Cellular and Tissue Selectivity of AAV Serotypes for Gene Delivery to Chondrocytes and Cartilage.

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.

Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis.

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis.

scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints.

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 10(11) vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

HIV infection of human cartilage.

Infection of human cartilage with HIV in vivo has not previously been reported. Specimens of articular cartilage taken at postmortem from ten patients who were HIV-positive were examined. Two had AIDS and eight were believed to have stage-2 disease. The standard polymerase chain reaction (PCR) protocol was modified to allow semiquantitative analysis of the samples. Oligonucleotide primers labelled with 32P gamma-ATP were used to detect a segment of HIV DNA and a control DNA gene segment (HLA genome) to estimate the ratio of infected cells. The 32P-labelled PCR products were separated on acrylamide gels and visualised directly by autoradiography and computer densitometry. Infection of human cartilage in vivo was demonstrated in nine of the ten samples in which the PCR analysis was positive. The other did not react sufficiently to produce detectable radiolabelled PCR product despite repeated DNA digestion and extraction. Cartilage infected with HIV could be a potential source of HIV when used in operations.

Comparative study by computed radiography, histology, and scanning electron microscopy of the articular cartilage of normal goats and in chronic infection with caprine arthritis-encephalitis virus.

In the northeast of Brazil, caprine arthritis-encephalitis (CAE) is one of the key reasons for herd productivity decreasing that result in considerable economic losses. A comparative study was carried out using computed radiography (CR), histological analysis (HA), and scanning electronic microscopy (SEM) of the joints of CAE infected and normal goats. Humerus head surface of positive animals presented reduced joint space, increased bone density, and signs of degenerative joint disease (DJD). The carpal joint presented no morphological alterations in CR in any of the animals studied. Tarsus joint was the most affected, characterized by severe DJD, absence of joint space, increased periarticular soft tissue density, edema, and bone sclerosis. Histological analysis showed chronic tissue lesions, complete loss of the surface zone, absence of proteoglycans in the transition and radial zones and destruction of the cartilage surface in the CAE positive animals. Analysis by SEM showed ulcerated lesions with irregular and folded patterns on the joint surface that distinguished the limits between areas of normal and affected cartilage. The morphological study of the joints of normal and CAE positive goats deepened understanding of the alteration in the tissue bioarchitecture of the most affected joints. The SEM finding sustained previous histological reports, similar to those found for rheumatoid arthritis, suggesting that the goat infected with CAE can be considered as a potential model for research in this area.

In vivo evaluation of gene transfer into mesenchymal cells (in view of cartilage repair).

Gene transfer of specific growth factors is suitable for inducing chondrogenic differentiation of -mesenchymal cells to be used for cartilage regeneration. However, extent and quality of repair tissue formation also depend on biomechanical and metabolic influences that can only be studied in vivo. We describe three methods to evaluate viral gene transfer into mesenchymal cells in animal models of articular cartilage defects, e.g., mouse, rat and miniature pig models, focussing on the repair of hyaline cartilage tissue.

The synergistic effects of microfracture, perforated decalcified cortical bone matrix and adenovirus-bone morphogenetic protein-4 in cartilage defect repair.

We reported a technique for articular cartilage repair, consisting of microfracture, a biomaterial scaffold of perforated decalcified cortical bone matrix (DCBM) and adenovirus-bone morphogenetic protein-4 (Ad-BMP4) gene therapy. In the present study, we evaluated its effects on the quality and quantity for induction of articular cartilage regeneration. Full-thickness defects were created in the articular cartilage of the trochlear groove of rabbits. Four groups were assigned: Ad-BMP4/perforated DCBM composite (group I); perforated DCBM alone without Ad-BMP4 (group II); DCBM without perforated (group III) and microfracture alone (group IV). Animals were sacrificed 6, 12 and 24 weeks postoperation. The harvested tissues were analyzed by magnetic resonance image, scanning electron microscope, histological examination and immunohistochemistry. Group I showed vigorous and rapid repair leading to regeneration of hyaline articular cartilage at 6 weeks and to complete repair of articular cartilage and subchondral bone at 12 weeks. Groups II and III completely repaired the defect with hyaline cartilage at 24 weeks, but group II was more rapid than group III in the regeneration of repair tissue. In group IV the defects were concave and filled with fibrous tissue at 24 weeks. These findings demonstrated that this composite biotechnology can rapidly repair large areas of cartilage defect with regeneration of native hyaline articular cartilage.

Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.

OBJECTIVE: To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno-associated virus (rAAV) transduction in human articular chondrocytes. METHODS: Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0-1,000 J/m(2)) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence-activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full-thickness explants were exposed to UV light (0-200 J/m(2)), infected with rAAV-eGFP, and analyzed for transduction via immunohistochemistry. RESULTS: Toxicity from UV exposure was observed at doses > or =500 J/m(2) and > or =200 J/m(2) in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m(2) increased the mean fluorescence intensity (MFI) of the transduced cells 4-fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached approximately 100% after exposure to 100 J/m(2) of UV light and 10(3) MOIs for 8 days. Under the same conditions, 200 J/m(2) of UV light enhanced the MFI 7-fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m(2) of UV light increased the transduction efficiency 2-fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m(2) of UV light increased the transduction efficiency 3-fold (to 100%). CONCLUSION: UV light at doses of up to 200 J/m(2) (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV-infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV-activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.

