Histological Observation of Regions around Bone Tunnels after Compression of the Bone Tunnel Wall in Ligament Reconstruction.

The objectives of this study were to investigate the time-course of influence of compression of bone tunnel wall in ligament reconstruction on tissue around the bone tunnel and to histologically examine the mechanism of preventing the complication of bone tunnel dilation, using rabbit tibia. A model in which the femoral origin of the extensor digitorum longus tendon was cut and inserted into a bone tunnel made proximal to the tibia was prepared in the bilateral hind legs of 20 Japanese white rabbits. In each animal, a tunnel was made using a drill only in the right leg, while an undersized bone tunnel was made by drilling and then dilated by compression using a dilator to the same tunnel size as that in the right leg. Animals were sacrificed at 0, 2, 4, 8 and 12 weeks after surgery (4 animals at each time point). Observation of bone tunnels by X-ray radiography showed osteosclerosis in the 2- and 4-week dilation groups. Osteosclerosis appeared as white lines around the bone tunnel on X-ray radiography. This suggests that dilation promotes callus formation in the bone tunnel wall and prevents the complication of bone tunnel enlargement after ligament reconstruction.

Development of a systematic telesimulation curriculum for laparoscopic inguinal hernia repair.

Purpose: Telesimulation, whereby learners telecommunicate and use simulator resources to receive education at an off-site location, has been used to educate surgical trainees about how to perform basic surgical procedures. However, it has not yet been used for advanced surgical procedures. We aimed to develop a telesimulation curriculum to teach a common laparoscopic procedure called transabdominal preperitoneal (TAPP) repair and to explore the feasibility of its use. Methods: Learning objectives were created to develop a telesimulation curriculum that included didactic telelecture and telesimulation training. Pre-tests and post-tests to assess the didactic materials were developed and assessed among surgeons with various experiences. We assessed the feasibility of the telelecture and telesimulation separately. Pre-tests, post-tests, and questionnaires were used to assess the telelectures. We created a TAPP repair telesimulation system and checked for problems during training. Trainees were assessed to determine their skill improvement using previously published assessment tools and questionnaires. Results: A didactic telelecture was developed based on the learning objectives using an expert consensus and pilot-tested among five participants. After the lecture, their test scores improved and they expressed positive opinions about the usefulness of telelectures. The TAPP repair telesimulation training was pilot-tested among three trainees. No technical problems occurred during training. All trainees improved their skills after the telesimulation training and agreed that the training was useful for learning the TAPP repair procedure. Conclusions: We systematically developed a telesimulation curriculum for the TAPP repair procedure and demonstrated its feasibility among learners.

Effect of chondroitinase on dermatan sulfate-facilitated arginine amidase released from rabbit ear artery.

We examined the effects of chondroitinases on the release of dermatan sulfate (DS)-induced arginine amidase (AA) from rabbit ear artery. DS-induced AA release was significantly decreased by treatment with chondroitinase ABC (ABCase) in the rabbit ear artery. On the other hand, Chondroitinase ACII (ACIIase) enhanced spontaneous and DS-induced AA release. Heat-inactivated ABCase and ACIIase did not affect spontaneous and DS-induced AA release. Furthermore, ABCase, but not ACIIase and heat-inactivated chondroitinases, degraded DS. These results indicate that the facilitatory effect of DS-induced AA release from the rabbit ear artery is affected by the molecular size of DS.

Analytical method to determine keratan sulfate in the serum using HPLC.

There is an established method to assess the extent of articular cartilage damage that is based on the determination of keratan sulfate (KS) in synovial fluid by high performance liquid chromatography (HPLC). However, since it is easier to collect serum than synovial fluid, and the serum concentration of KS might reflect the extent of degeneration and injury of the articular cartilage, we aimed at developing a method to determine KS in serum by HPLC. Human serum was spiked with low-sulfated KS or poly-sulfated KS. After digestion of serum protein with a protease, the KS fraction was extracted by anion exchange column chromatography and then desalted. The KS fraction was digested with keratanase II and determined by HPLC. The recovery of KS added to the serum was 98% or more and did not depend on the degree of KS sulfation. A good recovery rate was obtained within a range of 50-100,000 ng/ml. The inter-day assay variation was 6.0% for 10 days. This method enables the determination of serum KS concentration without being influenced by the degree of KS sulfation.

[Absolute risk for fracture and WHO guideline. Biological markers for osteoarthritis (OA)].

