Chondrogenic potentials of human synovium-derived cells sorted by specific surface markers.

OBJECTIVE: We aimed to evaluate expression levels of nine candidate surface markers for chondrogenic potential in human synovial cells and to determine whether cell pellets positively sorted by each specific marker would have valuable chondrogenic potential. METHODS: The expression levels of the selected nine leading surface markers in synovial cells from knee joints in 15 patients with primary knee osteoarthritis were evaluated at the stage of isolation and after cultivation using flow cytometry. We obtained positive and negative cells for each surface marker using a magnetically activated cell sorting method and compared chondrogenic potentials between the positive and the negative cell pellets. RESULTS: CD29, CD44, CD73, and CD90 were expressed on the most synovial cells at the isolation stage and on almost all cells at stage of P0 and P1. CD133 was rarely expressed at any stages of the evaluated cells. CD166 was expressed in 7.1% of cells at the isolation stage on average, but this expression increased after cell passages. The expressions of CD10 and CD105 also increased after cell passages while the expression of CD49a made no significant difference at progressive stages of isolation and passage. Comparison of chondrogenic potentials between positive and negative cell pellets for each marker revealed that only CD105- and CD166-positive cell pellets showed better chondrogenic potentials (type II collagen gene expression, cartilage matrix formation, and GAG expression) than the corresponding negative cell pellets. CONCLUSION: Our study suggests that CD105 and CD166 would be valuable surface markers associated with chondrogenic potential; thus, CD105- and CD166-enriched cells derived from human synovium would be practical and valuable sources for cartilage regeneration.

Patient expectations of total knee replacement and their association with sociodemographic factors and functional status.

We aimed to document the pre-operative expectations in Korean patients undergoing total knee replacement using an established survey form and to determine whether expectations were influenced by sociodemographic factors or pre-operative functional status. Expectations regarding 17 items in the Knee Replacement Expectation Survey form were investigated in 454 patients scheduled for total knee replacement. The levels and distribution patterns of summated expectation and of five expectation categories (relief from pain, baseline activity, high flexion activity, social activity and psychological well-being) constructed from the 17 items were assessed. Univariate analyses and multivariate logistic regression were performed to examine the associations of expectations with the sociodemographic factors and the functional status. The top three expectations were relief from pain, restoration of walking ability, and psychological well-being. Of the five expectation categories, relief from pain was ranked the highest, followed by psychological well-being, restoration of baseline activity, ability to perform high flexion activities and ability to participate in social activities. An age of < 65 years, being employed, a high Western Ontario and McMaster Universities osteoarthritis index function score and a low Short-form 36 social score were found to be significantly associated with higher overall expectations.

What should be considered in using standard knee radiographs to estimate mechanical alignment of the knee?

OBJECTIVE: Anatomical tibiofemoral angle (anatomical TFA) of the knee measured on standard knee radiographs is still widely used as proxy for mechanical tibiofemoral angle (mechanical TFA), because of the practical and economic limitations in using full-limb radiographs. However, reported differences between anatomical and mechanical TFAs show wide variations. The first aim of this study was to determine whether gender, the presence of advanced osteoarthritis (OA), and history of total knee arthroplasty (TKA) influence the differences between anatomical and mechanical TFAs. The second aim was to identify anatomical features that cause divergences between anatomical and mechanical TFAs, and the final aim was to determine whether anatomical TFA measured using reference points more distant from the knee provides more accurate estimates of mechanical TFA. DESIGN: In 102 knees with advanced OA before and after TKAs and 99 control knees with no/minimal OA, we assessed the differences between two anatomical TFAs, namely, anatomical TFA1 and anatomical TFA2, which were based on conventional or more distant proximal and distal reference points on standard knee radiographs, respectively, and the mechanical TFA measured on full-limb radiographs. These differences were investigated for women vs men, no/minimal OA vs advanced OA, and for knees before vs after TKA. Regression analyses were performed to determine associations between femoral and tibial anatomical characteristics and the differences between mechanical and anatomical TFAs. RESULTS: The OA group showed significantly greater differences between mechanical and anatomical TFAs than the control group for both genders. In OA and TKA group, women were more likely to have greater mean differences between mechanical and anatomical TFAs than men. However, TKA did not significantly affect these differences. Femoral and tibial bowing angles, particularly of the femur, were found to be the major contributors to divergences between mechanical and two anatomical TFAs. Furthermore, anatomical TFA2 was found to provide more accurate estimates of mechanical TFA. CONCLUSIONS: We found that the differences between mechanical and anatomical TFAs depend on gender and the presence of advanced OA, but not on a history of TKA. These findings indicate that prediction of mechanical TFA based on anatomical TFA is dependent on study population characteristics. This study also shows that the presence of lateral bowing of the femur is a major cause of mechanical TFA to anatomical TFA variations associated with gender and advanced OA. To reduce the adverse effects of anatomical variations on estimations of mechanical TFA based on an anatomical TFA method, more distant proximal and distal reference points are recommended to determine anatomical TFA value on standard knee radiographs.

