Degeneration of the intervertebral disc (IVD), and subsequent low back pain, is an almost inevitable cause of disability. The underlying mechanisms are complex and current therapeutic strategies mainly focus on symptomatic relief rather than on the intrinsic regeneration of the IVD. This talk will provide an overview of special anatomical features and the composition of the IVD as well as its cellular microenvironment. Selected promising conceptional regenerative approaches will be discussed.

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity.

Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining.

HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts.

Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity.

AM specifically allows for cost-efficient production of patient-specific Orthopaedic medical devices with unusual designs and properties. A porous design allows to adjust the stiffness of metallic implants to that of the host bone. Beyond traditional metals, like titanium alloys, this talk will review the present state-of-the-art of directly printed absorbable metal families. Physicochemical, mechanical and biological properties of standardized design prototypes from all currently available metal families will be compared and their clinical application potential discussed. The impact of in vitro test environments on comparative corrosion behavior, post manufacturing aspects, and the recent status quo in biocompatibility testing and present knowledge gaps will be addressed.

Articular cartilage is a relatively hypoxic tissue with a unique extracellular matrix that is enriched with cations, resulting in an elevated interstitial fluid osmolarity. Several biomechanical and physicochemical stimuli are reported to influence chondrocyte metabolism. For regenerative in vitro applications, increasing the extracellular osmolarity above plasma level to more physiological valuesinduces chondrogenic marker expression and the differentiation of chondroprogenitor cells. Calcineurin inhibitor FK506 modulates the differentiation of primary chondrocytes under such conditions and its effect on cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling will be described. Supraphysiological osmolarity compromises chondrocyte proliferation, while physosmolarity or FK506 did not. Rather, the combination of the latter increased proteoglycan and collagen expression in chondrocytesin vitro and in situ, affecting expression of TGF-β-inducible protein TGFBI and chondrogenic (SOX9, Col2) as well as terminal differentiation markers (e.g., Col10). Surprisingly, expression of particularly minor collagens (e.g., Col9, Col11) was improved. Physiological osmolarity seems to promote terminal chondrogenic differentiation of progenitor cells through sensitization of TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 seems to enhance signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our data help explaining seemingly contradictory earlier findings and potentially benefit future cell-based cartilage repair strategies.

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.

In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.

While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE₁, ARE₂) were identified in the SOX9 promoter region. ARE₂ mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE₁ did not. A functional ARE₂ site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.

Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

Many age-related diseases affect our skeletal system, but bone health-targeting drug development strategies still largely rely on 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings.

MC3T3-E1 pre-osteoblast spheroids were cultured in V-shaped plates for 28 days in alpha-MEM (10% FCS, 1% L-Gln, 1X NEAA) with 1% pen/strep, changed every two days, and differentiation was induced by 10mM b-glycerophosphate and 50µg/ml ascorbic-acid. Osteogenic cell differentiation was assessed through profiling mRNA expression of selected osteogenic markers by efficiency corrected normalized 2^DDCq RT-qPCR. Biomineralization in spheroids was evaluated by histochemistry (Alizarin Red/von Kossa staining), Alkaline phosphatase (Alp) activity, Fourier transform infrared spectroscopy (FTIR) analyses, micro-CT analyses, and scanning electron microscopy on critical point-dried samples. GraphPad Prism 9 analyses comprised Shapiro-Wilk and Brown-Forsythe tests as well as 2-way ANOVA with Tukey post-hoc and non-parametric Kruskal-Wallis with Dunn post-hoc tests.

During mineralization, as opposed to non-mineralizing conditions, characteristic mRNA expression profiles of selected early and late osteoblast differentiation markers (e.g., RunX, Alp, Col1a1, Bglap) were observed between day 0 and 28 of culture; Alp was strongly upregulated (p<0.001) from day 7 on, followed by its enzymatic activity (p<0.001). Bglap and Col1a1 expression peaked on (p<0.001) and from day 14 on (p<0.05), respectively. IHC revealed osteocalcin staining in the spheroid core regions at day 14, while type I collagen staining of the cores was most prominent from day 21 on. Alizarin Red and Von Kossa confirmed central and radially outwards expanding mineralization patterns between day 14 and day 28, which was accompanied by a steady increase in extracellular calcium deposition over time (p<0.001). Micro-CT analyses allowed quantitative appreciation of the overall increase in mineral density over time (day21, p<0.05; d28, p<0.001), while SEM-EDX and FTIR ultimately confirmed a bone-like hydroxyapatite mineral deposition in 3D.

