The use of mesenchymal stem cell (MSCs) for intervertebral disc (IVD) regeneration has been extensively explored in the last two decades. MSCs are potent cell types that can be easily and safely harvested due to their abundancy and availability. Moreover, they are characterized by the capacity to differentiate towards IVD cells as well as release growth factors to support resident cell metabolism and recruit local progenitor cells to induce endogenous repair of degenerated IVDs. This talk will outline the characteristics of the main MSC sources and their effect towards IVD regeneration based on available preclinical and clinical evidence. In addition, innovative aspects of MSC-derived cell-free therapies will also be discussed

Osteoarthritis (OA) is a common age-related degenerative joint disease, affecting 7% of the global population, more than 500 million people worldwide. Exosomes from mesenchymal stem cells (MSCs) showed promise for OA treatment, but the insufficient biological targeting weakens its efficacy and might bring side effects. Here, we report the chondrocyte-targeted exosomes synthesized via click chemistry as a novel treatment for OA. Exosomes are isolated from human umbilical cord-derived MSCs (hUC-MSCs) using multistep ultracentrifugation process, and identified by electron microscope and nanoparticle tracking analysis (NTA). Chondrocyte affinity peptide (CAP) is conjugated on the surface of exosomes using click chemistry. For tracking, nontagged exosomes and CAP-exosomes are labeled by Dil, a fluorescent dye that highlights the lipid membrane of exosomes. To verify the effects of CAP-exosomes, nontagged exosomes and CAP-exosomes are added into the culture medium of interleukin (IL)-1β-induced chondrocytes. Immunofluorescence are used to test the expression of matrix metalloproteinase (MMP)-13. CAP-exosomes, compared with nontagged exosomes, are more easily absorbed by chondrocytes. What's more, CAP-exosomes induced lower MMP-13 expression of chondrocytes when compared with nontagged exosomes (p<0.001). CAP-exosomes show chondrocyte-targeting and exert better protective effect than nontagged exosomes on chondrocyte extracellular matrix. Histological and in vivo validation are now being conducted

Due to the presence of megakaryocytes, platelets and clotting factors, bone marrow aspirate (BMA) tends to coagulate. For the first time, starting from our previous studies on mesenchymal vertebral stem cells, it has been hypothesized that coagulated BMA represents a safe and effective autologous biological scaffold for bone regeneration in spinal surgery. The present research involved advanced preclinical in vitro models and the execution of a pilot clinical study. Evaluation of cell morphology, growth kinetics, immunophenotyping, clonogenicity, trilineage-differentiation, growth-factors and HOX and TALE gene expression were analyzed on clotted- and un-clotted human V-BMA. In parallel, a pilot clinical study on ten patients with degenerative spine diseases submitted to instrumented posterior arthrodesis, is ongoing to assess the ability of clotted-V-BMA to improve spinal fusion at 6- and 12-months follow-up. Results demonstrated that clotted-V-BMA have significantly higher growth-factor expression and mesenchymal stem cell (MSCs) viability, homogeneity, clonogenicity, and ability to differentiate towards the osteogenic phenotype than un-clotted-V-BMA. Clotted-V-BMA also highlighted significant reduced expression of PBX1 and of MEIS3 genes negatively involved in osteoblast maturation and differentiation. From December 2020, eight patients have already been enrolled with first promising results that will be finally evaluated in the next two months. The application of V-BMA-clot as carrier of progenitors and cytokines and as natural scaffold with a structural texture represents a point-of-care orthobiologic product to improve spinal fusion. Clinical application seems to be efficacy, and we will confirm and strengthen these data with the final results of the pilot clinical study

