Intraocular kinetics of pathological ATP after photoreceptor damage in rhegmatogenous retinal detachment.

PURPOSE: Extracellular Adenosine triphosphate (ATP) released by dying cells may cause a secondary cell death in neighboring cells in retinal degeneration. We investigated intraocular ATP kinetics to gain mechanical insights into the pathology in rhegmatogenous retinal detachment (RRD). STUDY DESIGN: Retrospective clinical study. METHODS: Vitreous or subretinal fluids (SRF) were obtained from patients with RRD (n=75), macular hole (MH; n=20), and epiretinal membrane (ERM; n=35) during vitrectomy. ATP levels in those samples were measured by luciferase assay. RESULTS: Mean ATP levels in the vitreous from RRD patients were significantly higher compared to those from MH and ERM patients (2.3 and 0.3 nM, respectively. P<0.01). Mean ATP levels in the SRF from RRD (11.7 nM) were higher than those in the vitreous from RRD (P<0.01). Mean ATP levels in the vitreous with short durations (1-8 days) of RRD were higher compared to those with long durations (>8 days) (3.2 and 1.4 nM, respectively. P<0.05). Similarly, ATP in SRF with short durations were higher than those with long durations (23.8 and 3.6 nM, respectively. P<0.05). Furthermore, the concentrations of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), a major ATP degradative enzyme, in the vitreous from RRD were higher than those from MH/ERM (1.2 and 0.2 ng/ml, respectively. P<0.01). ENTPD1 expression was localized in the cytoplasm of CD11b-positive infiltrating cells in the vitreous and retinal cells. CONCLUSION: ATP increased in the vitreous and SRF in RRD and decreased over time with an upregulation of ENTPD1. The kinetics indicate the pathological mechanism of the excessive extracellular ATP after RRD.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

Association of Stability and Size of Unhealed Area With Failure After Internal Fixation for Osteochondritis Dissecans Lesions of the Knee: Radiological Evaluation Using Computed Tomography.

BACKGROUND: Open reduction and internal fixation (ORIF) has been widely performed because the osteochondral component of the osteochondritis dissecans (OCD) lesion is the most suitable for reconstructing the joint structure. PURPOSE: To evaluate radiological healing in terms of reconstructed bony structure after ORIF with bone graft by computed tomography (CT), to identify preoperative prognostic factors for failure, and to determine the cutoff value of radiological healing for risk of failure. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A retrospective cohort study of 42 patients (44 knees) who underwent internal fixation with bone graft for OCD lesions of the knee from 2004 to 2018 was conducted. All patients were evaluated 6 months postoperatively, and if not healed 6 months after surgery, they were evaluated by CT periodically thereafter. Radiological healing was judged according to the following 3 criteria: (1) reossification of the OCD lesion, (2) bony continuity between the OCD lesion and basal floor, and (3) reconstructed bony surface of the femoral condyle reconstructed to match the normal joint. Then, the percentage of the radiological healing area was calculated as the ratio of the healing length to the total lesion length. The nonhealing area was calculated by multiplying the sum of the total nonhealing length. Clinical failure was defined as any definitive reoperation for the same OCD lesion, such as fragment excision, or a cartilage restoration procedure. After 6 months, all eligible patients underwent arthroscopy to check for protrusion of the absorbable pin into the joint; the removal of an absorbable pin protruding into the joint was not considered a failure. RESULTS: Clinical failure was recorded for 4 cases (9.1%). The mean overall percentage of the radiological healing area of OCD 6 months after ORIF with bone graft was 79.5% ± 24.4%, and the mean overall nonhealing area at 6 months was 87.8 ± 107.9 mm2. The percentages of radiological healing area of stable (International Cartilage Regeneration & Joint Preservation Society OCD II) lesions and femoral condylar (lateral femoral condyle + medial femoral condyle) lesions were significantly lower than unstable lesions and femoral groove lesions, respectively (P = .01 and P = .03, respectively). On receiver operating characteristic curve analysis, the cutoff points for predicting a significantly increased risk of failure were 33.9% (sensitivity, 100%; specificity, 100%; area under the curve, 1) for the percentage of radiological healing area and 222.9 mm2 (sensitivity, 95%; specificity, 100%; area under the curve, 0.956) for the nonhealing area 6 months postoperatively. CONCLUSION: A stable lesion and a femoral condylar lesion were the predictors of poor radiological healing on CT images 6 months after ORIF with bone graft. The risk of failure was increased significantly in cases with only approximately one-third of the lesion healed or in cases with large nonhealing areas at 6 months postoperatively.