A novel and thoroughly characterized versatile bone-like 3D biomineralization in vitro model was established, which allows for studying effects of pharmacological interventions on bone mineralization ex vivo under physiomimetic conditions. Ongoing studies currently aim at elucidating in how far it specifically recapitulates intramembranous ossification.

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a crucial transcription factor to maintain cellular redox homeostasis, but is also affecting bone metabolism. As the association between Nrf2 and osteoporosis in elderly females is not fully elucidated, our aim was to shed light on the potential contribution of Nrf2 to the development of age-dependent osteoporosis using a mouse model.

Female wild-type (WT, n=18) and Nrf2-knockout (KO, n=12) mice were sacrificed at different ages (12 weeks=young mature adult, and 90 weeks=old), morphological cortical and trabecular properties of femoral bone analyzed by micro-computed tomography (µCT), and compared to histochemistry. Mechanical properties were derived from quasi-static compression tests and digital image correlation (DIC) used to analyze full-field strain distribution. Bone resorbing cells and aromatase expression by osteocytes were evaluated immunohistochemically and empty osteocyte lacunae counted in cortical bone. Wilcoxon rank sum test was used for data comparison and differences considered statistically significant at p<0.05.

When compared to old WT mice, old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), and cortical bone fraction (Ct.Ar/Tt.Ar). Surprisingly, these parameters were not different in skeletally mature young adult mice. Metaphyseal trabeculae were thin but present in all old WT mice, while no trabecular bone was detectable in 60% of old KO mice. Occurrence of empty osteocyte lacunae did not differ between both groups, but a significantly higher number of osteoclast-like cells and fewer aromatase-positive osteocytes were found in old KO mice. Furthermore, female Nrf2-KO mice showed an age-dependently reduced fracture resilience when compared to age-matched WT mice.

Our results confirmed lower bone quantity and quality as well as an increased number of bone resorbing cells in old female Nrf2-KO mice. Additionally, aromatase expression in osteocytes of old Nrf2-KO mice was compromised, which may indicate a chronic lack of estrogen in bones of old Nrf2-deficient mice. Thus, chronic Nrf2 loss seems to contribute to age-dependent progression of female osteoporosis.

As compared to magnesium (Mg) and iron (Fe), solid zinc (Zn)-based absorbable implants show better degradation rates. An ideal bone substitute should provide sufficient mechanical support, but pure Zn itself is not strong enough for load-bearing medical applications. Modern processing techniques, like additive manufacturing (AM), can improve mechanical strength of Zn. To better mimic the in vivo situation in the human body, we evaluated the degradation behavior of porous Zn implants in vitro under dynamic conditions. Our study applied selective laser melting (SLM) to build topographically ordered absorbable Zn implants with superior mechanical properties. Specimens were fabricated from pure Zn powder using SLM and diamond unit cell topological design. In vitro degradation was performed under both static and dynamic conditions in a custom-built set-up under cell culture conditions (37 °C, 20% O2 and 5% CO2) for up to 28 days. Mechanical properties of the porous structures were determined according to ISO 13314: 2011 at different immersion time points. Modified ISO 10993 standards were used to evaluate biocompatibility through direct cell seeding and indirect extract-based cytotoxicity tests (MTS assay, Promega) against identically designed porous titanium (Ti-6Al-4V) specimens as reference material. Twenty-four hours after cell seeding, its efficacy was evaluated by Live-Dead staining (Abcam) and further analyzed using dual channel fluorescent optical imaging (FOI) and subsequent flow cytometric quantification. Porous Zn implants were successfully produced by means of SLM with a yield strength and Young's modulus in the range of 3.9–9.6 MPa and 265–570 MPa, respectively. Dynamic flow significantly increased the degradation rate of AM porous Zn after 28 days. Results from Zn extracts were similar to Ti-6Al-4V with >95% of cellular activity at all tested time points, confirming level 0 cytotoxicity (i.e., This study clearly shows the great potential of AM porous Zn as a bone substituting material. Moreover, we demonstrate that complex topological design permits control of mechanical properties and degradation behavior.

