Impaired fracture healing in macrophage migration inhibitory factor-deficient mice.

UNLABELLED: This study investigated the role of macrophage migration inhibitory factor (MIF) in fracture repair using MIF gene-deficient mice (MIF KO). Fracture healing was delayed in MIF KO, and this was mainly due to the delay in the mineralization of osteoid within the fracture callus. INTRODUCTION: We previously reported that the expression of macrophage migration inhibitory factor (MIF) was up-regulated during the fracture healing process in rats. However, its role in the pathophysiology of this process remained unclear. The aim of the present study was to clarify the role of MIF in the fracture healing process using MIF gene-deficient mice (MIF KO). METHODS: Bone repair in wild-type mice (WT) and MIF KO (n = 70, respectively) was investigated using a tibia fracture model. Radiographic, biomechanical, histological, bone histomorphometric, and molecular analyses were performed. RESULTS: Post-fracture biomechanical testing showed that maximum load and stiffness were significantly lower in MIF KO than in WT on day 42. However, similar levels were observed between the two groups on day 84. Bone histomorphometric analysis revealed significantly higher osteoid volume, a lower mineral apposition rate, and smaller numbers of osteoclasts in the MIF KO callus compared to the WT callus. The messenger ribonucleic acid expressions of matrix metalloproteinase (MMP)-2, membranous type 1-MMP, cathepsin K, and tissue nonspecific alkaline phosphatase were found to be significantly suppressed in the MIF KO callus. CONCLUSION: The results of the present study suggest that delayed fracture healing in MIF KO was mainly attributable to a delay in osteoid mineralization.

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 novel DNA vaccine-targeting macrophage migration inhibitory factor improves the survival of mice with sepsis.

Sepsis is a common and frequently fatal condition and there is an urgent need for new therapies that will further reduce sepsis-induced mortality. Macrophage migration inhibitory (MIF) factor is important in the regulation of innate and adaptive immunity and is believed to play a key regulatory role in sepsis and autoimmune disease. As MIF deficiency or immunoneutralization protects mice or rats from fatal endotoxic shock or other inflammatory diseases, we examined whether DNA vaccination against this molecule would also be protective. DNA vaccines can stimulate both humoral and cellular immunity simultaneously and have been shown to be effective against a variety of pathogens or cytokine-driven pathologies. Mice were immunized with a MIF/tetanus toxin (TTX) DNA vaccine and sepsis was then induced by lipopolysaccharide or cecal ligation and puncture. The MIF/TTX DNA-vaccinated mice were protected from the lethal effect of sepsis compared with control-vaccinated mice in both models. Compared with the control-vaccinated mice, the MIF/TTX DNA-vaccinated mice also showed significantly lower serum tumor necrosis factor (TNF)-alpha protein levels and reduced mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, macrophage inflammatory protein-2 and Toll-like receptor-4 in the lungs. Thus, the MIF/TTX DNA vaccine may be useful for the prophylaxis of septic shock.

The responses of extrinsic fibroblasts infiltrating the devitalised patellar tendon to IL-1beta are different from those of normal tendon fibroblasts.

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1beta, PDGF-BB, and TGF-beta1 in fibroblasts infiltrating the frozen-thawed patellar tendon. Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1beta than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1beta was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-beta1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1beta had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.

Interaction of human platelet membrane glycoproteins with collagen and lectins.

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, an in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with 125I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.

Growth kinetics and integrin expression of fibroblasts infiltrating devitalised patellar tendons are different from those of intrinsic fibroblasts.

We compared the biological characteristics of extrinsic fibroblasts infiltrating the patellar tendon with those of normal, intrinsic fibroblasts in the normal tendon in vitro. Infiltrative fibroblasts were isolated from the patellar tendons of rabbits six weeks after an in situ freeze-thaw treatment which killed the intrinsic fibroblasts. These intrinsic cells were also isolated from the patellar tendons of rabbits which had not been so treated. Proliferation and invasive migration into the patellar tendon was significantly slower for infiltrative fibroblasts than for normal tendon fibroblasts. Flow-cytometric analysis indicated that expression of alpha5beta1 integrin at the cell surface was significantly lower in infiltrative fibroblasts than in normal tendon fibroblasts. The findings suggest that cellular proliferation and invasive migration of fibroblasts into the patellar tendon after necrosis are inferior to those of the normal fibroblasts. The inferior intrinsic properties of infiltrative fibroblasts may contribute to a slow remodelling process in the grafted tendon after ligament reconstruction.

