Functional Anatomy of the Craniolateral and Caudomedial Bundles of the Caudal Cruciate Ligament in Beagle Dogs.

OBJECTIVE: The aim of this study was to investigate functional anatomy of the craniolateral bundle (CrLB) and caudomedial bundle (CdMB) of caudal cruciate ligament in normal Beagles. MATERIALS AND METHODS: Twelve stifle joints of 12 Beagle cadavers that were euthanatized for reasons other than musculoskeletal disease were used. Anatomical characteristics of femoral and tibial attachment regions of the caudal cruciate ligament were described, and location and area of the footprints of the CrLB and CdMB were objectively determined. The CrLB was artificially divided into two equal fibre bundle units and the CdMB was artificially divided into four equal bundle units. Changes in the fibre arrangement were recorded during stifle range of motion, and the tension of each fibre bundle unit at various stifle joint angles was objectively measured. RESULTS: The CrLB was attached to the intercondylar fossa of the femur, and the CdMB was attached to the medial surface of the medial condyle of the femur. The centre of the CrLB attachment site was located craniomedial to that of the CdMB on the tibia. During stifle range of motion, each divided fibre bundle unit in the CrLB and CdMB was twisted externally. The tension of entire CdMB tended to be higher than that of entire CrLB at 180 to 135 degrees of the stifle angles. In full extension angle, the tension of entire CdMB was 1.55 times higher than that of entire CrLB. CLINICAL SIGNIFICANCE: This study contributes to understanding of the biomechanics and the function of the caudal cruciate ligament, which help to elucidate the pathophysiology of caudal cruciate ligament tear and to develop stifle joint stabilization surgery.

Functional Anatomy of the Craniomedial and Caudolateral Bundles of the Cranial Cruciate Ligament in Beagle Dogs.

OBJECTIVE: The aim of this study was to investigate detailed functional anatomy of the craniomedial bundle (CrMB) and caudolateral bundle (CdLB) of cranial cruciate ligament (CrCL) for application of intra-articular anatomical reconstruction in dogs. MATERIALS AND METHODS: Twelve stifle joint of 12 Beagle cadavers were used. Anatomical characteristics of femoral attachment regions of the CrCL were determined. Then, the location and area of the footprints of the CrMB and CdLB were objectively measured. Each bundle was equally divided into four fibre bundles, and changes in the fibre arrangement were recorded during stifle range of motion. Additionally, the tension of each fibre bundle at various stifle joint angles was objectively measured. RESULTS: The attachment region of the CrCL clearly differed from that reported in humans. The centre of the CdLB attachment site was located craniodistal to that of the CrMB on the femur and slightly caudal to that of the CrMB on the tibia. The area of the CrMB on the femoral and tibial attachment sites was larger than that of the CdLB. During stifle range of motion, each divided fibre bundle in the CrMB and CdLB was twisted intricately. The tension of the CrMB was higher than that of CdLB. The central part of the CrMB was always tense during stifle range of motion. CLINICAL SIGNIFICANCE: These results may contribute to the application of anatomical reconstruction surgery for CrCL deficient stifle in dogs.

Biomechanical and histological evaluations of the doubled semitendinosus tendon autograft after anterior cruciate ligament reconstruction in sheep.

BACKGROUND: A sheep or goat anterior cruciate ligament (ACL) reconstruction model with the semitendinosus tendon autograft has not as of yet been established. HYPOTHESIS: The semitendinosus tendon graft may be necrotized after ACL reconstruction until 12 weeks. Structural properties of the femur-graft-tibia complex may not be restored to the normal ACL level even at 12 months after surgery. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-eight mature sheep were used. In each animal, the right knee underwent ACL reconstruction using the semitendinosus tendon autograft. Two of them were not tested because of pneumonia. Twenty of 36 sheep were used for biomechanical evaluations. Five randomly selected animals were sacrificed at 0, 12, 24, or 52 weeks after surgery. In each animal, both the knees were harvested, and the left knee was used to obtain the control data. The remaining 16 sheep were used for histological evaluation. Two or 3 randomly selected animals were sacrificed at 0, 2, 6, 12, 24, or 52 weeks after surgery. RESULTS: The tendon graft was predominantly acellular at 2 weeks. Although cell infiltration increased at 6 weeks, the core portion remained necrotic even at 12 weeks. At 24 and 52 weeks, the necrotic lesion disappeared in the core portion. In each period, the anteroposterior translation of the reconstructed knee remained significantly greater than that of the control (P < .0001). At 52 weeks, the maximum load of the femur-graft-tibia complex was significantly lower than that of the femur-ACL-tibia complex (P < .0001), although there was no significant difference in the cell density, the tissue dimensions, the stiffness, and the elongation at failure between the 2 complexes. CONCLUSION: The sheep ACL reconstruction model showed predominant intrinsic fibroblast necrosis in the tendon autograft until 12 weeks. Although the structural properties of the femur-graft-tibia complex gradually improved, they were not completely restored to the femur-ACL-tibia complex level even at 52 weeks. CLINICAL RELEVANCE: Remodeling of the semitendinosus tendon autograft after ACL reconstruction is not different from that of the bone-tendon-bone graft. This study has suggested that vigorous activity should not be permitted for patients in the early periods after ACL reconstruction using semitendinosus tendon autografts, which are necrotized and weakened after surgery.

