Katsumasa Tei

SIRT1 regulation of apoptosis of human chondrocytes.

OBJECTIVE SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA). METHODS The expression of SIRT1 in human chondrocytes was examined by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO)-induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction. RESULTS SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2. CONCLUSION These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA.

A prospective randomised study of anatomical single-bundle versus double-bundle anterior cruciate ligament reconstruction: quantitative evaluation using an electromagnetic measurement system

We conducted a prospective randomised study of anatomical single-bundle (A-SB group) versus double-bundle (A-DB group) anterior cruciate ligament (ACL) reconstruction using the hamstrings tendons. Twenty patients with unilateral ACL deficiency were randomised into two groups. We created the bone tunnels at the position of the original insertion of the anteromedial bundle footprint and posterolateral bundle footprint in the A-DB group and at the central position between these two bundles in the A-SB group. All of the patients were tested before ACL reconstruction and one year after surgery. The KT-1000 measurements, isokinetic muscle peak torque and heel-height difference were evaluated and the general knee condition was assessed by Lysholm score. For pre- and postoperative stability assessment, we used the six-degrees-of-freedom of knee kinematic measurement system using an electromagnetic device (the EMS) for quantitative assessment during the Lachman test and the pivot shift test. There were no significant differences in the KT-1000 measurements, isokinetic muscle peak torque, heel-height difference, and Lysholm score at one-year follow-up between these two groups. The EMS data showed there were significant differences in the acceleration of the pivot shift test between the operated knee and the contralateral normal knees in the A-SB group. In conclusion, clinical outcomes were equally good in both groups. However, the EMS data showed the anatomical double-bundle ACL reconstruction tended to be biomechanically superior to the single-bundle reconstruction.

Enhancement of Tendon-Bone Osteointegration of Anterior Cruciate Ligament Graft Using Granulocyte Colony-Stimulating Factor

Background Whereas anterior cruciate ligament rupture usually requires reconstruction, the attachment between the tendon and the bone is the weakest region in the early posttransplantation period. In this process, the acquisition of appropriate vascularity is a key for early bone-tendon healing. Hypothesis Granulocyte colony-stimulating factor has an effect on the maturation of bone-tendon integration of anterior cruciate ligament reconstruction. Study Design Controlled laboratory study. Methods Twenty-eight healthy adult beagle dogs underwent bilateral anterior cruciate ligament reconstruction using the ipsilateral flexor digitorum superficialis tendon and were divided into 2 groups. A granulocyte colony-stimulating factor-incorporated gelatin surrounded the graft in the granulocyte colony-stimulating factor group, and the same gelatin without granulocyte colony-stimulating factor was used as the control group. Assessment was done at 2 and 4 weeks. Results Histological analysis at week 2 demonstrated that, in addition to more Sharpey fibers, microvessels were significantly enhanced in the granulocyte colony-stimulating factor groups grafts. Computed tomography at week 4 showed a significantly smaller tibial bone tunnel in the granulocyte colony-stimulating factor group. Real-time polymerase chain reaction revealed significantly elevated messenger ribonucleic acid expression levels of vascular endothelial growth factor and osteocalcin in the tibial bone tunnel and graft compared with controls. Furthermore, biomechanical testing of force during loading to ultimate failure at week 4 demonstrated a significant increase in strength in the granulocyte colony-stimulating factor group. Conclusion This study demonstrated that a local application of granulocyte colony-stimulating factor-incorporated gelatin significantly accelerates bone-tendon interface strength via enhanced angiogenesis and osteogenesis. Clinical Relevance Granulocyte colony-stimulating factor has therapeutic potential in promoting an environment conductive to angiogenesis and osteogenesis in bone tunnels.

Administrations of Peripheral Blood CD34‐Positive Cells Contribute to Medial Collateral Ligament Healing via Vasculogenesis

Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor‐enriched cell population, have been reported to contribute to neoangiogenesis; however, the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte colony‐stimulating factor‐mobilized peripheral blood (GM‐PB) CD34+ cells with atelocollagen (CD34+ group), GM‐PB mononuclear cells (MNCs) with atelocollagen (MNC group), or atelocollagen alone (control group) was locally transplanted after the creation of medial collateral ligament injury in immunodeficient rats. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunohistochemical staining at the injury site demonstrated that molecular and histological expression of human‐specific markers for endothelial cells was higher in the CD34+ group compared with the other groups at week 1. Endogenous effect, assessed by capillary density and mRNA expression of vascular endothelial growth factor, was significantly higher in CD34+ cell group than the other groups. In addition to the observation that, as assessed by real‐time RT‐PCR, gene expression of ligament‐specific marker was significantly higher in the CD34+ group than in the other groups, ligament healing assessed by macroscopic, histological, and biomechanical examination was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data strongly suggest that local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.