The large bone defects with high risk of delayed bone union and pseudoarthrosis remain significant clinical challenge. Aim of the present study was the investigation of the critical size fracture healing process in transgenic mice using a novel beta-TCP scaffold. The luciferase transgenic mice strains (BALB/C-Tg(NF-kappaB-RE-luc)-Xen) and FVB/N-Tg(Vegfr2-luc)-Xen were used. Critical size fracture on femur was performed and stabilized using external fixation (RISystem). The fracture was bridged with a synthetic scaffold with and without Strontium. In consequence, the expression levels of NF-kappaB and VEGFR2 could be monitored in a longitudinal fashion using the Xenogen imaging system for two months. Animals were euthanized, serial section of femur were prepared, and the fracture sites were histologically examined. Sr reduced inflammation in the early phase of healing (15th days), but it was increased in the late healing stage. The level of VEGFR2 activity increases in the Sr doped beta-TCP group at the 15th day, the luciferase activity starts to decrease in this group and show significantly less activity compared to other groups in the second half. In the group without scaffold a connective tissue formation were observed. In both, beta-TCP and beta-TCP+Sr, the connection of newly formed tissue within integrated canals in scaffold was visible. Tissue formation in beta-TCP+Sr group was significantly higher than in the beta-TCP group, whereas the percentage of osseous tissue in relation to the newly formed tissue was in beta-TCP scaffold much more than in beta-TCP+ Sr groups. This study presents the first data regarding VEGFR2 and NF-kappB and angiogenesis activity profiles during fracture healing. The collected longitudinal data reduces the number of experimental animals in the study. Addition of strontium in scaffolds influenced the inflammation in different stage of the healing. This effect might influence the healing process and may prove to be advantageous for osteoporosis fracture healing.

Direct metal printed (DMP) porous iron implants possess promising mechanical and corrosion properties for various clinical application. Nevertheless, there is a requirement for better co-relation between in vitro and in vivo corrosion and biocompatibility behaviour of such biomaterials. Our present study evaluates absorption of porous iron implants under both static and dynamic conditions. Furthermore, this study characterizes their cytocompatibility using fibroblastic, osteogenic, endothelial and macrophagic cell types.

In vitro degradation was performed statically and dynamically in a custom-built set-up placed under cell culture conditions (37 °C, 5% CO2 and 20% O2) for 28 days. The morphology and composition of the degradation products were analysed by scanning electron microscopy (SEM, JSM-IT100, JEOL). Iron implants before and after immersion were imaged by μCT (Quantum FX, Perkin Elmer, USA). Biocompatibility was also evaluated under static and dynamic in vitro culture conditions using L929, MG-63, HUVEC and RAW 264.7 cell lines. According to ISO 10993, cytocompatibility was evaluated directly using live/dead staining (Live and Dead Cell Assay kit, Abcam) in dual channel fluorescent optical imaging (FOI) and additionally quantified by flow cytometry. Furthermore, cytotoxicity was indirectly quantified using ISO conform extracts in proliferation assays. Strut size of DMP porous iron implants was 420 microns, with a porosity of 64% ± 0.2% as measured by micro-CT. After 28 days of physiological degradation in vitro, dynamically tested samples were covered with brownish degradation products. They revealed a 5.7- fold higher weight loss than statically tested samples, without significant changes in medium pH. Mechanical properties (E = 1600–1800 MPa) of these additively manufactured implants were still within the range of the values reported for trabecular bone, even after 28 days of biodegradation. Less than 25% cytotoxicity at 85% of the investigated time points was measured with L929 cells, while MG-63 and HUVEC cells showed 75% and 60% viability, respectively, after 24 h, with a decreasing trend with longer incubations. Cytotoxicity was analysed by two-way ANOVA and post-hoc Tukey's multiple comparisons test. Under dynamic culture conditions, live-dead staining and flow cytometric quantification showed a 2.8-fold and 5.7-fold increase in L929 and MG-63 cell survival rates, respectively, as compared to static conditions.

Therefore, rationally designed and properly coated iron-based implants hold potential as a new generation of absorbable Orthopaedic implants.