Evaluations of radiographic joint space--do they adequately predict cartilage conditions in the patellofemoral joint of the patients undergoing total knee arthroplasty for advanced knee osteoarthritis?

OBJECTIVE: To determine whether assessments of patellofemoral (PF) joint space reliably predict the cartilage conditions in the PF joint of the patients undergoing total knee arthroplasty (TKA) for advanced knee osteoarthritis. METHODS: Radiographic joint spaces and gross cartilage conditions in the PF joint were assessed in 151 osteoarthritic knees undergoing TKA. Minimum joint space widths (MJSWs) in the medial and lateral compartments of the PF joint were measured separately on Merchant radiographs. Degrees of joint space were graded as normal, narrowed and obliterated, and expected locations of significant cartilage lesions were classified as medial, lateral, and global. Cartilage lesions were grossly assessed in terms of their severity (depth and size) and location. Cross-table analyses and kappa statistics were used to determine the level of agreements between radiographic and gross assessments and the diagnostic accuracies of the radiographic assessments. RESULTS: The agreements between the radiographic assessments and the gross assessments on the lesion severities and locations were only fair [kappa coefficient (k)=0.288 and 0.211, respectively]. The cross-table analyses demonstrated that 45 (47.4%) of 95 knees with a normal radiographic joint space had moderate or severe cartilage degeneration of the PF joint identified with gross assessments. In the radiographic assessments, the lateral compartment of the PF joint was the most frequent location of joint space narrowing (71.4%) whereas in the gross assessments, the medial compartment of the PF joint was the most frequent location of significant cartilage lesion (48.1%). Diagnostic accuracies on the lesion severities and locations were generally poor. CONCLUSION: This study demonstrates that prediction of the cartilage conditions of the PF joint by the radiographic joint space can be inaccurate.

The relationship of the medially-offset stem of the tibial component to the medial tibial cortex in total knee replacements in Korean patients.

We examined the placement of the stem in relation to the medial tibial cortex when using total knee replacements (TKRs) with medially-offset tibial stems in Korean patients. Measurements were performed on the pre- and post-operative radiographs of 246 osteoarthritic knees replaced between January 2005 and May 2006 using the Genesis II or E-motion TKR with a medially-offset stem. Pre-operatively, we measured the distance between the mechanical axis and that of the tibial shaft and post-operatively, that between the midline of the tibial stem and the axis of the shaft. Knees were identified in which there was radiological contact between the tip of the stem and the medial tibial cortex. The mechanical axis was located medial to the axis of the shaft in 203 knees (82.5%). Post-operatively, the midline of the tibial stem was located medial to the tibial shaft axis in 196 knees (79.7%). In 16 knees (6.5%) there was radiological contact between the tibial stem or cement mantle and the medial tibial cortex. Our study has shown that the medially-offset stem in the tibial component may not be a good option for knees undergoing replacement for advanced osteoarthritis in some Korean patients.

The epidemiology of total knee replacement in South Korea: national registry data.

