Ex vivo organotypic bone slice culture reveals preferential chondrogenesis after sustained growth plate injury.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 µm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

Continuous iontronic chemotherapy reduces brain tumor growth in embryonic avian in vivo models.

Local and long-lasting administration of potent chemotherapeutics is a promising therapeutic intervention to increase the efficiency of chemotherapy of hard-to-treat tumors such as the most lethal brain tumors, glioblastomas (GBM). However, despite high toxicity for GBM cells, potent chemotherapeutics such as gemcitabine (Gem) cannot be widely implemented as they do not efficiently cross the blood brain barrier (BBB). As an alternative method for continuous administration of Gem, we here operate freestanding iontronic pumps - "GemIPs" - equipped with a custom-synthesized ion exchange membrane (IEM) to treat a GBM tumor in an avian embryonic in vivo system. We compare GemIP treatment effects with a topical metronomic treatment and observe that a remarkable growth inhibition was only achieved with steady dosing via GemIPs. Daily topical drug administration (at the maximum dosage that was not lethal for the embryonic host organism) did not decrease tumor sizes, while both treatment regimes caused S-phase cell cycle arrest and apoptosis. We hypothesize that the pharmacodynamic effects generate different intratumoral drug concentration profiles for each technique, which causes this difference in outcome. We created a digital model of the experiment, which proposes a fast decay in the local drug concentration for the topical daily treatment, but a long-lasting high local concentration of Gem close to the tumor area with GemIPs. Continuous chemotherapy with iontronic devices opens new possibilities in cancer treatment: the long-lasting and highly local dosing of clinically available, potent chemotherapeutics to greatly enhance treatment efficiency without systemic side-effects. SIGNIFICANCE STATEMENT: Iontronic pumps (GemIPs) provide continuous and localized administration of the chemotherapeutic gemcitabine (Gem) for treating glioblastoma in vivo. By generating high and constant drug concentrations near the vascularized growing tumor, GemIPs offer an efficient and less harmful alternative to systemic administration. Continuous GemIP dosing resulted in remarkable growth inhibition, superior to daily topical Gem application at higher doses. Our digital modelling shows the advantages of iontronic chemotherapy in overcoming limitations of burst release and transient concentration profiles, and providing precise control over dosing profiles and local distribution. This technology holds promise for future implants, could revolutionize treatment strategies, and offers a new platform for studying the influence of timing and dosing dependencies of already-established drugs in the fight against hard-to-treat tumors.

Measuring the global mechanical properties of the human thorax: Costo-vertebral articulation.

Biomechanical simulation of the human thorax, e.g. for 3D-printed rib implant optimisation, requires an accurate knowledge of the associated articulation and tissue stiffness. The present study is focusing on determining the stiffness of the costo-vertebral articulations. Specimens of rib segments including the adjacent thoracic vertebrae and ligaments were obtained from two human post-mortem bodies at four different rib levels. The rib samples were loaded with a tensile force in the local longitudinal, sagittal and transverse direction and the resulting displacement was continuously measured. The moment-angle response of the rib articulations was also determined by applying a load at the rib end in the cranial - caudal direction and measuring the resulting displacement. The torsional load response of the costo-vertebral articulations at an applied moment between -0.1 Nm and 0.1 Nm corresponded to a median range of motion of 13.2° (6.4° to 20.9°). An almost uniform stiffness was measured in all tensile loading directions. The median displacement at the defined force of 28 N was 1.41 mm in the longitudinal, 1.55 mm in the sagittal, and 1.08 mm in the transverse direction. The measured moment-angle response of the costo-vertebral articulation is in line with the data from literature. On the contrary, larger displacements in longitudinal, sagittal and transverse directions were measured compared to the values found in literature.

Proton and Carbon Ion Irradiation Changes the Process of Endochondral Ossification in an Ex Vivo Femur Organotypic Culture Model.

