Physicochemical stimuli to enhance the quality of human engineered cartilage: the role of osmolarity and calcium

Due to the low regenerative capacity of articular cartilage, regenerative approaches are needed to treat cartilage defects and to restore the function of the tissue in the joint. However, a general drawback of current cartilage replacement tissues is an insufficient deposition of its main molecular components, type II collagen and proteoglycan. As a result, the tissue cannot withstand the demanding mechanical conditions in the joint. Recent studies of our group achieved an acute stimulation of cartilage matrix synthesis by a defined mechanical loading protocol which depended on the tissue’s glycosaminoglycan (GAG)-content and its associated fixed charge density (FCD). However, to what extend mechano-induced physicochemical sub-stimuli contribute to cartilage matrix production remains unclear. Identifying the decisive sub-parameter that contributes to load-induced stimulation of cartilage matrix synthesis would provide an easily applicable stimulus to optimize the quality of cartilage replacement tissues. Due to the essential role of osmotic pressure for cartilage function, hyperosmotic challenge appears as an important sub-parameter of the loading-response. However, the contribution of acute hyperosmotic pressure to cartilage homeostasis is unclear and models that take a cartilage typical FCD into consideration are required. Interestingly, long-term maturation of animal chondrocytes under hyperosmotic conditions enhanced the matrix content of engineered cartilage but this was so far never investigated for human 3D-cultured chondrocytes. Thus, the aim of this this study was to elucidate whether acute hyperosmotic stimulation, as a sub-parameter of mechanical compression, regulates cartilage matrix synthesis in a human engineered cartilage model at low and high FCD. In parallel, it was investigated whether long-term hyperosmotic stimulation can enhance the matrix synthesis and deposition of cartilage replacement tissue. To achieve these aims, human engineered cartilage was pre-matured for 3 or 35 days to develop a cartilage-like matrix of low or high FCD. Acute hyperosmotic stimulation on day 3 and on day 35 for 3 to 24 hours indicated that the known mechano-response markers ERK1/2, p38, NFAT5, FOS and FOSB are also immediate osmo-response markers, irrespective of the FCD content of the tissue. Opposite to previous results from mechanical loading studies, a downregulation of pro-chondrogenic SOX9 protein and BMP pathway activity indicated an anti-chondrogenic effect of short-term hyperosmotic stimulation on chondrocytes. However, this did not lead to changes in cartilage matrix synthesis at low and at high FCD. Thus, although acute hyperosmotic stimulation and mechanical compression partly triggered similar response pathways, short-term hyperosmotic pressure was no major player to influence the regulation of cartilage matrix synthesis. In the context of long-term hyperosmotic stimulation, previous studies suggested a role of the extracellular calcium microenvironment for cartilage matrix synthesis and deposition. Since articular chondrocytes (AC) and mesenchymal stromal cells (MSC) are often used cell types for the design of cartilage replacement tissues, the response of both cell types to long-term hyperosmotic stimulation was investigated using extracellular calcium. Interestingly, the here obtained data revealed that long-term hyperosmotic calcium stimulation for 35 days compromised cartilage matrix formation in AC-based cartilage replacement tissue but promoted the cartilage matrix formation in neocartilage generated from MSC. Investigation of pro- and anti-chondrogenic signaling pathways after long-term calcium stimulation indicated a specific induction of catabolic S100A4 and PTHLH expression in AC. Stimulating AC with recombinant human PTHrP1-34 peptide partly reproduced the calcium-mediated reduction of cartilage matrix deposition, suggesting a role of PTHrP for impaired cartilage matrix formation. Importantly, the inverse regulation of GAG synthesis in AC and MSC-derived chondrocytes was calcium-specific and not caused by general hyperosmotic effects. Long-term extracellular calcium stimulation, therefore, provides a novel means to enhance the cartilage matrix content of MSC-based engineered cartilage whereas such conditions should be avoided during AC neocartilage formation. Overall, this study provides important information on the role of physicochemical stimuli for cartilage matrix formation in human engineered cartilage. It was demonstrated that acute hyperosmotic pressure was no effective stimulus to influence cartilage matrix synthesis, and further studies are now needed to determine the contribution other load-induced sub-parameter for GAG synthesis. Furthermore, this study indicated that long-term high extracellular calcium treatment provides a novel means to enhance the quality of MSC-based cartilage replacement tissue by stimulating GAG synthesis and GAG deposition. For the application in osteochondral tissue engineering approaches, this implies that MSC should be the first choice for cartilage matrix deposition in the vicinity of resorbable calcified bone replacement materials. However, studies are now needed to confirm the here observed effects of soluble extracellular calcium using resorbable bone replacement material as a potential calcium source

