Osteogenesis and osteoclastogenesis on a chip: Engineering a self-assembling 3D coculture.

Healthy bone is maintained by the process of bone remodeling. An unbalance in this process can lead to pathologies such as osteoporosis which are often studied with animal models. However, data from animals have limited power in predicting the results that will be obtained in human clinical trials. In search for alternatives to animal models, human in vitro models are emerging as they address the principle of reduction, refinement, and replacement of animal experiments (3Rs). At the moment, no complete in vitro model for bone-remodeling exists. Microfluidic chips offer great possibilities, particularly because of the dynamic culture options, which are crucial for in vitro bone formation. In this study, a scaffold free, fully human, 3D microfluidic coculture model of bone remodeling is presented. A bone-on-a-chip coculture system was developed in which human mesenchymal stromal cells differentiated into the osteoblastic lineage and self-assembled into scaffold free bone-like tissues with the shape and dimensions of human trabeculae. Human monocytes were able to attach to these tissues and to fuse into multinucleated osteoclast-like cells, establishing the coculture. Computational modeling was used to determine the fluid flow induced shear stress and strain in the formed tissue. Furthermore, a set-up was developed allowing for long-term (35 days) on-chip cell culture with benefits including continuous fluid-flow, low bubble formation risk, easy culture medium exchange inside the incubator and live cell imaging options. This on-chip coculture is a crucial advance towards developing in vitro bone remodeling models to facilitate drug testing.

Local variations in mechanical properties of human hamstring tendon autografts for anterior cruciate ligament reconstruction do not translate to a mechanically inferior strand.

A ruptured anterior cruciate ligament (ACL) is often reconstructed with a multiple-strand autograft of a semitendinosus tendon alone or combined with a gracilis tendon. Up to 10% of patients experience graft rupture. This potentially results from excessive local tissue strains under physiological loading which could either result in direct mechanical failure of the graft or induce mechanobiological weakening. Since the original location in the hamstring tendon cannot be traced back from an autograft rupture site, this study explored whether clinical outcome could be further improved by avoiding specific locations or regions of human semitendinosus and/or gracilis tendons in ACL grafts due to potential mechanical or biochemical inferiority. Additionally, it examined numerically which clinically relevant graft configurations experience the lowest strains - and therefore the lowest rupture risk - when loaded with equal force. Remnant full-length gracilis tendons from human ACL reconstructions and full-length semitendinosus- and ipsilateral gracilis tendons of human cadaveric specimens were subjected to a stress-relaxation test. Locations at high risk of mechanical failure were identified using particle tracking to calculate local axial strains. As biochemical properties, the water-, collagen-, glycosaminoglycan- and DNA content per tissue region (representing graft strands) were determined. A viscoelastic lumped parameter model per tendon region was calculated. These models were applied in clinically relevant virtual graft configurations, which were exposed to physiological loading. Configurations that provided lower stiffness - i.e., experiencing higher strains under equal force - were assumed to be at higher risk of failure. Suitability of the gracilis tendon proper to replace semitendinosus muscle-tendon junction strands was examined. Deviations in local axial strains from the globally applied strain were of similar magnitude as the applied strain. Locations of maximum strains were uniformly distributed over tendon lengths. Biochemical compositions varied between tissue regions, but no trends were detected. Viscoelastic parameters were not significantly different between regions within a tendon, although semitendinosus tendons were stiffer than gracilis tendons. Virtual grafts with a full-length semitendinosus tendon alone or combined with a gracilis tendon displayed the lowest strains, whereas strains increased when gracilis tendon strands were tested for their suitability to replace semitendinosus muscle-tendon junction strands. Locations experiencing high local axial strains - which could increase risk of rupture - were present, but no specific region within any of the investigated graft configurations was found to be mechanically or biochemically deviant. Consequently, no specific tendon region could be indicated to provide a higher risk of rupture for mechanical or biochemical reasons. The semitendinosus tendon provided superior stiffness to a graft compared to the gracilis tendon. Therefore, based on our results it would be recommended to use the semitendinosus tendon, and use the gracilis tendon in cases where further reinforcement of the graft is needed to attain the desired length and cross-sectional area. All these data support current clinical standards.

