3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Primary Human Chondrocytes Affected by Cigarette Smoke-Therapeutic Challenges.

Although several researchers have attested deleterious effects of smoking to the musculoskeletal system, the association between smoking and the onset of osteoarthritis (OA) remains unclear. Here, we investigate the effect of cigarette smoke extract (CSE) on primary human chondrocytes. The present study demonstrates that physiological concentrations of CSE (0.1%-10%) inhibit the viability, proliferation, and matrix formation of chondrocytes in a dose- and time-dependent manner. Significant amounts of free radicals were generated by 10% of CSE and led to cell death. A clinical dosage (4 mg/mL) of dexamethasone (Dex) showed toxic effects on chondrocytes, and the long-time treatment by lower doses (4-400 µg/mL) induced hypertrophic changes in the chondrocytes. To substitute Dex, diclofenac (Dic, 1 µg/mL) and acetaminophen (Ace, 10 µg/mL) were tested and did not worsen the metabolic activity of CSE-exposed chondrocytes. Hyaluronic acid (HA, 5 mg/mL) combined with Dic or Ace significantly inhibited the oxidative stress and enhanced the viability and matrix formation of CSE-exposed chondrocytes. This study shows for the first time that CSE mediates the disruption of cartilage through inducing cell death by increasing oxidative stress, and that this effect is fortified by Dex. The deleterious effects of CSE on chondrocytes could be reversed by treatment with HA combined with first-line analgesic/anti-inflammatory agents.

Resveratrol protects primary cilia integrity of human mesenchymal stem cells from cigarette smoke to improve osteogenic differentiation in vitro.

Several studies have explored the negative effects of cigarette smoke on bone healing; however, the complex pathogenesis still remains unclear. One crucial and primary factor determining effective fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) into bone-forming cells. Recently, primary cilia, microtubule-based sensory organelles, have been shown to be critical in lineage commitment and differentiation of MSCs. Our present study indicates that exposure to cigarette smoke extract (CSE 0.1-10%) impaired osteogenic differentiation of human mesenchymal stem cell line (SCP-1) and interestingly, also affected primary cilia distribution and integrity in these cells during the differentiation. Furthermore, significant amounts of free radicals generated by CSE could be causative of primary cilia loss since treatment with 0.01% of hydrogen peroxide, a prime free radical in CSE, destroyed primary cilia in these cells. The debilitated differentiation of CSE-exposed SCP-1 cells also correlated with the significantly reduced expression of transcription factor and target genes of primary cilia-specific hedgehog signalling, a key player in osteogenic differentiation. As a treatment strategy, co-incubation of the CSE-exposed SCP-1 cells with the antioxidant resveratrol (1 µM) had a protective effect as it significantly reduced free radical production, protected the primary cilia and enhanced osteogenic differentiation. The current study shows for the first time that cigarette smoke affects primary cilia in human MSCs during osteogenic differentiation and treatment with resveratrol could reverse the effects and enhance differentiation, thus opening up potential therapeutic alternatives to treat fracture healing in smokers, in particularly, when delayed fracture healing is assumed.

Impact of Four Protein Additives in Cryogels on Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet-rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds' porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad-MSCs were osteogenically differentiated on these scaffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients' PRP.

[Implantation of a Modular Distal Femoral Replacement with Compressive Osseointegration as a Salvage Procedure in a Complex Femoral Posttraumatic Setting]. / Implantation einer modularen Knietotalendoprothese mit distalem Femurersatz mit kompressiver Osteointegration als Rückzugsoption bei komplexer femoraler posttraumatischer Ausgangssituation.