Human Autoantibody Silencing by Immunoglobulin Light Chains

Several newly arising human antibodies are polyreactive, but in normal individuals the majority of these potentially autodestructive antibodies are removed from the repertoire by receptor editing or B cell deletion in the bone marrow. To determine what proportion of naturally arising autoantibodies can be silenced by immunoglobulin (Ig) light chain receptor editing, we replaced the light chains in 12 such antibodies with a panel of representative Igκ and Igλ chains. We found that most naturally arising autoantibodies are readily silenced by light chain exchange. Thus, receptor editing may account for most autoreactive antibody silencing in humans. Light chain complementarity determining region (CDR) isoelectric points did not correlate with silencing activity, but Igλ genes were more effective than Igκ genes as silencers. The greater efficacy of Igλ chains as silencer of autoreactivity provides a possible explanation for the expansion and altered configuration of the Igλ locus in evolution

Defective B cell tolerance checkpoints in systemic lupus erythematosus

A cardinal feature of systemic lupus erythematosus (SLE) is the development of autoantibodies. The first autoantibodies described in patients with SLE were those specific for nuclei and DNA, but subsequent work has shown that individuals with this disease produce a panoply of different autoantibodies. Thus, one of the constant features of SLE is a profound breakdown in tolerance in the antibody system. The appearance of self-reactive antibodies in SLE precedes clinical disease, but where in the B cell pathway tolerance is first broken has not been defined. In healthy humans, autoantibodies are removed from the B cell repertoire in two discrete early checkpoints in B cell development. We found these checkpoints to be defective in three adolescent patients with SLE. 25–50% of the mature naive B cells in SLE patients produce self-reactive antibodies even before they participate in immune responses as compared with 5–20% in controls. We conclude that SLE is associated with abnormal early B cell tolerance

Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase

Funding Information: Brendan M. Gabriel was supported by fellowships from the Novo Nordisk Foundation ( NNF19OC0055072 ) & the Wenner-Gren Foundation , an Albert Renold Travel Fellowship from the European Foundation for the Study of Diabetes , and an Eric Reid Fund for Methodology from the Biochemical Society . Abdalla D. Mohamed was funded initially by Sarcoma UK (grant number SUK09.2015 ), then supported by funding from Postdoctoral Fellowship Program ( Helmholtz Zentrum München, Germany ), and currently by Cancer Research UK . Publisher Copyright: © 2023 The AuthorsPeer reviewedPublisher PD

Cell death in denervated skeletal muscle is distinct from classical apoptosis

Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. The purpose of the present study was to investigate the occurrence of cell death and its mechanism in rat skeletal muscle undergoing post-denervation atrophy. We studied the expression of specific markers of apoptosis and necrosis in experimentally denervated tibialis anterior, extensor digitorum longus and soleus muscles of adult rats. Fluorescent staining of nuclear DNA with propidium iodide revealed the presence of nuclei with hypercondensed chromatin and fragmented nuclei typical of apoptotic cells in the muscle tissue 2, 4 and to a lesser extent 7 months after denervation. This finding was supported by electron microscopy of the denervated muscle. We found clear morphological manifestations of muscle cell death, with ultrastructural characteristics very similar if not identical to those considered as nuclear and cytoplasmic markers of apoptosis. With increasing time of denervation, progressive destabilization of the differentiated phenotype of muscle cells was observed. It included disalignment and spatial disorganization of myofibrils as well as their resorption and formation of myofibril-free zones. These changes initially appeared in subsarcolemmal areas around myonuclei, and by 4 months following nerve transection they were spread throughout the sarcoplasm. Despite an increased number of residual bodies and secondary lysosomes in denervated muscle, we did not find any evidence of involvement of autophagocytosis in the resorption of the contractile system. Dead muscle fibers were usually surrounded by a folded intact basal lamina; they had an intact sarcolemma and highly condensed chromatin and sarcoplasm. Folds of the basal lamina around the dead cells resulted from significant shrinkage of cell volume. Macrophages were occasionally found in close proximity to dead myocytes. We detected no manifestations of inflammation in the denervated tissue. Single myocytes expressing traits of the necrotic phenotype were very rare. A search for another marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, revealed the presence of multiple DNA fragments in cell nuclei in only a very small number of cell nuclei in 2 and 4 month denervated muscle and to less extent in 7 month denervated muscle. Virtually no TUNEL reactivity was found in normal muscle. Double labeling of tissue denervated for 2 and 4 months for genome fragmentation with the TUNEL method and for total nuclear DNA with propidium iodide demonstrated co-localization of the TUNEL-positive fragmented DNA in some of the nuclei containing condensed chromatin and in fragmented nuclei. However, the numbers of nuclei of abnormal morphology containing condensed and/or irregular patterns of chromatin distribution, as revealed by DNA staining and electron microscopy, exceeded by 33–38 times the numbers of nuclei positive for the TUNEL reaction. Thus, we found a discrepancy between the frequences of expression of morphological markers of apoptosis and DNA fragmentation in denervated muscle. This provides evidence that fragmentation of the genomic DNA is not an obligatory event during atrophy and death of muscle cells, or, alternatively, it may occur only for a short period of time during this process. Unlike classical apoptosis described in mammalian thymocytes and lymphoid cells, non-inflammatory death of muscle fibers in denervated muscle occurs a long time after the removal of myotrophic influence of the nerve and is preceded by the progressive imbalance of the state of terminal differentiation. Our results indicate that apoptosis appears to be represented by a number of distinct isotypes in animals belonging to different taxonomic groups and in different cell lineages of the same organism. Anat Rec 258:305–318, 2000. © 2000 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34287/1/10_ftp.pd

