Effect of suprascapular nerve injury on muscle and regenerated enthesis in a rat rotator cuff tear model.

BACKGROUND: Massive rotator cuff tears (RCTs) are complicated by muscle atrophy, fibrosis, and intramuscular fatty degeneration, which are associated with postoperative tendon-to-bone healing failure and poor clinical outcomes. We evaluated muscle and enthesis changes in large tears with or without suprascapular nerve (SN) injury in a rat model. METHODS: Sixty-two adult Sprague-Dawley rats were divided into SN injury (+) and SN injury (-) groups (n=31 each), comprising tendon (supraspinatus [SSP]/infraspinatus [ISP]) and nerve resection and tendon resection only cases, respectively. Muscle weight measurement, histological evaluation, and biomechanical testing were performed 4, 8, and 12 weeks postoperatively. Ultrastructural analysis with block face imaging was performed 8 weeks postoperatively. RESULTS: SSP/ISP muscles in the SN injury (+) group appeared atrophic, with increased fatty tissue and decreased muscle weight, compared to those in the control and SN injury (-) groups. Immunoreactivity was only positive in the SN injury (+) group. Myofibril arrangement irregularity and mitochondrial swelling severity, along with number of fatty cells, were higher in the SN injury (+) group than in the SN injury (-) group. The bone-tendon junction enthesis was firm in the SN injury (-) group; this was atrophic and thinner in the SN injury (+) group, with decreased cell density and immature fibrocartilage. Mechanically, the tendon-bone insertion was significantly weaker in the SN injury (+) group than in the control and SN injury (+) groups. CONCLUSIONS: In clinical settings, SN injury may cause severe fatty changes and inhibition of postoperative tendon healing in large RCTs. Level of evidence: Basic research, controlled laboratory study.

Improved intervertebral bone union in ALIF rat model with porous hydroxyapatite/collagen combined with platelet-rich plasma.

BACKGROUND CONTEXT: Platelet-rich plasma (PRP) can accelerate bone union in spinal fusion surgery with an autogenous bone graft. However, it is unclear whether bone union can be obtained by using artificial bone and PRP together in spinal interbody fusion surgery. PURPOSE: This study aimed to determine whether interbody fusion can be achieved by transplanting porous hydroxyapatite/collagen(HAp/Col) which is an artificial bone material frequently used in spinal fusion surgery, together with PRP in the intervertebral disc space in rats. STUDY DESIGN AND SETTING: A controlled laboratory study. METHODS: A total of fourty 10-week old Sprague-Dawley rats were used in this study and assigned to three groups as follow: disc curettage only (control group, n=10), disc curettage + HAp/Col transplant (H group, n=10), and disc curettage + HAp/Col + PRP transplant (H+P group, n=10). The other 10 rats were sacrificed as blood donors for acquisition of PRP. Microcomputed tomography (µCT) examinations were performed to evaluate bone union, bone volume (BV), and bone mineral density (BMD) at 4, 8, and 12 weeks following surgery. Twelve weeks postoperatively, each group of three of L4-L5 spines was harvested to perform histological examination (hematoxylin & eosin stain) and the others were subjected to biomechanical testing (compression properties). RESULTS: The platelet count in PRP was approximately 4.1 times greater than that in whole blood (260.6±26.2 × 104 mg/dL and 64.3±2.9 × 104 mg/dL in PRP and whole blood, respectively). All the L4-L5 lumbar discs were fused in the H+P group, whereas only one case was fused in the H group and none in the control group at 12 weeks after surgery. BV was significantly higher in the H+P group than in the H group or control groups (both p<.01), although BMD was not significantly different among the three groups. Upon histological analysis, mature bone formation was observed at the transplanted space in all cases in the H+P group, whereas fibrous tissue was observed at the location in the H and control groups. Regarding biomechanical properties, the ultimate load to failure was significantly higher in the H+P group than in the H group or control group (p=.021 and .013, respectively), although stiffness was not significantly different between the three groups. CONCLUSION: The combination of porous HAp/Col and PRP at an appropriate concentration can promote bone union in the intervertebral disc space without using an autologous bone graft in the rat model. Bone tissue formation was histologically confirmed, and it was mechanically strong. CLINICAL SIGNIFICANCE: This preclinical study showed that porous HAp/Col, when combined with PRP at an appropriate concentration, can induce bone union without autologous bone grafts. The results may eliminate the need for autologous bone collection for spinal fusion surgery in the future.

