Padilla, Consuelo M. López de

Aging is a major risk factor for osteoarthritis (OA), but the specific mechanisms connecting aging and OA remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro, offering a model to study aging-related changes under controlled conditions. OA cartilage was obtained from an 80-year-old male and a 72-year-old female, while normal cartilage was sourced from a 26-year-old male. Chondrocyte cultures were established and sub-cultured to their Hayflick limit. Bulk RNA sequencing on early- and late-passage human articular chondrocytes identified transcriptomic changes associated with cellular aging. Early-passage OA chondrocytes already showed senescent phenotypes, unlike normal chondrocytes. All three cultures underwent 30 population doublings before replicative exhaustion, at which point all cells displayed senescence. During this process, cells lost their ability to form cartilaginous pellets. Differential gene expression analysis revealed distinct transcriptomic profiles between early- and late-passage chondrocytes and between normal and OA-derived cells. Genes related to matrix synthesis, degradation, inflammation, and the senescence-associated secretory phenotype (SASP) showed significant expression changes. Despite being a small pilot study, these findings suggest that further research into the molecular and metabolic changes during chondrocyte senescence could provide valuable insights into OA pathobiology.

Abstract Objective Aging is a major risk factor for OA, but the specific mechanisms underlying this connection remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro which provides an opportunity to study changes related to aging under controlled laboratory conditions. Methods Cartilage was recovered from two knees with OA and one normal knee. Chondrocyte cultures were established and sub-cultured until their Hayflick limit. Bulk RNA sequencing on early- and late-passage human articular chondrocytes allowed identification of transcriptomic changes associated with cellular aging. Results One male (80 years old) and one female (72 years old) patient provided OA cartilage. The donor of normal chondrocytes was a 26-year old male. Early passage chondrocytes from the two OA samples already had the phenotype of senescing cells, unlike normal chondrocytes. Nevertheless, all three chondrocyte cultures underwent 30 population doublings before replicative exhaustion, by which point all cells displayed a senescent phenotype. During this process, the cells lost their ability to form cartilaginous pellets. Differentially expressed genes (DEGs) analysis confirmed distinct transcriptomic profiles between early- and late-passage chondrocytes, as well as between cells isolated from normal and OA cartilage. Various changes in expression of genes related to cartilage matrix synthesis, degradation, inflammation and the senescence-associated secretory phenotype (SASP) were noted. Conclusions Although only a small pilot study, its data suggest that a larger and deeper study of the molecular and metabolic events accompanying the senescence of chondrocytes could provide important insights into the pathobiology of OA.

AbstractThis study defined and compared the course of native, impaired and growth factor‐stimulated bone regeneration in a rat femoral defect model. A mid‐diaphyseal defect with rigid internal fixation was surgically created in the right femur of male Fischer rats and serially analyzed over 36 weeks. Native bone regeneration was modeled using a sub‐critical, 1 mm size defect, which healed uneventfully. Critical size defects of 5 mm were used to analyze impaired bone regeneration. In a third group, the 5 mm defects were filled with 11 µg of recombinant human bone morphogenetic protein 2 (rhBMP2) impregnated onto an absorbable collagen sponge, modeling its clinical use. Native bone regeneration was characterized by endochondral ossification with progressive remodeling to ultimately resemble intact femora. An endochondral response was also observed under conditions of impaired bone regeneration, but by week 8 medullary capping occurred with fibrofatty consolidation of the tissue within the defect, resembling an atrophic non‐union. rhBMP2 treatment was associated with prolonged inflammatory cytokine expression and rapid intramembranous bone formation occurring with reduced expression of cartilage‐associated collagens. Between weeks 4 and 36, rhBMP2‐treated bones demonstrated decreased trabecular number and increased trabecular separation, which resulted in inferior mechanical properties compared with bones that healed naturally. Clinical Significance: Recombinant human bone morphogenetic protein 2 (rhBMP2) is used clinically to promote healing of long bones. Our data suggest that it drives intramembraneous ossification producing an inferior regenerate that deteriorates with time. Clinical outcomes would be improved by technologies favoring endochondral regenerative ossification.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin–eosin and immunostaining:

ABSTRACT Previously, we demonstrated that intratumoral delivery of adenoviral vector encoding single‐chain (sc) IL‐23 (Ad.scIL‐23) was able to induce systemic antitumor immunity. Here, we examined the role of IL‐23 in diabetes in nonobese diabetic mice. Intravenous delivery of Ad.scIL‐23 did not accelerate the onset of hyperglycemia but instead resulted in the development of psoriatic arthritis. Ad.scIL‐23–treated mice developed erythema, scales, and thickening of the skin, as well as intervertebral disc degeneration and extensive synovial hypertrophy and loss of articular cartilage in the knees. Immunological analysis revealed activation of conventional T helper type 17 cells and IL‐17–producing γδ T cells along with a significant depletion and suppression of T cells in the pancreatic lymph nodes. Furthermore, treatment with anti–IL‐17 antibody reduced joint and skin psoriatic arthritis pathologies. Thus, these Ad.scIL‐23–treated mice represent a physiologically relevant model of psoriatic arthritis for understanding disease progression and for testing therapeutic approaches.—Flores, R. R., Carbo, L., Kim, E., Van Meter, M., De Padilla, C. M. L., Zhao, J., Colangelo, D., Yousefzadeh, M. J., Angelini, L. A., Zhang, L., Pola, E., Vo, N., Evans, C. H., Gambotto, A., Niedernhofer, L. J., Robbins, P. D. Adenoviral gene transfer of a single‐chain IL‐23 induces psoriatic arthritis–like symptoms in NOD mice. FASEB J. 33, 9505–9515 (2019). www.fasebj.org

Objective: We investigated ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) messenger RNA levels as a biomarker of disease features in systemic lupus erythematosus. Methods: We measured and compared messenger RNA (mRNA) levels of ADAMTS13 in peripheral blood cells in patients with systemic lupus erythematosus and healthy control subjects by whole-genome microarray. We retrospectively analyzed the correlations of ADAMTS13 mRNA expression with clinical features, laboratory parameters, therapeutic features, and disease activity (according to the Systemic Lupus Erythematosus Disease Activity Index). We also examined the association of three single nucleotide polymorphisms (rs4962145, rs2285467, and rs685523) of the ADAMTS13 gene with patient characteristics. Results: In 309 patients, the median ADAMTS13 mRNA expression levels were significantly higher in blood cells of systemic lupus erythematosus patients than in 23 healthy controls ( p = .03). Notably, ADAMTS13 mRNA expression levels were significantly higher in systemic lupus erythematosus patients with a history of stroke ( p = .02) or transient ischemic attack ( p = .02). Among the three single nucleotide polymorphisms analyzed, rs2285467 was significantly associated with stroke ( p = .03) and anticardiolipin antibodies ( p = .04). Conclusions: Increased expression of ADAMTS13 mRNA in blood cells is associated with the presence of ischemic cerebrovascular disease in systemic lupus erythematosus patients and suggests a potential role for ADAMTS13 in the pathogenesis of ischemic cerebrovascular disease in these patients.