Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan.

BACKGROUND: New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. METHODS: At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. RESULTS: The Lysholm score significantly increased from 60.0 +/- 13.7 points to 89.8 +/- 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. CONCLUSIONS: We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

A study of the native-denatured (N in equilibrium with D) transition in lysozyme. II. Kinetic analysis of protease digestion.

Kinetic analyses of the protease digestion of several chemical derivatives of lysozyme [EC 3.2.1.17] showed that only the D(denatured) state of the protein is digested and that the reaction velocity is proportional to the equilibrium constant (KD) of the N in equilibrium with D transition of the protein. Alteration of the net charge of lysozyme by acetylation caused a shift of the N in equilibrium with D transition to the right (ten-fold increase in KD compared to that of native enzyme). Both the formation of a lysozyme-inhibitor complex and the introduction of a covalent bond in the lysozyme molecule restricted the transition. The magnitude of the N in equilibrium with D transition is related to the susceptibility of lysozyme to protease digestion and it is estimated that the N in equilibrium with D transition in proteins is generally important in the intracellular catabolism of proteins.

A study of the native-denatured (N in equilibrium with D) transition in lysozyme. III. Effect of alteration of net charge by acetylation.

Measurement of the enzymic activity and fluorescence properties showed that the gross conformation of acetylated lysozyme [EC 3.2.1.17] is very similar to that of the native enzyme. On the other hand, protease digestion, t-butyl hypochloride modification and thermal denaturation experiments performed on native, acetylated, and guanidinated lysozymes showed that acetylation caused a small but significant shift of the N in equilibrium with D transition to the right. Thus it can be concluded that charge balance in a protein plays an important role in maintaining its conformation. The difference between equilibrium and kinetic methods of monitoring protein denaturation was also clarified.

Significance of the single-legged hop test to the anterior cruciate ligament-reconstructed knee in relation to muscle strength and anterior laxity.

We investigated the significance of the single-legged hop test to the anterior cruciate ligament-reconstructed knee as it specifically relates to knee muscle strength recovery and residual anterior laxity. The hop test was conducted on 107 patients with unilateral anterior cruciate ligament-reconstructed knees (78 semitendinosus tendon autografts and 29 bone-patellar tendon-bone autografts). Patients were tested an average of 2 years after surgery. Correlation coefficient analyses determined whether the relationship between knee muscle strength recovery and the hop test was invariant across all levels of residual anterior laxity and whether the relationship between residual anterior laxity and the hop test was invariant across levels of quadriceps muscle strength recovery. Furthermore, we examined the effect of graft harvest site on the hop index. Positive correlations were found between the hop index and muscle strength index at all levels of residual anterior laxity (P < 0.05), but these correlation coefficients were relatively low. There were no apparent correlations between the hop index and residual anterior laxity at all levels of quadriceps muscle strength recovery. There was no significant difference in the average hop index between the semitendinosus tendon autograft group and the bone-patellar tendon-bone autograft group.

Effects of tetraacetyl-bis-dehydroascorbic acid, a derivative of ascorbic acid, on Ehrlich cells and HeLa cells (human carcinoma cells).

Derivatives of ascorbic acid were synthesized, and the studies were made on their effects in Ehrlich ascites carcinoma cells, in regard to the inhibition and the prolongation of survival time as well as on the morphological degeneration in HeLa cells. In a model infection study carried out by using tetraacetyl-bis-dehydroascorbic acid in dd mice infected with Ehrlich cells, it was proved that the prolongation of survival time was nearly double in comparison to the control group mice. Also, it was noted that hypertrophy due to abdominal dropsy and body weight were reduced much more than in the control group. From these results, the inhibiting effect of tetraacetyl-bis-dehydroascorbic acid was confirmed. While in the case of DHA and other derivatives, almost no inhibition and prolongation of survival time were observed. As for HeLa cells in a tissue culture, tetraacetyl-bis-DHA, in a dosage of 125-250 mug/ml, demonstrated definitely its morphological degeration. After 125 mug/ml of tetraacetyl-bis-DHA was added to a tissue culture solution of HeLa cells, the cells were washed and recultured. No growth of the cells was observed. Consequently, this substance was confirmed to be anti-HeLa substance with a low toxicity.

Studies on synergisidin.

