Citations

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.

Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (ClinicalTrials.gov Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (ClinicalTrials.gov Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress.Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540.

MicroRNAs (miRNAs) are short non-coding RNA sequences with the ability to inhibit the expression of a target mRNA at the post-transcriptional level, acting as modulators of both the degenerative and regenerative processes. Therefore, these molecules constitute a potential source of novel therapeutic tools. In this study, we investigated the miRNA expression profile that presented in enthesis tissue upon injury. For this, a rodent enthesis injury model was developed by creating a defect at a rat’s patellar enthesis. Following injury, explants were collected on days 1 (n = 10) and 10 (n = 10). Contra lateral samples (n = 10) were harvested to be used for normalization. The expression of miRNAs was investigated using a “Fibrosis” pathway-focused miScript qPCR array. Later, target prediction for the aberrantly expressed miRNAs was performed by means of the Ingenuity Pathway Analysis, and the expression of mRNA targets relevant for enthesis healing was confirmed using qPCRs. Additionally, the protein expression levels of collagens I, II, III, and X were investigated using Western blotting. The mRNA expression pattern of EGR1, COL2A1, RUNX2, SMAD1, and SMAD3 in the injured samples indicated their possible regulation by their respective targeting miRNA, which included miR-16, -17, -100, -124, -133a, -155 and -182. Furthermore, the protein levels of collagens I and II were reduced directly after the injury (i.e., day 1) and increased 10 days post-injury, while collagens III and X showed the opposite pattern of expression.

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.

Purpose of review To assess the present status of gene therapy for osteoarthritis (OA). Recent findings An expanding list of cDNAs show therapeutic activity when introduced into the joints of animals with experimental models of OA. In vivo delivery with adenovirus or adeno-associated virus is most commonly used for this purpose. The list of encoded products includes cytokines, cytokine antagonists, enzymes, enzyme inhibitors, growth factors and noncoding RNA. Elements of CRISPR-Cas have also been delivered to mouse knees to ablate key genes. Several human trials have been initiated, using transgenes encoding transforming growth factor-β 1 , interleukin-1 receptor antagonist, interferon-β, the NKX3.2 transcription factor or variant interleukin-10. The first of these, using ex vivo delivery with allogeneic chondrocytes, gained approval in Korea which was subsequently retracted. However, it is undergoing Phase III clinical trials in the United States. The other trials are in Phase I or II. No gene therapy for OA has current marketing approval in any jurisdiction. Summary Extensive preclinical data support the use of intra-articular gene therapy for treating OA. Translation is beginning to accelerate and six gene therapeutics are in clinical trials. Importantly, venture capital has begun to flow and at least seven companies are developing products. Significant progress in the future can be expected.

The use of multiphasic scaffolds to treat injured tendon-to-bone entheses has shown promising results in vitro. Here, we used two versions of a biphasic silk fibroin scaffold to treat an enthesis defect created in a rat patellar model in vivo. One version presented a mixed transition between the bony and the tendon end of the construct (S-MT) while this transition was abrupt in the second version (S-AT). At 12 weeks after surgery, the S-MT scaffold promoted better healing of the injured enthesis, with minimal undesired ossification of the insertion area. The expression of tenogenic and chondrogenic markers was sustained for longer in the S-MT-treated group and the tangent modulus of the S-MT-treated samples was similar to the native tissue at 12 weeks while that of the S-AT-treated enthesis was lower. Our study highlights the important role of the transition zone of multiphasic scaffolds in the treatment of complex interphase tissues such as the tendon-to-bone enthesis.