Generation of vestibular tissue-like organoids from human pluripotent stem cells using the rotary cell culture system

Hair cells are specialized mechanosensitive cells responsible for mediating balance and hearing within the inner ear. In mammals, hair cells are limited in number and do not regenerate. Human pluripotent stem cells (hPSCs) provide a valuable source for deriving human hair cells to study their development and design therapies to treat and/or prevent their degeneration. In this study we used a dynamic 3D Rotary Cell Culture System (RCCS) for deriving inner ear organoids from hPSCs. We show RCCS-derived organoids recapitulate stages of inner ear development and give rise to an enriched population of hair cells displaying vestibular-like morphological and physiological phenotypes, which resemble developing human fetal inner ear hair cells as well as the presence of accessory otoconia-like structures. These results show that hPSC-derived organoids can generate complex inner ear structural features and be a resource to study inner ear development

Accelerating functional gene discovery in osteoarthritis

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Quantitative Evaluation of Mechanical Properties in Tissue-Engineered Auricular Cartilage

ISSN:1937-3368ISSN:1937-337

Multimodal imaging demonstrates concomitant changes in bone and cartilage after destabilisation of the medial meniscus and increased joint laxity.

OBJECTIVE: Alterations in joint mechanics can cause osteoarthritis, which results in degeneration of both cartilage and bone tissue. The objective of this work is to measure changes in the laxity of the mouse knee joint after destabilisation of the medial meniscus (DMM) and to visualise and quantify the resulting three-dimensional changes in the bone and cartilage. METHODS: Skeletally mature C57Bl6 male mice underwent DMM surgery in the right leg. Animals were sacrificed immediately 0 weeks (n=15), 4 weeks (n=11) or 8 weeks (n=12) after surgery. For the 0-week group, the anterior-posterior (AP) and varus-valgus laxity of the DMM limb were compared to the contralateral limb. For 4 and 8-week groups, tibiae were scanned with micro-computed tomography (μCT) to quantify and visualise bone changes and with confocal scanning laser microscopy (CSLM) to measure changes in cartilage. RESULTS: Laxity testing measured an increase in AP range of motion, particularly in the anterior direction. The DMM limbs showed a decrease in epiphyseal trabecular bone at 8 weeks and a decrease in cartilage volume, primarily on the posterior medial plateau, compared to the contralateral limb. Significant bone remodelling was observed at the periphery of the joint and in severe cases, osteolysis extended through the growth plate. CONCLUSION: Multimodal imaging allowed quantifiable 3D assessment of bone and cartilage and indicated extensive changes in the tissues. The increase in AP laxity suggests that DMM surgery redistributes loading posteriorly on the medial plateau, resulting in bone and cartilage loss primarily on the posterior portion of the medial plateau

Cartilage Regeneration in the Head and Neck Area: Combination of Ear or Nasal Chondrocytes and Mesenchymal Stem Cells Improves Cartilage Production

Background: Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. Methods: Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. Results: In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. Conclusions: By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required. CLINICAL QUESTIONS/LEVEL OF EVIDENCE: Therapeutic, V

High-Resolution Peripheral Quantitative Computed Tomography for Bone Evaluation in Inflammatory Rheumatic Disease.

High resolution peripheral quantitative computed tomography (HR-pQCT) is a 3-dimensional imaging modality with superior sensitivity for bone changes and abnormalities. Recent advances have led to increased use of HR-pQCT in inflammatory arthritis to report quantitative volumetric measures of bone density, microstructure, local anabolic (e.g., osteophytes, enthesiophytes) and catabolic (e.g., erosions) bone changes and joint space width. These features may be useful for monitoring disease progression, response to therapy, and are responsive to differentiating between those with inflammatory arthritis conditions and healthy controls. We reviewed 69 publications utilizing HR-pQCT imaging of the metacarpophalangeal (MCP) and/or wrist joints to investigate arthritis conditions. Erosions are a marker of early inflammatory arthritis progression, and recent work has focused on improvement and application of techniques to sensitively identify erosions, as well as quantifying erosion volume changes longitudinally using manual, semi-automated and automated methods. As a research tool, HR-pQCT may be used to detect treatment effects through changes in erosion volume in as little as 3 months. Studies with 1-year follow-up have demonstrated progression or repair of erosions depending on the treatment strategy applied. HR-pQCT presents several advantages. Combined with advances in image processing and image registration, individual changes can be monitored with high sensitivity and reliability. Thus, a major strength of HR-pQCT is its applicability in instances where subtle changes are anticipated, such as early erosive progression in the presence of subclinical inflammation. HR-pQCT imaging results could ultimately impact decision making to uptake aggressive treatment strategies and prevent progression of joint damage. There are several potential areas where HR-pQCT evaluation of inflammatory arthritis still requires development. As a highly sensitive imaging technique, one of the major challenges has been motion artifacts; motion compensation algorithms should be implemented for HR-pQCT. New research developments will improve the current disadvantages including, wider availability of scanners, the field of view, as well as the versatility for measuring tissues other than only bone. The challenge remains to disseminate these analysis approaches for broader clinical use and in research

The influence of matrix elasticity on chondrocyte behavior in 3D

Cells actively probe the stiffness of their surrounding and respond to it. The authors recently found that maintenance of the chondrogenic phenotype was directly influenced by this property in 2D. Since studies about this process in 3D are still largely absent, this study aimed to transfer this knowledge into a 3D environment. Agarose was modified with RGD to allow active stiffness sensing or RGE as a control. Hydrogels with different mechanical properties were produced by using different concentrations of agarose. Primary chondrocytes were incorporated into the gel, cultured for up to two weeks, and then constructs were analyzed. Cells were surrounded by their own ECM from an early stage and maintained their chondrogenic phenotype, independent of substrate composition, as indicated by a high collagen type II and a lack of collagen type I production. However, softer gels showed higher DNA and GAG content and larger cell clusters than stiff gels in both RGD- and RGE-modified agarose. The authors hypothesize that matrix elasticity in the tested range does not influence the maintenance of the chondrogenic phenotype in 3D but rather the size of the formed cell ECM clusters. The deviation of these findings from previous results in 2D stresses the importance of moving towards 3D systems that more closely mimic in vivo conditions. © 2011 John Wiley & Sons, Ltd