Aarhus Regenerative Orthopaedics Symposium (AROS).

The combination of modern interventional and preventive medicine has led to an epidemic of ageing. While this phenomenon is a positive consequence of an improved lifestyle and achievements in a society, the longer life expectancy is often accompanied by decline in quality of life due to musculoskeletal pain and disability. The Aarhus Regenerative Orthopaedics Symposium (AROS) 2015 was motivated by the need to address regenerative challenges in an ageing population by engaging clinicians, basic scientists, and engineers. In this position paper, we review our contemporary understanding of societal, patient-related, and basic science-related challenges in order to provide a reasoned roadmap for the future to deal with this compelling and urgent healthcare problem.

Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes.

BACKGROUND AND PURPOSE: In vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments. Native chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. We compared monolayer culture to combined 3D and hypoxic culture using quantitative gene expression analysis. METHODS: Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were used for either monolayer or scaffold culture. The scaffolds were clinically available MPEG-PLGA scaffolds (ASEED). After harvesting of cells for baseline investigation, the remainder was divided into 3 groups for incubation in conditions of normoxia (21% oxygen), hypoxia (5% oxygen), or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2, and 6 days after the baseline time point, respectively. Quantitative RT-PCR was performed using assays for RNA encoding collagen types 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyde-3-phosphate dehydrogenase relative to 2 hypoxia-stable housekeeping genes. RESULTS: Sox9, aggrecan, and collagen type 2 RNA expression increased with reduced oxygen. On day 6, the expression of collagen type 2 and aggrecan RNA was higher in 3D culture than in monolayer culture. INTERPRETATION: Our findings suggest that there was a combined positive effect of 3D culture and hypoxia on cartilage-specific gene expression. The positive effects of 3D culture alone were not detected until day 6, suggesting that seeding of chondrocytes onto a scaffold for matrix-assisted chondrocyte implantation should be performed earlier than 2 days before implantation.

Combined Bone Marrow Aspirate and Platelet-Rich Plasma for Cartilage Repair: Two-Year Clinical Results.

PURPOSE: To evaluate the clinical and biological outcome of combined bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP) on a collagen scaffold for treating cartilage lesions in the knee. METHODS AND MATERIALS: Ten patients (mean age 29.4 years, range 18-36) suffering from large full-thickness cartilage in the knee were treated with BMAC and PRP from January 2015 to December 2016. In a 1-step procedure autologous BMAC and PRP was seeded onto a collagen scaffold and sutured into the debrided defect. Patients were evaluated by clinical outcome scores (IKDC [International Knee Documentation Committee Subjective Knee Form], KOOS [Knee Injury and Osteoarthritis Outcome Score], and pain score using the Numeric Rating Scale [NRS]) preoperatively, after 3 months, and after 1 and 2 years. Second-look arthroscopies were performed (n = 7) with biopsies of the repair tissue for histology. All patients had magnetic resonance imaging (MRI) preoperatively, after 1 year, and after 2 to 3.5 years with MOCART (magnetic resonance observation of cartilage repair tissue) scores evaluating cartilage repair. RESULTS: After 1 year significant improvements were found in IKDC, KOOS symptoms, KOOS ADL (Activities of Daily Living), KOOS QOL (Quality of Life), and pain at activity. At the latest follow-up significant improvements were seen in IKDC, KOOS symptoms, KOOS QOL, pain at rest, and pain at activity. MRI MOCART score for cartilage repair improved significantly from baseline to 1-year follow-up. Histomorphometry of repair tissue demonstrated a mixture of fibrous tissue (58%) and fibrocartilage (40%). CONCLUSION: Treatment of cartilage injuries using combined BMAC and PRP improved subjective clinical outcome scores and pain scores at 1 and 2 years postoperatively. MRI and histology indicated repair tissue inferior to the native hyaline cartilage.

Mesenchymal Stem Cell Extracellular Vesicles as Adjuvant to Bone Marrow Stimulation in Chondral Defect Repair in a Minipig Model.