Penicillium sp. No. Y-11930 was isolated from soil sample collected at Shimouma, Setagaya, Tokyo in September 1978. Synergisidin produced by the strain was obtained with high yield in starch-corn steep liquor medium, extracted with ethyl acetate at pH 5.0 and crystallized from ethyl acetate after decolorization with active charcoal. The antibacterial activities of synergisidin against Gram-positive and Gram-negative bacteria and Mycobacterium were almost nothing but synergisidin showed weak activities against eumycetes with MICs of 6.25-100 microgram/ml. However, synergidisin was confirmed and discovered to show 30-125-fold strong synergistic effects against Candida sp. in particular with addition to extremely small quantities of imidazole antimycotics such as econazole, miconazole and clotrimazole. The acute toxicity was LD50 smaller than or equal to 250 mg/kg in mice (i.p.). the morphological degenerative effect on HeLA cells was observed in concentrations of more than 0.122 microgram/ml. The chemical structure of synergisidin was estimated as 7, 16-dihydroxy-2-methyl-4-oxo-3-oxabicyclo [10. 3. 1] hexadeca-5, 10-diene or 2, 15-dihydroxy-7-methyl-5-oxo-6-oxabicyclo (11. 3. 0] hexadeca-3, 11-diene (the same structure as decumbin, brefeldin A and ascotoxin) from various physiochemical properties but later, comparison with brefeldin A and ascotoxin revealed that synergisidin was identical with those.

Studies on the mechanism of synergistic action with synergisidin and imidazole antimycotics.

The investigation of mechanism of synergistic action with SYN and ECZ was performed using C. albicans SC5314 so that SYN was confirmed to show strong synergistic effects against Candida sp. in particular with addition of extremely small quantities under the MICs of imidazole antimycotics such as ECZ, MCZ and CTZ. The synergistic effect of antifungal activity against C. albicans SC5314 with a combination of SYN and ECZ (SYN + ECZ) showed fungistatic action. Effect of SYN + ECZ on osmotic resistance was not recognized and protoplast was not observed under a microscope. Accordingly, SYN + ECZ was considered not to take part in cell wall synthesis directly. For effect of SYN + ECZ on release of intracellular components, slow release of 260 nm-absorbing substances was occurred, so that SYN + ECZ was seemed not to affect cytoplasmic membrane damage directly. Also, it was suggested clearly that SYN + ECZ affected lipid metabolism and glycolysis including TCA cycle from the investigation on antagonism by growth recovery of C. albicans SC5314 by 106 kinds of substances such as fatty acids, isoprenoids, phospholipids, vitamins, amino acids, nucleic acid-related substances and TCA cycle-related substances. From the above results, it was suggested that the mechanism of synergistic action with SYN and ECZ against C. albicans SC5314 was due to affect the different reactions in lipid metabolism and the similar reactions in glycolysis including TCA cycle, respectively, in consideration of respective mechanism of actions of SYN alone and ECZ alone. A part of this work was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, 1981 (Kyoto).

Studies on terreic acid.

It was found that Aspergillus sp. No. Y-8980 which was isolated from a soil sample collected at Yoron Island in Kagoshima Prefecture belonged to Aspergillus terreus group by morphological observation. The active substance produced by the strain was obtained with a high yield in sucrose-yeast extract medium and extracted by chloroform, ethyl acetate and n-butanol at pH 2.4 approximately 2.6 from the culture broth. The substance was crystallized from chloroform and ethyl acetate after charcoal treatment of the crude crystal. From various physico-chemical properties, it was found that the substance was identical to terreic acid. Terreic acid showed MICs of 25 approximately 100 mcg/ml, 12.5 mcg/ml and 50 mcg/ml against Gram-positive and Gram-negative bacteria, Xanthomonas oryzae and Xanthomonas citri, respectively, but it did not control Pseudomonas, fungi and yeast. The LD50 was 75 mg/kg i.p. and i.v. in mice. With regards to the anti-tumor effect, the morphological degeneration on HeLa cells (human carcinoma cells) was observed in the concentrations of more than 6.25 mcg/ml of terreic acid. An increase of body weight of mice caused by Ehrlich ascites carcinoma cells was not definitely observed by the daily administration of 150 mcg of terreic acid per mouse for 8 consecutive days. Above showed the enough survival effect in dd mice implanted with Ehrlich ascites carcinoma cells, and the effect also was demonstrated by anatomies of mice.

The relationship between postoperative ligament balance and preoperative varus deformity in total knee arthroplasty.

We hypothesize that in knees with severe varus deformities, varus-valgus ligament imbalance tends to remain postoperatively after total knee arthroplasty. The purpose of this study was to evaluate the correlation between preoperative varus deformity and postoperative ligament balance quantitatively measured by stress radiograph using Telos SE in total knee arthroplasty. In this study, 44 knee arthroplasties in 32 patients were evaluated. We defined the angle in varus and valgus stress as "varus angle" and "valgus angle," and the sum of varus angle and valgus angle as "sum of varus-valgus angle." There was a significant correlation between preoperative lateral femoral angle and varus angle (p < 0.0001, r = 0.56), which means that postoperative ligament imbalance tends to remain in knees with preoperative varus deformities. There was a significant correlation between the postoperative period and the sum of varus-valgus angle, which suggests that knees with total knee arthroplasty have potential to increase laxity postoperatively.