Comparison of Structural Properties Between Postnatal and Adult Tendon Insertion with FIB/SEM Tomography in Rat.

OBJECTIVE: The repaired tendon-bone interface after rotator cuff (RC) repair has been identified as a mechanical weak point, which may contribute to re-tearing. Analyzing the postnatal development of a normal tendon insertion in detail may be useful in helping to promote the regeneration of a normal tendon insertion. We verified the morphological differences between postnatal and adult tendon insertions in terms of the cellular structural properties using FIB/SEM tomography. MATERIALS AND METHOD: SPostnatal and adult Sprague-Dawley rats were used as a model of tendon insertion. The morphological structure of the insertion was evaluated using hematoxylin and eosin (HE) staining, and the 3D ultrastructure of the cells in the insertion was evaluated using FIB/SEM tomography. Additionally, the volume of the cell bodies, nuclei, and cytoplasm were measured and compared in a quantitative analysis. RESULTS: On conventional histology, the boundary line between the fibrocartilage and mineralized cartilage was flat in the adult insertions; however, the boundary line between the mineralized cartilage and bone formed deep interdigitations. The morphology of the cells among the collagen bundles in the adult insertions was completely different from those in the postnatal insertions at the 3D ultrastructural level. The cellular structural properties were statistically different between the postnatal and adult insertions. CONCLUSIONS: In the present study, the morphological differences between postnatal and adult tendon insertion in terms of the ultrastructural cellular properties were clarified. These findings may aid in determining how to regenerate a clinically stable tendon insertion at the tendon-bone interface after RC repair.

Distribution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells in the urethra and periurethral genital organs in mice.

The aim of this study is to clarify the disibution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells (SIR-NECs) in mouse prostate and in the surrounding genital organs by histological and immunohistochemical analysis of the light microscopic serial sections of urethra. We collected lower urinary tracts from 13-week-old mice and observed the distribution pattern and shape of the SIR-NECs by serial light microscopy. The organs on the sections were divided into three anatomical zones to clarify the distribution pattern of SIR-NECs: (1) zone A, the ducts near the prostatic urethra; (2) zone B, the ducts outside the urethral sphincter; and (3) zone C, the acinus areas. Sections were double immune-stained with antibodies against serotonin and one of neuroendocrine-related factors (NRFs), including 10 neural cell markers and eight neurotransmitters, and also 4',6-diamino-2-phenylindole (DAPI). In addition, SIR-NECs were double immune-stained with antibodies against cytokeratin 5 (CK5) and p63, together with DAPI. SIR-NECs were mostly localized in zone A, and no SIR-NECs were observed in zone C. The proportion of flask-shaped SIR-NECs was approximately 15% in zones A and B. No flask-shaped SIR-NECs were observed in urethral epithelia. The NRFs co-localized with SIR-NEC were calcitonin gene-related peptide, CD56, chromogranin A, neuron-specific enolase, neuron cytoplastic protein 9.5, and synaptophysin (72.3%, 73.2%, 88.9%, 92.3%, 91.7%, and 81.9%, respectively). CK5 and p63 were not co-localized with SIR-NECs.　In this study, SIR-NEC of the urethra and the surrounding genital organs was ubiquitous in the urethra and the ducts near the urethra and co-expressed specific nerve-related NRFs.

Ectopic subcutaneous transplantation of fetal rat urogenital sinus and seminal vesicle promotes the organ growth and formation.

PURPOSE: The fate of subcutaneously transplanted urogenital sinus (UGS) and seminal vesicle (SV) was investigated in the present study. MATERIALS AND METHODS: Fetal UGS and SV extracted from 20-embryonic-day-old male normal and GFP transgenic rats were subcutaneously transplanted into 7-week-old male immunologically inhibited rats. The transplants were then examined at 2, 4, 8, and 16 weeks after transplantation. We analyzed the survival ratio, weight, and histopathology as well as the immunohistochemical characteristics of the transplanted tissues. For control experiments, 2-, 4-, 8-, and 16-week-old normal male rats were used. RESULTS: Almost all of the transplanted tissues survived under the skin, and the tissue weights increased over time after transplantation. The histopathological characteristics and immunohistochemical staining pattern with certain antibodies of the transplanted tissues were similar to those of normal adult rat prostate and seminal vesicle. The transplanted GFP transgenic tissues demonstrated spontaneous growth and organ formation under the skin, showing distribution and movement of transplanted cells and tissues. CONCLUSION: Subcutaneously transplanted fetal UGS and SV were able to develop into mature adult organs.