OBJECTIVE: This study evaluated the effects of mesenchymal stem cell-extracellular vesicles (MSC-EVs) on chondrocyte proliferation in vitro and on cartilage repair in vivo following bone marrow stimulation (BMS) of focal chondral defects of the knee. METHODS: Six adult Göttingen minipigs received 2 chondral defects in each knee. The pigs were randomized to treatment with either BMS combined with MSC-EVs or BMS combined with phosphate-buffered saline (PBS). Intraarticular injections MSC-EVs or PBS were performed immediately after closure of the surgical incisions, and at 2 and 4 weeks postoperatively. Repair was evaluated after 6 months with gross examination, histology, histomorphometry, immunohistochemistry, and micro-computed tomography (µCT) analysis of the trabecular bone beneath the defect. RESULTS: Defects treated with MSC-EVs had more bone in the cartilage defect area than the PBS-treated defects (7.9% vs. 1.5%, P = 0.02). Less than 1% of the repair tissue in both groups was hyaline cartilage. International Cartilage and Joint Preservation Society II histological scoring showed that defects treated with MSC-EVs scored lower on "matrix staining" (20.8 vs. 50.0, P = 0.03), "cell morphology" (35.4 vs. 53.8, P = 0.04), and "overall assessment" (30.8 vs. 52.9, P = 0.03). Consistently, defects treated with MSC-EVs had lower collagen II and higher collagen I areal deposition. Defects treated with MSC-EVs had subchondral bone with significantly higher tissue mineral densities than PBS-treated defects (860 mg HA/cm3 vs. 838 mg HA/cm3, P = 0.02). CONCLUSION: Intraarticular injections of MSC-EVs in conjunction with BMS led to osseous ingrowth that impaired optimal cartilage repair, while enhancing subchondral bone healing.

Bosworth fracture dislocation of the ankle: - Two case reports with perioperative illustration.

The Bosworth fracture dislocation of the ankle is rare and present difficulties in treatment if not immediately recognized. Here we present two cases with pre- and postoperative x-rays and perioperative image of the dislocation. The fracture dislocations were further complicated by talocrural dislocations and were treated with open reduction and internal fixation.

Corrigendum to 'Regenerative technologies to bed side: Evolving the regulatory' [J Orthop Translat 9 (2017) 1-7].

[This corrects the article DOI: 10.1016/j.jot.2017.02.001.].

Regenerative technologies to bed side: Evolving the regulatory framework.

There are high expectations for the clinical application of regenerative medicine technologies to treat musculoskeletal disorders. However, there are still big hurdles in bringing cell-based products to the market, mainly due to strict regulatory frameworks to approve these. Recently, the Japanese Pharmaceuticals and Medical Devices Agency adopted new regulations under legislature. The translational potential of this article is to inform on the regulations to bring experimental phase regenerative concepts to market approval in the United States and Europe, and highlight the opportunities granted by Japanese regulatory framework. Furthermore, we discuss the perspectives on the quickly evolving regulatory environment.

Distribution of pericellular matrix molecules in the temporomandibular joint and their chondroprotective effects against inflammation.

The objectives of this study were to (1) determine the distribution and synthesis of pericellular matrix (PCM) molecules (collagen VI, collagen IV and laminin) in rat temporomandibular joint (TMJ) and (2) investigate the effects of PCM molecules on chondrocytes against inflammation in osteoarthritis. Four zones (fibrous, proliferating, mature and hypertrophic) of condylar cartilage and three bands (anterior, intermediate and posterior) of disc were analysed by immunohistochemistry for the presence of PCM molecules in rat TMJs. Isolated chondrocytes were pre-treated with PCM molecules before being subjected to interleukin (IL)-1ß treatment to stimulate inflammation. The responses of the chondrocytes were analysed using gene expression, nitric oxide release and matrix metalloproteinase (MMP)-13 production measures. Histomorphometric analyses revealed that the highest areal deposition of collagen VI (67.4%), collagen IV (45.7%) and laminin (52.4%) was in the proliferating zone of TMJ condylar cartilage. No significant difference in the distribution of PCM molecules was noted among the three bands of the TMJ disc. All three PCM molecules were expressed intracellularly by chondrocytes cultured in the monolayer. Among the PCM molecules, pre-treatment with collagen VI enhanced cellular proliferation, ameliorated IL-1ß-induced MMP-3, MMP-9, MMP-13 and inducible nitric oxide synthase gene expression, and attenuated the downregulation of cartilage matrix genes, including collagen I, aggrecan and cartilage oligomeric matrix protein (COMP). Concurrently, collagen VI pretreatment inhibited nitric oxide and MMP-13 production. Our study demonstrates for the first time the distribution and role of PCM molecules, particularly collagen VI, in the protection of chondrocytes against inflammation.

