IntroductionAngiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection.MethodsFirst, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group).ResultsHistologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group.ConclusionsConsidering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0427-y) contains supplementary material, which is available to authorized users.

BackgroundThere is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical.ResultsHere we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples.ConclusionsWe have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy.

The effect of using vitrified-thawed chondrocyte sheets on articular cartilage repair was examined because the methods for storing chondrocyte sheets are essential for allogeneic chondrocyte sheet transplantation. Six Japanese white rabbits were used as sources of articular chondrocytes and synovial cells. Chondrocytes were harvested from the femur, and synovial cells were harvested from inside the knee joints. After coculture of the chondrocytes with synovial cells, triple-layered chondrocyte sheets were fabricated. Eighteen rabbits were used, with six rabbits in each of three groups: osteochondral defect only (control, group A); chondrocyte sheets (group B); and vitrified-thawed chondrocyte sheets (group C). An osteochondral defect was created on the femur. After transplantation, the weight distribution ratio of the undamaged and damaged limbs was measured as a pain-alleviating effect. The rabbits were euthanized at 12 weeks, and the transplanted tissues were evaluated for histology (Safranin O staining and immunostaining) using the International Cartilage Repair Society grading system. For both evaluations, significant differences were observed between groups A and B, and between groups A and C (p < 0.05). No significant differences were observed between groups B and C. Thus, pain-alleviating effects and tissue repair were achieved using vitrified-thawed chondrocyte sheets. Copyright © 2017 John Wiley & Sons, Ltd.

We studied the ability of collagen vitrigel material to repair cartilage in vivo when used alone or with transforming growth factor-β (TGF-β). We measured the time course and quantity of TGF-β1 released from the collagen vitrigel in vitro to quantify the controlled release of TGF-β1. Over 14 days, 0.91 ng of TGF-β was released from the collagen vitrigel. Osteochondral defects were made in the femoral trochlear groove in 36 Japanese white rabbits, which were divided into three groups: untreated group (group A), collagen vitrigel-implanted group (group B), and TGF-β1-incorporated collagen vitrigel-implanted group (group C). The weight distribution ratio between the affected and unaffected limbs served as an indicator of pain. Animals were sacrificed at 4 and 12 weeks after surgery, and their tissues were assessed histologically. The weight distribution ratio increased in all groups and did not differ significantly between groups at 12 weeks. Group A needed 6 weeks to attain maximum improvement, and groups B and C showed near-maximum improvement at 4 and 2 weeks, respectively. The International Cartilage Repair Society II score improved significantly in group C relative to the other groups. These findings suggest that sustained, slow release of TGF-β caused early pain mitigation and cartilage repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2592-2602, 2017.

Platelet-activated serum (PAS) was collected from rabbits. This contains high concentrations of growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, and transforming growth factor-beta (TGF-β). PAS was injected into the knee joints of Japanese White rabbits subjected to anterior cruciate ligament transection (ACL-T) to investigate its therapeutic effects on articular cartilage. The effect of Avastin (an anti-VEGF monoclonal antibody) on VEGF expression was also investigated. The levels of VEGF, PDGF-BB, and TGF-β in PAS, platelet-rich plasma (PRP) and autologous serum from untreated rabbits were analysed by enzyme-linked immunosorbent assays. The samples (n = 24 rabbits) were divided into control (C), PAS (S), Avastin (A) and PAS + Avastin (S + A) treatment groups. Intra-articular injections were administered weekly for 7 weeks after ACL-T, during which the weight distribution ratios of the damaged limbs were evaluated. Histological evaluation was performed 12 weeks after ACL-T using Mankin score. The VEGF, PDGF-BB and TGF-β expression levels were significantly higher (P < 0.05) in the PAS than in the PRP or autologous serum samples. The weight distribution ratios of damaged limbs improved significantly after ACL-T in all treatment groups (P < 0.05). The proximal medial, distal medial and lateral aspects of joints in the treatment groups showed significant differences in Mankin scores compared with controls (P < 0.05). The damaged limb weight distribution ratios, Mankin scores and articular cartilage structure did not differ significantly among the three treatment groups, which all showed significant improvements in structure compared with controls. Copyright © 2017 John Wiley & Sons, Ltd.