Notochordal cell matrix as a bioactive lubricant for the osteoarthritic joint.

Notochordal cell derived matrix (NCM) can induce regenerative effects on nucleus pulposus cells and may exert such effects on chondrocytes as well. Furthermore, when dissolved at low concentrations, NCM forms a viscous fluid with potential lubricating properties. Therefore, this study tests the feasibility of the use of NCM as a regenerative lubricant for the osteoarthritic joint. Chondrocyte-seeded alginate beads were cultured in base medium (BM), BM with NCM (NCM), or BM with TGF-ß1 (TGF), as well as BM and NCM treated with IL-1ß. NCM increased GAG deposition and cell proliferation (stronger than TGF), and GAG/DNA ratio and hydroxyproline content (similar to TGF). These effects were maintained in the presence of IL-1ß. Moreover, NCM mitigated expression of IL-1ß-induced IL-6, IL-8, ADAMTS-5 and MMP-13. Reciprocating sliding friction tests of cartilage on glass were performed to test NCM's lubricating properties relative to hyaluronic acid (HA), and showed a dose-dependent reduction in coefficient of friction with NCM, similar to HA. NCM has anabolic and anti-inflammatory effects on chondrocytes, as well as lubricating properties. Therefore, intra-articular NCM injection may have potential as a treatment to minimize pain while restoring the affected cartilage tissue in the osteoarthritic joint.

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Potential application of notochordal cells for intervertebral disc regeneration: an in vitro assessment.

Recent studies suggest that notochordal cells (NCs) might be involved in intervertebral disc homeostasis, a role exploitable to counteract matrix degradation as observed during degeneration. This study aimed to evaluate the potential of NCs to promote matrix production by nucleus pulposus cells (NPCs) and to compare it to the currently proposed addition of bone marrow stromal cells (BMSCs). Using alginate beads, bovine NPCs were exposed for 28 d to porcine NC conditioned medium (NCCM); direct co-culture with porcine NCs or bovine BMSCs; or the combination of BMSCs and NCCM. Effects on cell proliferation, disc matrix production (proteoglycans, collagens) and disc matrix protein expression (aggrecan, collagen 1 and 2, SOX9) were determined and compared to TGFß stimulation. NCCM strongly promoted NPC proliferation (x 2.2) and matrix production (x 3.9) to levels similar to that with TGFß, whereas the direct addition of NCs had no effect. Co-culture of NPCs and BMSCs led to proteoglycan synthesis similar to NPCs alone, which was slightly improved by NCCM (x 1.5). Histological analysis confirmed biochemical data. Gene expression of analysed proteins remained stable for all groups and unaffected by medium conditions. NCs could substantially stimulate NPCs through factors secreted into conditioned medium and in levels similar to the addition of BMSCs. This study showed that molecular agents secreted by NCs constitute a promising alternative to the proposed "standard" injection of BMSCs for disc repair: their effects are similar, do not require the injection of a large number of cells and can be further amplified when the factors are identified.

Professionals' attitudes after a seclusion reduction program: anything changed?

Changing professionals' attitudes toward seclusion is seen as an important condition to reduce its use. The purpose of this study was to determine whether professionals from a mental health institute in the Netherlands changed in their attitudes toward seclusion after implementation of a multifaceted seclusion reduction program. Professionals working on four acute admission wards filled in the Professional Attitudes Toward Seclusion Questionnaire (PATS-Q) before and after a seclusion reduction program. Changes were analyzed by comparing mean scores on the PATS-Q. After the program, professionals scored significantly higher on 'ethics' and 'more care'. As expected, no change occurred on 'reasons' for the use of seclusion. In addition, no significant changes were found on 'confidence', 'better care' and 'other care'. Significant changes in professional attitudes concerning the ethics of using seclusion and involving issues of more care were observed after a seclusion reduction program. Mental health professionals moved in the direction of 'transformers', indicating an increased criticism of the practice of seclusion and increased willingness to change their own use of seclusion.