Objectives. Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration. Methods. MSCs from young, adult and OVX rats were put in a Boyden chamber to establish their migration towards SDF-1. This was compared with MSCs transfected with CXCR4, as well as MSCs differentiated to osteoblasts. Results. MSCs from OVX rats migrate significantly (p < 0.05) less towards SDF-1 (9%, . sd. 5%) compared with MSCs from adult (15%, . sd. 3%) and young rats (25%, . sd. 4%). Cells transfected with CXCR4 migrated significantly more towards SDF-1 compared with non-transfected cells, irrespective of whether these cells were from OVX (26.5%, . sd. 4%), young (47%, . sd. 17%) or adult (21%, . sd. 4%) rats. Transfected MSCs differentiated to osteoblasts express CXCR4 but do not migrate towards SDF-1. Conclusions. MSC migration is impaired by age and osteoporosis in rats, and this may be associated with a significant reduction in bone formation in osteoporotic patients. The migration of stem cells can be ameliorated by upregulating CXCR4 levels which could possibly enhance fracture healing in osteoporotic patients. Cite this article: A. Sanghani-Kerai, M. Coathup, S. Samazideh, P. Kalia, L. Di Silvio, B. Idowu, G. Blunn. Osteoporosis and ageing affects the migration of stem cells and this is ameliorated by transfection with CXCR4. Bone Joint Res 2017;6:–365. DOI: 10.1302/2046-3758.66.BJR-2016-0259.R1

To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model. 5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10. 6. and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls. All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings. The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested

A promising application of Mesenchymal stem cells (MSCs) is the treatment of non-unions. Substituting bone grafts, MSCs are directly injected into the fracture gap. High cell viability seems to be a prerequisite for therapeutic success. Administration of the MSCs via injection creates shear stresses possibly damaging or destroying the cells. Aim of this study was to investigate the effect of the injection process on cell viability. MSCs were isolated and cultivated from femoral tissue of five subjects undergoing arthroplasty. Prior to injection, the cells were identified as MSCs. After dissolving to a concentration of 1 Million cells/ml, 1 ml of the suspension was injected through a cannula of 200 mm length and 2 mm diameter (14 G) with flow rates of 38 and 100 ml/min. The viability of the MSCs at different flow rates was evaluated by staining to detect the healthy cell fraction. It was analyzed statistically against a control group via the Kruskal-Wallis-test and for equivalence via the TOST procedure. Significance level was set to 5 %, equivalence margin to 20 %. The healthy cell fraction of the control group was 85.88 ± 2.98 %, 86.04 ± 2.53 % at 38 ml/min and 85.48 ± 1.64 % at 100 ml/min. There was no significant difference between the fraction of healthy cells (p = 0.99) for different volume flows, but a significant equivalence between the control group and the two volume flows (38 ml/min: p = 0.002, 100 ml/min: p = 0.001). When injecting MSC solutions, e.g. into a non-union, the viability of the injected cells does not deterioriate significant. The injecting technique is therefore feasible

As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. The aim of this research was to create bioinks that can be injected or 3D bioprinted to aid osteochondral defect repair using human cells. Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA). Chondrocytes or mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also produced via 3D culture and then bioprinted to accelerate cell growth and development of ECM in bioprinted constructs. Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture over 28 days, with accelerated cell growth seen with inclusion of MSC or chondrocyte spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects (OCDs) and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period. In conclusion we developed novel composite AlgMA/GelMA bioinks that can be triple-crosslinked, facilitating dense chondrocyte and MSC growth in constructs following 3D bioprinting. The bioink can be injected or 3D bioprinted to successfully repair in vitro OCDs, offering hope for a new approach to treating AC defects

As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. The aim of this research was to create bioinks that can be injected or 3D bioprinted to aid osteochondral defect repair using human cells. Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA). Chondrocytes or mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also bioprinted to accelerate cell growth and development of ECM in bioprinted constructs. Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture over 28 days, with accelerated cell growth seen with inclusion of MSC or chondrocyte spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects (OCDs) and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period. In conclusion we developed novel composite AlgMA/GelMA bioinks that can be triple-crosslinked, facilitating dense chondrocyte and MSC growth in constructs following 3D bioprinting. The bioink can be injected or 3D bioprinted to successfully repair in vitro OCDs, offering hope for a new approach to treating AC defects