Bioabsorbable metals hold a lot of potential as orthopaedic implant materials. Three metal families are currently being investigated: iron (Fe), magnesium (Mg) and zinc (Zn). Currently, however, biodegradation of such implants is poorly predictable. We thus used Direct Metal Printing to additively manufacture porous implants of a standardized bone-mimetic design and evaluated their mechanical properties and degradation behaviour, respectively, under in vivo-like conditions.

Atomized powder was manufactured to porous implants of repetitive diamond unit cells, using a ProX DMP 320 (Layerwise, Belgium) or a custom-modified ReaLizer SLM50 metal printer. Degradation behaviour was characterized under static and dynamic conditions in a custom-built bioreactor system (37ºC, 5% CO₂ and 20% O₂) for up of 28 days. Implants were characterized by micro-CT before and after in vivo-like degradation. Mechanical characterization (according to ISO 13314: 2011) was performed on an Instron machine (10kN load cell) at different immersion times in simulated body fluid (r-SBF). Morphology and composition of degradation products were analysed (SEM, JSM-IT100, JEOL). Topographically identical titanium (Ti-6Al-4V, Ti64) specimen served as reference.

Micro-CT analyses confirmed average strut sizes (420 ± 4 μm), and porosity (64%), to be close to design values. After 28 days of in vivo-like degradation, scaffolds were macroscopically covered by degradation products in an alloy-specific manner. Weight loss after cleaning also varied alloy-specifically, as did the change in pH value of the r-SBF. Corrosion time-dependent changes in Young's moduli from 1200 to 800 MPa for Mg, 1000 to 700 MPa for Zn and 48-8 MPa for iron were statistically significant.

In summary, DMP allows to accurately control interconnectivity and topology of implants from all three families and micro-structured design holds potential to optimize their degradation speed. This first systematic report sheds light into how design influences degradation behaviour under in vivo-like conditions to help developing new standards for future medical device evaluation.

Biodegradable metals as orthopaedic implant materials receive substantial scientific and clinical interest. Marketed cardiovascular products confirm good biocompatibility of iron. Solid iron biodegrades slowly in vivo and has got supra-physiological mechanical properties as compared to bone and porous implants can be optimized for specific orthopaedic applications. We used Direct Metal Printing (DMP)3 to additively manufacture (AM) scaffolds of pure iron with fine-tuned bone-mimetic mechanical properties and improved degradation behavior to characterize their biocompatibility under static and dynamic 3D culture conditions using a spectrum of different cell types.

Atomized iron powder was used to manufacture scaffolds with a repetitive diamond unit cell design on a ProX DMP 320 (Layerwise/3D Systems, Belgium). Mechanical characterization (Instron machine with a 10kN load cell, ISO 13314: 2011), degradation behavior under static and dynamic conditions (37ºC, 5% CO2 and 20% O2) for up of 28 days, with μCT as well as SEM/energy-dispersive X-ray spectroscopy (EDS) (SEM, JSM-IT100, JEOL) monitoring under in vivo-like conditions. Biocompatibility was comprehensively evaluated using a broader spectrum of human cells according to ISO 10993 guidelines, with topographically identical titanium (Ti-6Al-4V, Ti64) specimen as reference. Cytotoxicity was analyzed by two-way ANOVA and post-hoc Tukey's multiple comparisons test (α = 0.05).

By μCT, as-built strut size (420 ± 4 μm) and porosity of 64% ± 0.2% were compared to design values (400 μm and 67%, respectively). After 28 days of biodegradation scaffolds showed a 3.1% weight reduction after cleaning, while pH-values of simulated body fluids (r-SBF) increased from 7.4 to 7.8. Mechanical properties of scaffolds (E = 1600–1800 MPa) were still within the range for trabecular bone, then. At all tested time points, close to 100% biocompatibility was shown with identically designed titanium (Ti64) controls (level 0 cytotoxicity). Iron scaffolds revealed a similar cytotoxicity with L929 cells throughout the study, but MG-63 or HUVEC cells revealed a reduced viability of 75% and 60%, respectively, already after 24h and a further decreased survival rate of 50% and 35% after 72h. Static and dynamic cultures revealed different and cell type-specific cytotoxicity profiles. Quantitative assays were confirmed by semi-quantitative cell staining in direct contact to iron and morphological differences were evident in comparison to Ti64 controls.

