European cells & materials最新文献

Extracellular matrix (ECM) biomaterials have shown promise for treating small artucular-joint defetcs. However, ECM-based biomaterials generally lack appropriate mechanical properties to support physiological loads and are prone to delamination in larger cartilage defects. To overcome these common mechanical limitations, a collagen hyaluronic-acid (CHyA) matrix, with proven regenerative potential, was reinforced with a bioabsorbable 3D-printed framework to support physiological loads. Polycaprolactone (PCL) was 3D-printed in two configurations, rectilinear and gyroid designs, that were extensively mechanically characterised. Both scaffold designs increased the compressive modulus of the CHyA matrices by three orders of magnitude, mimicking the physiological range (0.5-2.0 MPa) of healthy cartilage. The gyroid scaffold proved to be more flexible compared to the rectilinear scaffold, thus better contouring to the curvature of a femoral condyle. Additionally, PCL reinforcement of the CHyA matrix increased the tensile modulus and allowed for suture fixation of the scaffold to the subchondral bone, thus addressing the major challenge of biomaterial fixation to articular joint surfaces in shallow defects. In vitro evaluation confirmed successful infiltration of human mesenchymal stromal cells (MSCs) within the PCL-CHyA scaffolds, which resulted in increased production of sulphated glycosaminoglycans (sGAG/DNA; p = 0.0308) compared to non-reinforced CHyA matrices. Histological staining using alcian blue confirmed these results, while also indicating greater spatial distribution of sGAG throughout the PCL-CHyA scaffold. These findings have a great clinical importance as they provide evidence that reinforced PCL-CHyA scaffolds, with their increased chondroinductive potential and compatibility with joint fixation techniques, could be used to repair large-area chondral defects that currently lack effective treatment options.

Osteoarthritis (OA) is a leading cause of chronic pain and disability, for which there is no cure. Mesenchymal stromal cells (MSCs) have been used in clinical trials for treating OA due to their unique ability to generate paracrine anti-inflammatory and trophic signals. Interestingly, these studies have shown mainly short-term effects of MSCs in improving pain and joint function, rather than sustained and consistent benefits. This may reflect a change or loss in the therapeutic effects of MSCs after intra-articular injection. The present study aimed to unravel the reasons behind the variable efficacy of MSC injections for OA using an in vitro co-culture model. Osteoarthritic human synovial fibroblasts (OA-HSFs) were co-cultured with MSCs to investigate their reciprocal effects on cell responses and whether a short-term exposure of OA cells to MSCs was sufficient for reducing their diseased characteristics in a sustained manner. Gene expression and histological analyses were performed. OA-HSFs exposed to MSCs showed short-term downregulation of inflammatory markers. However, the MSCs showed upregulation of inflammatory markers and impaired ability to undergo osteogenesis and chondrogenesis in the presence of OA-HSFs. Moreover, short-term exposure of OA-HSFs to MSCs was found to be insufficient for inducing sustained changes to their diseased behaviour. These findings suggested that MSCs may not provide long-term effects in correcting the OA joint environment due to them adopting the diseased phenotype of the surrounding tissues, which has important implications for the future development of effective stem-cell-based OA treatments with long-term therapeutic efficacy.

Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.

Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells' phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

Recent studies highlighted the crucial contribution of subchondral bone to OA development. Yet, only limited data have been reported on the relation between alteration to cartilage morphology, structural properties of the subchondral bone plate (SBP) and underlying subchondral trabecular bone (STB). Furthermore, the relationship between the morphometry of the cartilage and bone in the tibial plateau and the OA-induced changes in the joint's mechanical axis remains unexplored. Therefore, a visualisation and quantification of cartilage and subchondral bone microstructure in the medial tibial plateau was performed. End stage knee-OA patients with varus alignment and scheduled for total knee arthroplasty (TKA) underwent preoperative fulllength radiography to measure the hip-knee-ankle angle (HKA) and the mechanical-axis deviation (MAD). 18 tibial plateaux were μ-CT scanned (20.1 μm/voxel). Cartilage thickness, SBP, and STB microarchitecture were quantified in 10 volumes of interest (VOIs) in each medial tibial plateau. Significant differences (p < 0.001) were found for cartilage thickness, SBP, and STB microarchitecture parameters among the VOIs. Closer to the mechanical axis, cartilage thickness was consistently smaller, while SBP thickness and STB bone volume fraction (BV/TV) were higher. Moreover, trabeculae were also more superior-inferiorly oriented, i.e. perpendicular to the transverse plane of the tibial plateau. As cartilage and subchondral bone changes reflect responses to local mechanical loading patterns in the joint, the results suggested that region-specific subchondral bone adaptations were related to the degree of varus deformity. More specifically, subchondral sclerosis appeared to be most pronounced closer to the mechanical axis of the knee.