Muscle Contraction and Force: the Importance of an Ancillary Network, Nutrient Supply and Waste Removal

Muscle contraction studies often focus solely on myofibres and the proteins known to be involved in the processes of sarcomere shortening and cross-bridge cycling, but skeletal muscle also comprises a very elaborate ancillary network of capillaries, which not only play a vital role in terms of nutrient delivery and waste product removal, but are also tethered to surrounding fibres by collagen ”wires”. This paper therefore addresses aspects of the ancillary network of skeletal muscle at both a microscopic and functional level in order to better understand its role holistically as a considerable contributor to force transfer within muscular tissue

Study on the clinical application of pulsed DC magnetic technology for tracking of intraoperative head motion during frameless stereotaxy

BACKGROUND: Tracking of post-registration head motion is one of the major problems in frameless stereotaxy. Various attempts in detecting and compensating for this phenomenon rely on a fixed reference device rigidly attached to the patient's head. However, most of such reference tools are either based on an invasive fixation technique or have physical limitations which allow mobility of the head only in a restricted range of motion after completion of the registration procedure. METHODS: A new sensor-based reference tool, the so-called Dynamic Reference Frame (DRF) which is designed to allow an unrestricted, 360° range of motion for the intraoperative use in pulsed DC magnetic navigation was tested in 40 patients. Different methods of non-invasive attachment dependent on the clinical need and type of procedure, as well as the resulting accuracies in the clinical application have been analyzed. RESULTS: Apart from conventional, completely rigid immobilization of the head (type A), four additional modes of head fixation and attachment of the DRF were distinguished on clinical grounds: type B1 = pin fixation plus oral DRF attachment; type B2 = pin fixation plus retroauricular DRF attachment; type C1 = free head positioning with oral DRF; and type C2 = free head positioning with retroauricular DRF. Mean fiducial registration errors (FRE) were as follows: type A interventions = 1.51 mm, B1 = 1.56 mm, B2 = 1.54 mm, C1 = 1.73 mm, and C2 = 1.75 mm. The mean position errors determined at the end of the intervention as a measure of application accuracy were: 1.45 mm in type A interventions, 1.26 mm in type B1, 1.44 mm in type B2, 1.86 mm in type C1, and 1.68 mm in type C2. CONCLUSION: Rigid head immobilization guarantees most reliable accuracy in various types of frameless stereotaxy. The use of an additional DRF, however, increases the application scope of frameless stereotaxy to include e.g. procedures in which rigid pin fixation of the cranium is not required or desired. Thus, continuous tracking of head motion allows highly flexible variation of the surgical strategy including intraoperative repositioning of the patient without impairment of navigational accuracy as it ensures automatic correction of spatial distortion. With a dental cast for oral attachment and the alternative option of non-invasive retroauricular attachment, flexibility in the clinical use of the DRF is ensured