The effect of phlebotomy on serum erythropoietin levels in normal healthy subjects.

We evaluated endogenous serum erythropoietin (Epo) levels in 14 normal subjects (eight males and six females) after a single 400-ml phlebotomy. The subjects were followed up for 56 days. The hemoglobin (Hb) values of both males and females decreased to a nadir on days 3 to 7 post-phlebotomy. Hb values gradually increased, but did not completely recover to pre-phlebotomy levels by day 56. Serum Epo levels increased after 6 h post-phlebotomy, to 20.1 +/- 5.4 (mU/ml) in males and 20.7 +/- 7.0 in females, from the pre-phlebotomy levels of 14.6 +/- 4.0 in males and 13.4 +/- 4.1 in females, respectively. Epo levels continued to increase to peak levels of 25.5 +/- 6.3 in males and 28.7 +/- 11.5 in females on days 7 to 14 and thereafter decreased until day 56. Thus, the Epo response to a 400-ml phlebotomy was relatively small in magnitude and was not sufficient to initiate a significant increase in erythropoiesis. This finding suggests that the administration of recombinant human erythropoietin (rHu-Epo) may be effective for the prompt correction of anemia induced by autologous blood donation and for increasing the volume of predonated autologous blood.

Laxity and flexibility of the ankle following reconstruction with the Chrisman-Snook procedure.

The effect of reconstruction of the anterior talofibular ligament with the Chrisman-Snook procedure on neutral zone laxity (anterior-posterior displacement at low loads) and flexibility (a measure of the nonlinear load-displacement response) of the ankle was investigated in vitro during the anterior drawer test. Neutral zone laxity was defined as the magnitude of anterior-posterior displacement of the ankle joint at +/- 2.5 N of applied load. The flexibility parameter was defined as the slope of a line between the natural logarithm of the anterior load applied to the ankle and the resulting displacement. After reconstruction with the Chrisman-Snook procedure, the values for neutral zone laxity of the ankle were significantly less than normal at 0 degree of plantar flexion, whereas the flexibility values were significantly greater than normal. This study revealed that, after the Chrisman-Snook procedure, values for ankle flexibility are not restored to normal even if those for neutral zone laxity are reduced to less than normal. The findings suggest that this nonanatomical reconstruction procedure does not reproduce normal kinematics of the ankle joint. This may help explain some of the adverse clinical reports associated with the Chrisman-Snook reconstruction procedure.

Determination of a zero strain reference for the anteromedial band of the anterior cruciate ligament.

The objective of this study was to verify a method previously used to determine a reference length for calculations of anterior cruciate ligament strain. In nine knee specimens, an arthroscopic force probe and a Hall effect transducer were placed in the anteromedial band of the ligament. Anteroposterior-directed shear loads then were applied to the knee joint with the knee flexed to 30 degrees. From the sigmoidal curve for shear load versus displacement of the anterior cruciate ligament midsubstance, the length of the transducer at the inflection point was determined graphically by two independent examiners. Previous studies suggested that the inflection point corresponds to the slack-taut transition of the anteromedial band. The force probe was used to determine the actual length of the transducer when the anteromedial band became load bearing. No significant differences were found between the reference lengths determined by the inflection point method and the force probe. The force probe demonstrated that the anterior cruciate ligament became load bearing when an anterior shear load of 8.8 N was applied to the tibia with the knee at 30 degrees of flexion. Furthermore, multiple cycles of anteroposterior shear loading did not influence these values. The force probe verified that the inflection method provides a reasonable estimate of the absolute strain reference (within 0.7% strain).

Biomechanical analysis of the ankle anterior drawer test for anterior talofibular ligament injuries.

The effect of sectioning the anterior talofibular ligament on the load-displacement behavior of the ankle was evaluated in vitro during the anterior drawer test using the flexibility approach. Controlled forces were applied across the ankle joint in the anterior-posterior direction, and the resulting displacements were measured at four flexion angles (10 degrees of dorsiflexion, neutral, and 10 degrees and 20 degrees of plantar flexion). The anterior talofibular ligament then was sectioned, and the anterior-posterior loadings were repeated at the four flexion angles. Two parameters were developed to describe the nonlinear load-displacement response of the ankle joint: neutral zone laxity (joint displacement between +/- 2.5 N) and flexibility (a measure of the nonlinear load-displacement response of the ankle between 10 and 50 N of anterior drawer loading). After sectioning the anterior talofibular ligament, significant increases in neutral zone laxity were observed at all angles of ankle flexion. The largest increases in neutral zone laxity were found with the ankle in 10 degrees of plantar flexion (76.3% increase) and 20 degrees of plantar flexion (89.7% increase). After sectioning the ligament, a significant increase (19.3%) in flexibility of the ankle was observed at 10 degrees of dorsiflexion, but no change in flexibility was observed with the ankle in the neutral and plantar flexed positions. These findings indicate that anterior drawer testing of the anterior talofibular ligament-deficient ankle between 10 degrees and 20 degrees of plantar flexion results in the largest increase in neutral zone laxity compared with the normal ankle with intact ligaments.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon.