Local administration of autologous synovium-derived cells improve the structural properties of anterior cruciate ligament autograft reconstruction in sheep.

BACKGROUND: The structural properties of a tendon autograft deteriorate during the remodeling phase after anterior cruciate ligament (ACL) reconstruction. HYPOTHESIS: A local application of autologous synovium-derived cells cultured in medium supplemented with transforming growth factor ß (TGFß) may inhibit the deterioration of structural properties of the tendon graft after ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: Fifty-two mature sheep were used. In each animal, the right knee underwent ACL reconstruction using the semitendinosus tendon autograft; then, the animals were divided into 5 groups of 10. No additional treatments were applied in group 1, whereas fibrin sealant was applied around the graft in group 2. In group 3, autologous synovium-derived cells cultured in standard medium were applied around the graft with fibrin sealant, whereas autologous synovium-derived cells cultured in TGFß-supplemented medium were applied with fibrin sealant in group 4. In group 5, fibrin sealant containing 20 ng of TGFß was applied around the graft. Each animal was sacrificed at 12 weeks after the surgery. In each group, 7 and 3 sheep were used for biomechanical and histologic evaluations, respectively. The remaining 2 sheep were used to confirm whether the applied cells infiltrated the graft at 1 week after surgery. RESULTS: Confocal microscope observations showed that the applied cells that were labeled before implantation infiltrated into the superficial portion of the graft at 1 week. Biomechanically, the maximum load and the stiffness of groups 4 and 5 were significantly greater than those of groups 1, 2, and 3. Histologically, necrotic lesions were observed in the core portion of the midsubstance in groups 1 and 2. In groups 3, 4, and 5, no necrotic lesions were found in the midsubstance. CONCLUSION: A local application of autologous synovium-derived cells cultured in TGFß-supplemented medium or a fibrin sealant containing TGFß significantly inhibits the natural deterioration of the structural properties of the tendon graft after ACL reconstruction. CLINICAL RELEVANCE: Administration of autologous synovium-derived cells cultured in TGFß-supplemented medium or TGFß and fibrin glue alone can be a potential cell-based therapy to prevent graft deterioration after transplantation or accelerate mechanical restoration of the deteriorated graft.

Implantation of autogenous meniscal fragments wrapped with a fascia sheath enhances fibrocartilage regeneration in vivo in a large harvest site defect.

BACKGROUND: Concerning meniscal tissue regeneration, many investigators have studied the development of a tissue-engineered meniscus. However, the utility still remains unknown. HYPOTHESIS: Implantation of autogenous meniscal fragments wrapped with a fascia sheath into the donor site meniscal defect may significantly enhance fibrocartilage regeneration in vivo in the defect. STUDY DESIGN: Controlled laboratory study. METHODS: Seventy-five mature rabbits were used in this study. In each animal, an anterior one-third of the right medial meniscus was resected. Then, the animals were divided into the following 3 groups of 25 rabbits each: In group 1, no treatment was applied to the meniscal defect. In group 2, the defect was covered with a fascia sheath. In group 3, after the resected meniscus was fragmented into small pieces, the fragments were grafted into the defect. Then, the defect with the meniscal fragments was covered with a fascia sheath. In each group, 5 rabbits were used for histological evaluation at 3, 6, and 12 weeks after surgery, and 5 rabbits were used for biomechanical evaluation at 6 and 12 weeks after surgery. RESULTS: Histologically, large round cells in group 3 were scattered in the core portion of the meniscus-shaped tissue, and the matrix around these cells was positively stained by safranin O and toluisin blue at 12 weeks. The histological score of group 3 was significantly higher than that of group 1 and group 2. Biomechanically, the maximal load and stiffness of group 3 were significantly greater than those of groups 1 and 2. CONCLUSION: This study clearly demonstrated that implantation of autogenous meniscal fragments wrapped with a fascia sheath into the donor site meniscal defect significantly enhanced fibrocartilage regeneration in vivo in the defect at 12 weeks after implantation in the rabbit. CLINICAL RELEVANCE: This study proposed a novel strategy to treat a large defect after a meniscectomy.

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases.

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.

Local administration of interleukin-1 receptor antagonist inhibits deterioration of mechanical properties of the stress-shielded patellar tendon.

