Tomonoshin Kanazawa

An immunohistological study of the integration at the bone-tendon interface after reconstruction of the anterior cruciate ligament in rabbits

We studied bone-tendon healing using immunohistochemical methods in a rabbit model. Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification.

Risk factors for shoulder re-dislocation after arthroscopic Bankart repair

BackgroundRecent studies have shown effective clinical results after arthroscopic Bankart repair (ABR) but have shown several risk factors for re-dislocation after surgery. We evaluated whether patients are at a risk for re-dislocation during the first year after ABR, examined the recurrence rate after ABR, and sought to identify new risk factors.MethodsWe performed ABR using bioabsorbable suture anchors in 102 consecutive shoulders (100 patients) with traumatic anterior shoulder instability. Average patient age and follow-up period was 25.7 (range, 14–40) years and 67.5 (range, 24.5–120) months, respectively. We evaluated re-dislocation after ABR using patient telephone interviews (follow-up rate, 100%) and correlated re-dislocation with several risk factors.ResultsRe-dislocation after ABR occurred in nine shoulders (8.8%), of which seven sustained re-injuries within the first year with the arm elevated at 90° and externally rotated at 90°. Of the remaining 93 shoulders without re-dislocation, 8 had re-injury under the same conditions within the first year. Thus, re-injury within the first year was a risk for re-dislocation after ABR (P < 0.001, chi-squared test). Using multivariate analysis, large Hill-Sachs lesions (odds ratio, 6.77, 95% CI, 1.24–53.6) and <4 suture anchors (odds ratio, 9.86, 95% CI, 2.00–76.4) were significant risk factors for re-dislocation after ABR.ConclusionsThe recurrence rate after ABR is not associated with the time elapsed and that repair strategies should augment the large humeral bone defect and use >3 anchors during ABR.

Effects of corticosteroids and hyaluronic acid on torn rotator cuff tendons in vitro and in rats.

Corticosteroids (CS) or hyaluronic acid (HA) is used in subacromial injection for the conservative treatment of rotator cuff tears (RCT); this study addresses the question of how CS and HA affect the tendon tissue and fibroblasts in vitro and in rats. Cell proliferation assays were performed in human tendon fibroblasts from RCT. Rats underwent surgery to create RCT, and the surgical sites were injected with CS or HA. The rotator cuff tendons were subjected to biomechanical testing, microscopic and immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), and ultrastructural analysis. Cell proliferation was significantly decreased with CS in vitro (p < 0.05). Maximal load of CS‐treated tendons was significantly decreased compared with that of HA‐treated tendons (p < 0.05), as well as PCNA+ cells at 2 weeks (p < 0.05). Ultrastructural observations of the CS‐treated rats detected apoptosis of tendon fibroblasts 24 h after surgery. Histological and biomechanical data 4 weeks after surgery were not significant among the three groups. Unlike HA, CS caused cell death, and inhibition of the proliferation of tendon fibroblasts, leading to a delay of tendon healing involved and a subsequent decrease of biomechanical strength at the surgical site.

Histomorphometric and ultrastructural analysis of the tendon-bone interface after rotator cuff repair in a rat model

Successful rotator cuff repair requires biological anchoring of the repaired tendon to the bone. However, the histological structure of the repaired tendon-bone interface differs from that of a normal tendon insertion. We analysed differences between the normal tendon insertion and the repaired tendon-bone interface after surgery in the mechanical properties, histomorphometric analysis, and 3-dimensional ultrastructure of the cells using a rat rotator cuff repair model. Twenty-four adult Sprague-Dawley (SD) rats underwent complete cuff tear and subsequent repair of the supraspinatus tendon. The repaired tendon-bone interface was evaluated at 4, 8, and 12 weeks after surgery. At each time point, shoulders underwent micro-computed tomography scanning and biomechanical testing (N = 6), conventional histology and histomorphometric analysis (N = 6), and ultrastructural analysis with focused ion beam/scanning electron microscope (FIB/SEM) tomography (N = 4). We demonstrated that the cellular distribution between the repaired tendon and bone at 12 weeks after surgery bore similarities to the normal tendon insertion. However, the ultrastructure of the cells at any time point had a different morphology than those of the normal tendon insertion. These morphological differences affect the healing process, partly contributing to re-tearing at the repair site. These results may facilitate future studies of the regeneration of a normal tendon insertion.