OBJECTIVE: Knee osteoarthritis (OA) is prevalent among the elderly in Asian countries; however, the utilization of total knee replacement (TKR) is unknown. Using data from a national registry, we sought to estimate the incidence of TKR by age and sex, and the trend for TKR utilization from 2002 to 2005 in South Korea. METHODS: Reimbursement records from all hospitals in South Korea were extracted from the Health Insurance Review Agency (HIRA) database. Records with both the procedure code corresponding to a TKR and the diagnosis code of knee OA were selected. We estimated the age- and sex-specific rates of TKR. To compare the rate of TKR between men and women, we calculated age-standardized risk ratios. A TKR registry from a single centre containing more clinical information was analysed, and the differences in the clinical features between men and women were compared. RESULTS: From 2002 to 2005, 47 961 TKRs were performed in subjects over the age of 40 yrs due to OA. The rate of TKR has increased over the 4 yrs and was much higher in women than in men. Compared with men, the age-standardized rate ratios for TKR in women ranged from 7.4 to 8.0. The single-centre registry data revealed that there was no difference in age, disease duration and the Kellgren-Lawrence grade at the time of surgery between men and women. CONCLUSION: Using a national database, we found that the rate of TKR increased steadily from 2002 to 2005 in South Korea. The TKR rate in women was much higher than that in men. Risk factors that account for such disparity in TKR utilization need to be further investigated.

Association between radiological findings and symptoms at the patellofemoral joint in advanced knee osteoarthritis.

We investigated the association between the radiological findings and the symptoms arising from the patellofemoral joint in advanced osteoarthritis (OA) of the knee. Four radiological features, joint space narrowing, osteophyte formation, translation of the patella and focal attrition were assessed in 151 consecutive osteoarthritic knees in 107 patients undergoing total knee replacement. The symptoms which were assessed included anterior knee pain which was scored, the ability to rise from a chair and climb stairs, and quadriceps weakness. Among the radiological features, only patellar translation and obliteration of the joint space had a statistically significant association with anterior knee pain (odds ratio (OR) 4.85; 95% confidence interval (CI) 1.83 to 12.88 and OR 11.23; 95% CI 2.44 to 51.62) respectively. Patellar translation had a statistically significant association with difficulty in rising from a chair (OR 9.06; 95% CI 1.75 to 45.11). Other radiological features, including osteophytes, joint space narrowing, and focal attrition had no significant association. Our study indicates that the radiological findings of patellar translation and significant loss of cartilage are predictive of patellofemoral symptoms and functional limitation in advanced OA of the knee.

Correlation of maximum flexion with clinical outcome after total knee replacement in Asian patients.

This study aimed to determine the correlation between the amount of maximum flexion and the clinical outcome in 207 Koreans (333 knees) undergoing total knee replacement. The association of maximum flexion with clinical outcome was evaluated one year postoperatively using three scoring systems; the American Knee Society score, Western Ontario McMaster Universities Osteoarthritis index and the Short Form-36. The mean maximum flexion decreased post-operatively at 12 months from 140.1 degrees (60 degrees to 160 degrees ) to 133.0 degrees (105 degrees to 150 degrees ). Only the social function score of the Short Form-36 correlated significantly with maximum flexion (correlation coefficient = 0.180, p = 0.039). In comparative analyses of subgroups divided by a maximum flexion of 120 degrees , we found no significant differences in any parameters except the social function score of the Short Form-36 (41.9 vs 47.3, p = 0.031). Knees with a maximum flexion of more than 135 degrees had a better functional Western Ontario McMasters Universities Osteoarthritis index score than knees with maximum flexion of 135 degrees or less (17.5 vs 14.3, p = 0.031). We found only weak correlation between the postoperative maximum flexion and the clinical parameters for pain relief, function and quality of life, even in Korean patients. Efforts to increase post-operative maximum flexion should be exercised with caution until concerns relating to high-flexion activities are sufficiently resolved.

Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium.