Particle therapy (PT) that utilizes protons and carbon ions offers a promising way to reduce the side effects of radiation oncology, especially in pediatric patients. To investigate the influence of PT on growing bone, we exposed an organotypic rat ex vivo femur culture model to PT. After irradiation, histological staining, immunohistochemical staining, and gene expression analysis were conducted following 1 or 14 days of in vitro culture (DIV). Our data indicated a significant loss of proliferating chondrocytes at 1 DIV, which was followed by regeneration attempts through chondrocytic cluster formation at 14 DIV. Accelerated levels of mineralization were observed, which correlated with increased proteoglycan production and secretion into the pericellular matrix. Col2α1 expression, which increased during the cultivation period, was significantly inhibited by PT. Additionally, the decrease in ColX expression over time was more pronounced compared to the non-IR control. The chondrogenic markers BMP2, RUNX2, OPG, and the osteogenic marker ALPL, showed a significant reduction in the increase in expression after 14 DIV due to PT treatment. It was noted that carbon ions had a stronger influence than protons. Our bone model demonstrated the occurrence of pathological and regenerative processes induced by PT, thus building on the current understanding of the biological mechanisms of bone.

Influence of the Print Envelope Temperature on the Morphology and Tensile Properties of Thermoplastic Polyolefins Fabricated by Material Extrusion and Material Jetting Additive Manufacturing.

Additive manufacturing (AM) nowadays has become a supportive method of traditional manufacturing. In particular, the medical and healthcare industry can profit from these developments in terms of personalized design and batches ranging from one to five specimens overall. In terms of polymers, polyolefins are always an interesting topic due to their low prices, inert chemistry, and crystalline structure resulting in preferable mechanical properties. Their semi-crystalline nature has some advantages but are challenging for AM due to their shrinkage and warping, resulting in geometrical inaccuracies or even layer detaching during the process. To tackle these issues, process parameter optimization is vital, with one important parameter to be studied more in detail, the print envelope temperature. It is well known that higher print envelope temperatures lead to better layer adhesion overall, but this investigation focuses on the mechanical properties and resulting morphology of a semi-crystalline thermoplastic polyolefin. Further, two different AM technologies, namely material jetting (ARBURG plastic freeforming-APF) and filament-based material extrusion, were studied and compared in detail. It was shown that higher print envelope temperatures lead to more isotropic behavior based on an evenly distributed morphology but results in geometrical inaccuracies since the material is kept in a molten state during printing. This phenomenon especially could be seen in the stress and strain values at break at high elongations. Furthermore, a different crystal structure can be achieved by setting a specific temperature and printing time, also resulting in peak values of certain mechanical properties. In comparison, better results could be archived by the APF technology in terms of mechanical properties and homogeneous morphology. Nevertheless, real isotropic part behavior could not be managed which was shown by the specimen printed vertically. Hence, a sweet spot between geometrical and mechanical properties still has to be found.

Disruption of Endochondral Ossification and Extracellular Matrix Maturation in an Ex Vivo Rat Femur Organotypic Slice Model Due to Growth Plate Injury.

Postnatal bone fractures of the growth plate (GP) are often associated with regenerative complications such as growth impairment. In order to understand the underlying processes of trauma-associated growth impairment within postnatal bone, an ex vivo rat femur slice model was developed. To achieve this, a 2 mm horizontal cut was made through the GP of rat femur prior to the organotypic culture being cultivated for 15 days in vitro. Histological analysis showed disrupted endochondral ossification, including disordered architecture, increased chondrocyte metabolic activity, and a loss of hypertrophic zone throughout the distal femur. Furthermore, altered expression patterns of Col2α1, Acan, and ColX, and increased chondrocyte metabolic activity in the TZ and MZ at day 7 and day 15 postinjury were observed. STEM revealed the presence of stem cells, fibroblasts, and chondrocytes within the injury site at day 7. In summary, the findings of this study suggest that the ex vivo organotypic GP injury model could be a valuable tool for investigating the underlying mechanisms of GP regeneration post-trauma, as well as other tissue engineering and disease studies.