Acceleration of Tendon-Bone Healing of Anterior Cruciate Ligament Graft Using Autologous Ruptured Tissue

Background: It has been recently reported that human anterior cruciate ligament (ACL) ruptured tissue contains abundant vascular stem cells that contribute to tendon-bone healing in an immunodeficient rat model of ACL reconstruction. Hypothesis: Autologous ruptured ACL tissue has an effect on the maturation of bone-tendon integration in anterior cruciate ligament reconstruction. Study Design: Controlled laboratory study. Methods: Twenty healthy adult beagle dogs underwent bilateral ACL reconstruction using the ipsilateral flexor digitorum superficialis tendon and were divided into 2 groups: right knee (a tissue-treated group) and left knee (a control group). The tissue-treated group received autologous ruptured ACL tissue, which was obtained 2 days after resection and sutured to the tibial side of the graft. Histological, radiographic, and biomechanical assessments were performed. In addition, immunohistochemical staining was performed to assess angiogenesis and osteogenesis. Results: Histological assessment and staining for osteoblasts and endothelial cells at week 2 demonstrated early healing, inducing endochondral ossification-like integration with enhanced angiogenesis and osteogenesis in the tissue-treated group’s grafts. Computed tomography at week 4 showed a significantly smaller tibial bone tunnel in the tissue-treated group (tissue, 19.0 ± 4.4 mm2; control, 42.6 ± 4.7 mm2; P = .009, n = 5). Furthermore, biomechanical testing of force during loading to ultimate failure at week 4 demonstrated a significantly higher strength in the tissue-treated group (tissue, 66.4 ± 10.1 N; control, 30.5 ± 10.3 N; P = .009, n = 5). Conclusion: In the present study, the authors elucidated that transplantation of ACL-ruptured tissue, which was sutured to the tibial side of the graft, contributed to early tendon-bone healing in a canine model of ACL reconstruction. Clinical Relevance: Anterior cruciate ligament ruptured tissue has a therapeutic potential in promoting an appropriate environment for tendon-to-bone healing in bone tunnels of ACL reconstruction.

Bone Regeneration Properties of Granulocyte Colony-Stimulating Factor via Neovascularization and Osteogenesis

OBJECTIVES It has been well recognized that appropriate vascularization is emerging as a prerequisite for bone development and regeneration. The aim of this study was to test the hypothesis that locally applied granulocyte colony-stimulating factor (G-CSF) enhances bone regeneration via revascularization and osteogenesis. METHODS A segmental bone defect (20mm) was created at the diaphysis of the rabbit ulna. The defects were treated with cationized gelatin hydrogel, which was the drug delivery system, with G-CSF, and then bone regeneration, neovascularization, and osteogenesis properties with G-CSF were assessed. RESULTS Radiographic, computed tomography, and histological findings revealed that bone formation was significantly promoted in G-CSF-treated group as early as 2 weeks. Immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and flow cytometry studies indicated that angiogenesis/vasculogenesis, which are regulated by mobilization and incorporation of CD34+/G-CSF receptor (CSFR+) cells, and osteogenesis, which is regulated by osteocalcin+/G-CSFR+ cells, were also significantly enhanced in the G-CSF group. CONCLUSIONS This study suggests that locally applied G-CSF contributes to an ideal local environment for fracture healing by supplying adequate blood flow and stimulating osteogenesis. G-CSF may have the therapeutic potential for bone regeneration.

Soft tissue balance measurement in minimal incision surgery compared to conventional total knee arthroplasty.

PurposesMinimal incision surgery (MIS) total knee arthroplasty (TKA) is widely promoted as a possible improvement over conventional TKA, and accurate implantations have recently been reported using navigation systems. However, soft tissue balance during MIS-TKA remains challenging. Therefore, in this report, joint gap (component gap) and ligament balance (varus angle) were assessed during MIS-TKA using a tensor, which enables soft tissue balance assessment with a reduced patellofemoral joint and femoral component in place.MethodsResults were compared to those of conventional TKA. Posterior stabilized TKA were performed in 50 knees (25 knees: MIS-TKA using quadriceps-sparing approach; 25 knees: conventional TKA using medial parapatellar approach) with varus osteoarthritis. Component gap and varus angle were measured using the tensor with a reduced patellofemoral joint at 0, 10, 45, 90, and 135°.ResultsWhereas the component gap in MIS-TKA was significantly larger through the entire arc of flexion compared with conventional TKA, the pattern of joint looseness showed no difference between the two procedures. The varus angle in MIS-TKA was significantly larger than that in conventional TKA at 0, 90, and 135° of knee flexion.ConclusionsMIS-TKA may lead to ligament imbalance due to the difficulties induced by a limited working space. Understanding this pattern allows surgeons to be able to adjust the soft tissue balance more accurately and thereby expect a better post-operative outcome even in MIS-TKA.