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. Previous studies suggested that the regenerative activity of stem cells can be enhanced by exposure to tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells-derived mesenchymal-like progenitors (hiPSCs-MPs) can enhance the regenerative potential of human bone marrow mesenchymal stromal cells (hBMSCs). ECM was engineered from hiPSC-MPs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. hBMSCs were cultured on the engineered ECM, and differentiated into osteogenic, chondrogenic and adipogenic lineages. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularization of ECM resulted in efficient cell elimination, as observed in our previous studies. Cultivation hBMSCs on the ECM in osteogenic medium significantly increased hBMSC growth, collagen deposition and alkaline phosphatase activity. Furthermore, expression of osteogenic genes and matrix mineralization were significantly higher compared to plastic controls. Chondrogenic micromass culture on the ECM significantly increased cell growth and expression of chondrogenic markers, including glycosaminoglycans and collagen type II. Adipogenic differentiation of hBMSCs on the ECM resulted in significantly increased hBMSC growth, but significantly reduced lipid vacuole deposition compared to plastic controls. Together, our studies suggest that BMSCs differentiation into osteogenic and chondrogenic lineages can be enhanced, whereas adipogenic activity is decreased by the culture on engineered ECM. Contribution of specific matrix components and underlying mechanisms need to be further elucidated. Our studies suggest that the three-lineage differentiation of aged BMSCs can be modulated by culture on hiPSC-engineered ECM. Further studies are aimed at scaling-up to three-dimensional ECM constructs for osteochondral tissue regeneration

Osteoarthritis (OA) is a disabling disease depriving the quality of life of patients. Mesenchymal stem cells (MSCs) are recently used to modify the inflammatory and degenerative cascade of the disease. Source of MSCs could change the progression and symptoms of OA due to their different metabolomic activities. We asked whether MSCs derived from the infrapatellar fat (IPF), synovium (Sy) and subcutaneous (SC) tissues will decrease inflammatory and degenerative markers of normal and OA chondrocytes and improve regeneration in culture. Tissues were obtained from three male patients undergoing arthroscopic knee surgery due to sports injuries after ethical board approval. TNFa concentration decreased in all MSC groups (Sy=156,6±79, SC=42,1±6 and IPF=35,5±3 pg/ml; p=0,036) on day 14 in culture. On day seven (Sy=87,4±43,7, SC=23±8,9 and IPF=14,7±3,3 pg/ml, p=0,043) and 14 (Sy=29,1±11,2, SC=28,3±18,5 and IPF=20,3±16,2 pg/ml, p=0,043), MMP3 concentration decreased in all groups. COMP concentration changes however were not significant. Plot scores of tissues for PC2-13,4% were significantly different. Based on the results of liquid chromatography-mass spectrometry (LC-MS) metabolomics coupled with recent data processing strategies, clinically relevant seven metabolites (L-fructose, a-tocotrienol, coproporphyrin, nicotinamide, bilirubin, tauro-deoxycholic acid and galactose-sphingosine) were found statistically different (p<0.05 and fold change>1.5) ratios in tissue samples. Focusing on these metabolites as potential therapeutics could enhance MSC therapies. Acknowledgment: Hacettepe University, Scientific Research Projects Coordination Unit (#THD-2020-18692) and Turkish Society of Orthopedics and Traumatology (#TOTBID-89) funded this project. Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA)

Introduction. Articular cartilage has a low self-regeneration capacity. Cartilage defects have to be treated to minimize the risk of the onset of osteoarthritis. Bioactive glass (BG) is a promising source for cartilage tissue engineering. Until now, conventional BGs (like BG1393) have been used, mostly for bone regeneration, as they are able to form a hydroxyapatite layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to study the effect of 3D printed hydrogel scaffolds supplemented with spheres of the BG CAR12N to improve the chondrogenesis of mesenchymal stem cells (MSCs). Method. Based on our new glass composition (CAR12N), small BG spheres (25-40 µm) were produced and mixed with hydrogel and primary human (h) MSCs. Grid printed scaffolds were cultivated up to 21 days in expansion or chondrogenic differentiation medium. Macroscopical images of the scaffolds were taken to observe surface changes. Vitality, DNA and sulfated glycosaminoglycan (GAG) content was semiquantitatively measured as well as extracellular matrix gene transcription. Result. It was possible to print grid shaped hydrogel scaffolds with BG spheres and hMSCs. No significant changes in scaffold shape, surface or pore size were detected after 21 days in culture. The BG spheres were homogeneously distributed inside the grids. Vitality was significantly higher in grids with CAR12N spheres in comparison to those without. The DNA content remained constant over three weeks, but was higher in the sphere containing scaffolds than in those without BG spheres. GAG content in the grids increased not only with additional cultivation time but especially in grids with BG spheres in chondrogenic medium. Aggrecan and type II collagen gene expression was significantly higher grids cultured in the chondrogenic differentiation medium. Conclusion. This developed 3D model, is very interesting to study the effect of BG on hMSCs and to understand the influence of leaking ions on chondrogenesis

