Yu Mochizuki

Beneficial effect of basic fibroblast growth factor on the repair of full-thickness defects in rabbit articular cartilage

Abstract The effects of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness cartilage defects were examined. Four-millimeter diameter, cylindrical defects were made in rabbit articular cartilage and were filled with human recombinant bFGF. The addition of bFGF to the defect induced the formation of a thick cartilage layer composed of chondrocytes and a metachromatic-stained matrix after 6 weeks. The score of the bFGF-treated tissue, as evaluated by a semiquantitative histological scale, was significantly higher than that of the untreated tissue. At 24 weeks, the cartilage-like matrix that contained the proteoglycans and type II collagen was thicker in the bFGF-treated tissue than in the untreated tissue. Immunohistochemical analysis of the tissues at 6–12 weeks with an anti-bFGF monoclonal antibody suggested that a single application of bFGF increased the number of differentiating chondrocytes that synthesized bFGF at a high level. In contrast, immunostaining of the tissues at 6–12 weeks with a monoclonal antibody against proliferating cell nuclear antigen showed that the number of proliferating cells in the bFGF-treated tissue was fewer than in the untreated tissue. These findings suggest that administration of bFGF into cartilagenous defects promotes the differentiation of chondrocytes and their matrix synthesis, and that this growth factor is useful for improving cartilage repair.

Induction of apoptosis in the synovium of mice with autoantibody-mediated arthritis by the intraarticular injection of double-stranded microRNA-15a

OBJECTIVE MicroRNA is a family of noncoding RNAs that exhibit tissue-specific or developmental stage-specific expression patterns and are associated with human diseases. MicroRNA-15a (miR-15a) is reported to induce cell apoptosis by negatively regulating the expression of Bcl-2, which suppresses the apoptotic processes. The purpose of this study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by cells and could induce Bcl-2 dysfunction and cell apoptosis in the synovium of arthritic mice in vivo. METHODS Autoantibody-mediated arthritis was induced in male DBA/1J mice. In the experimental group, double-stranded miR-15a labeled with FAM-atelocollagen complex was injected into the knee joint. In the control group, control small interfering RNA-atelocollagen complex was injected into the knee joint. Synovial expression of miR-15a was analyzed by quantitative polymerase chain reaction, FAM by fluorescence microscopy, Bcl-2 by Western blotting, and Bcl-2 and caspase 3 by immunohistochemistry. RESULTS The expression of miR-15a in the synovium of the experimental group was significantly higher than that in the control group. Green fluorescence emission of FAM was observed in the synovium of the experimental group. Bcl-2 protein was down-regulated and the expression of caspase 3 was increased as compared with that in the control group. CONCLUSION These results indicate that the induction of cell apoptosis after intraarticular injection of double-stranded miR-15a occurs through inhibition of the translation of Bcl-2 protein in arthritic synovium.

Rotator Cuff Regeneration Using a Bioabsorbable Material With Bone Marrow–Derived Mesenchymal Stem Cells in a Rabbit Model

Background: Rotator cuff regeneration using tissue engineering techniques is a challenging treatment in elderly patients with irreparable rotator cuff tears. Hypothesis: A polyglycolic acid sheet scaffold with seeded mesenchymal stem cells (MSCs) may enhance the expression of type I collagen products and increase the mechanical strength of the regenerated tendon in vivo. Study Design: Controlled laboratory study. Methods: A surgically created defect of infraspinatus tendons of rabbits was reconstructed with 2 different materials, a polyglycolic acid (PGA) sheet alone (PGA group) (n = 34) and a PGA sheet seeded with autologously cultured MSCs (MSC group) (n = 34). The authors created a tendon defect model without embedding any graft as the control model (control group) (n = 34). The rabbits were sacrificed at 4, 8, and 16 weeks after the operation and then were histologically evaluated. The rabbits were also biomechanically evaluated by measuring the ultimate failure loads and Young’s modulus at 4 and 16 weeks following implantation. Results: In the MSC group, the fibrocartilage layers and Sharpey fibers were found regularly in the insertion site at 8 weeks compared with the PGA group. In control group, thin membranes with many fibroblasts arranged in an irregular pattern linked the end of the torn cuff to the bone without any Sharpey fibers and type I collagen. A large volume of type I collagen was found in comparison with type III collagen at 16 weeks in the MSC group, whereas type III collagen was more prevalent than type I in the PGA group. The tendon maturing score in the MSC group had higher values than the PGA and control groups at 8 and 16 weeks (mean values were 21.0 ± 0.89, 24.0 ± 2.53 in the MSC group; 16.7 ± 2.25, 21.3 ± 2.42 in the PGA group; and 10.2 ± 0.98, 12.2 ± 1.72 in the control group, respectively) (P < .05). The results of the mechanical analysis revealed that the regenerated tendons in the MSC group had better tensile strength than in the PGA and control groups at 16 weeks (mean values were 3.04 ± 0.54 in the MSC group, 2.38 ± 0.63 in the PGA group, and 1.58 ± 0.13 in the control group) (P < .05). Conclusion: Bone marrow–derived MSCs were able to regenerate tendon-bone insertions and the tendon belly, including the production of type I collagen, and increased the mechanical strength of the regenerated rotator cuff tendon. Clinical Relevance: Rotator cuff regeneration using MSCs is a promising treatment for massive rotator cuff defects.