Correlative imaging of collagen fibers and fibroblasts using CLEM optimized for picrosirius red staining and FIB/SEM tomography.

Conventional imaging for three-dimensional (3D) ultra-architectural analysis of collagen fibers and fibroblasts is time-consuming and requires numerous ultrathin sections to search the target area. Currently, no method allows 3D ultra-architectural analysis of predetermined areas including spatial relationships between collagen fibers and fibroblasts in vitro. Herein, we developed a new method for in vitro analysis of the 3D ultrastructure of fibroblasts and collagen fibers using CLEM optimized for picrosirius red staining and FIB/SEM tomography. Collagen fibers were observed between, rather than on top of, stacked cells. This method offers the advantage of mesoscopic and ultrastructural analysis, thus minimizing bias and ensuring accurate observation.

Cellular network across cementum and periodontal ligament elucidated by FIB/SEM tomography.

Cementocytes in cementum form a lacuna-canalicular network. However, the 3D ultrastructure and range of the cementocyte network are unclear. Here, the 3D ultrastructure of the cementocyte network at the interface between cementum and periodontal ligament (PDL) was investigated on the mesoscale using FIB/SEM tomography. The results revealed a cellular network of cementocytes and PDL cells. A previous histomorphological study revealed the osteocyte-osteoblast-PDL cellular network. We extended this knowledge and revealed the cementum-PDL-bone cellular network, which may orchestrate the remodeling and modification of periodontal tissue, using a suitable method for imaging of complex tissue.

Three-dimensional ultrastructural imaging and quantitative analysis of the periodontal ligament.

The periodontal ligament (PDL) is a unique connective tissue mainly comprising collagen fiber bundles and cells between the roots of teeth and inner walls of the alveolar-bone socket. PDL fiber bundles are arrayed between teeth and bone, with both ends embedded in the cementum or alveolar bone as Sharpey's fiber. These bundles, synthesized by PDL fibroblasts (PDLFs), form several distinct groups within the PDL which has important functions besides tooth anchoring including tooth nutrition, proprioception, sensory detection, homoeostasis, and repair of damaged tissue. However, little is known about how the regular-PDL fiber bundle arrays are formed, maintained, and remodeled over large distances from cementum to alveolar bone. Recently, novel instruments and 3D-imaging methods have been developed that have been applied to the investigation of hard tissues including the PDL. Work from our laboratory has revealed the three-dimensional (3D) ultrastructure of PDLFs and PDL collagen bundles by focused ion beam/scanning electron microscope tomography. We have shown that PDLFs have a flat shape with long processes or a wing-like shape, while PDL bundles are a multiple-branched structure wrapped in thin sheets of PDLF cytoplasm. Furthermore, PDLFs form an extensive cellular network between the cementum and alveolar bone. The PDL cellular network is presumed to synchronize PDL fiber bundles and regulate arrays of PDL fiber bundles via gap junctions. In this review, we summarize and discuss our current 3D-histomorphometric studies of the PDL at the mesoscale level.

Three-dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. MATERIAL AND METHODS: The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. RESULTS: Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. CONCLUSION: These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

The periodontal ligament (PDL) maintains the environment and function of the periodontium. The PDL has been remodelled in accordance with changes in mechanical loading. Three-dimensional (3D) structural data provide essential information regarding PDL function and dysfunction. However, changes in mechanical loading associated with structural changes in the PDL are poorly understood at the mesoscale. This study aimed to investigate 3D ultrastructural and histomorphometric changes in PDL cells and fibres associated with unloading condition (occlusal hypofunction), using focused ion beam/scanning electron microscope tomography, and to quantitatively analyse the structural properties of PDL cells and fibres. PDL cells formed cellular networks upon morphological changes induced via changes in mechanical loading condition. Drastic changes were observed in a horizontal array of cells, with a sparse and disorganised area of collagen bundles. Furthermore, collagen bundles tended to be thinner than those in the control group. FIB/SEM tomography enables easier acquisition of serial ultrastructural images and quantitative 3D data. This method is powerful for revealing 3D architecture in complex tissues. Our results may help elucidate architectural changes in the PDL microenvironment during changes in mechanical loading condition and regeneration, and advance a wide variety of treatments in dentistry.