Anti-NF-κB peptide derived from nuclear acidic protein attenuates ovariectomy-induced osteoporosis in mice.

NF-κB is a transcription factor that is activated with aging. It plays a key role in the development of osteoporosis by promoting osteoclast differentiation and inhibiting osteoblast differentiation. In this study, we developed a small anti-NF-κB peptide called 6A-8R from a nuclear acidic protein (also known as macromolecular translocation inhibitor II, Zn2+-binding protein, or parathymosin) that inhibits transcriptional activity of NF-κB without altering its nuclear translocation and binding to DNA. Intraperitoneal injection of 6A-8R attenuated ovariectomy-induced osteoporosis in mice by inhibiting osteoclast differentiation, promoting osteoblast differentiation, and inhibiting sclerostin production by osteocytes in vivo with no apparent side effects. Conversely, in vitro, 6A-8R inhibited osteoclast differentiation by inhibiting NF-κB transcriptional activity, promoted osteoblast differentiation by promoting Smad1 phosphorylation, and inhibited sclerostin expression in osteocytes by inhibiting myocyte enhancer factors 2C and 2D. These findings suggest that 6A-8R has the potential to be an antiosteoporotic therapeutic agent with uncoupling properties.

Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6-CXCR2 pathway.

OBJECTIVE: To investigate the efficacy of basic fibroblast growth factor (bFGF) in promoting meniscus regeneration by cultivating synovial mesenchymal stem cells (SMSCs) and to validate the underlying mechanisms. METHODS: Human SMSCs were collected from patients with osteoarthritis. Eight-week-old nude rats underwent hemi-meniscectomy, and SMSCs in pellet form, either with or without bFGF (1.0 × 106 cells per pellet), were implanted at the site of meniscus defects. Rats were divided into the control (no transplantation), FGF (-) (pellet without bFGF), and FGF (+) (pellet with bFGF) groups. Different examinations, including assessment of the regenerated meniscus area, histological scoring of the regenerated meniscus and cartilage, meniscus indentation test, and immunohistochemistry analysis, were performed at 4 and 8 weeks after surgery. RESULTS: Transplanted SMSCs adhered to the regenerative meniscus. Compared with the control group, the FGF (+) group had larger regenerated meniscus areas, superior histological scores of the meniscus and cartilage, and better meniscus mechanical properties. RNA sequencing of SMSCs revealed that the gene expression of chemokines that bind to CXCR2 was upregulated by bFGF. Furthermore, conditioned medium derived from SMSCs cultivated with bFGF exhibited enhanced cell migration, proliferation, and chondrogenic differentiation, which were specifically inhibited by CXCR2 or CXCL6 inhibitors. CONCLUSION: SMSCs cultured with bFGF promoted the expression of CXCL6. This mechanism may enhance cell migration, proliferation, and chondrogenic differentiation, thereby resulting in superior meniscus regeneration and cartilage preservation.

Changes in patellar height after anatomical ACL reconstruction with BTB autograft with a focus on patellar tendon removal volume.