Effects of lidocaine on torn rotator cuff tendons.

We determined lidocaines action on torn rotator cuff tendons in vitro and in vivo. For in vitro experiments, cell proliferation and viability assays were performed using tenocytes derived from human torn rotator cuff tendons. For in vivo experiments, acute rotator cuff tears were made on the supraspinatus tendons in the rats’ bilateral shoulders; before closure, lidocaine was injected into the shoulder and saline into the contralateral shoulder (control). After sacrifice, the specimens underwent biomechanical testing or histological analysis at 24 h and at 2, 4, and 8 weeks after surgery. The extent of collagen organization and apoptosis were semi‐quantitatively evaluated using collagen picrosirius red staining. Apoptosis was examined using TUNEL staining and electron microscopy. Cell proliferation decreased dose‐dependently. After exposure to 0.1% lidocaine for 24 h, cell viability decreased. Two and 4 weeks after surgery, the ultimate load to failure decreased more in the lidocaine group than in the control group, with significantly reduced stiffness in the lidocaine group 2 weeks after surgery. Collagen organization significantly decreased in the lidocaine group by 4 weeks after surgery but returned to baseline at 8 weeks. TUNEL staining detected numerous apoptotic tenocytes at the torn tendon edge exposed to lidocaine 24 h after surgery; electron microscopy confirmed the condensed cell nuclei. These changes were not observed in controls. Lidocaine caused cytotoxicity to tenocytes under both conditions, decreased biomechanical properties, and induced apoptosis and delay of collagen organization in this model. Subacromial lidocaine injections in patients with rotator cuff tears should be performed carefully.

Three‐dimensional ultrastructural analysis of development at the supraspinatus insertion by using focused ion beam/scanning electron microscope tomography in rats

To obtain a successful outcome after rotator cuff repair, the repaired tendon must be biologically anchored to the bone. However, the histological structure at the repaired tendon‐bone interface differs from that of the site of normal tendon insertion. Therefore, analyzing postnatal development in detail will contribute to understanding the repaired tendon‐bone interface after rotator cuff repair. In this study, we analyzed postnatal development at the tendon‐bone insertion in terms of temporal changes in SOX9/SCX expression and three‐dimensional (3D) ultrastructure with FIB/SEM tomography, a new scanning electron microscopic method. Sixteen postnatal Sprague–Dawley rats were used for the study. One‐, two‐, three‐, and four‐week‐old rats were sacrificed and both right and left shoulders were removed; eight normal supraspinatus tendon insertions were isolated for each time point. At each time point, four specimens were evaluated with fluorescent immunostaining for SOX9/SCX expression, and the remaining four specimens were evaluated with FIB/SEM tomography. Even in postnatal development, SOX9+/SCX+ expression was observed at the tendon insertion; expression gradually decreased with postnatal development at the normal tendon insertion. In 3D ultrastructure, the morphology of the cells and the number/orientation of the cell processes drastically changed by postnatal week 4. The pattern of SOX9/SCX expression and 3D ultrastructural changes obtained in this study contribute to an understanding of the complicated development of normal tendon‐bone insertion. Therefore, this study helps elucidate the pathophysiology of tendon‐bone insertion, especially in cases of rotator cuff tear and repair.

Inducement of semitendinosus tendon regeneration to the pes anserinus after its harvest for anterior cruciate ligament reconstruction-A new inducer grafting technique

