased in spheroid pellets to 21.2±3.4 mg/ml and in single cell pellets to 20.8±6.6 mg/ml on day 21. Comparing the levels of hypertrophic markers, single cell pellets showed 7-fold and 20-fold higher expression of COL1A1 and COL10A1 than spheroid pellets on day 21. The encapsulation of hMSC spheroids in THA-Col resulted in an upregulation of chondrogenic-associated markers and GAG content in all hydrogels with differences in cell differentiation related to the Col and THA polymer ratio, while level of hypertrophy was comparable in all groups with values similar to the spheroid pellet group. Spheroids embedded in hydrogels with lower THA content (group 1 and 3) resulted in more pronounced chondrogenic phenotype marked by upregulation of COL2A1 (3’200–4’500-fold) and ACAN (152–179-fold) relative to the respective samples on day 1. Spheroids embedded in higher THA content hydrogels (group 2 and 4) showed less pronounced chondrogenesis marked by lower upregulation of COL2A1 (980–1800-fold) and ACAN (25–68-fold, relative to day 1 samples). This was confirmed by quantification of GAGs, increasing from 2.5±1.9 and 2.5±1.7 mg/ml (day 1) to 11.4±2.5 and 9.9±3.8 mg/ml on day 21 for groups 1 and 4, respectively. (Immuno-) histological stainings resulted in a more homogenous staining in lower THA content hydrogels compared to a more local matrix deposition in samples with higher THA content. Conclusion. The reduced level of hypertrophy in hMSC pellets prepared from cell spheroids compared to single cell pellets at same cell count might be related to the packing density of the cells with cells being more densely packed in single cell pellets compared to pellets from spheroids. Investigating the effect of polymer ratios on chondrogenesis, it seems that the THA content is the driving factor influencing hMSC chondrogenesis rather than Col content in THA-Col composites at comparable mechanical properties. This study highlights the feasibility to use hMSC spheroids as alternative approach to study in vitro chondrogenic differentiation and the suitability to investigate the effect of biomaterial composition on chondrogenesis and hMSC hypertrophy

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

Objective. Early cell loss of up to 50% is common to in vitro chondrogenesis of mesenchymal stromal cells (MSC) and stimulation of cell proliferation could compensate for this unwanted effect and improve efficacy and tissue yield for cartilage tissue engineering. We recently demonstrated that proliferation is an essential requirement for successful chondrogenesis of MSC, however, how it is regulated is still completely unknown. We therefore aimed to identify signaling pathways involved in the regulation of proliferation during in vitro chondrogenesis and investigated, whether activation of relevant pathways could stimulate proliferation. Design. Human MSC were subjected to in vitro chondrogenesis for up to 42 days under standard conditions in the presence of 10 ng/ml TGF-β. Cells were or were not additionally treated with inhibitors of bone morphogenetic protein (BMP), insulin-like growth factor (IGF) IGF/PI3K, fibroblast growth factor (FGF) or indian hedgehog (IHH) pathways for two or four weeks. To investigate the stimulation of proliferation by exogenous factors, cells were treated with BMP-4, IGF-1, FGF-18 or purmorphamine (small molecule hedgehog agonist). Proliferation was determined by [3H]-thymidine incorporation. Results and Discussion. Quantitative assessment of proliferation revealed that proliferation arrest occurred during condensation up to day 3 and cell division was re-initiated thereafter with a peak on day 28. To test which pathways are relevant for the restart of proliferation, BMP, IGF/PI3K, FGF or IHH signaling was inhibited up to day 14. All treatments significantly reduced proliferation > 50% and, thus, seemed to participate in the re-entry into the cell cycle. To study whether the same pathways are relevant to maintain cells in a proliferative state later on, inhibitors were supplemented from day 14–28. This resulted in a significant decrease of proliferation in the groups treated with inhibitors of BMP (67% decrease), FGF (70%) and IHH (30%) signaling, while inhibition of IGF/PI3K did not influence late proliferation. Although BMP-4, IGF-1 or FGF-18 are known mitogenic factors in the growth plate, stimulation of cells by exogenous addition of these factors did not enhance proliferation in any differentiation phase. In contrast, stimulation of IHH signaling from day 14–28 significantly increased proliferation by 44%. This is in line with the documented strong mitogenic activity of hedgehog signaling in the proliferative zone of the growth plate. Thus, our data demonstrated that BMP, IGF/PI3K, FGF and IHH essentially participate in the regulation of proliferation during in vitro chondrogenesis. Early or late activation of single pathways by exogenous factors was, however, not sufficient to increase proliferation significantly with the exception of late activation of hedgehog signaling. Optimization of stimulation of the hedgehog pathway with a focus on increased tissue yield will now be the next step

Tissue engineering is a rapidly expanding field of research. Bone and cartilage engineering are being undertaken in an attempt to treat osteoarthritis and repair bone defects. In spite of extensive research little successful clinical application of this work has been seen. There are however many advances in the field that one day may have therapeutic interest. One particular area of interest is the potential for using osteophyte tissue in repairing osteoarthritic defects. Osteophytes represent an attempt by the body to regenerate bone and cartilage. They present an obvious source of cells for tissue engineering. Research ay QUT has shown that cells within the osteophytes are a better source of bone and cartilage regeneration in the laboratory than matched patient’s bone marrow stem cells. Osteoarthritis remains the ultimate challenge for orthopaedic tissue engineering. Understanding the chemical and mechanical signals occurring in osteoarthritis presents opportunities for targeted drug delivery and potential slowing of disease. We have identified changes within the MMP profile of cells at the osteochondral junction. Subchondral sclerosis appears to be associated with changes in the nature of chondrocytes deep within the cartilage layer. This transformation of chondrocytes into osteoblast-like tissue in many ways mimics the changes seen in the growth plate once maturity is reached. Understanding the parallels between these processes may help answer some of the mechanisms of the development of osteoarthritis. This talk will discuss the above topics as well as other areas of interest to an orthopaedic surgeon working within a group of 10 cell biologists