Biological markers for osteoarthritis (OA) are indicators of articular tissue metabolism, measuring the levels of molecules derived from joint structures into synovial fluids, serum and urine. Radiological findings are the basis of diagnosis of OA, but have a weakpoint visualizing the figures that have already occurred. For assessing disease activity or monitoring disease progression, estimation of articular metabolism using biological markers is very important. Synovial fluid reflects specifically the status of the punctured joint, however invasive. Less-invasive newly developed serum and urine markers appear to be also useful for the evaluation of OA.

Development and validation of a checklist for assessing recorded performance of laparoscopic inguinal hernia repair.

BACKGROUND: Despite a need for video assessment for the performance transabdominal preperitoneal procedure (TAPP), the present assessment tools have not been validated for the use of evaluation of the recorded performance. We aimed to develop a checklist for the evaluation of the recorded performance of TAPP. METHODS: The TAPP checklist was developed by hernia experts from multiple institutes. Thirty unedited TAPP videos were rated by 3-blinded hernia experts. Inter-rater reliability and construct and concurrent validities were evaluated. RESULTS: The inter-rater reliability for 3 raters was .75 (95% confidence interval .60 to .86). The median total score of each group demonstrated a significant difference among experienced (>50 TAPP), intermediate (≥10 TAPP, <50), and novice (<10 TAPP) surgeons (P < .001). The checklist score showed a high correlation with TAPP experience and previously validated global scale for laparoscopic inguinal hernia repair. CONCLUSIONS: The TAPP checklist is a valid metrics for the assessment of the recorded TAPP performance.

Histomorphometric Evaluation of Cartilage Degradation using Rabbit Articular Chondrocytes Cultured in Alginate Beads - Effects of Hyaluronan.

OBJECTIVE: A 3-dimensional alginate bead culturing method using rabbit articular chondrocytes was studied for the screening of the effectiveness of drugs for articular diseases. DESIGN: The beads cultured with IL-1ß, TGF-ß, and Hyaluronan (HA) were evaluated histochemically with Alecian blue and immunohistochemically with CS-56 antibody. Chondrocytes in alginate beads were arbitrarily classified into four groups: 1) chodrocyte surrounded with cell-associated matrix (CAM) in which proteoglycan (PG) was positively stained (PG-possitive chondrocyte); 2) chondrocyte with PG-negative CAM; 3) PG-positive CAM alone, and 4) PG-negative CAM alone. Total sulfated GAG concentrations in the culture media were quantitated by dimethylmethylene blue (DMMB) assay. ProMMP-3, TIMP-1 and -2 concentrations in the culture media were determined by sandwich enzyme immunoassays. RESULTS: Significant increase of PG-nagative cells were immunohistochemically found by IL-1ß stimulation. The pretreatment with TGF-ß almost fully suppressed those increase of PG-negative cells by IL-1ß. Both GAG and proMMP-3 concentrations in the culture media were significantly increased after IL-1ß stimulation. There were no significant differences in both TIMP-1 and TIMP-2 concentrations in the culture media with or without IL-1ß stimulation. 800-kDa HA reduced significantly the number of PG-negative cells and proMMP-3 concentration in the culture media, but showed no effects on the concentrations of both TIMPs. CONCLUSIONS: Because this 3-dimensional chondrocyte culture in alginate beads is close to in vivo conditions, this method can be used for evaluation of the effectiveness of novel drugs for articular diseases.

Acute stress augments innate immunity in the liver and increases hyaluronan levels in the tissues and blood of mice.

The effect of acute stress on the immune system was examined in mice. Restraint stress decreased the number of lymphocytes in the liver, whereas the number of lymphocytes remained unchanged in the spleen and thymus. In the liver, the decrease in number appeared at 1.5 h and fell to a third of he control level at 3 h. The proportions of IL-2Rbeta(+)CD3(int) cells, NKT cells, CD44(+) T cells and B cells were changed in the liver. The absolute numbers of IL-2Rbeta(+)CD3(int) cells, NKT cells and CD3(+)CD44(+) cells remained constant in the liver under the stress, while those of total T cells and NK cells decreased. The levels of hyaluronan (HA) in various tissues and sera were then examined. The expression of hyaluronan binding protein (HABP) was found to increase in the skin, liver and kidney as shown by immunohistochemical staining. An increase of HA in sera due to stress was seen at 3 h. The present results suggest that the activation of CD44(+) T cells and unconventional T cells (i.e., innate immunity) in the blood and the elevated levels of HA (ligand for CD44) in the tissues and blood are crucial responses to acute stress exposure.