PurposeTo investigate the usefulness of the “inducer grafting” technique for regeneration of the semitendinosus (ST) tendon after its harvest for anterior cruciate ligament (ACL) reconstruction.MethodsTwenty knees of 20 patients (mean age at the time of surgery, 23.1 years) underwent ACL reconstruction with a double bundle autograft using the ST tendon (7 patients) and the ST + the gracilis (G) tendons (13 patients).“Inducer grafting” techniqueAfter harvesting the ST tendon, a passing pin with a loop thread is inserted along with the tendon stripper. The passing pin is pulled out from the medial thigh and the loop thread retained. As an inducer graft, the ST tendon branch is used. After the ACL graft has been secured, the inducer graft is sutured to the pes anserinus and the proximal end passed through by pulling the thread out. Then the inducer graft is placed within the tendon canal. The mean follow-up period was 15 months. The presence and morphology of the regenerated ST tendon were examined by MRI. And the isometric hamstring strength was examined at 45°, 90° and 120° of knee flexion.ResultsOne month after the operation in all the patients, MRI demonstrated a low-intensity structure at the anatomical location of the ST, at the level of the superior pole of the patella and the joint line, apparently representing the regenerated ST tendon. Four months after the operation, the distal portion of the regenerated ST tendon had reached the pes anserinus in all patients. Twelve months after the operation, the regenerated ST tendon was hypertrophic in 19 of the 20 patients (95%). The isometric knee flexion torque of the ACL-reconstructed limb was significantly lower at 90° and 120° compared with the contralateral limb.ConclusionThese results suggest that the “inducer grafting” technique is able to improve the regeneration rate of the harvested ST tendon and promote hypertrophy of the regenerated ST tendon, extending all the way to the pes anserinus. However, this technique couldn’t improve the deficits in knee flexion torque after ACL reconstruction.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Regeneration of ring-shaped lateral meniscus after partial resection of discoid meniscus with anterior cruciate ligament reconstruction

INTRODUCTION The ring-shaped lateral meniscus is very rare. Although it is essentially known as a congenital anomaly, a central tear in an incomplete discoid meniscus or an old bucket-handle tear in a meniscus may be easily mistaken for a ring-shaped meniscus. We experienced a ring-shaped lateral meniscus that regenerated after partial resection of a discoid meniscus together with anterior cruciate ligament (ACL) reconstruction. PRESENTATION OF CASE A 37-year-old female patient still experienced unrelenting knee pain 6 months after ACL reconstruction and partial meniscectomy of a discoid lateral meniscus. A repeat arthroscopy was performed. The lateral tibial plateau was covered in the form of a ring by meniscus-like tissue. The meniscus-like tissue appeared to have regenerated inward toward the center from the stump after the partial meniscectomy and was connected from the anterior to posterior horn, forming an interhorn bridge. Partial meniscectomy was repeated. Histologically, the regenerated tissue was not meniscal, but comprised mature fibrocartilage; macroscopically; however, it was very similar to meniscal tissue. Two years after the initial operation, the patient had no complaints and experienced full return of function. DISCUSSION The reason for such regeneration is unknown, but may have been attributed to the specific intra-articular environment that developed after the ACL reconstruction. CONCLUSION This is the first report of regenerative development of a ring-shaped lateral meniscus. When a ring-shaped lateral meniscus is diagnosed, we must accurately determine whether it is a true congenital anomaly in consideration of the present case.

Chronological changes in the collagen-type composition at tendon-bone interface in rabbits.

Objectives The purpose of this study was to evaluate chronological changes in the collagen-type composition at tendon–bone interface during tendon–bone healing and to clarify the continuity between Sharpey-like fibres and inner fibres of the tendon. Methods Male white rabbits were used to create an extra-articular bone–tendon graft model by grafting the extensor digitorum longus into a bone tunnel. Three rabbits were killed at two, four, eight, 12 and 26 weeks post-operatively. Elastica van Gieson staining was used to colour 5 µm coronal sections, which were examined under optical and polarised light microscopy. Immunostaining for type I, II and III collagen was also performed. Results Sharpey-like fibres comprised of type III collagen in the early phase were gradually replaced by type I collagen from 12 weeks onwards, until continuity between the Sharpey-like fibres and inner fibres of the tendon was achieved by 26 weeks. Conclusions Even in rabbits, which heal faster than humans, an observation period of at least 12 to 26 weeks is required, because the collagen-type composition of the Sharpey-like fibre bone–tendon connection may have insufficient pullout strength during this period. These results suggest that caution is necessary when permitting post-operative activity in humans who have undergone intra-bone tunnel grafts.