BACKGROUND: Although anatomical anterior cruciate ligament reconstruction (ACLR) can provide satisfactory outcomes, little is known about how this procedure impacts patellar height. Since harvesting bone-patellar tendon-bone (BTB) autografts is a potential risk factor for decreased patellar height, we examined changes in patellar height after anatomical ACLR with BTB autograft with a focus on the size of the harvested graft. METHODS: Subjects were 84 patients (49 males, 35 females; mean age, 23 years) who underwent primary anatomical ACLR with central third BTB autograft. Preoperative to postoperative Caton-Deschamps index (CDI) ratio was calculated using lateral knee radiographs before and 6 months after surgery. The length and cross-sectional area (CSA) of the graft were measured intraoperatively, and the CSA of the contralateral patellar tendon was measured by ultrasound 6 months postoperatively. The difference in graft CSA relative to the contralateral tendon CSA, expressed as a percentage (gCSA:ctCSA percentage), was also calculated. RESULTS: Patellar height decreased slightly after surgery (preoperative CDI: 0.856 ± 0.113; postoperative CDI: 0.841 ± 0.113), with a mean difference between preoperative and postoperative CDIs of -0.015 (range: -0.293 to 0.101). Although the CDI of male subjects significantly decreased after surgery (preoperative: 0.852 ± 0.117; postoperative: 0.827 ± 0.115), no significant changes were noted in female subjects (preoperative: 0.862 ± 0.108; postoperative: 0.861 ± 0.108). Graft length and CSA did not significantly impact the CDI ratio (r = -0.138 and r = -0.038, respectively). Moreover, no significant relationship was observed between the gCSA:ctCSA percentage and CDI ratio (r = 0.118). CONCLUSIONS: Although patellar height slightly, but significantly, decreased at 6 months after anatomical ACLR with BTB autograft, it was not affected by the length and CSA of harvested grafts. The decrease in postoperative patellar height was observed only in male subjects, suggesting the potential importance of sex differences in soft tissue healing during the postoperative period.

The Wound Dressing Influenced Effectiveness of Cryotherapy After Anterior Cruciate Ligament Reconstruction: Case-Control Study Comparing Gauze Versus Film Dressing.

Purpose: To compare the clinical effectiveness of cryotherapy after anterior cruciate ligament reconstruction using 2 different wound dressings, conventional postoperative gauze dressings and polyurethane semipermeable transparent film dressings. Methods: In total, 60 patients who had undergone arthroscopic anterior cruciate ligament reconstruction with an autogenous patellar tendon were assigned to 2 groups. The surgical wound was covered with 5 sheets of gauze with an elastic bandage (control group) in 30 patients and film dressing was used (film group) in the remaining 30 patients. Silicone drainage catheters were inserted at the intercondylar notch, beside the distal outlet of the tibial tunnel for 2 days. After 1 hour of cooling using the device, the knee was chilled with an ice bag every 2 hours until the next morning. The severity of pain was evaluated by the number of times an analgesic, 50 mg of diclofenac sodium suppositories, had to be administered in the 24 hours after surgery. The amount of drainage during the following 2 days, the range of motion at 21 days, the change of hemoglobin concentration at 1 and 7 days, and C-reactive protein (CRP) at 1 and 7 days were examined. Results: The number of patients who used an analgesic was 18 in the control group and 7 in the film group (P = .003). The amount of drainage was 165.2 ± 72.9 mL in the control group and 289.7 ± 77.6 mL in the film group (P < .001). The postoperative CRP value was 0.77 ± 0.65 mg/dL at 1 day in the control group and 0.39 ± 0.42 mg/dL in the film group (P = .009). No statistical difference was seen for hemoglobin concentration at 1 or 7 days, CRP at 7 days or range of motion at 21 days. Conclusions: In this study, we found that film dressing enhanced the effect of cryotherapy with respect to pain control, wound drainage, and inflammation immediately after surgery compared with traditional gauze dressing with elastic wrap. Level of Evidence: III, case-control study.

Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro.