Serum keratan sulfate transiently increases in the early stage of osteoarthritis during strenuous running of rats: protective effect of intraarticular hyaluronan injection.

INTRODUCTION: Osteoarthritis is influenced by genetic and environment factors, including mechanical stress; however, the relationship between running and the development of osteoarthritis remains a matter of controversy. We investigated whether osteoarthritic change could be obtained in a rat strenuous running model, whether serum keratan sulfate in rats could be detected by HPLC and was associated with onset or progression of osteoarthritis, and whether hyaluronan injection suppressed development of osteoarthritis and elevation of serum keratan sulfate. METHODS: Wistar rats were forced to run 30 km in 6 weeks on a treadmill machine. Articular cartilage of the knees was evaluated macroscopically and immunohistologically. Serum keratan sulfate was examined every week by HPLC. The effect of weekly knee injection of hyaluronan was also investigated. RESULTS: Cartilage surfaces stained with India ink became irregular, metachromasia by safranin-O staining appeared to be almost lost, and Mankin's score significantly worsened after 30 km of running. Serum keratan sulfate in rats was detected by HPLC and transiently increased (peaked at 3 to 4 weeks) along with depletion of keratan sulfate in cartilage tissue. Hyaluronan treatment suppressed morphological progression of osteoarthritis and elevation of serum keratan sulfate. CONCLUSION: Rat strenuous running induced osteoarthritis. Serum keratan sulfate was associated with progression of osteoarthritis. Weekly intraarticular injection of hyaluronan controlled the development of osteoarthritis, and the effect was reflected by serum keratan sulfate.

Intraarticular injection of high molecular weight hyaluronan for osteoarthritis of the knee - prediction of effectiveness with biological markers.

OBJECTIVE: The possibility of predicting the effectiveness of intraarticular injection of high molecular weight hyaluronan (HA) was investigated using biological markers. METHODS: In 32 patients with osteoarthritis (OA) of the knee, 38 knees were treated with HA injection, and the clinical symptoms were evaluated using the Japanese Orthopaedic Association (JOA) score and pain visual analog scale (VAS). The concentrations of chondroitin 6-sulfate, 4-sulfate (C6S, C4S), and aggrecan were measured in synovial fluid collected at the time of initiation of injection. The relationship between the biological markers and the improvement of clinical symptoms after injection for 1 month was investigated. RESULTS: C6S/C4S and concentration of aggrecan decreased after injection, although these decreases were not significant. Positive correlations were noted between the concentrations of C6S and aggrecan before HA injection and the improvement of the JOA score after injection; however, radiological OA stage had no significant relation with improvement both of the JOA score and VAS. CONCLUSION: It has been reported that the concentration of aggrecan-derived fragments in synovial fluid decreases with advancement of the OA stage, reflecting decreases in the amount of residual cartilage and suppression of chondrocyte metabolism. Our findings suggested that HA injection exhibits a greater clinical effect in cases with a high intraarticular aggrecan fragment concentration, i.e., cases in which a high amount of residual cartilage and chondrocyte metabolic activity remain. The biological markers were useful in predicting the effectiveness of HA injection for OA of the knee.

In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel.

Graft implantation is one of the more popular procedures for repairing cartilage defects; however, sacrifices of the donor site have been an issue. Mesenchymal stem cells (MSCs) are a fascinating source for regenerative medicine because they can be harvested in a less invasive manner and are easily isolated and expanded, with multi-potentiality including chondrogenesis. MSCs can be isolated from various adult mesenchymal tissues including synovium. Here, we attempted to form cartilage from the composites of synovium-derived MSCs with collagen gel in vitro. After 21 days of culture, the composites had increased their cartilage matrix, as demonstrated by toluidine blue staining and immunohistochemistry for type II collagen. The composites consisting of 5 x 10(7) and 10(8) cells/ml in gel were richer in proteoglycans than those consisting of lower cell densities. After 1 day, MSCs/gel composites contracted and the diameter decreased by 30%; however, they were stable thereafter. Round cells with short processes producing collagen fibrils showing a similar morphology to that of chondrocytes were seen in the composites by transmission electron microscopy. During composite culture, chondroitin sulfate and mRNA expression for cartilage-related genes increased, demonstrating cartilage maturation. Using an optimized method, we obtained cartilage discs with a diameter of 7 mm and a thickness of 500 microm. Our procedure should thus make it possible to produce a large cartilage matrix in vitro. The tissue engineering of autologous cartilage from the composites of synovium-derived MSCs with collagen gel in vitro for transplantation may be a future alternative to graft implantation for patients with cartilage defects.

