Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2⁺ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.Purpose: Here, we present a comprehensive protocol for sorting of Tie2⁺ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2⁺ NPCs.Methods: We present flow cytometry protocols to isolate the Tie2⁺ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2⁺ NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented.Results: Our protocols are efficient towards labeling and isolation of Tie2⁺ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.Conclusions: Current identification of Tie2⁺ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2⁺ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2⁺ fraction is the subject of future research.

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

It is not fully understood how cells detect external mechanical forces, but mechanosensitive ion channels play important roles in detecting and translating physical forces into biological responses (mechanotransduction). With the “OoClamp” device, we developed a tool to study electrophysiological processes, including the gating properties of ion channels under various gravity conditions. The “OoClamp” device uses an adapted patch clamp technique and is operational during parabolic flight and centrifugation up to 20 g. In the framework of the REXUS/BEXUS program, we have further developed the “OoClamp” device with the goal of conducting electrophysiological experiments aboard a flying sounding rocket. The aim of such an experiment was first to assess whether electrophysiological measurements of Xenopus laevis oocytes can be performed on sounding rocket flights, something that has never been done before. Second, we aimed to examine the gating properties of ion channels under microgravity conditions. The experiment was conducted in March 2016 on the REXUS 20 rocket. The post-flight analysis showed that all recording chambers were empty as the rocket reached the microgravity phase. A closer analysis of the flight data revealed that the oocytes were ripped apart a few seconds after the rocket launch. This first attempt at using sounding rockets as a research platform for electrophysiological recordings was therefore limited. Our modified “OoClamp” hardware was able to perform the necessary tasks for difficult electrophysiological recordings aboard a sounding rocket; however, the physical stresses during launch (acceleration and vibrations) did not support viability of Xenopus oocytes.

Fragility hip fractures in older adults are a major burden for the individual and the society, as they result in increased morbidity, mortality and substantial health care expenditure. Most fragility hip fractures are caused by falls, resulting in an impact on the postero-lateral aspect of the hip, inducing a force that exceeds the femoral bone strength. The risk of fracture depends on the rate of falling, the fall-induced impact force, and the femoral bone strength. This article presents the framework of a novel fragility hip fracture risk calculator based on a combined mechanistic and stochastic modelling approach. The model integrates the stochastic aspects of a fall and its dynamics together with a 1D mechanical model predicting the impact force in the hip to calculate a one-year absolute fracture risk. The required input parameters are based on QCT images, but can be substituted with more accessible anthropometric and densitometric data. To demonstrate how the parameters are extracted from clinical data and how the model is calibrated, data from the AFFIRM-CT cohort were used. A sensitivity analysis was conducted, confirming that the fall rate, the trochanteric soft tissue thickness and the bone strength are the dominating parameters influencing the risk of fragility hip fractures. Furthermore, output variables such as the predicted impact velocity and impact force aligned well with experimental data. Thus, the model is able to reflect observations from empirical data, indicating that it can capture the intrinsic aspects that define the risk of fragility hip fractures.