INTRODUCTION: Glucocorticoids are widely used to treat various diseases including rheumatoid arthritis (RA); however, one of the most frequent and severe adverse effects is glucocorticoid-induced osteoporosis (GIOP). Iguratimod (IGU) is a novel conventional synthetic disease-modifying anti-rheumatic drug developed in Japan. The aim of this study is to investigate the effects of IGU on glucocorticoid-induced disorder of bone metabolism in vitro. MATERIALS AND METHODS: In osteoclastogenesis of mouse bone marrow-derived cells, tartrate-resistant acid phosphatase staining, resorption pit assay, western blotting, real-time polymerase chain reaction (PCR), and mRNA sequencing were performed. In osteoblastogenesis of MC3T3-E1 cells, alkaline phosphatase (ALP) staining and activity, alizarin red staining, and mRNA sequencing were performed, and real-time PCR and western blotting were conducted in MC3T3-E1 cells and murine osteocyte-like cell line MLO-Y4 cells. RESULTS: IGU significantly suppressed a dexamethasone-induced increase in osteoclasts, differentiation, and bone resorption activity by inhibition of the receptor activator of the nuclear factor kappa-B (RANK)/tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)/nuclear factor kappa-B (NFκB)-p52 pathway. In MC3T3-E1 cells, IGU significantly upregulated dexamethasone-induced downregulation of ALP activity, bone mineralization, and osteoblast-related gene and protein expression. In MLO-Y4 cells, IGU significantly upregulated dexamethasone-induced downregulation of the gene expression of ALP and osteocalcin, and also downregulated receptor activator of NFκB ligand (RANKL)/osteoprotegerin gene expression ratio without dexamethasone. CONCLUSION: These results suggest that IGU may improve glucocorticoid-induced disorder of bone metabolism and may exhibit positive effects against GIOP associated with RA.

Combined effect of teriparatide and an anti-RANKL monoclonal antibody on bone defect regeneration in mice with glucocorticoid-induced osteoporosis.

OBJECTIVE: The purpose of this study was to examine the effect of single or combination therapy of teriparatide (TPTD) and a monoclonal antibody against the murine receptor activator of nuclear factor κB ligand (anti-RANKL Ab) on cancellous and cortical bone regeneration in a mouse model of glucocorticoid-induced osteoporosis (GIOP). METHODS: C57BL/6 J mice (24 weeks of age) were divided into five groups: (1) the SHAM group: sham operation + saline; (2) the prednisolone (PSL) group: PSL + saline; (3) the TPTD group: PSL + TPTD; (4) the Ab group: PSL + anti-RANKL Ab; and (5) the COMB group: PSL + TPTD + anti-RANKL Ab (n = 8 per group). With the exception of the SHAM group, 7.5 mg of PSL was inserted subcutaneously into mice, to generate a mouse model of GIOP. Four weeks after insertion, bone defects with a diameter of 0.9 mm were created to assess bone regeneration on both femoral metaphysis (cancellous bone) and diaphysis (cortical bone). After surgery, therapeutic intervention was continued for 4 weeks. Saline (200 µl) or TPTD (40 µg/kg) was injected subcutaneously five times per week, whereas the anti-RANKL Ab (5 mg/kg) was injected subcutaneously once on the day after surgery. Subsequently, the following analyses were performed: microstructural assessment of bone regeneration and bone mineral density (BMD) measurement via micro-computed tomography, and histological, histomorphometrical, and biomechanical analyses with nanoindentation. RESULTS: The COMB group showed the highest lumbar spine BMD increase (vs. the PSL, TPTD, and Ab groups). The volume of regenerated cancellous bone at the bone defect site was higher in the COMB group compared with the PSL, TPTD, and Ab group. The volume of the regenerated cortical bone was significantly higher in the COMB group compared with the PSL group, and its hardness was significantly higher in the COMB group compared with the PSL and TPTD groups. CONCLUSION: In a mouse model of glucocorticoid-induced osteoporosis, the combination therapy of TPTD plus the anti-RANKL Ab increased bone mineral density in the lumbar spine and regenerated cancellous bone volume compared with single administration of each agent, and also increased regenerated cortical bone strength compared with single administration of TPTD.

Promoting Effect of Basic Fibroblast Growth Factor in Synovial Mesenchymal Stem Cell-Based Cartilage Regeneration.