Effects of transforming growth factor-β 1 on the early stages of healing of the Achilles tendon in a rat model

We studied the effects of transforming growth factor-β 1 (TGF-β 1) on the genetic expression of procollagen type I and III and its effects on structural properties in the early stages of healing in rat Achilles tendon. The Achilles tendons in 90 rats were transsected and repaired immediately. TGF-β 1 dissolved in phosphate-buffered saline was injected locally at the repair site using two different doses, and outcomes in both groups were compared to that in the control group given phosphate-buffered saline only. Five animals in each group were killed at one, two, and four weeks postoperatively, and the healing tendon was evaluated. A dose-dependent increase in the expression of procollagen type I and III mRNA was found one week postoperatively. The failure load and stiffness of the healing tendon were increased by TGF-β 1 at two and four weeks.

Tendon-Bone Insertion Repair and Regeneration Using Polyglycolic Acid Sheet in the Rabbit Rotator Cuff Injury Model

Background The quality of tendons has considerable limitations regarding torn rotator cuff tendons. Tissue-engineering techniques using a biodegradable scaffold offer potential alternatives for recreating a valid tendon-to-bone interface. Hypothesis A polyglycolic acid (PGA) sheet could facilitate the regeneration of the rotator cuff tendon insertion in vivo. Study Design Controlled laboratory study. Methods An implant consisting of a PGA sheet, a rapidly absorbable material, was used to replace a completely resected infraspinatus tendon insertion in 33 adult Japanese white rabbits. The contralateral infraspinatus tendon was replaced by poly-L-lactate-epsilon-caprolactone (PLC), a slowly absorbable material, by the same methods based on the results of the pilot study. Histological comparisons were made at 4, 8, and 16 weeks, and mechanical evaluations were performed at 4 and 16 weeks in both groups. Unrepaired defects were created in a control group. Results: In the control group, the rotator cuff defects were covered with thin fibrous membranes with many fibroblasts arranged in an irregular pattern. In the PLC group, some chondrocytes were seen in the tendon insertion; however, these were not arranged along the long axis for a 16-week period. In the PGA group, a well-arranged fibrocartilage layer could be found in the regenerated tendon insertions; however, these tendon insertions were mainly regenerated by type III collagen. In mechanical examinations, the PGA group had significantly higher values in the maximum failure load, tensile strength, and Youngs modulus for the 4-week and 16-week periods. These 3 categories statistically improved from 4 to 16 weeks postoperatively in both groups except for the Youngs modulus in the PGA group (E = 5.66 at 4 weeks to 5.53 at 16 weeks). Conclusion The PGA sheet scaffold material allows for tendon insertion regeneration with a fibrocartilage layer but displays mechanical properties inferior to those of the normal tendon in an animal model. Clinical Relevance The PGA sheet represent a possible alternative scaffold material for tendon regeneration in rotator cuff repair.

Augmented meniscal healing with free synovial autografts: an organ culture model

We examined whether or not free synovial autografts can augment the healing of lesions artificially created in the human knee meniscus in an organ culture model. The effectiveness of free synovial autografts was also compared with that of fibrin glue (Tisseel). In the control group, the accumulation of cellular components increased at the edge of a 4-mm meniscal defect following culture, although no cells were found to invade the defect. In the fibrin glue group, an increased migration of cells into fibrin glue packed into the defect was observed until 4 weeks following cultivation. However, newly formed collagen produced by the cells in the fibrin glue was minimal even 4 weeks after cultivation. In contrast, in the synovial group, newly formed collagen production, as well as cellular proliferation, increased in the grafted synovium with time after cultivation. By 4 weeks, the gap between the edge of the defect and the grafted synovium was bridged by newly formed collagen tissue, indicating improved histologic repair. Our results suggest that free autogenous synovial tissue grafts in meniscal lesions are superior to fibrin glue, and that interpositional synovial autografts to augment repair of meniscal tears in the avascular zone may be clinically useful.