BACKGROUND: Synovium is the only tissue that can produce hyaline cartilage in benign conditions, such as synovial chondromatosis and osteoarthritis, suggesting potential advantages in chondrogenesis using mesenchymal stromal cells. We performed surface characterization of cells isolated from the synovium of patients with osteoarthritis after different passages and induced chondrogenic differentiation. METHODS: Using cells obtained from synovium, colony-forming unit fibroblast assay and characterization of cell-surface markers by flow cytometry using 22 different Ab at different passages were performed. Cells were cultured under chondrogenic conditions and evaluated grossly, histologically, immunohistochemically and by [(35)S]sulfate incorporation and reverse transcription-PCR. RESULTS: The positive cell-surface markers of immediately isolated cells were CD10, CD13, CD14, CD34, CD44, CD45, CD49a, CD62e, CD73 and HLA-DR. After the first passage (P), CD14, CD34, CD45, CD62e and HLA-DR disappeared, whereas CD105 and CD166 appeared and CD10, CD13, CD44, CD49a and CD73 showed increased expression levels. The surface marker expression level did not vary much after P1 through to P8. The chondrogenic differentiation potential of cells from the synovium was confirmed using various evaluation methods. DISCUSSION: We have demonstrated that cells from synovium contain a mesenchymal stromal cell population capable of chondrogenic differentiation, which seems to increase with passage under our culture conditions. The cell-surface markers were found to change remarkably after the first passage and then remained stable. The results of this study may be helpful for sorting mesenchymal stromal cells from heterogeneous synovial cells for future studies.

Correlation of the condition of the patellar articular cartilage and patellofemoral symptoms and function in osteoarthritic patients undergoing total knee arthroplasty.

We sought to determine the degree of correlation between the condition of the patellar articular cartilage and patellofemoral symptoms and function in osteoarthritic patients undergoing total knee arthroplasty. The depth of the osteoarthritic lesion, as graded by the Outerbridge classification and its size and location were assessed to determine the condition of the patellar cartilage in 80 consecutive osteoarthritic knees undergoing total knee arthroplasty. The association between the condition of the cartilage and patellofemoral symptoms and function was investigated by correlation analysis. The depth and size of the lesion had a significant but weak correlation with anterior knee pain (r = -0.300 and -0.289; p = 0.007 and 0.009, respectively), whereas location had no significant association (p > 0.05). None had a significant association with patellofemoral functional parameters (chair-rising, stair-climbing, and quadriceps power) (p > 0.05). Our study indicates that patellofemoral symptoms and function are not completely determined by the condition of the cartilage. Caution should be taken when the symptoms and functional limitations are attributed to a lesion in the patellofemoral joint in making a decision regarding patellar resurfacing in total knee arthroplasty.

The in vitro effects of dehydroepiandrosterone on human osteoarthritic chondrocytes.

OBJECTIVE: To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on human osteoarthritic chondrocytes. DESIGN: Chondrocytes isolated from human osteoarthritic knee cartilage were three-dimensionally cultured in alginate beads, except for cell proliferation experiment. Cells were treated with DHEA in the presence or absence of IL-1beta. The effects on chondrocytes were analyzed using a 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (for chondrocyte proliferation), a dimethylmethylene blue (DMB) assay (for glycosaminoglycan (GAG) synthesis), and an indole assay (for DNA amount). Gene expressions of type I and II collagen, metalloproteinase-1 and -3 (MMP-1 and -3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as the IL-1beta-induced gene expressions of MMP-1 and -3 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein synthesis of MMP-1 and -3 and TIMP-1 was determined by Western blotting. RESULTS: The treatment of chondrocytes with DHEA did not affect chondrocyte proliferation or GAG synthesis up to 100 micro M of concentration. The gene expression of type II collagen increased in a dose-dependent manner, while that of type I decreased. DHEA suppressed the expression of MMP-1 significantly at concentrations exceeding 50 micro M. The gene expression of MMP-3 was also suppressed, but this was without statistical significance. The expression of TIMP-1 was significantly increased by DHEA at concentrations exceeding 10 micro M. The effects of DHEA on the gene expressions of MMP-1 and -3 were more prominent in the presence of IL-1beta, in which DHEA suppressed not only MMP-1, but also MMP-3 at the lower concentrations, 10 and 50 micro M, respectively. Western blotting results were in agreement with RT-PCR, which indicates that DHEA acts at the gene transcription level. CONCLUSIONS: Our study demonstrates that DHEA has no toxic effect on chondrocytes up to 100 micro M of concentration and has an ability to modulate the imbalance between MMPs and TIMP-1 during OA at the transcription level, which suggest that it has a protective role against articular cartilage loss.

In vitro release of vancomycin from vancomycin-loaded blood coated demineralised bone.

