Osteoarthritis (OA) is the most prevalent degenerative joint disease that is a leading cause of disability worldwide. Existing therapies of OA only address the symptoms. Liraglutide is a well-known anti-diabetic medication that is used to treat type 2 diabetes and obesity. In inflammatory and post-traumatic OA animal models, liraglutide has demonstrated anti-inflammatory, pain-relieving, and cartilage-regenerating effects1 . The objective of this study is to investigate liraglutide's ability to reduce inflammation and promote anabolism in human OA chondrocytes in vitro. Pellets formed with human OA chondrocytes were cultured with a chondrogenic medium for one week to form cartilage tissue. Afterward, pellets were cultured for another 2 weeks with a chondropermissive medium. The OA group was treated with IL-1β to mimic an inflammatory OA condition. The drug group was treated with 0.5 or 10 µM liraglutide. On days 0, 1, and 14, pellets were collected. Conditioned medium was collected over the 2 weeks culture period. The gene and protein expression levels of regenerative and inflammatory biomarkers were evaluated and histological analyzes were performed. Results showed that the nitric oxide release of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were lower than the OA group. The DNA content of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were higher than the OA group on day 14. The RT-qPCR results showed that the anabolism (ACAN, COMP, and COL2) markers were higher expressed in the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups when compared with the OA group. The inflammation (CCL-2 and IL-8) markers and catabolism markers (MMP-1, MMP-3, ADAMTS4, and ADAMTS5) had lower expression levels in the OA + liraglutide groups compared to the OA group. The histomorphometric analysis (Figure 1) supported the RT-qPCR results. The results indicate that liraglutide has anabolic and anti-inflammatory effects on human OA chondrocyte pellets.

Acknowledgments: This project has received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program. The funding agencies supporting this work are (in alphabetical order of participating countries): France: BPI France; Germany: Project Management Agency (DLR), which acts on behalf of the Federal Ministry of Education and Research (BMBF); The Netherlands: Netherlands Enterprise Agency (RVO); Switzerland: Innosuisse (the Swiss Innovation Agency).

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A novel ex vivo intervertebral disc (IVD) organ model and corresponding sample holder were developed according to the requirements for six degrees of freedom loading and sterile culture in a new generation of multiaxial bioreactors. We tested if the model can be maintained in long-term IVD organ culture and validated the mechanical resistance of the IVD holder in compression, tension, torsion, and bending.

An ex vivo bovine caudal IVD organ model was adapted by retaining 5-6 mm of vertebral bone to machine a central cross and a hole for nutrient access through the cartilaginous endplate. A counter cross was made on a customized, circular IVD holder. The new model was compared to a standard model with a minimum of bone for the cell viability and height changes after 3 weeks of cyclic compressive uniaxial loading (0.02-0.2 MPa, 0.2 Hz, 2h/ day; n= 3 for day 0, n= 2 for week 1, 2, and 3 endpoints). Mechanical tests were conducted on the assembly of IVD and holder enhanced with different combinations of side screws, top screws, and bone adhesive (n=3 for each test).

The new model retained a high level of cell viability after three weeks of in vitro culture (outer annulus fibrosus 82%, inner annulus fibrosus 69%, nucleus pulposus 75%) and maintained the typical values of IVD height reduction after loading (≤ 10%). The holder-IVD interface reached the following highest average values in the tested configurations: 320.37 N in compression, 431.86 N in tension, 1.64 Nm in torsion, and 0.79 Nm in bending.

The new IVD organ model can be maintained in long-term culture and when combined with the corresponding holder resists sufficient loads to study IVD degeneration and therapies in a new generation of multiaxial bioreactors.

Successful application of patient derived cells to engineer vascularized bone grafts is often hampered by low cell numbers and lengthy in vitro expansion. With sound induced morphogenesis (SIM), local cell density enhancement was shown to improve microvasculature formation at lower cell concentration than conventional methods [1]. SIM takes advantage of hydrodynamic forces that act on cells to arrange them within a hydrogel. Following, we are evaluating the potential of cell-hydrogel biografts with high local cell density to improve the therapeutic efficacy in clinical scenarios such as anastomosis or bone formation within non-union fractures.