The purpose of the present study is to determine the effect of the stress enhancement and intrinsic fibroblasts on the extracellular matrix of the patellar tendon. Thirty-two female Japanese White rabbits were divided into four groups. In Group 1, the patellar tendon underwent the in situ freeze-thaw treatment to kill intrinsic fibroblasts of the patellar tendon and the patellar tendon underwent the wrapping treatment with nylon membrane filters to inhibit extrinsic cell infiltration. In Group 2, the medial and the lateral portions of the frozen-thawed patellar tendon were resected to enhance the stress, and then the central two-thirds of the patellar tendon underwent the wrapping treatment. In Group 3, the patellar tendon without the freeze/thaw treatment underwent the wrapping treatment. In Group 4, the patellar tendon was narrowed and wrapped in the same manner. All rabbits were killed 6 weeks after surgery. While the elastic modulus and the tensile strength of the patellar tendon in Group 2 were significantly less than those in Group 1, we could not find any significant differences in these parameters between Groups 3 and 4. Histologically, while no fibroblasts were observed in Groups 1 and 2, fibroblasts were found in Groups 3 and 4. This study revealed that stress enhancement decreases the elastic modulus and the tensile strength of the extracellular matrix of the patellar tendon and that intrinsic fibroblasts prevent the detrimental effect of stress enhancement on mechanical properties of the patellar tendon.

The effect of cyclic displacement on the biomechanical characteristics of anterior cruciate ligament reconstructions.

This study was conducted to clarify the influence of cyclic displacement on the structural properties of four types of femur-graft-tibia complexes used to reconstruct the anterior cruciate ligament. Forty hindlimbs from pigs were used. In two groups, bone-patellar tendon-bone grafts were secured with interference screws (group A) or the suture-post technique (group B). In two groups, multistrand flexor tendons were fixed using the tape-staple technique (group C) or the sutures-tied-over-a-button technique (group D). In each group, five femur-graft-tibia complexes underwent tensile failure tests without cyclic displacement. The other five complexes underwent 5000 cycles of cyclic elongation for 2 mm, and then underwent the tensile failure tests. The initial stiffness significantly decreased after cyclic displacement in each group, although there were no significant differences in the linear stiffness and the ultimate failure load between the tests with and without cyclic displacement. These findings suggest that 5000 cycles of repetitive elongation of the femur-graft-tibia complex by 2 mm does not jeopardize the graft fixed with the procedures used in this study, despite a slight but significant increase of an anterior-posterior laxity of the knee.

The effect of anterior cruciate ligament graft elongation at the time of implantation on the biomechanical behavior of the graft and knee.

This investigation determined the effect that anterior cruciate ligament graft elongation at the time of surgical reconstruction has on the long-term biomechanical behavior of the graft and knee joint. We chose the canine model for anterior cruciate ligament reconstruction, using the medial third of the patellar tendon with attached proximal bone block. Elongation of the graft was measured immediately after graft fixation during passive knee flexion using the Hall effect transducer. The dogs were divided into either Group 1 (graft elongation behavior within the 95% confidence limits of the normal anterior cruciate ligament) or Group 2 (graft elongation behavior more than the 95% confidence limits of the normal anterior cruciate ligament). All of the dogs were sacrificed 18 months postoperatively, and we evaluated anteroposterior load displacement (i.e., anteroposterior laxity) of the knee and the structural properties of the graft. The anteroposterior laxity behavior of the reconstructed knees in Group 2 was significantly more than that of Group 1. Group 2 had significantly less linear stiffness of the graft than Group 1. There was no difference in the ultimate failure load and absorbed energy at failure values of the grafts between Groups 1 and 2. The findings from this investigation indicate that the graft elongation behavior at the time of reconstruction is a critical factor that influences the long-term success of anterior cruciate ligament reconstruction.

A new indole N-glycoside antibiotic SF-2140 from an Actinomadura. I. Taxonomy and fermentation of producing microorganism.