Synovial mesenchymal stem cell (SMSC) is the promising cell source of cartilage regeneration but has several issues to overcome such as limited cell proliferation and heterogeneity of cartilage regeneration ability. Previous reports demonstrated that basic fibroblast growth factor (bFGF) can promote proliferation and cartilage differentiation potential of MSCs in vitro, although no reports show its beneficial effect in vivo. The purpose of this study is to investigate the promoting effect of bFGF on cartilage regeneration using human SMSC in vivo. SMSCs were cultured with or without bFGF in a growth medium, and 2 × 105 cells were aggregated to form a synovial pellet. Synovial pellets were implanted into osteochondral defects induced in the femoral trochlea of severe combined immunodeficient mice, and histological evaluation was performed after eight weeks. The presence of implanted SMSCs was confirmed by the observation of human vimentin immunostaining-positive cells. Interestingly, broad lacunae structures and cartilage substrate stained by Safranin-O were observed only in the bFGF (+) group. The bFGF (+) group had significantly higher O'Driscoll scores in the cartilage repair than the bFGF (-) group. The addition of bFGF to SMSC growth culture may be a useful treatment option to promote cartilage regeneration in vivo.

Autologous Osteochondral Transplantation in Full-thickness Patella Chondral Lesion: A Case Series.

PURPOSE: Autologous osteochondral transplantation (AOT) in the focal cartilage lesion of the patella has been reported with less successful results compared with other sites. The purposes were to investigate the clinical outcomes of AOT for focal patellar chondral lesion without patellofemoral instability. METHODS: Between 2001 and 2007, six patients (five males and one female) with a focal patellar cartilage lesion without patellofemoral malalignment and instability were treated with AOT. The mean age was 38 (27-51) years. Intraoperatively, the size and location of lesion were assessed by international cartilage repair society classification. Lysholm score was investigated preoperatively, at 6 months, 1- and 2-year, and final follow-up. Mean follow-up period was 51 months (24-101). Transplanted grafts were evaluated by magnetic resonance imaging (MRI) and second-look arthroscopy. RESULTS: The mean size was 133mm2(78-225). All six cases improved at final follow-up (Lysholm score 79-100). Although immediate pain relief obtained in four cases, severe pain was persistent in remaining two cases during the 1styear and gradually relieved by 2 years following surgery. The size of these two cases was significantly larger (over 170 mm2) than that of four cases (100 mm2 in average) (P<0.05), and their locations were apart from center of the patella inspite of four cases localized centrally (P<0.05). Repaired cartilage did not show any difference by MRI and arthroscopically. CONCLUSION: AOT in focal patellar chondral lesions without patellofemoral malalignment showed excellent results. In cases of large off-centeredlesions, however, it took longer for pain relief following AOT.

Relationship between bone plug position and morphological changes of tunnel aperture in anatomic rectangular tunnel ACL reconstruction.

PURPOSE: In animal studies after ACL reconstruction (ACL-R) using the bone-patellar tendon-bone (BTB), the graft-healing pattern was found to depend on the relationship between bone plug and the tunnel wall. This difference of graft-healing pattern could influence the postoperative morphological changes of the tunnel. However, no study has assessed the relationship between bone plug position and the change of tunnel morphology. Therefore, the main purpose of this study was to investigate the relationship between the bone plug position within femoral or tibial tunnel and morphological changes of each tunnel aperture in ACL-R using computed tomography. METHODS: Subjects were 30 consecutive patients (six females and 24 males; mean age, 20.4 ± 5.4 years) who underwent primary ACL-R using BTB. The distance from the tunnel aperture to the tendon-bone junction (TBJ) at 2 weeks postoperatively, and tunnel aperture enlargement and tunnel wall migration from 2 weeks to 6 months postoperatively, were evaluated. RESULTS: The distance from the femoral tunnel aperture to the TBJ in most cases was less than 2 mm, whereas the TBJ was located within the tibial tunnel. Femoral tunnel aperture was significantly enlarged (17.0 ± 11.7%) distally, and the tibial tunnel aperture was significantly enlarged (19.6 ± 12.5%) posterolaterally. Only the position at distal portion of femoral bone plug was correlated with femoral tunnel aperture enlargement (r = 0.454, p = 0.0015). CONCLUSION: Both femoral and tibial tunnel aperture were significantly enlarged distally and posterolaterally 6 months postoperatively. Only correlation between the position at distal portion of femoral bone plug and femoral tunnel enlargement were found, suggesting the deep plug position in the tunnel is a risk factor for femoral tunnel enlargement, highlighting the importance of accurately locating the TBJ just at the femoral tunnel aperture. Another option is to deviate the harvest site in the patellar tendon to match the shape of the TBJ and the tunnel aperture. LEVEL OF EVIDENCE: 4 (case series).