[A study on the morphological characters of immortalized mandibular condylar chondrocyte].

OBJECTIVE: The purpose of this study was to compare the morphological character between immortalized mandibular condylar chondrocyte (IMCC) and primarily cultured mandibular condylar chondrocyte (MCC). METHODS: The phase contrast microscope, photomicroscope and transmission electron microscope were used to observe the morphological character of IMCC and MCC. The highresolution pathological image and word report system-1000 (HPIAS-1000) was used to compare the size of IMCC and MCC. RESULTS: The phase contrast micrography showed that MCCs in primary culture underwent distinct morphological changes with respect to shape, size, and density of the cells. The majority of MCCs were in polygonal shape earlier in culture, while more fusi-form and spindle-shaped cells were found after 4-5 passages. While IMCCs were polygonal-shaped, similar to MCCs. Subculture, freezing and recovering had no effect on cellular shape of IMCC. Transmission electron microscopy indicated that MCC had chondrocyte-like phenotype, while IMCC looked like prechondroblast or immature chondrocyte. Some of IMCCs had irregular nucleus, and the proportion of nucleus/cytoplasm changed. By analysis of HPIAS-1000, the diameter and area of IMCC were obvious smaller than those of MCC (P < 0.01). CONCLUSION: IMCC retain the main morphological character of MCC, and also keep a stable phenotype, which belong to immature chondrocytes, similar to cells in the proliferative zone.

In vivo direct gene transfer into articular cartilage by intraarticular injection mediated by HVJ (Sendai virus) and liposomes.

OBJECTIVE: To establish a system for efficient, direct in vivo gene transfer into joints. METHODS: A hemagglutinating virus of Japan (HVJ; Sendai virus)-liposome suspension containing SV40 large T antigen (SVT) gene was injected intraarticularly into knee joints of 6-week-old female Lewis rats. Rats were killed at various times for immunohistochemical analysis of the expression of SVT gene. RESULTS: The expression of SVT gene was detected immunohistochemically in chondrocytes in the superficial and middle zones of articular cartilage in the knee joints. The average percentage of SVT-positive cells was estimated to be approximately 30% on days 3, 7, 14, and 21 after transfection. Moreover, no pathologic change caused by HVJ-liposome injection was observed in the joints. CONCLUSION: The transfection frequency and stability of expression recognized in this study indicate the possibility of a strategy for treatment of joint disorders, including arthritis, using direct gene transfer.

Induction of an invasive phenotype by human parvovirus B19 in normal human synovial fibroblasts.

OBJECTIVE: To investigate the possible role of human parvovirus B19 as an etiologic agent in rheumatoid arthritis (RA), with particular emphasis on its ability to induce invasiveness in human synovial fibroblasts. METHODS: We established an experimental in vitro system in which normal primary human synovial fibroblasts were treated with or without parvovirus B19-containing human sera for 7 days. The fibroblasts were then tested for their ability to degrade reconstituted cartilage matrix using a well-characterized cartilage invasion assay system. RESULTS: Incubation with parvovirus B19-containing serum induced an invasive phenotype in normal human synovial fibroblasts. B19 serum-treated synovial fibroblasts exhibited an increase in invasion of up to 248% compared with the activity of fibroblasts in media alone, in contrast to B19-negative sera-treated synovial fibroblasts, which exhibited no significant change compared with that in media alone. In addition, preincubation of viremic serum with a neutralizing antibody to B19 abrogated the observed effect. CONCLUSION: These results provide direct evidence regarding the ability of parvovirus B19 to induce invasive properties in normal human synovial fibroblasts. Parvovirus B19 has been proposed as an etiologic agent of RA, and our data provide the first biologic link between exposure to B19 and phenotypic changes in normal human synovial fibroblasts.

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment.

A chondrogenic cell line, TC6, was established by using cells derived from articular cartilage of transgenic mice harboring a temperature-sensitive mutant simian virus (SV) 40 large T-antigen gene. TC6 cells express genes encoding proteins related to cartilage phenotypes such as type II collagen. To examine the in vivo behavior of the TC6 cells, these cells were implanted into cavity-shaped full-thickness defects made in the articular cartilage of the central part of the patellar grooves of mouse femora. One week after implantation, the morphology of the cells was still fibroblastic but these cells were just about to start to form a cartilage-like matrix. By 6 weeks after implantation, the cells had produced abundant cartilaginous matrix and their morphology became closer to that of authentic chondrocytes. This was in sharp contrast to the fibroblastic morphology of these cells in an in vitro environment even after long-term culture. These observations indicate that a cartilage-matrix environment provides a scaffold for the TC6 cells to form cartilage tissues. Our data show that the genetically engineered chondrocytic cell line, TC6, can form a cartilage-like matrix in vivo.