Optimization and manufacture of polyetheretherketone patient specific cranial implants by material extrusion - A clinical perspective.

Polyetheretherketone (PEEK) is a high performing thermoplastic that has established itself as a 'gold-standard' material for cranial reconstruction. Traditionally, milled PEEK patient specific cranial implants (PSCIs) exhibit uniform levels of smoothness (excusing suture/drainage holes) to the touch (<1 µm) and homogenous coloration throughout. They also demonstrate predictable and repeatable levels of mechanical performance, as they are machined from isotropic material blocks. The combination of such factors inspires confidence from the surgeon and in turn, approval for implantation. However, manufacturing lead-times and affiliated costs to fabricate a PSCI are high. To simplify their production and reduce expenditure, hospitals are exploring the production of in-house PEEK PSCIs by material extrusion-based additive manufacturing. From a geometrical and morphological perspective, such implants have been produced with good-to-satisfactory clinical results. However, lack of clinical adoption persists. To determine the reasoning behind this, it was necessary to assess the benefits and limitations of current printed PEEK PSCIs in order to establish the status quo. Afterwards, a review on individual PEEK printing variables was performed in order to identify a combination of parameters that could enhance the aesthetics and performance of the PSCIs to that of milled implants/cranial bone. The findings from this review could be used as a baseline to help standardize the production of PEEK PSCIs by material extrusion in the hospital.

Measurement of global mechanical properties of human thorax: Costal cartilage.

Surgical resection of chest wall tumours may lead to a loss of ribcage stability and requires reconstruction to allow for physical thorax functioning. When titanium implants are used especially for larger, lateral defects, they tend to break. Implant failures are mainly due to specific mechanical requirements for chest-wall reconstruction which must mimic the physiological properties and which are not yet met by available implants. In order to develop new implants, the mechanical characteristics of ribs, joints and cartilages are investigated. Rib loading is highly dependent on the global thorax kinematics, making implant development substantially challenging. Costal cartilage contributes vastly to the entire thorax load-deformation behaviour, and also to rib loading patterns. Computational models of the thoracic cage require mechanical properties on the global stiffness, to simulate rib kinematics and evaluate stresses in the ribs and costal cartilage. In this study the mechanical stiffness of human costal cartilage is assessed with bending, torsion and tensile tests. The elastic moduli for the bending in four major directions ranged from 2.2 to 60.8 MPa, shear moduli ranged from 5.7 to 24.7 MPa for torsion, and tensile elastic moduli ranging from 5.6 to 29.6 MPa. This article provides mechanical properties for costal cartilage. The results of these measurements are used for the development of a whole thorax finite element model to investigate ribcage biomechanics and subsequently to design improved rib implants.

MUG500+: Database of 500 high-resolution healthy human skulls and 29 craniotomy skulls and implants.

In this article, we present a skull database containing 500 healthy skulls segmented from high-resolution head computed-tomography (CT) scans and 29 defective skulls segmented from craniotomy head CTs. Each healthy skull contains the complete anatomical structures of human skulls, including the cranial bones, facial bones and other subtle structures. For each craniotomy skull, a part of the cranial bone is missing, leaving a defect on the skull. The defects have various sizes, shapes and positions, depending on the specific pathological conditions of each patient. Along with each craniotomy skull, a cranial implant, which is designed manually by an expert and can fit with the defect, is provided. Considering the large volume of the healthy skull collection, the dataset can be used to study the geometry/shape variabilities of human skulls and create a robust statistical model of the shape of human skulls, which can be used for various tasks such as cranial implant design. The craniotomy collection can serve as an evaluation set for automatic cranial implant design algorithms.

Automatic skull defect restoration and cranial implant generation for cranioplasty.