First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions.

BACKGROUND: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. PURPOSE: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image-based estimation of morphologic and compositional quality of the repair tissue. RESULTS: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. CONCLUSION: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation.

Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

BACKGROUND: Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. RESULTS: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. CONCLUSIONS: Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Anterior knee symptoms after double-bundle ACL reconstruction with hamstring tendon autografts: an ultrasonographic and power Doppler investigation.

PURPOSE: Anterior knee pain related to the donor site is a frequent complication of anterior cruciate ligament reconstruction (ACLR) with bone-patellar tendon-bone autograft tissue. Even when hamstring tendon (HT) grafts are used instead, symptoms such as mild pain and discomfort can still occur. The purpose of this study was to elucidate the pathophysiology of anterior knee symptoms after ACLR with HT autografts. METHODS: Fifty-seven patients (22 men and 35 women; mean age, 24.7 years) who underwent anatomic double-bundle ACLR with HT autografts were examined 6 months post-operatively. The presence of anterior knee symptoms, anterior knee laxity, range of motion, and muscle strength were assessed. Changes in patellar tendon and infrapatellar fat pad (IFP) morphology and blood flow were also evaluated using ultrasound. Potential variables affecting the presence of anterior knee symptoms were subjected to univariate analysis and multivariate logistic regression analysis to identify independent risk factors. RESULTS: Six months post-operatively, the total incidence of anterior knee symptoms was 56.1 % (32/57). According to univariate analysis, age, quadriceps strength, and increased blood flow in the IFP were significantly associated with the presence of anterior knee symptoms. Multivariate logistic regression analysis revealed that increased blood flow in the IFP was an independent factor for the presence of anterior knee symptoms (odds ratio 5.0; 95 % confidence interval 1.3-19.9). There were no significant findings inside the patellar tendon. CONCLUSIONS: Increased blood flow in the IFP was identified as an independent factor for the presence of anterior knee symptoms 6 months after ACLR with HT autografts. The ultrasound evaluation can help to define precisely the origin of anterior knee symptoms after ACLR with HT autografts. LEVEL OF EVIDENCE: Case series with no comparison groups, Level IV.

Morphological changes in femoral tunnels after anatomic anterior cruciate ligament reconstruction.

PURPOSE: Few studies investigated the enlargement inside the tunnel as well as the morphological change at the aperture after anterior cruciate ligament (ACL) reconstruction, whereas the tunnel enlargement has been well documented. The purposes were to evaluate the change in the cross-sectional area along the femoral tunnel and to morphologically clarify the enlargement at the femoral tunnel aperture after anatomic triple-bundle (ATB) ACL reconstruction. METHODS: The study included 15 patients with unilateral ACL rupture. ATB ACL reconstruction was performed using semitendinosus tendon autografts. Three-dimensional computer models of the femur and bone tunnels were reconstructed from computed tomography images obtained 3 weeks and 1 year postoperatively. The cross-sectional area at the aperture as well as inside the tunnel was compared between the two periods. Likewise, the location of tunnel walls and center in the tunnel footprint were evaluated. RESULTS: The cross-sectional area enlarged by 22.7 % for anteromedial/intermediate graft (P = 0.002) and 28.6 % for posterolateral graft (P = 0.002) at the aperture, while decreased by 36.2 % at 10 mm from the aperture for anteromedial/intermediate graft (P = 0.004). Both the anterior and posterior walls shifted anteriorly, while the distal wall shifted distally in both tunnels. Consequently, the center in the footprint significantly shifted anteriorly (4.9-6.6 %) and distally (2.2-2.6 %) in both tunnels. CONCLUSIONS: The femoral tunnel enlargement occurred at the aperture after ATB ACL reconstruction, but did not occur in the middle of the femoral tunnel. The morphology at the aperture changed with time after surgery as the tunnel walls translated anteriorly and distally. LEVEL OF EVIDENCE: Case series, Level IV.

