Localized Rotator Cuff Tendon Degeneration for Cadaveric Shoulders with and Without Tears Isolated to the Supraspinatus Tendon

Localized differences in tissue degeneration throughout intact and torn rotator cuff tendons have not been well quantified. The objective of this study was to investigate histological differences in localized degeneration in tendons with and without rotator cuff tears isolated to the supraspinatus tendon. Four intact shoulders and four shoulders with rotator cuff tears isolated to the supraspinatus tendon were dissected down to the infraspinatus and supraspinatus tendons. Biopsies were taken throughout the tendon insertion, mid-substance, myotendinous junction, and around the tear if present. Samples were stained with hematoxylin and eosin and tendon degeneration was graded based on collagen fiber organization, nuclei shape, cellularity, and lipoid degeneration. Comparisons in degeneration parameters were made based on the tendon type (supraspinatus vs. infraspinatus), location within the tendon, and presence of a tear. Supraspinatus tendons exhibited more degeneration than the infraspinatus tendon (P < 0.05). Significant increases in lipoid degeneration were found near the myotendinous junction compared to the rest of the tendon (P < 0.001). Tendons with rotator cuff tears showed greater amounts of lipoid degeneration compared to intact tendons (P = 0.03). A strong negative correlation was found between lipoid degeneration and collagen fiber organization (r = −0.922, P = 0.001). No differences in degeneration were found between medial, anterior, and posterior edges of the tear. The study highlights specific factors of tendon degeneration contributing to the local differences in tendon degeneration. By understanding local differences in tendon degeneration, surgical protocols for repair can be improved. Clin. Anat., 33:1007–1013, 2020. © 2019 Wiley Periodicals, Inc.     

Type VI collagen in mouse masseter tendon,from osseous attachment to myotendinous junction

Background and Methods: The association of masseter tendon type VI collagen with other extracellular matrix (ECM) components was examined from osseous attachment to myotendinous junction by immunohistochemistry and transmission electron microscopy with ATP treatment and enzyme digestion. Results: In the tendon proper, fibrocytes extended their processes among bundles of striated collagen fibrils and associated with adjacent cells through amorphous materials, thus forming a three-dimensional network. The amorphous or filamentous material was observed around the fibrocyte cell body and along the cell processes, where the localization of type VI collagen was confirmed by immunohistochemistry using anti-type VI collagen antibody. After treatment with 20 mM adenosine 5′-triphosphate (ATP), 100 nm periodic fibrils, an aggregated form of type VI collagen, were formed in the place where amorphous or filamentous material was present before the treatment. In myotendinous junction, the ATP-aggregated periodic fibrils were observed to associate with the external lamina of the muscle cells as well as among junctional tendon collagen fibrils. In the tendonbone boundary, ATP-aggregated periodic fibrils were observed around fibrocartilage-like cells in the uncalcifying area but not in the calcification front. Prolonged ATP treatment or hyaluronidase predigestion caused the formation of type VI collagen periodic fibrils in the area near the calcified matrix. Conclusions: The distribution of type VI collagen in mouse masseter tendon is different in different anatomical position. This may reflect the different functional demand for this collagen. © 1995 Wiley-Liss, Inc.     

Immunohistological study on collagenous proteins of benign and malignant human cartilaginous tumours of bone

Summary The immunohistological distribution of collagen types I, II, III, V and VI in human benign and malignant cartilaginous tumours of bone was examined with regard to their aggressiveness. The matrix of enchondromas consisted of type II collagen distributed diffusely, and type VI predominantly localized in the immediate surroundings of the cells. Types I, III and V collagen were not found. These findings were similar to the distribution of collagenous proteins in normal hyaline cartilage where each lobule was consistently rimmed by types I and V collagen. In grade 1 chondrosarcomas, the main collagenous components of matrix were also types II and VI collagen. Type II was sometimes found in the cytoplasm of tumour cells and type VI tended to lose territorial localization. In addition, type I collagen was demonstrated consistently and type V in some cases. In grade 2 chondrosarcomas, type II collagen was demonstrated not only in the matrix but occasionally in the cytoplasm of tumour cells. Type VI was dispersed in the intercellular areas. The other collagenous proteins such as types I, III and V were also present in the matrix. In grade 3 chondrosarcomas, type II collagen was localized predominantly in the cytoplasm of tumour cells and in the adjacent matrix. Type VI was markedly decreased with complete loss of pericellular distribution, whereas types I, III and V were constantly present in the matrix. Those alterations in the distribution of collagen types correlated well with the aggressive behaviour of the tumours. The findings suggest that distribution of different collagen types in cartilaginous tumours reflects the immaturity of the tumour cells and is a useful indicator of their aggressiveness.     

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies.     