Effects of Estrogen-Deficient State on Rotator Cuff Healing.

BACKGROUND: Rotator cuff retears after surgical repair are a concern, despite advances in operative techniques, but few studies have investigated the effects of the estrogen-deficient state on tendon-to-bone healing at the repair site. PURPOSE: We evaluated the effect of the estrogen-deficient state on tendon-to-bone healing after rotator cuff repair in an ovariectomized rat model. STUDY DESIGN: Controlled laboratory study. METHODS: Female Sprague Dawley rats underwent detachment and immediate repair of the supraspinatus tendon. Surgery was performed in 24 rats at 4.5 weeks of age 17 weeks after ovariectomy (OVX group) and in 24 age-matched control rats without ovariectomy (control group). Animals were sacrificed at 2, 4, 8, and 12 weeks after surgery for biomechanical and histological evaluations of reattachment. Bone mineral density (BMD) at the insertion site and cancellous bone in the humeral head was assessed by micro-computed tomography. RESULTS: BMD was significantly lower both at the insertion site and in cancellous area in the OVX group than in the control group at weeks 2 to 12. Ultimate load to failure, ultimate stress, linear stiffness, and the Young modulus were significantly lower in the OVX group than in the control group at 2 and 4 weeks, but the difference was no longer significant at 8 and 12 weeks. At 2 and 4 weeks, relatively immature granulation tissue was observed in the OVX group compared with the control group. At 8 and 12 weeks after surgery, there were differences in the tendon-bone interface in the 2 groups: Direct insertion with well-established chondroid tissue was seen in the control group, and indirect insertion without chondroid tissue was seen in the OVX group. Consistently, the amount of chondroid tissue was greater and collagen organization was better in the control group than in the OVX group. Cells expressing cathepsin K were significantly more numerous both at the insertion site and in cancellous bone in the OVX group than in the control group. CONCLUSION: The estrogen-deficient state by ovariectomy, compared with control rats, led to decreased biomechanical properties and poor development of chondroid tissue that influenced the repair of the tendon insertion after surgery. CLINICAL RELEVANCE: Agents that modulate bone metabolism might improve tendon-to-bone healing in patients with an estrogen-deficient state, such as postmenopausal women who undergo rotator cuff surgery.

Histological study of costal cartilage after transplantation and reasons for avoidance of postoperative resorption and retention of cartilage structure in rats.

BACKGROUND: Limited information is available on the biological status of transplanted cartilage from which the perichondrium has been removed. This article describes the histological and three-dimensional structural picture of cartilage, using green fluorescent protein (GFP) transgenic rats and normal wild rats. METHODS: Three sections of costal cartilage were harvested from 10-week-old wild rats. One section was used as a specimen while two were subcutaneously collected from the dorsal region of 10-week-old GFP rats at 4 and 8 weeks post-transplant. The experiment was performed in two randomized groups. The perichondrium was removed from transplanted cartilage in the first group and perichondrium of transplanted cartilage remained intact in the second group. Histology and focused ion beam/scanning electron microscope (FIB/SEM) tomography were used to evaluate the transplanted cartilage. RESULTS: All 40 transplanted sections were harvested and no infections, exposure or qualitative change of cartilage matrix were seen following transplant. Histological analyses showed that the surface layer of the GFP-negative transplanted cartilage was replaced with GFP-positive chondrocytes 8 weeks post-transplant in the first group. A three-dimensional layer of perichondrium-like tissue reconstructed around the cartilage at 8 weeks was confirmed, resembling normal perichondrium. However, the GFP-positive chondrocytes were not replaced in the second group. CONCLUSIONS: The cell renewal of chondrocytes is necessary for subcutaneously transplanted cartilage to maintain its tissue composition over a long period of time. The histological and ultrastructural analyses revealed that cells from recipient tissue generated new chondrocytes even when cartilage was implanted after removing the perichondrium.