In vitro and in vivo studies have demonstrated the possibility that cancellous bone could be used as a carrier of antibiotics for local delivery. However, the release of antibiotics from the loaded cancellous bone is too rapid and uncertain. We hypothesised that demineralisation of cancellous bone would increase the amount of antibiotic adsorbed, and coating of the freeze-dried antibiotic-impregnated cancellous bone with bio-compatible material would prolong antibiotic release. Bovine cancellous bone blocks of equal size were demineralised using a 0.5 N HCl solution and loaded with vancomycin solution under vacuum. The loaded bone blocks were then freeze-dried. To obtain a bio-compatible coating, the vancomycin-impregnated bone blocks were soaked in fresh human venous blood for 3 h. The release of impregnated antibiotic from the bone blocks was evaluated in phosphate-buffered saline and foetal bovine serum. It was found that significantly larger amounts of vancomycin were adsorbed into the demineralised bone blocks than into the un-demineralised blocks. The blood coating was found to increase the duration of vancomycin release from the blocks. With demineralisation and blood coating, the blocks eluted vancomycin higher than therapeutic concentration for a 5-week period.

A comparative study of the healing of tendon autograft and tendon-bone autograft using patellar tendon in rabbits.

In order to compare the healing of tendon to bone and the healing of bone to bone in a rabbit model, the lateral 4 mm of patellar tendons were detached from their insertion into the tibia either subperiosteally (group I) or with a bone block (group II) and implanted into drill holes in the proximal articular surface of the tibia. The histological and biomechanical features of the graft incorporation were observed at 2, 4, 8 and 12 weeks. Histological patterns similar to normal tendon-bone attachment were seen at the tendon-bone interface in group I by 12 weeks, while direct bony union was seen in group II by 8 weeks. The maximum tensile load and stiffness were significantly greater in group II at 4 and 8 weeks while the difference between the two groups was not significant at 2 and 12 weeks. These findings show that more rapid incorporation of the graft occurs in group II although no significant difference in biomechanical parameters was noted once healing was complete.

Dehydroepiandrosterone-dependent induction of peroxisomal proliferation can be reduced by aspartyl esterification without attenuation of inhibitory bone loss in ovariectomy animal model.

The purpose of this study was to determine whether esterification of dehydroepiandrosterone with aspartate (DHEA-aspartate) could reduce peroxisomal proliferation induced by DHEA itself, without loss of antiosteoporotic activity. Female Sprague-Dawley rats were ovariectomized, then DHEA or DHEA-aspartate was administered intraperitoneally at 0.34 mmol/kg BW 3 times a week for 8 weeks. DHEA-aspartate treatment in ovariectomized rats significantly increased trabeculae area in tibia as much as DHEA treatment. Urinary Ca excretion was not significantly increased by DHEA or DHEA-aspartate treatment in ovariectomized rats, while it was significantly increased by ovariectomy. Osteocalcin concentration and alkaline phosphatase activity in serum and cross linked N-telopeptide type I collagen level in urine were not significantly different between DHEA-aspartate and DHEA treated groups. DHEA-aspartate treatment significantly reduced liver weight and hepatic palmitoyl-coA oxidase activity compared to DHEA treatment. DHEA-aspartate treatment maintained a nearly normal morphology of peroxisomes, while DHEA treatment increased the number and size of peroxisomes in the liver. According to these results, it is concluded that DHEA-aspartate ester has an inhibitory effect on bone loss in ovariectomized rats with a marked reduction of hepatomegaly and peroxisomal proliferation compared to DHEA.

Indirect signs of anterior cruciate ligament injury on SPET: comparison with MRI and arthroscopy.

Indirect signs of anterior cruciate ligament (ACL) injury on magnetic resonance imaging (MRI) include bony contusions in the lateral femoral condyle and the posterior portion of the lateral tibial plateau. This study was undertaken to assess the value of single photon emission tomography (SPET) in the diagnosis of ACL injury by examining the uptake pattern in the distal femur and the proximal tibia. Thirty-five patients were examined using SPET, MRI and arthroscopy. Seventeen patients were found to have ACL tears on arthroscopy. The duration of symptoms was 4 days to 10 years (mean 26.4 months). MRI and SPET images were analysed retrospectively without information from arthroscopic examination. Radionuclide uptake in the lateral femoral condyle and the posterior lateral tibial plateau was considered an indirect sign of ACL injury on SPET. We evaluated the diagnostic value of indirect signs of ACL injury obtained on SPET by comparing these findings with arthroscopic and MRI results. Fifteen of 17 patients with ACL injury showed indirect signs on SPET. The sensitivity, specificity, positive predictive value and negative predictive value for indirect signs of ACL injury were 88%, 56%, 65% and 83% on SPET and 59%, 94%, 91% and 71% on MRI, respectively. However, despite the higher sensitivity of indirect signs on SPET than on MRI, the overall diagnostic value of MRI is better than that of SPET. In the clinical setting, indirect signs of ACL injury may be of value in interpreting incidental findings on SPET.