This first report confirms that DMP allows accurate control of interconnectivity and topology of iron scaffold structures. While microstructure and chemical composition influence degradation behavior - so does topology and environmental in vitro conditions during degradation. While porous magnesium corrodes too fast to keep pace with bone remodeling rates, our porous and micro-structured design just holds tremendous potential to optimize the degradation speed of iron for application-specific orthopaedic implants. Surprisingly, the biological evaluation of pure iron scaffolds appears to largely depend on the culture model and cell type. Pure iron may not yet be an ideal surface for osteoblast- or endothelial-like cells in static cultures. We are currently studying appropriate coatings and in vivo-like dynamic culture systems to better predict in vivo biocompatibility.

Onset and progression of osteoarthritis (OA) is affected by a plethora of factors, including joint injury, obesity, aging, and heredity. This multi-factorial etiology obstructs our understanding of driving molecular mechanisms, which likely comprise an interplay between systemic and local factors. Next to biomechanical factors and cytokines, the course of OA appears to be altered by microenvironmental oxidative stress: cumulative evidence now suggests a prominent participation of cell signalling mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of cellular protective processes, in this process. Nrf2 activation through phosphorylation of mitogen-activated protein kinases (MAPKs) regulates Nrf2 target genes, like hemeoxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), or NAD(P)H Quinone Dehydrogenase 1 (NQO1) in OA chondrocytes. Maintaining high levels of HO-1 appears to be beneficial against OA development. Experimental manipulation of putative antioxidant response element (ARE) binding sites alters the in vitro expression of key transcription factors of chondrocyte markers in promoter-reporter assays. Potentially, Nrf2 is involved in autophagy, intermediary metabolism and unfolded protein response. RNAi-mediated depletion of Nrf2 further significantly abrogated anti-inflammatory and chondroprotective effects and epigenetics link transcriptional pathways of ‘N-factors’, Nrf2 and NFATs, to micro-RNA signalling. Current findings thus reveal novel mechanisms regulating extracellular matrix synthesis by chondrocytes. A further understanding of these pathways and their regulation will lead to important novel targets to slow OA progression.

The ideal bone substituting biomaterials should possess bone-mimicking mechanical properties; have of porous interconnected structure, and adequate biodegradation behaviour to enable full recovery of bony defects. Direct metal printed porous scaffolds hold potential to satisfy all these requirements and were additively manufactured (AM) from atomized WE43 magnesium alloy powder with grain sizes between 20 and 60 μm. Their micro-structure, mechanical properties, degradation behavior and biocompatibility was then evaluated in vitro. Firstly, post-processing values nicely followed design parameters. Next, Young's moduli were similar to that of trabecular bone (i.e., E = 700–800 MPa) even after 28 days of simulated in vivo-like corrosion by in vitro immersion. Also, a relatively moderate hydrogen evolution, corresponding to a calculated 19.2% of scaffold mass loss, was in good agreement with 20.7% volume reduction as derived from reconstructed μCT images. Finally, only moderate cytotoxicity (i.e., level 0, <25%), even after extensive ISO 10993-conform testing for 72 h using MG-63 cells, was determined using WE43 extracts (2 way ANOVA, post-hoc Tukey's multiple comparisons test; α = 0.05). Cytotoxicity was further evaluated by direct live-dead staining assays, revealing a higher cell death in static culture. However, intimate cell-metal contact was observed by SEM. In summary, while pure WE43 may not yet be an ideal surface for cell adhesion, this novel AM process allows for adjusting biodegradation through topological design. Our approach holds tremendous potential to develop functional and biodegradable implants for orthopaedic applications.

Introduction

In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions.

Background

Treatment of cartilage defects requires in vitro expansion of human articular chondrocytes (HACs) for autologous chondrocyte implantation (ACI). During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype (i.e. collagen type II (COL2) expression). This de-differentiation makes them inappropriate for ACI. Physiological osmolarity (i.e. 380 mOsm) improves COL2 expression in vitro, but the underlying reason is unknown. However, an accepted key regulator of chondrocyte differentiation, transforming growth factor beta (TGFβ), is known to stimulate COL2 production. In this study we aimed to elucidate if TGFβ signaling could potentially be driving the COL2 expression under physiological culture conditions.