A fast and fully automatic design of 3D printed patient-specific cranial implants is highly desired in cranioplasty - the process to restore a defect on the skull. We formulate skull defect restoration as a 3D volumetric shape completion task, where a partial skull volume is completed automatically. The difference between the completed skull and the partial skull is the restored defect; in other words, the implant that can be used in cranioplasty. To fulfill the task of volumetric shape completion, a fully data-driven approach is proposed. Supervised skull shape learning is performed on a database containing 167 high-resolution healthy skulls. In these skulls, synthetic defects are injected to create training and evaluation data pairs. We propose a patch-based training scheme tailored for dealing with high-resolution and spatially sparse data, which overcomes the disadvantages of conventional patch-based training methods in high-resolution volumetric shape completion tasks. In particular, the conventional patch-based training is applied to images of high resolution and proves to be effective in tasks such as segmentation. However, we demonstrate the limitations of conventional patch-based training for shape completion tasks, where the overall shape distribution of the target has to be learnt, since it cannot be captured efficiently by a sub-volume cropped from the target. Additionally, the standard dense implementation of a convolutional neural network tends to perform poorly on sparse data, such as the skull, which has a low voxel occupancy rate. Our proposed training scheme encourages a convolutional neural network to learn from the high-resolution and spatially sparse data. In our study, we show that our deep learning models, trained on healthy skulls with synthetic defects, can be transferred directly to craniotomy skulls with real defects of greater irregularity, and the results show promise for clinical use. Project page: https://github.com/Jianningli/MIA.

Targeted Chemotherapy of Glioblastoma Spheroids with an Iontronic Pump.

Successful treatment of glioblastoma multiforme (GBM), the most lethal tumor of the brain, is presently hampered by (i) the limits of safe surgical resection and (ii) "shielding" of residual tumor cells from promising chemotherapeutic drugs such as Gemcitabine (Gem) by the blood brain barrier (BBB). Here, the vastly greater GBM cell-killing potency of Gem compared to the gold standard temozolomide is confirmed, moreover, it shows neuronal cells to be at least 104-fold less sensitive to Gem than GBM cells. The study also demonstrates the potential of an electronically-driven organic ion pump ("GemIP") to achieve controlled, targeted Gem delivery to GBM cells. Thus, GemIP-mediated Gem delivery is confirmed to be temporally and electrically controllable with pmol min-1 precision and electric addressing is linked to the efficient killing of GBM cell monolayers. Most strikingly, GemIP-mediated GEM delivery leads to the overt disintegration of targeted GBM tumor spheroids. Electrically-driven chemotherapy, here exemplified, has the potential to radically improve the efficacy of GBM adjuvant chemotherapy by enabling exquisitely-targeted and controllable delivery of drugs irrespective of whether these can cross the BBB.

Thickness accuracy of virtually designed patient-specific implants for large neurocranial defects.

The combination of computer-aided design (CAD) techniques based on computed tomography (CT) data to generate patient-specific implants is in use for decades. However, persisting disadvantages are complicated design procedures and rigid reconstruction protocols, for example, for tailored implants mimicking the patient-specific thickness distribution of missing cranial bone. In this study we used two different approaches, CAD- versus thin-plate spline (TPS)-based implants, to reconstruct extensive unilateral and bilateral cranial defects in three clinical cases. We used CT data of three complete human crania that were virtually damaged according to the missing regions in the clinical cases. In total, we carried out 132 virtual reconstructions and quantified accuracy from the original to the generated implant and deviations in the resulting implant thickness as root-mean-square error (RMSE). Reconstructions using TPS showed an RMSE of 0.08-0.18 mm in relation to geometric accuracy. CAD-based implants showed an RMSE of 0.50-1.25 mm. RMSE in relation to implant thickness was between 0.63 and 0.70 mm (TPS) while values for CAD-based implants were significantly higher (0.63-1.67 mm). While both approaches provide implants showing a high accuracy, the TPS-based approach additionally provides implants that accurately reproduce the patient-specific thickness distribution of the affected cranial region.

AutoImplant 2020-First MICCAI Challenge on Automatic Cranial Implant Design.