We previously found that interleukin (IL)-1beta is over-expressed in the fibroblasts of the stress-shielded patellar tendon using a stress-shielding model [Uchida, H., Tohyama, H., Nagashima, K., Ohba, Y., Matsumoto, H., Toyama, Y., Yasuda, K., 2005. Stress deprivation simultaneously induces over-expression of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta in fibroblasts and mechanical deterioration of the tissue in the patellar tendon. Journal of Biomechanics 38(4), 791-798.]. Therefore, IL-1beta may play a role in tendon deterioration in response to stress deprivation. This study was conducted to clarify the effects of local administration of interleukin-1 receptor antagonist (IL-1ra) on the mechanical properties of the stress-shielded patellar tendon as well as the tendon fascicles harvested from it. Twenty-six mature rabbits were equally divided into Groups IL-1ra and PBS after the right patellar tendon underwent the stress-shielding treatment, which completely released the patellar tendon from tension by stretching the flexible wire installed between the patella and the tibial tubercle. In Group IL-1ra, IL-1ra was injected between the patellar tendon and the infra-patellar fat pad. In Group PBS, phosphate-buffered saline was injected in the same manner as IL-1ra. All rabbits were evaluated at 3 weeks after the stress-shielding procedure. The tangent modulus and the tensile strength of the patellar tendons were significantly greater in Group IL-1ra than in Group PBS, while there was no significant difference in the strain at failure between Groups IL-1ra and PBS. Concerning the mechanical properties of the fascicles harvested from the patellar tendon, however, we could not detect any significant differences in the tangent modulus, tensile strength, or strain at failure between Groups IL-1ra and PBS. The present study suggested that IL-1 plays an important role in the deterioration of the mechanical properties of the patellar tendon in response to stress shielding and that IL-1 does not affect the fascicles themselves.

Effects of local administration of vascular endothelial growth factor on mechanical characteristics of the semitendinosus tendon graft after anterior cruciate ligament reconstruction in sheep.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis. HYPOTHESIS: An application of VEGF may enhance angiogenesis in the grafted tendon in anterior cruciate ligament (ACL) reconstruction, and the application may affect mechanical characteristics of the ACL graft. STUDY DESIGN: Controlled laboratory study. METHODS: Eighteen sheep were divided into groups I and II. In group I, the harvested semitendinosus tendon was soaked in VEGF solution, and the right knee then underwent ACL reconstruction using this tendon. In group II, the right knee underwent identical procedures to those of group I except that the harvested tendon was soaked in phosphate-buffered saline. All animals were sacrificed 12 weeks after ACL reconstruction. RESULTS: Histologic findings showed that newly formed vessels and infiltrative fibroblasts were more abundant in group I than in group II. The anterior-posterior translation of the knee during an anterior-posterior force of +/- 100 N was significantly larger in group I than in group II by 2.58 mm (95% confidence interval, -1.76 mm to 1.76 mm) (P = .002). The linear stiffness of the femur-graft-tibia complex in group I was significantly lower than that in group II by 41.5 N/mm (95% confidence interval, -32.2 N/mm to 32.2 N/mm) (P = .017). CONCLUSION: This study has revealed that VEGF as administered in this study promotes angiogenesis in the ACL graft and significantly reduces the stiffness of the ACL graft with increased knee laxity at 12 weeks after ACL reconstruction. CLINICAL RELEVANCE: Exogenous VEGF application for ACL reconstruction can induce an increase in knee laxity and a decrease in the stiffness of the grafted tendon at least temporarily after ACL reconstruction. These potentially negative mechanical effects need to be taken into account when considering clinical use of VEGF.

Effects of administration of transforming growth factor (TGF)-beta1 and anti-TGF-beta1 antibody on the mechanical properties of the stress-shielded patellar tendon.

Previous studies have shown that, in the stress-shielded patellar tendon, the mechanical properties of the tendon were dramatically reduced and TGF-beta was over-expressed in tendon fibroblasts. In the present study, therefore, we tested two supportive hypotheses using 40 rabbits: One was that an application of TGF-beta1 might significantly increase the tensile strength and the tangent modulus of the stress-shielded patellar tendon. The other one was that an administration of anti-TGF-beta1 antibody might significantly reduce the mechanical properties of the stress-shielded patellar tendon. In the results, an application of 4-ng TGF-beta1 significantly increased the tangent modulus of the stress-shielded patellar tendon at 3 weeks (p = 0.019), compared with the sham treatment. Concerning the tensile strength, the 4-ng TGF-beta1 application increased the average value, but a statistical significance was not reached. An application of 50-microg anti-TGF-beta1 antibody significantly reduced the tangent modulus and the tensile strength of the stress-shielded patellar tendon at 3 weeks (p = 0.0068 and p = 0.0355), compared with the sham treatment. Because the stress-shielding treatment used in this study dramatically reduces the tangent modulus and the tensile strength of the patellar tendon, the present study suggested that an administration of TGF-beta1 weakly but significantly inhibited the reduction of the mechanical properties of the stress-shielded patellar tendon, and that inactivation of TGF-beta1 with its antibody significantly enhanced the reduction of the mechanical properties that occurs in the stress-shielded patellar tendon. These results suggested that TGF-beta1 plays an important role in remodeling of the stress-shielded patellar tendon.

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog.

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.