Background

Transcription factor nuclear factor E2p45-related factor 2 (Nrf2) is crucial for controlling the antioxidant response and maintaining cellular redox homeostasis. Binding of Nrf2 to antioxidant response elements (ARE) promotes the expression of anti-oxidative stress enzymes. In osteoblasts, Nrf2 directly interacts with Runx2, a strong transcriptional activator of osteoblast-specific genes. Sox9, a key regulator of chondrocyte differentiation is dominant over Runx2 in mesenchymal chondrogenic precursors. We therefore aimed to elucidate the role of Nrf2, and its regulation of Sox9, in chondrocytes.

Adult chondrocytes experience a hypoxic environment in vivo. Culturing chondrocytes under oxygen tension that more closely resembles the in vivo situation, i.e. hypoxic conditions, has been shown to have positive effects on matrix synthesis. During redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances redifferentiation is still incompletely elucidated. We employed micro-bioreactor technology to elucidate the contribution of TGF-β superfamily ligands to the chondrocyte differentiation process under hypoxic conditions in vitro.

Dedifferentiated chondrocytes in alginate were cultured for 48 hours under hypoxic (1% pO2) or normoxic (20%) conditions, using specialized bioreactor technology. Gene expression of chondrocyte-specific markers (SOX9, COL2A1, COL1A1, AGC1 and MMP13) as well as established hypoxia-controlled genes (GDF1-, PHD3, HAS2, VEGF, COX2) and components of the TGF-β superfamily signaling pathways were analyzed by qPCR and protein expression after 48 hours in combination with TGF-β superfamily ligand-specific siRNA as well as selected TGF-β superfamily receptor inhibitors.

Hypoxic culture showed robust upregulation of the selected hypoxia-specific marker genes. In addition, well-established chondrocyte-specific markers like SOX9 and collagen type II were upregulated. TGF-β isoforms were selectively upregulated under hypoxia on both mRNA and protein level. In addition, both Activin receptor-like kinases, ALK1 and ALK5, were upregulated under hypoxia, while respective type II and III receptors were unresponsive. The hypoxia-induced COL2 expression was abrogated by TGF-β2 siRNA, as was ALK5 inhibition. Our data strongly indicates that TGF-β superfamily signaling pathways are involved in chondrocyte redifferentiation under low oxygen tension in vitro.

As cartilage has poor intrinsic repair capacity, in vitroexpansion of human articular chondrocytes (HACs) is required for cell-based therapies to treat cartilage pathologies. During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. It has been shown that physiological osmolarity (i.e. 380 mOsm) increases collagen type II (COL2) expression in vitro, but the underlying molecular mechanism is unknown. Transforming growth factor beta (TGFβ) super family members are accepted key regulators of chondrocyte differentiation and known to stimulate COL2 production. In this study we aimed to elucidate the role of TGFβ superfamily member signalling as a molecular mechanism potentially driving the COL2 expression under physiological (380 mOsm) culture conditions.

HACs from OA patients (p1) were cultured in cytokine-free medium of 280 or 380 mOsm, under standard 2D in vitroconditions, with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, BMPs and chondrocyte marker genes was evaluated by QPCR. TGFβ2 protein secretion was evaluated using ELISA and bioactivity was determined using an established reporter cell line. Involvement of BMP signaling was investigated by culturing OA HACs (p1) in the presence or absence of dorsomorphin (10 µM).

Physiological osmolarity increased TGFβ2 and TGFβ3 mRNA expression, TGFβ2 protein secretion as well as general TGFβ activity by 380 mOsm. Upon TGFβ2 isoform-specific knockdown COL2 mRNA expression was induced. TGFβ2 RNAi induced expression of several BMPs (e.g. BMP2,-4,-6) and this induction was enhanced in culture conditions with physiological osmolarity. Dorsomorphin inhibited physiological osmolarity induced COL2 mRNA expression.

TGFβ2 knockdown under 380 mOsm increases COL2 expression in human osteoarthritic chondrocytes in vitromost likely through a regulatory feedback loop via BMP signaling, which is involved in osmolarity-induced COL2 expression. Future studies will further elucidate the BMP-mediated regulatory feedback loop after TGF β2 knockdown and its influence on COL2 expression.