To assess anastomosis, human umbilical vein endothelial cells (HUVEC) and human mesenchymal stromal cells (MSC) were mixed at a 1:1 ratio in PEG-based or Dextran-based hydrogels at a final concentration of 2×10⁶ cells×mL^-1. For ectopic bone formation, MSC were resuspended in PEG-based hydrogels at 2×10⁶ or 5×10⁶ cells×mL^-1, with or without BMP-2. Cells were assembled into distinct patterns at a frequency of 60 Hz. Four biografts of 4 × 9 mm² were implanted at the back of nude mice (total of 7 animals) and harvested after 2 or 8 weeks. Explants were fixed and imaged as whole constructs or embedded in paraffin for histological analysis.

Upon explantation, microscopic evaluation indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks and formed a pre-vascular network. In the second study, ectopic bone formation was more pronounced in areas of higher local cell density based on visual inspection. Ongoing experiments are further characterizing bone formation by micro-CT and histological evaluation.

Our results indicate that local cell density enhancement by sound requires a lower initial cell concentration than conventional, static seeding methods to achieve comparable microvasculature structures or local osteogenesis.

Introduction and Objective

Low back pain (LBP) is a major cause of long-term disability in adults worldwide and it is frequently attributed to intervertebral disc (IVD) degeneration. So far, no consensus has been reached regarding appropriate treatment and LBP management outcomes remain disappointing. Spine unloading or traction protocols are common non-surgical approaches to treat LBP. These treatments are widely used and result in pain relief, decreased disability or reduced need for surgery. However, the underlying mechanisms -namely, the IVD unloading mechanobiology- have not yet been studied. The aim of this first study was to assess the feasibility of IVD unloading in a large animal organ culture set-up and evaluate its impact on mechanobiology.

Introduction and Objective

Up to 30% of thoracolumbar (TL) fractures are missed in the emergency room. Failure to identify these fractures can result in neurological injuries up to 51% of the casesthis article aimed to clarify the incidence and risk factors of traumatic fractures in China. The China National Fracture Study (CNFS. Obtaining sagittal and anteroposterior radiographs of the TL spine are the first diagnostic step when suspecting a traumatic injury. In most cases, CT and/or MRI are needed to confirm the diagnosis. These are time and resource consuming. Thus, reliably detecting vertebral fractures in simple radiographic projections would have a significant impact. We aim to develop and validate a deep learning tool capable of detecting TL fractures on lateral radiographs of the spine. The clinical implementation of this tool is anticipated to reduce the rate of missed vertebral fractures in emergency rooms.

Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra.

pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity.

We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active.

The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies.

The use of stem cells transplanted into the intervertebral disc (IVD) is a promising regenerative approach to treat intervertebral disc degeneration (IDD). The aim of this study was to assess the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) loaded with human mesenchymal stem cells (hMSCs), on IVD extracellular matrix synthesis and nucleus pulposus (NP) marker expression in a whole IVD culture model.

HA was blended with batroxobin (BTX), a gelling agent activated in presence of PRP to construct a hydrogel. Bovine IVDs (n=25) were nucleotomised and filled with 1×10⁶ or 2×10⁶ hMSCs suspended in ∼150 mL of the PRP/HA/BTX hydrogel. IVDs harvested at day 0 and nucleotomised IVDs with no hMSCs and/or hydrogel were used as controls. hMSCs alone or encapsulated in the hydrogel were also cultured in well plates to examine the effect of the IVD microenvironment on hMSCs. After 1 week, tissue structure, scaffold integration and gene expression of anabolic (collagen type I, collagen type II and aggrecan), catabolic (matrix metalloproteinase 3 – MMP-3 –, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs 4) and NP cell (cytokeratin 19, carbonic anhydrase 12, cluster of differentiation 24) markers were assessed.