A new indole N-glycoside antibiotic SF-2140 which shows antiviral and weak antibacterial activity has been obtained from the cultured broth of an actinomycete strain. Strain SF-2140, designated Actinomadura albolutea sp. nov., was isolated from a soil sample collected in Hyogo Prefecture, Japan.

Mode of antifungal action of benanomicin A in Saccharomyces cerevisiae.

The mechanism of fungitoxic action of an antifungal antibiotic benanomicin A was studied with intact cells and protoplasts of Saccharomyces cerevisiae as well as with its enzymic preparations. The results obtained are summarized as follows: (1) benanomicin A at relatively high concentrations (almost equal to MIC) was fungicidal and disrupted the cell permeability barrier, inducing leakage of intracellular K+ and ATP in growing cells, while the antibiotic had none of these effects in non-growing cells; (2) no biosynthesis of any of several major cellular constituents in yeast cells was inhibited markedly or selectively enough to explain its fungitoxic activity; (3) whereas benanomicin A induced lysis of metabolically active yeast protoplasts incubated in the presence of glucose, inactive yeast protoplasts incubated without glucose were refractory to the lytic action of the antibiotic; (4) osmotically shocked yeast cells became feasible to the cidal action of benanomicin A; (5) benanomicin A substantially inhibited uptake of 6-deoxy-glucose by yeast cells; (6) liposomes composed of phospholipids and cholesterol were not susceptible to benanomicin A; and (7) benanomicin A inhibited in vitro activity of H(+)-ATPase from yeast cell membranes to a greater extent than that for H(+)-ATPase from yeast mitochondria or H(+)-ATPase from yeast vacuolar membranes. Based on these and our previous data that benanomicin A preferentially binds to mannan or mannoproteins constituting the cell wall and cell membrane of yeasts, such binding of the antibiotic is suggested to deteriorate the normal structure and function of those cell membranes of yeasts which are in a growing or metabolically active state, ultimately leading to cell death.

Binding of benanomicin A to fungal cells in reference to its fungicidal action.

An antifungal antibiotic, benanomicin A, binds in the presence of Ca2+ to susceptible fungi and some bacteria, but not to antibiotic-resistant bacteria and mammalian cells. With the susceptible yeast Saccharomyces cerevisiae, benanomicin A binds similarly to whole cells and to protoplasts. Studies using benanomicin A and three structurally related derivatives suggested that a carboxylic acid in the D-alanine moiety and a sugar moiety in the benanomicin A molecule are essential for both binding and antifungal activities against growing S. cerevisiae. An amino substituent on the sugar moiety can be replaced with a hydroxyl group without the loss of activities. Benanomicin A binds to various yeast mannans which differ in glycosidic linkages. These results indicate that binding of benanomicin A to the mannan portion of fungal cells is essential for exertion of the antifungal activity.

The effect of increased stress on the patellar tendon.

We performed a biomechanical and histological study to clarify the effect of stress enhancement on the in situ frozen-thawed patellar tendon of the rabbit as a tendon autograft model. We used 48 Japanese White rabbits divided into three groups. In group 1, the patellar tendon underwent in situ freeze-thaw treatment with liquid nitrogen to kill intrinsic fibroblasts. In group 2, after similar treatment, the medial and lateral portions were resected so that the cross-sectional area was reduced by a third. In group 3, after treatment, the cross-sectional area was reduced by a half. In groups 2 and 3, the stress in the tendon was calculated theoretically to be 150% and 200% of the physiological stress during locomotion. Eight rabbits in each group were killed at three and six weeks, respectively. At three weeks, the mean values for the tensile strength of groups 2 and 3 were 113.7% and 75.7% of that of group 1, and at six weeks 101.2% and 57.4%, respectively. The tensile strength in group 3 was significantly lower than that in groups 1 and 2. The histological findings in group 2 were similar to those in group 1, although an acellular area appeared to be wider in the core portion compared with group 1 at each period. In group 3, the collagen bundles of the tendon were less organised than those of groups 1 and 2. Our findings showed that stress enhancement affects the remodelling of the frozen-thawed patellar tendon and that excessively high stress reduces the mechanical properties of the tendon. This indicates that high stress on the patellar tendon autograft should be avoided during ligament reconstruction.

Application of an artificial neural network and genetic algorithm for determination of process orbits in the koji making process.