Patellofemoral chondral status after medial patellofemoral ligament reconstruction using second-look arthroscopy in patients with recurrent patellar dislocation.

BACKGROUND: Most patients with recurrent patellar dislocation show cartilage damage in the patellofemoral joint. Medial patellofemoral ligament reconstruction has become one of the most important surgical techniques for treating recurrent patellar dislocation. However, patellofemoral chondral status after this reconstruction has not been elucidated. The purpose of this study was to investigate the effects of medial patellofemoral ligament reconstruction on articular cartilage in the patellofemoral joint by comparing the arthroscopic chondral status at the time of reconstruction with that at second-look arthroscopy. METHODS: Participants in the present study comprised 31 patients (22 females, 9 males; 32 knees) who underwent second-look arthroscopy at a median of 12 months (range 6-40 months) after dual tunnel medial patellofemoral ligament reconstruction using a double-looped autologous semitendinosus tendon graft. Median age at the time of initial surgery was 20 years (range 13-43 years). The patellofemoral joint was divided into six portions, comprising the medial facet of the patella, central ridge, lateral facet of the patella, anterior medial femoral condyle, femoral groove, and anterior lateral femoral condyle. Chondral status in each portion according to the International Cartilage Repair Society classification was retrospectively evaluated at the time of initial surgery and second-look arthroscopy. RESULTS: Before medial patellofemoral ligament reconstruction, chondral lesions were observed in the patellofemoral joint in 31 knees (97%). At the central ridge of the patella, chondral damage was observed in 22 knees (69%) at initial surgery and damaged cartilages showed recovery in 6 knees. No significant difference in the alteration of chondral status was seen for the medial facet, lateral facet of the patella, anterior medial femoral condyle, femoral groove, and anterior lateral femoral condyle. CONCLUSIONS: According to short-term results, the patellofemoral chondral status after medial patellofemoral ligament reconstruction was not altered at second-look arthroscopy in most part of patellofemoral joint. At the central ridge of the patella, significant improvement of the International Cartilage Repair Society grading was observed.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

MRI analysis of single-, double-, and triple-bundle anterior cruciate ligament grafts.

PURPOSE: The purpose of the study was to evaluate the entire course of ACL grafts on coronal oblique MR images, focusing on differences in graft morphology and graft-to-tunnel healing among single-bundle (SB), double-bundle (DB), and triple-bundle (TB) reconstructions. METHODS: Eighty-three patients underwent anatomical ACL reconstruction using the semitendinosus tendon. SB reconstruction was performed on 20 patients, DB on 29 patients, and TB on 34 patients. The anteromedial-bundle (AMB) and posterolateral-bundle (PLB) images were extracted from coronal oblique images of grafts at 6 months to visualize their entire course. Signal intensity of grafts was measured independently in three regions: (1) intra-femoral tunnel region, (2) intra-articular region, and (3) intra-tibial tunnel region, followed by calculation of the signal-to-noise quotient (SNQ). To evaluate graft-to-tunnel healing, T2-weighted images were examined for the presence of a high signal-intensity lesion between the graft and bone tunnel around the tunnel aperture. RESULTS: AMB images showed that SB graft was thick throughout the entire course, while DB graft was thinner than SB graft. TB graft showed a fan shape approaching the tibial tunnels. The SNQ in the femoral tunnel of SB graft was significantly lower than in the DB and TB grafts. High signal-intensity lesions were frequently observed around the femoral tunnel aperture in PLB images of DB and TB grafts compared to SB grafts. CONCLUSION: Gross morphology of TB grafts resembled that of the natural ACL. However, the graft-to-tunnel healing around the femoral tunnel seemed to be insufficient in PLB images of DB and TB compared to SB grafts.