Histological analysis showed a good integration of the scaffold within the NP area with cell repopulation. At the gene expression level, the hMSC-loaded hydrogels demonstrated to increase disc cell anabolic and catabolic marker expression and promoted hMSC differentiation towards a NP cell phenotype.

This study demonstrated that the HA/PRP/BTX may represent a valid carrier for hMSCs being capable of stimulating cell activity and NP marker expression as well as achieving a good integration with the surrounding tissues.

Background and Purpose

Intervertebral disc (IVD) degeneration is a prominent cause of low back pain. IVD cells expressing angiopoietin-1 receptor Tie2 represent a progenitor cell population which decreases with progression of IVD degeneration. Homing of mesenchymal stem cells (MSCs) is a physiological mechanism aiming to enhance the regenerative capacity of the IVD. The purpose of this study was to assess the effect of MSC homing on the Tie2 positive IVD progenitor cell population, the IVD cell viability, and the proliferative phenotype of the IVD cells.

Orbital floor (OF) fractures are commonly treated by implanting either bioinert titanium or polyethylene implants, or by autologous grafts. A personalized implant made of biodegradable and osteopromotive poly(trimethylene carbonate) loaded with hydroxyapatite (PTMC-HA) could be a suitable alternative for patients where a permanent implant could be detrimental. A workflow was developed from the implant production using stereolithography (SLA) based on patient CT scan to the implantation and assessment its performance (i.e. implant stability, orbit position, bone formation) compared to personalised titanium implants in a repair OF defect sheep model. Implants fabrication was done using SLA of photo-crosslinkable PTMC mixed with HA [1–3]. Preclinical study: (sheep n=12, ethic number 34_2016) was conducted by first scanning the OF bone of each sheep in order to design and to fabricate patient specific implants (PSI) made of PTMC-HA. The fabricated PSI was implanted after creating OF defect. Bone formation and defect healing was compared to manually shaped titanium mesh using time-laps X-ray analyses, histology (Giemsa-Eosin staining) and sequential fluorochrome staining over 3-months. Additionally, the osteoinductive property of the biomaterials was assessed by intramuscular implantation (IM). In this study, we showed that the composite PTMC-HA allowed for ectopic bone formation after IM implantation, without requiring any biotherapeutics. In addition, we could repair OF defect on sheep using SLA-fabricated PTMC-HA with a good shape fidelity (compared to the virtual implant) and a better bone integration compared to the titanium mesh. This study opens the field of patient-specific implants made of degradable and osteoinductive scaffolds fabricated using additive manufacturing to replace advantageously autologous bone and titanium implants.

Angiogenesis is a key factor in early stages of wound healing and is crucial for tissue regeneration. Gold standard for large bone defect treatment is the transplantation of autologous bone grafts, but is not entirely satisfying (e.g. limited amount). Cell therapies and tissue engineering approaches may overcome these problems by using cells and autologous blood components obtainable by less invasive procedures. Pre-clinical studies previously showed promising results combining endothelial progenitor cells (EPCs) and mesenchymal stem cell (MSCs) in polyurethane scaffolds in presence of PRP (1). A systemic investigation of the chemical and mechanical characteristics of different PRP gels formulations suggested their potential use as sustained autologous growth factor delivery system (2). Here we investigate PRP hydrogels as autologous injectable cell delivery systems for EPCs and MSCs and their efficacy in promoting fast neo-vascularization for bone repair applications.