Precipitant induced porosity augmentation of polystyrene preserves the chondrogenicity of human chondrocytes.

Cells constantly sense and receive chemical and physical signals from neighboring cells, interstitial fluid, and extracellular matrix, which they integrate and translate into intracellular responses. Thus, the nature of the surface on which cells are cultured in vitro plays an important role for cell adhesion, proliferation, and differentiation. Autologs chondrocyte implantation is considered the treatment of choice for larger cartilage defects in the knee. To obtain a sufficient number of chondrocytes for implantation multiple passaging is often needed, which raises concerns about the changes in the chondrogenic phenotype. In the present study, we analyzed the effect at cellular and molecular level of precipitant induced porosity augmentation (PIPA) of polystyrene surfaces on proliferation and differentiation of human chondrocytes. Human chondrocytes were isolated from healthy patients undergoing anterior cruciate ligament reconstruction and cultured on PIPA modified polystyrene surfaces. Microscopical analysis revealed topographically arranged porosity with micron pores and nanometer pits. Chondrocytes cultured on PIPA surfaces revealed no difference in cell viability and proliferation, but gene- and protein expressions of collagen type II were pronounced in the first passage of chondrocytes when compared to chondrocytes cultured on control surfaces. Additionally, an analysis of 40 kinases revealed that chondrocytes expanded on PIPA caused upregulated PI3K/mTOR pathway activation and inhibition of mTORC1 resulted in reduced sGAG synthesis. These findings indicate that PIPA modified polystyrene preserved the chondrogenicity of expanded human chondrocytes at gene and protein levels, which clinically may be attractive for the next generation of cell-culture surfaces for ex vivo cell growth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3073-3081, 2016.

Resveratrol has inhibitory effects on the hypoxia-induced inflammation and angiogenesis in human adipose tissue in vitro.

Hypoxia modulates the production of proteins involved in e.g. inflammation, angiogenesis and glucose utilization and hypoxia may therefore be an important factor underlying adipose tissue dysfunction in obesity. Resveratrol (RSV) is a natural polyphenolic compound and has been shown to have powerful anti-inflammatory effects and beneficial effects on several obesity-related complications. Thus, in the present study we investigated whether RSV has effects on hypoxic markers (GLUT-1, VEGF), hypoxia-induced key markers of inflammation (IL8, IL6), and leptin in human adipose tissue in vitro. Hypoxia was induced by incubating human adipose tissue fragments with 1% O2 for 24h as compared to 21% O2 The gene expressions were investigated by RT-PCR and protein release by Elisa. Hypoxia increases the expression of glucose transporter-1 (GLUT-1) (19-fold, p<0.001), vascular endothelial growth factor (VEGF) (10-fold, p<0.05), interleukin-8 (IL8) (8-fold, p<0.05), interleukin-6 (IL6) (5-fold, p<0.05) and leptin (9-fold). The protein levels of VEGF released to the medium was increased (8-fold, p<0.01) by hypoxia. RSV dose-dependently inhibited several of these hypoxia-induced expressions and at a concentration of 50 µM RSV almost completely inhibited the hypoxic responses at the above mentioned gene expression levels (p<0.05-p<0.001) and significantly attenuated the hypoxia-induced protein releases by 50-60%. These results demonstrate that hypoxia induces extensive changes in human adipose tissue in the expression and release of inflammation and angiogenesis-related adipokines. In addition the inhibition of hypoxia-mediated inflammation and angiogenesis might represent a novel mechanism of RSV in preventing obesity-related pathologies.