BACKGROUND: Large bone defects and losses play a crucial role in both tumour surgery and in complex primary and revision total knee replacement. The established options of cemented or uncemented long intramedullary stems are limited by large bone defects and are at risk from relatively high exposure to aseptic loosening. There is no general valid agreement on implant fixation of the distal femur. A further option is the cementless fixation method with compressive osseointegration, based on the so-called Wolff law of bone remodelling. This method was developed in order to reduce the high loosening rates in revision arthroplasty due to intense stress shielding and is intended to be applied in patients with huge bone losses. The so-called Compress® system (or CPS) allows such a distal femur reconstruction. It has mainly been applied and evaluated in tumor endoprosthetics. There are currently few data on the application of this system in complex distal femoral posttraumatic deformity or revision arthroplasty. PATIENTS: A case report of two male patients aged 59/56 years with a 1-year follow-up is presented. Both patients had a complex post-traumatic femoral deformity with bone loss, prior surgery and an ipsilateral hip replacement. Implantation was performed of a modular total knee replacement, consisting of a cemented modular tibia base plate and distal femoral replacement with cementless implant fixation by compressive osseointegration. Both patients were clinically and radiologically evaluated prospectively. RESULTS: Good clinical and radiological results were demonstrated in both patients after distal femoral replacement by compressive osseointegration. There was no need for further or revision surgery. Both patients were rapidly able to resume their jobs. The survival rates for CPS were comparable to published values with conventional procedures. There are yet no long-term results or extensive data for revision arthroplasty or posttraumatic cases. CONCLUSIONS: Besides distal femoral replacement with compressive osseointegration in oncological arthroplasty, the indication of complex distal femoral settings with large bone defects can be evaluated for daily clinical routine. Especially if there is ipsilateral total hip replacement, this option might be used to avoid interprosthetic stress risers.

Nicotine and Cotinine Inhibit Catalase and Glutathione Reductase Activity Contributing to the Impaired Osteogenesis of SCP-1 Cells Exposed to Cigarette Smoke.

Cigarette smoking has been identified as a major risk factor for osteoporosis decades ago. Several studies have shown a direct relationship between cigarette smoking, decreased bone mineral density, and impaired fracture healing. However, the mechanisms behind impaired fracture healing and cigarette smoking are yet to be elucidated. Migration and osteogenesis of mesenchymal stem/stromal cells (MSCs) into the fracture site play a vital role in the process of fracture healing. In human nicotine, the most pharmacologically active and major addictive component present in tobacco gets rapidly metabolized to the more stable cotinine. This study demonstrates that physiological concentrations of both nicotine and cotinine do not affect the osteogenic differentiation of MSCs. However, cigarette smoke exposure induces oxidative stress by increasing superoxide radicals and reducing intracellular glutathione in MSCs, negatively affecting osteogenic differentiation. Although, not actively producing reactive oxygen species (ROS) nicotine and cotinine inhibit catalase and glutathione reductase activity, contributing to an accumulation of ROS by cigarette smoke exposure. Coincubation with N-acetylcysteine or L-ascorbate improves impaired osteogenesis caused by cigarette smoke exposure by both activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and scavenging of ROS, which thus might represent therapeutic targets to support fracture healing in smokers.

Prenatal evidence of left-right asymmetries in auditory evoked responses using fetal magnetoencephalography.

Auditory evoked responses between right- and left-hemispheric derivations were investigated in 53 recordings from fetuses in the third trimester using fetal magnetoencephalography (fMEG). The side-different latency development of the component P2pm suggests an earlier maturation of certain right than homologous left hemispheric brain areas during fetal brain development.

Steroids that induce lung maturation acutely affect higher cortical function: a fetal magnetoencephalography study.

OBJECTIVE: To investigate whether steroids that induce lung maturation have acute effects on higher cortical function in the human fetus. METHODS: Cortical auditory-evoked responses (CAERs) were recorded from 10 singleton fetuses between 29 and 34 gestational weeks by fetal magnetoencephalography (fMEG) using transabdominal auditory stimulation prior to and within 3 hours after administering 2 × 12 mg of betamethasone, at an interval of 18 and 24 hours. RESULTS: The components of the CAER complex were categorized according to their latency: P2pm (186 + 20 ms, 90%), N2pm (260 + 34 ms, 50%), P3pm (474 + 36, 50%). In almost all of these cases the peak latencies of the fetal P2pm (P = .042) and P3pm (P = .043) were delayed after exposure to betamethasone (Wilcoxon rank test). The trend was also observable in N2pm (P = .08). CONCLUSION: Administration of betamethasone to expectant mothers was associated with acute change in higher cortical functions in the exposed fetuses. Implications regarding functional brain development need further evaluation.