In order to estimate alpha-amylase, glucoamylase, acid proteinase, and acid carboxypeptidase activities in koji from the process variables and initial conditions of the koji making process, artificial neural network (ANN) models (ANN-10, -11, -15, and -21) were constructed with 10, 11, 15, and 21 input variables, respectively. These models could estimate the enzyme activities with high accuracy. Temperature and humidity orbits were then acquired by a genetic algorithm searching in the reverse direction using ANN-10, -11, -15, and -21 (GA-10, -11, -15, and -21). The orbits acquired by GA-15 and -11 were almost identical to the actual orbits, but those acquired by GA-21 and -10 were different. Enzyme activities acquired by GA-15 had 1.3% errors compared with the target values, while those acquired by GA-11 had 9.7% errors. GA-15 was, therefore, selected as the most suitable algorithm and was used to determine temperature and humidity orbits for target enzyme activities. Test koji making was then carried out according to the orbits acquired. As a result, the enzyme activities of the koji produced were almost the same as the target values.

Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to compare intraosseous graft healing between the doubled flexor tendon (FT) graft and the bone-patellar tendon-bone (BPTB) graft in anterior cruciate ligament (ACL) reconstruction. TYPE OF STUDY: Randomized trial. METHODS: A biomechanical and histologic study was conducted with 24 adult beagle dogs. Bilateral ACL reconstructions were performed in each animal. Autogenous doubled FT and BPTB grafts were used for the left and right knees, respectively. Each end of the 2 grafts was tethered with a polyester suture to a screw post with a washer. The animals were then allowed unrestricted activities in their cages. Eight animals were killed at 3, 6, and 12 weeks, respectively. RESULTS: Histologically, the FT graft was anchored to the tunnel wall with newly formed collagen fibers resembling Sharpey's fibers by 12 weeks. These fibers were more abundant in the anterior (ventral) gap than in the posterior (dorsal) gap. In the BPTB graft, the bone plug was anchored with newly formed bone at 3 weeks, although osteocytes in the plug trabeculae were necrotic for 12 weeks. Degeneration of the tendon-bone junction in the plug progressed at 6 weeks. Tensile testing showed that the weakest site was different not only between the 2 grafts but also between the observation periods. In the FT graft, the weakest site was the graft-wall interface at 3 weeks and the intraosseously grafted tendon at 6 weeks. In the BPTB graft, the weakest site was the graft-wall interface at 3 weeks and the proximal site in the bone plug at 6 weeks. The ultimate failure load of the FT graft was significantly inferior (45.8%) to that of the BPTB graft at 3 weeks (P =.021). At 6 weeks, the load of the FT graft was 85% that of the BPTB graft without a significant difference (P =.395). CONCLUSIONS: As to the clinical relevance, the fixation device chosen for soft-tissue fixation appears to be more important than comparing it to the BPTB graft, although this has yet to be conclusively proven.

Interference screw fixation of doubled flexor tendon graft in anterior cruciate ligament reconstruction - biomechanical evaluation with cyclic elongation.

OBJECTIVE: To biomechanically evaluate interference screw fixation of the doubled flexor tendon graft in anterior cruciate ligament reconstruction using cyclic elongation. DESIGN: Biomechanical properties of the interference screw fixation of the flexor tendons were compared with those of three standard fixation techniques which had been commonly performed in anterior cruciate ligament reconstruction. BACKGROUND: The interference screw fixation of the flexor tendon graft has attracted notice because of various possible advantages. METHODS: Forty fresh frozen porcine hind limbs were divided into four groups of ten knees each. Anterior cruciate ligament reconstruction was carried out in each group using one of four different procedures. For each group, five femur-graft-tibia complexes underwent submaximal cyclic elongation of 5000 cycles after initial tension of 80 N was applied. Then, tensile testing was performed in the same manner for the complex with a tensile tester. The remaining five complexes were examined in the same tensile test without applying any cyclic elongation. RESULTS: The initial tension was more rapidly relaxed by cyclic elongation in the flexor tendon graft fixed with interference screws than in the bone-patellar tendon-bone graft fixed with two standard techniques. After cyclic elongation, while the ultimate failure load of the former was significantly lower than the latter, the linear stiffness of the former was significantly higher than the flexor tendon graft fixed with sutures. CONCLUSION: The present study has clarified that the advantage of the interference fixation for the doubled flexor tendon graft is the high linear stiffness of the FGT complex, and the disadvantage of this screw is the low ultimate failure load of the FGT complex. RELEVANCE: The present study has suggested that vigorous activities should not be permitted for the patients in the early period after anterior cruciate ligament reconstruction using this fixation technique, because of its low ultimate failure load.