Introduction. Homogenous and consistent preparations of mesenchymal stem cells (MSCs) can be acquired by selecting them for integrin α10β1 (integrin a10-MSCs). Safety and efficacy of intra-articular injection of allogeneic integrin a10-MSCs were shown in two post-traumatic osteoarthritis horse studies. The current study investigated immunomodulatory capacities of human integrin a10-MSCs in vitro and their cell fait after intra-articular injection in rabbits. Method. The concentration of produced immunomodulatory factors was measured after licensing integrin a10-MSCs with pro-inflammatory cytokines. Suppression of T-cell proliferation was determined in co-cultures with carboxyfluorescein N-succinimidyl ester (CFSE) labelled human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 and measuring the CFSE intensity of CD4+ cells. Macrophage polarization was assessed in co-cultures with differentiated THP-1 cells stimulated with lipopolysaccharide and analysing the M2 macrophage cell surface markers CD163 and CD206. In vivo homing and regeneration were investigated by injecting superparamagnetic iron oxide nanoparticles conjugated with Rhodamine B-labeled human integrin a10-MSCs in rabbits with experimental osteochondral defects. MSC distribution in the joint was followed by MRI and fluorescence microscopy. Result. The production of the immunomodulatory factors indoleamine 2,3-dioxygenase and prostaglandin E2 was increased after inflammatory licensing integrin a10-MSCs. Co-cultures with integrin a10-MSCs suppressed T-cell proliferation and increased the frequency of M2 macrophages. In vivo injected integrin a10-MSCs homed to osteochondral defects and were detected in the repair tissue of the defects up to 10 days after injection, colocalized with aggrecan and type II collagen. Conclusion. This study showed that human integrin a10-MSCs have immunomodulatory capacities and in vivo can home to the site of osteochondral damage and directly participate in cartilage regeneration. This suggests that human integrin α10β1-selected MSCs may be a promising therapy for osteoarthritis with dual mechanisms of action consisting of immunomodulation and homing to damage followed by early engraftment and differentiation into chondrocyte-like cells that deposit hyaline cartilage matrix molecules

The extracellular matrix (ECM)-based biomaterials provide a platform to mimic the disc microenvironment in facilitating stem cell transplantation for tissue regeneration. However, little is known about in vitro preconditioning human umbilical cord Wharton Jelly-derived mesenchymal stem cells (MSCs) on 3D hyaluronic acid (HA)/type II collagen (COLII) hydrogel for nucleus pulposus (NP) phenotype and pain modulation. We developed a tuneable 3D HA/COLII by fabricating HA/COLII hydrogel at 2 mg/ml COLII and various weight ratios of HA:COLII, 1:9 and 4.5:9. The hydrogel was characterized for degradability, stability, and swelling capacity. The viability of hWJ-MSC encapsulated on hydrogel supplemented with TGF-β3 was assessed. The implantation of HA/COLII hydrogel was done in surgically induced disc injury model of pain in the rat tail. The general health status in rats was monitored. The nociceptive behaviour in rats was performed for mechanical allodynia using von Frey test. The HA/COLII 4.5:9 hydrogel showed higher swelling capacity than weight ratio 1:9, suggesting that a higher amount of HA can absorb a large amount of water. Both HA/COLII 4.5:9 and 1:9 hydrogel formulations had a similar degradation profile, stable to the hydrolytic process. The hWJ-MSC-encapsulated on hydrogel marked higher cell viability with round morphology shape of cells in vitro. The surgically induced disc injury in the rat tail evoked mechanical allodynia, without affecting general health status in rats. The implantation of HA/COLII 1:9 hydrogel was observed to slightly alleviate injury-induced mechanical allodynia. Fine-tuning HA/COLII-based hydrogel provides the optimal swelling capacity, stability, degradability, and non-cytotoxic, mimicking the 3D NP niche in guiding hWJ-MSCs towards NP phenotype. The HA/COLII hydrogel could be employed as an advanced cell delivery system in facilitating stem cell transplantation for intervertebral disc regeneration targeting pain