The Use of an Electromagnetic Measurement System for Anterior Tibial Displacement During the Lachman Test

PURPOSE The purpose of this study was to assess quantitative anterior/posterior values during the Lachman test by an electromagnetic measurement system and to compare data with KT-1000 arthrometric measurements (MEDmetric, San Diego, CA), as well as the measurement of radiologic laxity by dynamic radiographs. METHODS We used an electromagnetic device to quantitatively evaluate anterior knee displacements. We tested 82 knees in 41 patients (30 isolated anterior cruciate ligament [ACL]-deficient, 11 ACL-reconstructed, and 41 contralateral ACL-intact knees). Anterior displacements during the Lachman test were calculated by the electromagnetic measurement system and fluoroscopic measurement, and anterior displacements were also measured by the KT-1000 arthrometer. Anterior/posterior displacements measured by these methods were compared, and correlations were assessed. RESULTS In ACL-deficient knees, mean anterior/posterior displacement (±SE) was 22.4 ± 0.8 mm in electromagnetic measurements, 22.0 ± 0.7 mm in fluoroscopic measurements, and 15.0 ± 0.6 mm in KT-1000 measurements. In contralateral ACL-intact knees, it was 15.7 ± 0.6 mm, 15.6 ± 0.5 mm, and 9.9 ± 0.4 mm, respectively. In ACL-reconstructed knees, it was 15.7 ± 0.7 mm, 16.2 ± 0.8 mm, and 11.2 ± 0.6 mm, respectively. In all knee conditions, significant differences between fluoroscopic measurements and KT-1000 measurements were detected (P < .01). Significant differences were also detected between electromagnetic measurements and KT-1000 measurements (P < .01). No significant differences were detected between fluoroscopic measurements and electromagnetic measurements. A strong correlation was obtained between KT-1000 measurements and fluoroscopic measurements (r = 0.62, P < .01) and between electromagnetic measurements and KT-1000 measurements (r = 0.64, P < .01). However, the strongest correlation was observed between electromagnetic measurements and fluoroscopic measurements (r = 0.96, P < .01). CONCLUSIONS An electromagnetic measurement system to test anterior/posterior tibial translation determined that quantification of the Lachman test could be performed as accurately as fluoroscopic measurements. LEVEL OF EVIDENCE Level II, development of diagnostic criteria on basis of consecutive patients with universally applied reference gold standard.

Novel Image-matching Software for Postoperative Evaluation After TKA

Although 2-dimensional assessments using postoperative plain radiographs have been used after total knee arthroplasty (TKA) in previous studies, a strong possibility exists that deviation can occur when assessing 3-dimensional (3-D) objects. The purpose of this study was to test the hypothesis that novel 3-D image-matching software could accurately assess the positioning of implants and could be a useful tool in postoperative evaluation after TKA.Total knee arthroplasty was performed in 30 consecutive patients. Intraoperatively, the thickness of each bone cut was measured. Postoperatively, the thickness of each part of the bone cut was measured using Athena Knee (SoftCube Co, Ltd, Osaka, Japan) 3-D image-matching software. The results revealed no significant differences in the medial compartment and significant differences of approximately 1 mm in the lateral compartment. The difference was possibly caused by the remaining cartilage in the lateral compartment. Linear regression analysis revealed a statistically excellent correlation between intra- and postoperative values in all parts of the bone cuts. Although the 3-D image-matching software used in this study was originally developed for preoperative planning in TKA, it is considered accurate enough to assess the positioning of implants with respect to the bone after TKA.

Leg Edema Due to a Mass in the Pelvis After a Large-Diameter Metal-On-Metal Total Hip Arthroplasty

We report the case of a patient with leg edema after large-diameter metal-on-metal total hip arthroplasty. At 1 year and 2 months after primary left large-diameter metal-on-metal total hip arthroplasty, the patient complained of left leg edema. At first, we suspected deep venous thrombosis. However, deep venous thrombosis was not detected by venous ultrasonographic examination. Computed tomography imaging revealed a mass in front of the iliac fossa. The mass compressed the left iliac artery and vein. We therefore believed that this lesion was the cause of the leg edema and performed resection of the mass. The resected mass consisted of necrotic tissue infiltrating inflammation cells, so it was diagnosed as pseudotumor. Unilateral leg edema disappeared gradually after the resection.