Hyaluronic Acid Accelerates Tendon-to-Bone Healing After Rotator Cuff Repair.

BACKGROUND: There is growing evidence that the subacromial injection of hyaluronic acid (HA) is effective for pain relief in rotator cuff tears; however, its effect on tendon-to-bone healing remains unknown. PURPOSE: To examine the effect of HA on the chondrogenesis of mesenchymal stem cells (MSCs) in vitro and on tendon-to-bone healing in a rotator cuff repair model. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral complete tears of the infraspinatus tendon were made in rabbits and subsequently repaired. Before closure, 1 mL HA was applied to the repaired site, and phosphate-buffered saline was used in the opposite side as a control. Biomechanical, histological, and immunohistochemical analyses were performed at 4, 8, and 12 weeks after surgery. After euthanizing each animal, the bone marrow was isolated from the femoral bone in the same rabbits. Then, MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. RESULTS: At 4 and 8 weeks after surgery, ultimate load-to-failure was significantly greater in the HA group than in the control group (45.61 ± 9.0 N vs 32.42 ± 9.4 N at 4 weeks, 90.7 ± 16.0 N vs 66.97 ± 10.0 N at 8 weeks; both P < .05) but not at 12 weeks after surgery (109.6 ± 40.2 N vs 108.1 ± 42.6 N, P > .05). Linear stiffness was not significant throughout the time point evaluation. The chondroid formation area at the tendon-bone interface stained by safranin O (control vs HA group) was 0.33% ± 0.7% versus 13.5% ± 12.3% at 4 weeks after surgery ( P < .05) and 3.0% ± 5.9% versus 12.9% ± 12.9% at 8 weeks after surgery ( P < .05), but there was no significant difference at 12 weeks after surgery. Maturity of collagen at the repaired site stained by PicroSirius Red (control vs HA group) was 16.2 ± 10.6 versus 43.5 ± 21.3 at 4 weeks after surgery ( P < .05), but there were no significant differences at 8 and 12 weeks after surgery. MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. The number of CD44-positive cells (control vs HA group) was 8.3% ± 1.4% versus 26.2% ± 5.2% at 3 days after surgery ( P < .05), 1.8% ± 1.1% versus 26.6% ± 11.6% at 4 weeks after surgery ( P < .05), 0.6% ± 0.9% versus 0.5% ± 0.6% at 8 weeks after surgery ( P > .05), and 1.8% ± 4.0% versus 5.4% ± 4.2% at 12 weeks after surgery ( P > .05). Compared with the control group, HA significantly increased the volume of cartilaginous pellet produced by MSCs (0.0016 ± 0.0015 mm3 at 0 mg/mL of HA, 0.0041 ± 0.0023 mm3 at 1.0 mg/mL, and 0.0041 ± 0.0018 mm3 at 4.0 mg/mL), with increased mRNA expression (relative ratio to control) of type 2 collagen (1.34 ± 0.38), SOX9 (1.58 ± 0.31), and aggrecan (1.30 ± 0.22) genes in the pellet ( P < .01). CONCLUSION: HA accelerated tendon-to-bone healing in the rotator cuff repair model, enhancing the biomechanical strength and increasing chondroid formation and tendon maturity at the tendon-bone interface. Based on the data of in vitro experiments, HA-activated MSCs may play a crucial role in the acceleration of tendon-to-bone healing. CLINICAL RELEVANCE: The data suggest the relevance of clinical application of HA to accelerate tendon-to-bone healing. It may decrease the number of retears after surgery.