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment. In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function. At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity. In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications. Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Objectives. Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods. A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results. This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion. This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model. Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls. Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression. Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects

Objectives. Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results. Methods. We hereby review the published literature on the use of MSCs in promoting bone consolidation during DO. Results. Studies differed in animal type (mice, rabbit, dog, sheep), bone type (femur, tibia, skull), DO protocols and cell transplantation methods. Conclusion. The majority of studies reported that the transplantation of MSCs enhanced bone consolidation or formation in DO. Many questions relating to animal model, DO protocol and cell transplantation regime remain to be further investigated. Clinical trials are needed to test and confirm these findings from animal studies. Cite this article: Y. Yang, S. Lin, B. Wang, W. Gu, G. Li. Stem cell therapy for enhancement of bone consolidation in distraction osteogenesis: A contemporary review of experimental studies. Bone Joint Res 2017;6:385–390. DOI: 10.1302/2046-3758.66.BJR-2017-0023

There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture. To assess the optimal concentration of MSCs for promoting fracture healing in a rat model. Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time polymerase chain reaction. Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post fracture, SDF-1, TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-β1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture. The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures

Deriving autologous mesenchymal stem cells (MSCs) from adipose tissues without using enzymes requires sophisticated biomedical instruments. Applied pressure on tissues and cells are adjusted manually although centrifugation and filtration systems are frequently used. The number of derived MSCs therefore could differ between instruments. We compared the number of MSCs obtained from four commercially available devices and our newly designed and produced instrument (A2, B3, L3, M2 and T3). Three-hundred mL of adipose tissue was obtained from a female patient undergoing liposuction using the transillumination solution. Obtained tissue was equally distributed to each device and handled according to the producers' guides. After handling, 3 mL stromal vascular fraction (SVF) was obtained from each device. Freshly isolated SVF was characterized using multi-color flow cytometry (Navios Flow Cytometer, Beckman Coulter, USA). Cell surface antigens were chosen according to IFATS and ISCT. CD31-FITC, CD34-PC5,5, CD73-PE, CD90-PB and CD45-A750 (Backman Coulter, USA) fluorochrome-labeled monoclonal antibodies were assessed. Markers were combined with ViaKrome (Beckman Coulter, USA) to determine cell viability. At least 10. 5. cells were acquired from each sample. A software (Navios EX, Beckman Coulter, USA) was used to create dot plots and to calculate the cell composition percentages. The data was analyzed in the Kaluza 2.1 software package (Beckman Coulter, USA). Graphs were prepared in GraphPad Prism. CD105 PC7/CD31 FITC cell percentages were 23,9%, 13,5%, 24,6%, 11,4% and 28,8% for the A2, B3, L3, M2 and T3 devices, respectively. We conclude that the isolated MSC percentage ranged from 11,4% to 28,8% between devices. The number of MSCs in SVF are key determinants of success in orthobiological treatments. Developing a device should focus on increasing the number of MSCs in the SVF while preserving its metabolic activity. Acknowledgments: Scientific and Technological Research Council of Türkiye (TÜBİTAK)- Technology and Innovation Funding Program Directorate (TEYDEB) funded this project (#321893). Servet Kürümoğlu and Bariscan Önder of Disposet Ltd., Ankara, Türkiye (. www.disposet.com. ) contributed to the industrial design and research studies. Ali Tuncel and Feza Korkusuz are members of the Turkish Academy of Sciences (TÜBA). Nilsu Baysal was funded by the STAR Program of TÜBITAK Grant # 3210893