Usefulness of 99Tcm-MDP knee SPET for pre-arthroscopic evaluation of patients with internal derangements of the knee.

The aim of this study was to ascertain whether knee SPET can localize lesion sites in patients with internal derangements of the knee. We performed knee SPET as a pre-arthroscopic examination in 63 consecutive patients. SPET imaging was performed with a triple-headed SPET camera 4 h after the injection of 99Tcm-methylene diphosphonate. Arthroscopic diagnoses were as follows: 28 medial meniscus injuries, 24 lateral meniscus injuries, 31 anterior cruciate ligament (ACL) injuries, three posterior cruciate ligament injuries and one medial collateral ligament injury. Of 30 patients with crescent-shaped increased activity at the medial tibial plateau, 22 had medial meniscus injuries (positive predictive value: PPV 73%); of 17 patients with crescent-shaped activity at the lateral tibial plateau, 13 had lateral meniscus injuries (PPV 76%). Of 18 patients with increased activity at ACL attachment sites (primary sign), 17 had ACL injuries (PPV 94%). Of 27 patients with increased activity at bone impaction sites of ACL injury (secondary sign), 22 had ACL injuries (PPV 81%). Of 32 patients who had either a primary or secondary sign, 26 had ACL injuries (PPV 81%). We conclude that knee SPET is very useful in the management of internal derangements of the knee, particularly in determining the need for arthroscopy by localizing lesion sites.

Apoptotic chondrocyte death in human osteoarthritis.

OBJECTIVE: To define apoptotic chondrocyte death and the expression of Bcl-2, Bax, and Fas in human osteoarthritis (OA) cartilage. METHODS: Cartilage samples were obtained from patients with knee OA at the time of joint replacement surgery and from normal autopsy cases. In OA, sections were obtained both from the lesional area, usually within 1 cm of bony exposure, and from the non-lesional area, which had macroscopically normal appearance or only mild surface irregularities. Apoptosis was verified by microscopic examination of hematoxylin and eosin stained specimens, TUNEL staining, electron microscopy, and DNA ladder analysis by electrophoresis. Immunohistochemistry was done to study the expression of Bcl-2, Bax, and Fas. Apoptotic cells and Bcl-2, Bax, and Fas positive cells were counted within defined microscopic fields. Expression of Bcl-2 and Bax was verified by Western blot. RESULTS: The percentage of apoptotic cells in the lesional area was significantly higher than in the non-lesional area in cartilage from the same patient with OA, while apoptotic cells were rarely seen in normal cartilage. This result was confirmed by TUNEL stain. Many chondrocytes with chromatin condensation were verified in electron microscopy, and DNA from OA lesional cartilage revealed a DNA ladder on electrophoresis. Bcl-2 and Fas expressions were significantly higher in the OA lesional area than in the non-lesional area. On the other hand, Bcl-2 expression in normal cartilage was significantly higher than in OA cartilage. There was no significant difference in Bax expression among normal, OA lesional, and OA non-lesional cartilage. CONCLUSION: These results show that apoptotic chondrocyte death occurs more frequently in OA compared to normal cartilage and in OA lesional compared to OA non-lesional cartilage. The different expression patterns of Bcl-2 and Fas in OA lesional and non-lesional cartilage suggest that they might be involved in the pathogenesis of OA.

Aging process is accompanied by increase of transglutaminase C.