Age-related changes and sex differences in chondroitin sulfate isomers and hyaluronic acid in normal synovial fluid.

Abstract The effects of factors such as age and sex on the metabolism of chondroitin sulfate (CS) and hyaluronic acid (HA) in knee joint tissues are believed to be profoundly important in the onset of joint diseases including osteoarthritis. To test whether age and sex influence CS isomers and HA in normal synovial fluid, we determined concentrations of chondroitin 6-sulfate (C6S), chondroitin 4-sulfate (C4S), and HA in healthy subjects of different ages. Synovial fluids were obtained from 187 healthy volunteers, 14-89 years of age. Chondroitin 6-sulfate, C4S, HA concentrations, and C6S : C4S ratio showed a significant negative correlation with age. There were no sex-related differences in HA concentration, but the concentrations of C6S and C4S and the C6S : C4S ratio were significantly lower in women than in men in most age groups.

The effects of age and sex on chondroitin sulfates in normal synovial fluid.

OBJECTIVE: To examine how age and sex influence chondroitin sulfates (CS) in normal synovial fluid, we measured the concentrations of chondroitin 6-sulfate (C6S), chondroitin 4-sulfate (C4S), and hyaluronic acid (HA) in healthy subjects of different ages. METHODS: Synovial fluid samples were obtained from 82 healthy volunteers, ages 20-79 years. RESULTS: The concentrations of CS and HA and the C6S:C4S ratio varied with age. Their values were highest between 20 and 30 years of age, and thereafter they showed a tendency to decrease. Statistically, the C6S concentration and the C6S:C4S ratio at ages 60-70 years were significantly lower than those at 20-30 years of age. There was also a clear between-sex difference, in which the CS concentrations and the C6S:C4S ratio in women were significantly lower than those in men (P = 0.0003 for C6S, P = 0.02 for C4S, P = 0.002 for C6S:C4S ratio). In sharp contrast, little between-sex difference was found in the HA concentration. In multiple regression analysis, age correlated strongly with the C6S concentration and the C6S:C4S ratio (r = -0.521 and r = -0.617, respectively), weakly with the C4S concentration (r = -0.202), and moderately with the HA concentration (r = -0.483). Sex showed a weak correlation with the concentrations of C6S and C4S and the C6S:C4S ratio (r = 0.307, r = 0.225, and r = 0.237, respectively), and little correlation was seen between sex and the HA concentration. CONCLUSION: The CS concentrations and the sulfation patterns in normal synovial fluid vary with age and sex, and these physiologic variations need to be taken into account when using synovial fluid CS as markers for arthritic conditions.

Induction of apoptosis after stent implantation in canine portal vein.

The purpose of this study was to analyze apoptosis in the vessel wall after stent implantation in the canine portal vein and also to investigate the expression of the p21 cyclin-dependent kinase inhibitor, which may regulate cellular proliferation after vascular injury. Uninjured, control veins had few detectable TUNEL-positive cells in the intima and media (0.829 +/- 0.413%). At 4 weeks after stent implantation, TUNEL-positive cells significantly increased to 50.5 +/- 4.639%. These cells were predominantly located around the stent struts, and appeared to be smooth muscle cells morphologically. At 12 weeks, 44.7 +/- 6.178% of the intimal and medial cells were still TUNEL positive, and there was no significant difference between 4 and 12 weeks. P21 was not detected in uninjured, normal veins. At 4 and 12 weeks after stent implantation, positive p21 immunostaining was sparsely expressed in the intima and media adjacent to the stent struts. Thus, stent implantation induced a prolonged apoptotic response and increased expression of p21 in the portal venous system. This prolonged apoptotic response, possibly regulated by p21, may have a significant role in modulating the cellularity of intimal formation.

Effect of denuded endothelial cells on arginine amidase activity released from rabbit arteries.

1. We examined the secretion of arginine amidase activity from rabbit aorta and ear arteries. 2. The amount of arginine amidase activity spontaneously secretion from the aorta was significantly less than that secreted from the ear artery. Dermatan sulphate significantly facilitated the secretion of arginine amidase activity from both the aorta and ear artery. 3. The dermatan sulphate-enhanced secretion of arginine amidase activity from the aorta and ear artery was reduced by denudation of the endothelium. 4. These findings may indicate that spontaneous release of arginine amidase activity from vascular smooth muscle and the enhancement of secretion of arginine amidase activity produced by dermatan sulphate depends on the endothelium.