PRP hydrogel and corresponding platelet lysate (PL) were produced from platelet concentrates as described before (3). MSCs were isolated by Ficoll-Paque centrifugation from human bone marrow (EK_regensburg12-101-0127), and cultured in alpha MEM containing 10% FCS and 5 ng/mL basic-FGF (GIBCO). EPCs (CD133+/CD34+) were isolated from MSC fractions using magnetic-activated cell sorting (MACS®) and further cultured in IMDM (GIBCO) containing 5% FCS and 5% PL. GFP positive HUVECs are from Angio-Proteomie, (Boston, USA). Prior to gel encapsulation, MSC and EPCs were pre-stained using PKH26-red® and PKH67-green® respectively. Cells in different proportions were encapsulated in 3D PRP gels, in FDA approved Fibrin gels and in Matrigel®. The gels were cultured in Ibidi microwells placed in an onstage incubator linked to an EVOS Auto Cell Imaging System. The cellular network formation capacity of HUVEC or EPCs and MSC in different proportions was analyzed for the 3 types of hydrogels using time lapse movies recorded over a period of 14 days. Parallel cultures were performed in a classical cell culture CO₂ incubator and sample gels were taken at different time points for additional immunostaining and gene expression analysis.

Preliminary results indicate high cell viability in all of the three tested gels. PRP hydrogels present a favorable environment for the formation of a 3 dimensional cellular network in cell co-culture. The formation of these networks was apparent as early as 4 days after seeding. Networks increase in complexity and branching over time. The same was observed when cells were embedded in Matrigel®, which is known for its pro-angiogenic properties. Further experiments are currently in process looking at the involvement of MSCs in this process and the effect of PRP 3D co-culture on their differentiation.

PRP was previously shown as a potent growth factor delivery system for tissue engineering. In the present work, the high cell viability together with the 3 dimensional capillary-like networks observed at early time points suggest that PRP can also be used as an autologous cell delivery and pro-angiogenic system for bone tissue repair.

Summary

Carriers for local delivery of stem cells into degenerative intervertebral discs need to be tested under physiological loading since stem cell viability, density and differentiation, as well as carrier stability are strongly affected by loading.

Summary Statement

To test regenerative therapies for the intervertebral disc it is necessary to create a cavity in the nucleus polposus mantaining the annulus fibrosus intact. The transpedicular mechanical nucleotomy represents the best method for this purpose.

Purpose: Since the human intervertebral disc (IVD) is loaded in compression for approximately 16h per day, we investigated the effect of 16h compression loading on the magnetic resonance imaging (MRI) parameters, biochemical contents, and mechanical properties, of IVDs.

Method: Bovine caudal discs (2–3 years-old; non-degenerated) from 3-disc motion segments were injected in the NP with either 5 mg of trypsin in 40 μl Tris buffer or with Tris buffer only. The motion segments were placed in bags containing saline solution and antibiotics and were kept at 37°C throughout the experiment. The motion segments were subjected to either 16h of cyclic compression loading or were left unloaded for 16h. The motion segments were then paraffin embedded for MRI examinations, which were carried out in a 1.5T machine. The IVDs were dissected and the NP and AF were separated for biochemical and mechanical analyses. The NP and AF tissues were analyzed for contents of water, glycosaminoglycan (GAG), total collagen, and denatured collagen. Swelling pressure, compressive modulus HA, and hydraulic permeability were also measured.

Results: Loading had a significant effect on the MRI parameters (T1, T2, T1rho, MTR, ADC) of both the NP and AF tissues. Loading had a greater effect on the MR parameters and biochemical composition of the NP than trypsin. In contrast, trypsin had a larger effect on the mechanical properties. Localized trypsin injection predominantly affected the NP. T1rho was sensitive to loading and correlated with the water content of the NP and AF but not with their proteoglycan content.

Conclusion: Few studies have been directed towards developing an objective and accurate diagnostic tool in the detection and quantification of matrix and mechanical changes in early IVD degeneration. In this report, we demonstrated that MR parameters were influenced by compression loading. We also show showed specific correlations between T1rho and the structural and compositional changes in the disc. Further studies are required to determine the potential of the T1rho technique to be used as a non-invasive diagnostic tool of the biochemical and mechanical changes occurring in disc degeneration.

Purpose: Quantitative MRI is currently being tested as an early and non-invasive diagnostic tool of disc problems prior to the appearance of symptoms. The aim of the present study was to determine the effects of cyclic loading and enzymatic digestion on quantitative MRI, biochemical composition, and mechanical properties of intervertebral disc tissue.