Crosslinking has been suggested as one of the mechanisms involved in the aging process. Among the various random or enzyme-mediated crosslinking reactions, transglutaminase (TGase)-catalyzed crosslinking activity has been proposed for its possible involvement in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. Moreover, recent findings of TGase C as a putative signal transducer and cell cycle regulator has renewed interest in the study of TGase C in relation to aging phenomena. The ubiquitous presence of TGase C compared to the organ-specific localization of other types of TGases has attracted special attention as a cellular aging device. In the present investigation for in vitro studies, we have compared the pattern of TGase C in young and old human red blood cells, separated by density differentiation, and in early and late-passage or hydrogen peroxide-treated human primary fibroblasts. For in vivo study, we monitored the age-dependent changes of TGase C in the liver and brain tissues of 4, 12, 18, and 24-month-old Sprague-Dawley rats. We obtained evidence that both the activity and protein levels of TGase C were high in old RBC and late-passage or hydrogen peroxide-treated fibroblasts. Similar findings were seen in liver and brain tissue such as age-dependent increases in TGase activity and protein level in an organ-specific pattern. These data suggest that TGase C might play an active role in the cellular process with age.

Effect of the distraction rate on the activity of the osteoblast lineage in distraction osteogenesis of rat's tibia. Immunostaining study of the proliferating cell nuclear antigen, osteocalcin, and transglutaminase C.

The purpose of this study was to investigate kinetics of the osteoblast lineage in the periosteum and endosteum according to different distraction rates in distraction osteogenesis of rat's tibia. An osteotomy was performed on 144 rats at the proximal diaphysis of the left tibia. The lengthening process was started after a latency period of 3 days, with varying distraction rates of 0.25 mm (group I), 0.5 mm (group II), 0.75 mm (group III), 1.0 mm (group IV) and proceeded until a 3.5 mm length gain was achieved. The animals that had an osteotomy alone, without lengthening, served as a control (group V). Immunohistochemical staining of proliferating cell nuclear antigen (PCNA), osteocalcin, and transglutaminase C (TGase C) were done on the four animals in each group sacrificed at post-distraction days 1, 3, 5, 7, 14, and 28 in order to observe the temporal changes among the experimental and control groups. Also, in order to compare the staining rates at a given length gain among the groups, animals in each group were additionally sacrificed 2 days post-distraction in group II; 2 and 4 days in group III; and 1.5, 2 and 2.5 days in group IV. The results of the expression rates of PCNA, osteocalcin, and TGase C in each group were analyzed quantitatively. The immunohistochemical study on callotasis of rat's tibia revealed that the osteoblast lineage in the periosteum is more activated than that in the endosteum for proliferation and differentiation by distraction, suggesting that the periosteum plays a more important role in neoosteogenesis in the distraction gap. Daily distraction rates ranging from 0.25 mm to 0.75 mm in two increments is appropriate for successful distraction osteogenesis of rat's tibia, but the rate of 0.25 mm a day is significantly better than that of 0.75 mm as was made evident in the immunohistochemical observations.

Modulation of transglutaminase expression in rat skeletal muscle by induction of atrophy and endurance training.

The persistence of muscle fiber number regardless of size reduction in muscle atrophy has not yet been fully explained. For the mechanism inherent in skeletal muscle tissues for preventing cellular death, the protective function of muscle tissue through transglutaminases has been tested, since the enzyme is responsible for structural stabilization and participates in signal transduction. In the present experiment, hindlimb suspension for two weeks caused a marked muscle atrophy in Wistar female rats. Comparison of muscle weight and histological analysis showed that suspension-induced atrophy in the hindlimb was more prominent in the soleus muscle, comprised mainly of type I fiber than that in the plantaris muscle of type II fibers. The immunohistochemical analysis with antitransglutaminase C antibody (anti TGase C Ab) showed that some atrophic bundles of soleus muscle were positively reacted with the antibody. The anti-TGase C Ab-reactive substances were observed to disappear significantly after endurance exercise, indicating their characteristic atrophy-dependency. The enzymatic analysis of transglutaminase showed the increase in activity in the atrophic soleus muscle tissue, compared with that in the normal or exercise-trained muscle tissues. From these results, the expression of TGase in the atrophic muscle is suggested to be the possible marker for muscle atrophy and its expression is probably related with the protective mechanism of the muscle tissue to prevent further cellular damage in the atrophic process.