Three-dimensional ultrastructural analysis of cells in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

The accurate comprehension of normal tissue provides essential data to analyse abnormalities such as disease and regenerative processes. In addition, understanding the proper structure of the target tissue and its microenvironment may facilitate successful novel treatment strategies. Many studies have examined the nature and structure of periodontal ligaments (PDLs); however, the three-dimensional (3D) structure of cells in normal PDLs remains poorly understood. In this study, we used focused ion beam/scanning electron microscope tomography to investigate the whole 3D ultrastructure of PDL cells along with quantitatively analysing their structural properties and ascertaining their orientation to the direction of the collagen fibre. PDL cells were shown to be in contact with each other, forming a widespread mesh-like network between the cementum and the alveolar bone. The volume of the cells in the horizontal fibre area was significantly larger than in other areas, whereas the anisotropy of these cells was lower than in other areas. Furthermore, the orientation of cells to the PDL fibres was not parallel to the PDL fibres in each area. As similar evaluations are recognized as being challenging using conventional two-dimensional methods, these novel 3D findings may contribute necessary knowledge for the comprehensive understanding and analysis of PDLs.

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.

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.

Interaction between Macrophages and Fibroblasts during Wound Healing of Burn Injuries in Rats.

Analysis of the structural changes and cell-to-cell interactions occurring during wound healing of burn injuries is essential to elucidate the morphological characteristics of the reconstitution of tissue architecture. However, conventional approaches do not provide sufficient information with respect to cell-to-cell interactions during wound healing. The aim of this study was to evaluate the interaction between bone marrow-derived cells and resident stromal cells throughout the wound healing of burn injuries, using immunohistochemistry and focused ion beam/scanning electron microscope tomography. We induced third-degree burn injuries on the backs of Wistar rats with a heated cylindrical aluminum block (2.0 cm in diameter). At 7 and 14 days after the burn injuries, the burned skin was immunostained with anti-Iba1 and anti-HSP47 antibodies for visualization of bone marrow-derived cells/macrophages and resident stromal cells/fibroblasts, respectively. Normal skin tissue was used as a control. Double-staining immunohistochemistry revealed frequent contacts between macrophages and fibroblasts and a higher contact ratio in the 3 normal skin compared with burned skin, particularly in the areas of granuloma. Three-dimensional ultrastructural analysis with focused ion beam/scanning electron microscope tomography revealed that macrophages and fibroblasts were located closer together in the normal skin than in the burned skin, confirming the analysis by light microscopic observations and ultrastructural analysis from single sections. These results highlight the importance of contact between macrophages and fibroblasts in the maintenance of skin tissue structure and during wound healing.

Factors Affecting Clinical Outcome in Patients With Structural Failure After Arthroscopic Rotator Cuff Repair.

PURPOSE: (1) To compare clinical outcomes between patients with large or massive rotator cuff tears who have healed cuffs and patients with postoperative structural failure (retear after complete or partial repair) and (2) to identify factors associated with clinical outcomes in patients with postoperative structural failure. METHODS: We conducted a retrospective study of consecutive patients with large or massive cuff tears who underwent arthroscopic repair at our institution between 2005 and 2012. On the basis of intraoperative findings and magnetic resonance imaging at final follow-up, the patients were divided into 3 groups: healed group, retear group (after complete repair), and partial-repair group. The outcome measures comprised the Japanese Orthopaedic Association (JOA) and University of California, Los Angeles (UCLA) scores; muscle strength; and range of motion. The tear length, tear width, muscle atrophy, and fatty degeneration were evaluated by preoperative and postoperative magnetic resonance imaging. The extent of tendon reattachment to the superior, middle, and inferior facets and the lesser tuberosity was examined on magnetic resonance images at final follow-up. RESULTS: In total, 74 patients (healed, 41; retear, 19; and partial repair, 14) were included in this study. The mean age was 63.8 ± 8.5 years, with a mean follow-up period of 3.6 ± 1.4 years. The postoperative JOA and UCLA scores significantly improved in all 3 groups, but the differences were not significant. In the retear and partial-repair groups, postoperative tendon preservation at the middle facet significantly affected the JOA and UCLA scores (P = .003 and P = .014, respectively). CONCLUSIONS: The JOA and UCLA scores were significantly improved in patients with structural failure. Under these conditions, the only clinical factor that affected patient outcome was tendon healing at the middle facet. Thus tendon preservation at the middle facet was a predictor of good clinical outcomes in patients who underwent arthroscopic rotator cuff repair of large or massive tears and had postoperative structural failure. LEVEL OF EVIDENCE: Level III, case-control study.

Effects of lidocaine on torn rotator cuff tendons.

We determined lidocaine's 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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1620-1627, 2016.

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. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:969-976, 2016.