Methods: Bovine tail segments consisting of three discs were subjected to 16h of cyclic compression loading (50N–300N–50N at 1Hz) or left unloaded for 16h while in saline solution at 37°C. Prior to loading, the nucleus pulposus were injected with either a trypsin or buffer solution. MR examinations were carried out in a 1.5T Siemens` Avanto system to measure T1 and T2 relaxation times, magnetization transfer ratio (MTR), and trace of the apparent diffusion coefficient (TrD). The nucleus pulposus and annulus fibrosus were dissected and analyzed for contents of water, glycosaminoglycan, total collagen, and denatured collagen. Cylindrical nucleus pulposus and annulus fibrosus tissue plugs were harvested, prepared, and tested under confined compression to measure compressive modulus (HA) and hydraulic permeability (k). ANOVA and linear regression analyses were performed (p< 0.05).

Results: Loading decreased the T1, T2, and TrD of NP while it increased MTR. Only water content in the nucleus pulposus was significantly influenced by loading. T1, water content, and k of the annulus fibrosus tissue were significantly reduced with loading.|Enzymatic treatment of the nucleus pulposus had no effect on its MR properties, but increased the percent of denatured collagen and thus decreased HA. None of the biochemical, mechanical, and MR parameters of the annulus fibrosus changed with trypsin treatment.

Conclusions: Dynamic loading of the disc segments for 16h decreased the permeability of both disc tissues. This was consistent with the measured drop in tissue hydration and was observed as a decrease in T1. Targeted trypsin digestion of the nucleus pulposus was confirmed with no detectable changes in the biochemical, biomechanical, or MR properties of the annulus fibrosus. Future studies will address additional quantitative MR parameters such as T1-rho, a higher strength magnet, and different enzymatic treatments. Funding: Other Education Grant Funding Parties: Canadian Institutes of Health Research, McGill William Dawson Scholar Award, and Whitaker Foundation

Purpose: Current research is focusing on the imitation of the optimal osteoconductive and osteoinductive properties of bone graft. We used in this study a β-trical-cium-phosphate scaffold impregnated with an osteogenetic transglutaminase (plasmatransglutaminase, FXIII, (Fibrogammin®)).

Methods: Release study: 27 & #946;-TCP discs (8x10mm) were impregnated with 10, 40 and 100 IU of FXIII. The release was measured with ELISA.|Animal study: A bilateral tibial metaphyseal defect (8.5 x 20 mm) was performed in eighteen adult sheep. The defects were filled with a & #946;-TCP cylinder (chronOS®, Synthes) in 16 sheep and left unfilled in 2 sheep (control group). The cylinders were impregnated with autologous venous blood, autologous bone marrow aspirate from the sternum and 125 IU FXIII in 4 sheep each or left unfilled and a daily dose of 1250 IU FXIII administered iv. over 14 days. After 6 and 12 weeks QCT, histology and histological analysis was performed.

Results: Release study: We found a linear release of FXIII with a plateau after 48 h. Until then on average 18% of the total dose was release from the scaffold. Animal study: The QCT analysis found unspecific changes in all group without any clear results regarding remodeling of the scaffold. The histological analysis showed the best bone ingrowth after 6 weeks in the bone marrow group and after 12 weeks in the local F XIII with on average 10% more bone ingrowth than in any other group. The best remodeling of the inner area in the scaffold was also seen in the local F XIII group.

Conclusions: FXIII is only partially released from a β-TCP scaffold. At least 80% of the dose remains in the scaffold after 48h. F XIII has a good osteogenetic property which is at least as good as bone marrow and better than venous blood; however a local application of F XIII is preferable to iv. administration. It stimulates osteoblast migration and proliferation in a β-TCP scaffold and causes a remodelling on the inside of the scaffold.

Funding : Commerical funding

Funding Parties : Grant by the AO Research Institute Davos, Switzerland and Synthes, Oberador, Switzerland