The aim of this paper is to provide a comprehensive overview of the MICCAI 2020 AutoImplant Challenge. The approaches and publications submitted and accepted within the challenge will be summarized and reported, highlighting common algorithmic trends and algorithmic diversity. Furthermore, the evaluation results will be presented, compared and discussed in regard to the challenge aim: seeking for low cost, fast and fully automated solutions for cranial implant design. Based on feedback from collaborating neurosurgeons, this paper concludes by stating open issues and post-challenge requirements for intra-operative use. The codes can be found at https://github.com/Jianningli/tmi.

Dietary spermidine improves cognitive function.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

Cell Morphology on Poly(methyl methacrylate) Microstructures as Function of Surface Energy.

Whilst the significance of substrate topography as a regulator of cell function is well established, a systematic analysis of the principles underlying this is still unavailable. Here we evaluate the hypothesis that surface energy plays a decisive role in substrate-mediated modulation of cell phenotype by evaluation of cell behaviour on synthetic microstructures exhibiting pronounced differences in surface energy. These microstructures, specifically cubes and walls, were fabricated from a biocompatible base polymer, poly(methyl methacrylate), by variotherm injection molding. The dimensions of the cubes were 1 µm x 1 µm x 1 µm (height x width x length) with a periodicity of 1:1 and 1:5 and the dimensions of the walls 1 µm x 1 µm x 15 mm (height x width x length) with a periodicity of 1:1 and 1:5. Mold inserts were made by lithography and electroplating. The surface energy of the resultant microstructures was determined by static contact angle measurements. Light scanning microscopy of the morphology of NT2/D1 and MC3T3-E1 preosteoblast cells cultured on structured PMMA samples in both cases revealed a profound surface energy dependence. "Walls" appeared to promote significant cell elongation, whilst a lack of cell adhesion was observed on "cubes" with the lowest periodicity. Contact angle measurements on walls revealed enhanced surface energy anisotropy (55 mN/m max., 10 mN/m min.) causing a lengthwise spreading of the test liquid droplet, similar to cell elongation. Surface energy measurements for cubes revealed increased isotropic hydrophobicity (87° max., H2O). A critical water contact angle of ≤ 80° appears to be necessary for adequate cell adhesion. A "switch" for cell adhesion and subsequently cell growth could therefore be applied by, for example, adjusting the periodicity of hydrophobic structures. In summary cell elongation on walls and a critical surface energy level for cell adhesion could be produced for NT2/D1 and MC3T3-E1 cells by symmetrical and asymmetrical energy barrier levels. We, furthermore, propose a water-drop model providing a common physicochemical cause regarding similar cell/droplet geometries and cell adhesion on the investigated microstructures.

A 300 µm Organotypic Bone Slice Culture Model for Temporal Investigation of Endochondral Osteogenesis.

Translational studies to elucidate the response of immature bone to biologic and physical stimuli have been held back by the lack of a viable long-term functional bone explant model. This study attempts to bridge this gap between cell culture and animal model studies. In this study, we describe a methodology to derive a 300 µm organotypic femur slice comprising physiological zones (epiphysis and meta-diaphysis) essential for endochondral bone development. The unique capability of slice culture model incorporating enhanced nutrient access to distinct bone tissue components associated with linear bone growth facilitates the investigation of the orchestrated cellular transition of chondrogenic and osteogenic cells involved in endochondral bone development in an ex vivo setup. Bone slices of 300 µm were prepared from 4-day-old postnatal rats and were viable in culture up to 21 days. On days 7 and 15, an increase in chondrogenic and osteogenic modulations was confirmed in epiphysis, metaphysis, and diaphysis. An increase in osteocytes, osteoblasts, and hypertrophic cells were found at these time points, as well as a noticeable increased expression of chondrogenic and osteogenic markers (collagen II, Runx2, and osteocalcin) confirmed endochondral progression. Osteoclast-mediated bone resorption was demonstrated on day 15 by tartrate-resistant acid phosphatase staining. Attenuated total reflection infrared spectroscopic analyses, furthermore, confirmed a time-dependent increase in phosphate levels, bone minerals, and hydroxyapatite for 15 days. Our establishment of a bone slice culture model closely mimicking the in vivo cellular transitions and endochondral microenvironment of a mineralizing bone provides a vital new tool for the elucidation of cellular and endochondral mechanisms of bone development, maturation, and growth plate modulations. The presented model has the potential to be utilized in implementation of preclinical, toxicological, and therapeutic investigations.