Chondroitinase ABC promotes corticospinal axon growth in organotypic cocultures

Study design:Organotypic coculture model using brain cortex and spinal cord of neonatal rats was used to test the effect of chondroitinase ABC (ChABC) on corticospinal axon growth.Objective:Chondroitin sulfate proteoglycan (CSPG) is neurite outgrowth inhibitory factor that combines with reactive astrocyte at the lesion site to form a dense scar that acts as a barrier to regenerating axons. ChABC is a bacteria enzyme that digests the glycosaminoglycan side chain of CSPG. We investigated the effect of ChABC on corticospinal axon growth quantitatively using the organotypic cocultures of brain cortex and spinal cord.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Method:We used organotypic cocultures with neonatal brain cortex and spinal cord as an in vitro assay system for assessing axon growth. After administering ChABC, we counted the number of axons passing through a reference line running parallel to the junction between the brain cortex and spinal cord 500 and 1000 μm from the junction. The immunoreactivity of CSPG was assessed.Result:The average number of axons after ChABC administration was significantly greater than in the control group. Administration of ChABC decreased CSPG expression in this coculture system.Conclusion:ChABC induces axonal regeneration by degrading CSPG after central nerve system injury. ChABC has great potential for future therapeutic use in spinal cord-injured patients.

Enhancement of bone formation in an experimental bony defect using ferumoxide-labelled mesenchymal stromal cells and a magnetic targeting system

We used interconnected porous calcium hydroxyapatite ceramic to bridge a rabbit ulnar defect. Two weeks after inducing the defect we percutaneously injected rabbit bone marrow-derived mesenchymal stromal cells labelled with ferumoxide. The contribution of an external magnetic targeting system to attract these cells into the ceramic and their effect on subsequent bone formation were evaluated. This technique significantly facilitated the infiltration of ferumoxide-labelled cells into ceramic and significantly contributed to the enhancement of bone formation even in the chronic phase. As such, it is potentially of clinical use to treat fractures, bone defects, delayed union and nonunion.

Is patella eversion during total knee arthroplasty crucial for gap adjustment and soft-tissue balancing?

INTRODUCTION Assessment of soft-tissue balance by the gap technique in Total Knee Arthroplasty (TKA) impacts femoral component rotation positioning. Proper femoral component rotation is a critical factor in TKA, both for adequate patellar tracking and in achieving a symmetrical flexion gap. Soft tissue balance assessment and gap measurements are performed at 90° flexion and with the patella everted in conventional TKA; during MIS-TKA, this step is performed with the patella in situ. We therefore investigated intraoperative joint gap parameters at 90° flexion with and without patellar eversion during conventional TKA, so as to better understand the influence of this intraoperative patellar position factor on final ligament balance. HYPOTHESIS Conducting TKA without patellar eversion increases both gap size and gap inclination. PATIENTS AND METHODS Twenty-four osteoarthritic knees were included in the study. Joint gap size and inclination were measured intraoperatively on a knee in 90° flexion, with and without patellar eversion. RESULTS The joint gap with patella in situ (17.0±3.4 mm) was significantly greater than with patellar eversion (15.4±3.0 mm), as was gap inclination at 90° flexion with the patella in situ (4.9±3.1°) compared to with patellar eversion (4.0±2.9°). DISCUSSION The flexion gap inclination obtained without patellar eversion was steeper than with patellar eversion. This induced more externally rotated femoral positioning in absence of patellar eversion. These results ought to be taken into account by surgeons considering switching from conventional to MIS-TKA.

Telomerase Activity in Giant Cell Tumors of Bone

BackgroundA giant cell tumor of bone (GCT) is a histologically benign neoplasma that has an unpredictable pattern of biological aggressiveness. In the present study, we investigated whether there was a correlation between telomere length or the levels of telomerase activity and other clinical features of GCTs, for the possible use of these factors as parameters of aggressiveness or prognosis.MethodsIn 16 surgically resected GCTs specimens, telomere length was assessed by terminal restriction fragments by Southern blot analysis. Telomerase activity was measured by a semiquantitative polymerase chain reaction–based telomeric repeat amplification protocol assay.ResultsTelomere length reduction was observed in 69% of the GCT samples. The telomere lengths of tumors were significantly shorter than those of normal tissue (P = .008). The mean telomere length of grade 3 tumors was significantly shorter than those of grade 1 and 2 tumors (P = .038). Telomerase activity was detected in 81% of tumor samples. The level of telomerase activity in tumors with local recurrence was significantly higher than in tumors without local recurrence (P = .011).ConclusionsThese results suggest that telomere length correlates with roentgenographic grade as a result of the frequency of cell division, and high telomerase activity indicates the aggressiveness of GCTs.