Intra-articular detachment of the Endobutton more than 18 months after anterior cruciate ligament reconstruction.

We report a case of detachment of an Endobutton (Acufex Microsurgical, Mansfield MA) used for femoral fixation of a reconstructed anterior cruciate ligament. The Endobutton, which was confirmed to be in place on the suprapatellar space of the femur by radiograph 18 months postoperatively, was found in the popliteal space by radiograph 25 months after surgery. This is a rare complication, but our case suggests that the Endobutton should not be fixed too distal close to the femoral groove.

Subtalar instability following lateral ligament injuries of the ankle.

A stress radiograph of the subtalar joint was taken using Telos equipment, while the X-ray beam was directed onto the posterior subtalar joint at angles of 30 degrees latero-medially and 40 degrees caudocranially. From this radiograph the subtalar tilt angle was measured as an index of stability of the subtalar joint. Intra-and interobserver evaluations of measurement of this angle were performed on 20 unstable ankles. The errors at a 95 per cent confidence level were 1.9 degrees for intraobserver A, 1.4 degrees for intraobserver B and 2 degrees in interobservation. Stability of the subtalar joint was evaluated on 46 lateral ligament injuries of the ankle (23 acute injuries and 23 chronic injuries) and 80 normal ankles. The subtalar tilt angle was 9.7 degrees +/ 3.2 degrees in acute injuries 10.3 degrees #/ 2.9 degrees in chronic injuries, and 5/2 degrees +/ 2/6 degrees in normal ankles. There were significant differences between the acute or chronic injury and the normal ankles. These results suggested that stability of the subtalar joint was disturbed following acute and chronic lateral ligament injuries of the ankle.

Effects of aggressive early rehabilitation on the outcome of anterior cruciate ligament reconstruction with multi-strand semitendinosus tendon.

To evaluate the effects of aggressive early rehabilitation on the clinical outcome of anterior cruciate ligament reconstruction using semitendinosus (and gracilis) tendon, 103 of 110 consecutive patients who underwent ACL reconstruction using multistrand semitendinosus tendon (ST) or the central one-third of patellar tendon with bony attachments (BTB) were analyzed prospectively. Subjectively, the Lysholm score was not different among the groups. The Lachman test indicated a trend of less negative grade in the ST men's group than that in the BTB men's group. On the patellofemoral grinding test, only women patients of both groups showed pain, with less positive crepitation in the ST group than in the BTB group. KT measurements at manual maximum showed more patients with more than 5 mm differences in the ST group than in the BTB group. The results of this study suggest that aggressive early rehabilitation after the ACL reconstruction using the semitendinosus (and gracilis) tendon has more risk of residual laxity than with the BTB.

Two-bundle reconstruction of the anterior cruciate ligament using semitendinosus tendon with endobuttons: operative technique and preliminary results.

We carried out two-bundle reconstruction of the anterior cruciate ligament (ACL) using multi-strand semitendinosus tendon for the purpose of getting better stability under aggressive rehabilitation by putting the graft in the normal anatomy with minimized notch-plasty. The drill guide position was determined using two-directional radiography in hyperextension. The two-bundle procedure was performed using the inside-out technique for grafting and two Endobuttons (Acufex Microsurgical, Mansfield, MA) for femoral graft fixation. The clinical outcomes 2 years postoperatively of 54 of 62 consecutive patients were reported. The preliminary results suggested that the two-bundle procedure showed a better trend with respect to anterior stability compared with those of our previous experience using the single-bundle technique under the same aggressive rehabilitation. The two-bundle procedures have several theoretical advantages over single-bundle with multi-strand tendon graft. The present two-bundle reconstruction is recommended to result in better anterior stability with minimized notch-plasty and with no serious complications.

Objective factors affecting overall subjective evaluation of recovery after anterior cruciate ligament reconstruction.

The goal of this prospective study was to determine how objective measurements affect the overall subjective evaluation of recovery after anterior cruciate ligament (ACL) reconstruction and to evaluate the effects of graft material, residual instability and sporting activity on patient satisfaction. Ninety-seven patients with unilateral ACL injuries whose ACL was reconstructed using either a multi-strand semitendinosus (ST) graft or a central-third bone-patellar tendon-bone (BTB) graft were followed for an average of 30 months. An "overall subjective recovery score" was derived from the patients' evaluation of function of the operated knee as a percentage of the unoperated knee. The mean overall subjective recovery score at follow-up was 80% (range, 20% to 100%). This study strongly suggested that integrity of the knee extension mechanism, that is, good recovery of knee extension strength with achievement of full extension and no problems in the patellofemoral joint, is the most important factor in patient satisfaction after ACL reconstruction.