Bio-artificial pleura using autologous dermal fibroblast sheets to mitigate air leaks during thoracoscopic lung resection.

Lung air leaks (LALs) due to visceral pleura injury during surgery are a difficult-to-avoid complication in thoracic surgery (TS). Reliable LAL closure is an important patient management issue after TS. We demonstrated both safeties of transplantation of a cultured human autologous dermal fibroblast sheet (DFS) to LALs. From May 2016 to March 2018, five patients who underwent thoracoscopic lung resection met all the inclusion criteria. Skin biopsies were acquired from each patient to source autologous dermal cells for DFS fabrication. During the primary culture, fibroblasts migrated from the dermal tissue pieces and proliferated to form cell monolayers. These fibroblasts were subcultured to confluence. Transplantable DFSs were fabricated from these subcultured fibroblasts that were trypsinized and seeded onto temperature-responsive culture dishes. After 10 days of fabrication culture, intact patient-specific DFS were harvested. DFSs were analyzed for fibroblast cell content and tissue contaminants prior to application. For closing intraoperative LAL, mean number of transplanted autologous DFS per patient was 6 ± 2 sheets. Mean chest drainage duration was 5.0 ± 4.8 days. The two patients with major LAL had a drainage duration of more than 7 days. All patients currently have no LAL recurrence after discharge. DFSs effectively maintain LAL closure via remodeling of the deposited extracellular matrix. The use of autologous DFSs to permanently close air leaks using a patient-derived source is expected to reduce surgical complications during high-risk lung resections.

Assessment of the Safety of Chondrocyte Sheet Implantation for Cartilage Regeneration.

We have previously studied the effects of chondrocyte sheets on the repair and regeneration of articular cartilage by using temperature-responsive culture inserts. On the basis of this work, we succeeded in rapid fabrication of chondrocyte sheets with the use of a coculture method in which inserts were placed between synoviocytes and chondrocytes. Treatment of cartilage defects using layered chondrocyte sheets promotes repair and regeneration; this method is compatible with in vivo osteoarthritis models that reproduce partial-thickness defects. In human stem cell clinical research guidelines, the Ministry of Health, Labour and Welfare (MHLW) approved several applications related to this technology. Indeed, its translation to a clinical setting is already yielding favorable results. In this study, we evaluated the risk of tumorigenesis associated with this treatment and characterized the dynamics of biological processes associated with the posttransplantation cell sheets in vivo. Furthermore, we also confirmed the safety of the procedure by using array comparative genomic hybridization (array CGH) and G-band staining to screen for deleterious genetic aberrations during prolonged subculture of cells. The safety of chondrocytes that were cultured for longer than normal was confirmed by the array CGH and G-band staining results. In addition, tumorigenicity testing confirmed that culture chondrocyte sheets are not tumorigenic. Furthermore, from the evaluation of bioluminescence imaging following implantation of the cell sheets, it was confirmed that the transplanted chondrocytes and synoviocytes remained in the knee joint and did not transfer elsewhere over time. We believe that the technique used in this study is a highly useful method for evaluating the safety of not only chondrocytes but also extensive subculturing in general.

Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts.

Conventional culture methods using temperature-responsive culture dishes require 4-5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co-culture) on temperature-responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real-time polymerase chain reaction and immunostaining of the three-layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan-1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A-disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin-1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co-cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd.

Repair of articular cartilage defect with layered chondrocyte sheets and cultured synovial cells.

In this study, we investigate the effects of treatment with layered chondrocyte sheets and synovial cell transplantation. An osteochondral defect was created of 48 Japanese white rabbits. In order to determine the effects of treatment, the following 6 groups were produced: (A) synovial cells (1.8 × 10(6) cells), (B)layered chondrocyte sheets (1.7 × 10(6) cells), (C) synovial cells (3.0 × 10(5) cells) + layered chondrocyte sheets, (D)synovial cells (6.0 × 10(5) cells) + layered chondrocyte sheets, (E)synovial cells (1.2 × 10(6) cells) + layered chondrocyte sheets, (F) osteochondral defect. Layered chondrocyte sheets and synovial cells were transplanted, sacrificed four and 12 weeks postoperatively. An incapacitance tester (Linton) was used to find trends in the weight distribution ratio of the damaged limbs after surgery. Sections were stained with Safranin-O. Repair sites were evaluated using ICRS grading system. In groups (A) to (E), the damaged limb weight distribution ratio had improved. The repair tissue stained positively with Safranin-O. Four and 12 weeks after surgery, groups (A) to (E) exhibited significantly higher scores than group (F), and groups (D) and (E) exhibited significantly higher scores than groups (A) and (B). This suggests the efficacy of combining layered chondrocyte sheets with synovial cells.

Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair.

INTRODUCTION: In this study, we investigate the efficacy of repairing an osteochondral defect in rabbit knee joints by administering bevacizumab, a humanized monoclonal anti-vascular endothelial growth factor (VEGF) antibody. METHODS: An osteochondral defect was created on the patellar groove of 20 Japanese white rabbits that were classified into two recipient groups: group B, administration of bevacizumab (100-mg intravenous injection on the day of surgery and 2 weeks later), and a control group (defect only). Rabbits were killed 1 and 3 months postoperatively. Sections were stained with safranin O. Repair sites were evaluated using the modified O'Driscoll International Cartilage Repair Society grading system. The expression of chondromodulin (ChM)-I and VEGF was evaluated using immunohistochemical analyses. RESULTS: At 1 month postoperatively, the repair site in group B was filled with cartilaginous tissue. At 3 months, the repair site retained this cartilage phenotype. At 1 month in the controls, the defects were mainly filled with fibrous tissue. At 3 months, the defect was replaced by fibrous tissue and bone. Over the 3-month period, histological scores were significantly higher in group B than in the controls. At 1 month, group B showed intense positive results for ChM-I in the bottom of the repair tissue. VEGF was also identified in the same area. In the controls, no ChM-I was observed in the repair tissue. Conversely, the remodeling hypertrophic chondrocyte layer stained intensely for VEGF. CONCLUSIONS: Intravenous administration of bevacizumab contributes to better repair of articular cartilage in an osteochondral defect model. We suggest the possibility of facilitating articular cartilage repair with anti-VEGF antibody rather than using cultured cells or artificial scaffolds.

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis.

BACKGROUND: We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically. RESULTS: In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups. CONCLUSION: At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.