Adjustable Polyurethane Foam as Filling Material for a Novel Spondyloplasty: Biomechanics and Biocompatibility.

OBJECTIVE: To investigate the biomechanics and biocompatibility of polyurethane (PU) foam with adjustable stiffness as a filling material for a novel spondyloplasty that is designed to reduce the risk of postoperative adjacent level fractures. METHODS: Sixty individual porcine lumbar vertebrae were randomly split into 4 groups: A, B, C, and D. Group A served as unmodified vertebral body controls. Groups B, C, and D consisted of hollowed vertebral bodies. Vertebrae of groups C and D were filled with adjustable PU foams of different stiffness. The compressive strength and stiffness of vertebrae from groups A-D were recorded and analyzed. 3T3 mouse fibroblasts were cultured with preformed PU foams for 4 days to test biocompatibility. RESULTS: The strength and stiffness of the hollowed groups were lower than in group A. However, the differences were not statistically significant between group A and group C (P > 0.05), and were obviously different between group A and group B or group D (P < 0.01 and <0.05, respectively). Moreover, the strength and stiffness after filling foams in group C or group D were significantly greater than in group B (P < 0.01 and <0.05, respectively). Live/dead staining of 3T3 cells confirmed the biocompatibility of the PU foam. CONCLUSIONS: The new PU foam shows adaptability regarding its stiffness and excellent cytocompatibility in vitro. The results support the clinical translation of the new PU foams as augmentation material in the development of a novel spondyloplasty.

Runx2 mediated Induction of Novel Targets ST2 and Runx3 Leads to Cooperative Regulation of Hypertrophic Differentiation in ATDC5 Chondrocytes.

Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2-/-) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.

Differential apoptotic response of MC3T3-E1 pre-osteoblasts to biodegradable magnesium alloys in an in vitro direct culture model.

The biodegradable magnesium-based implants have been widely utilized in medical orthopedic applications in recent years. We have recently shown that direct culture on Pure Mg and Mg2Ag alloys lead to a progressive differentiation impairment of MC3T3-E1 pre-osteoblasts. In this study, we aimed to analyze the apoptotic reaction of MC3T3-E1 cells in response to the direct culture on Pure Mg, Mg2Ag and Extreme High Pure Mg (XHP Mg) alloy samples. Our results demonstrated that long-term culturing of MC3T3-E1 cells on Pure Mg and Mg2Ag alloys induce time-dependent expression of active caspase-3 (active casp-3) and cleaved PARP-1 (cl. PARP-1), the hallmark of apoptosis reactions concomitant with a significant increase in the number of dead cells. However, direct culture on XHP Mg material results in a lower number of dead cells in comparison to Pure Mg and Mg2Ag alloys. Furthermore, XHP Mg materials influence expression of apoptotic markers in a process resembles that of observed in osteogenic condition apparently indicative of MC3T3-E1 osteodifferentiation. This study indicates that Mg alloy samples mediated differential apoptotic reactions of MC3T3-E1 cells can be ascribed to factors such as distinct topography and hydrophobicity features of Mg material surfaces, contrasting nature/composition of corrosion products as well as different impurities of these materials. Therefore, initial Mg alloys surface preparation, controlling the growth and composition of corrosion products and Mg alloys purity enhancement are necessary steps towards optimizing the Mg alloys usage in medical orthopedic applications.