Localization of type VI collagen in tissue-engineered cartilage on polymer scaffolds

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.     

Effects of oestrogen on the extracellular matrix in the endometrium of postmenopausal women.

AIM: To obtain insight into the effects of oestrogen on extracellular matrix (ECM) in the postmenopausal endometrium. METHODS: The distribution of the components of the ECM, including collagen types I, III, IV, and VI, and laminin, was investigated in the human postmenopausal endometrium by an indirect immunofluorescence method with specific monoclonal antibodies and a polyclonal antibody. Collagens were also extracted from the endometrial tissues of postmenopausal women who had or had not been treated with oestrogen for three weeks. RESULTS: Immunohistochemical studies demonstrated that type I collagen was the predominant interstitial collagen, and that types III and VI collagens were absent or very sparsely distributed in the stroma of the postmenopausal endometrium. However, types I, III, and VI collagens were diffusely localised in the stroma of the postmenopausal endometrium after administration of oestrogen. Even though type IV collagen was not seen in the basement membrane of the endometrial glands in the endometrium of postmenopausal women in the absence of oestrogen treatment, both type IV collagen and laminin were localised exclusively in the basement membrane of the endometrial glands in the postmenopausal endometrium after three weeks of oestrogen treatment. The level of type III collagen relative to that of type I collagen was significantly increased (p < 0.01) in the endometrium of oestrogen treated postmenopausal women compared with non-treated postmenopausal women. CONCLUSIONS: Conjugated equine oestrogen might induce changes in the distribution of components and in the composition of the ECM in the endometrium of postmenopausal women.     

肱二头肌长头肌腱病变与肩袖损伤的相关性及临床意义分析

目的 分析肱二头肌长头肌腱（long head of the biceps brachii tendon,LHBT）病变与肩袖损伤的相关性及临床意义。 方法　分析2010~2017年间西南医科大学附属中医医院经关节镜修复的126例肩袖损伤患者,术中对LHBT进行检查和评价,记录LHBT病变种类,分析LHBT病变与肩袖撕裂时间、撕裂大小和累及肌腱的相关性。 结果　发现LHBT I型病变（肌腱炎）51例（40.5%）,II型病变（半脱位）12例（9.5%）,III型病变（全脱位）12例（9.5%）,IV型病变（部分撕裂）15例（11.9%）,V型病变（完全撕裂）6例（4.8%）,30例（23.8%）无明显LHBT病理改变;病史超过12周的慢性肩袖撕裂患者均伴有LHBT病变;伴肩胛下肌（subscapularis,SSC）撕裂的肩袖损伤患者更易出现LHBT病变,且SSC撕裂程度越大,LHBT病变发生率越高,差异有统计学意义（P<0.05）。 结论 LHBT病变与肩袖损伤特别是合并SSC撕裂者密切相关,LHBT病变与SSC撕裂程度呈正相关;检查肩袖损伤的病例时,应注意LHBT的情况,避免漏诊;肩袖损伤的早期识别与修复可防止LHBT病变进一步恶化。     

A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The levels of four ECM components, i.e., collagens III, IV, VI, and laminin were markedly decreased in the sclerotic glomeruli as compared with week 8. In contrast, the amount of fibronectin in the sclerotic glomeruli increased dramatically. Immunoelectron microscopic examination showed fibronectin in the sclerotic lesions, in contrast to laminin, collagen type I, and collagen type IV. It is concluded that the sclerotic lesions in murine chronic GvHD contain fibronectin. The small amounts of the ECM components laminin, as well as collagens III, IV, and VI in the sclerotic glomeruli in GvHD, might represent remnants of mesangial material and collapsed capillary walls. These components are probably replaced by increased production and/or accumulation of collagen type I and fibronectin.     

The distribution of types I and III collagen and fibronectin in the healing equine tendon

During tissue response to injury the glycoproteins fibronectin and Type III collagen are synthesized in increased amounts. We have studied the distribution of these molecules in the healing tendon at various times after injury by comparison with that of the major constituent of normal tendon, Type I collagen. Immunofluorescent localization demonstrated the presence of fibronectin throughout the tendon within one week after injury. Staining was found in the matrix, both around capillaries and around fibroblast-like cells. Fibronectin was still apparent in the healing tendon at one month after injury, but after a further two months was no longer detectable. Type III collagen was present both in pericellular and matrix locations until three months after injury, and matrix staining was apparent during the entire fourteen-month period under study. Type III collagen was also found throughout the matrix of the contralateral superficial flexor tendon during this period.     

Immunohistochemical distribution patterns of collagen type II,chondroitin 4-sulfate,laminin and fibronectin in human nasal septal cartilage

Collagen type II, chondroitin 4-sulfate, laminin and fibronectin are major components of cartilage matrix. It is important to know their distribution patterns to evaluate relationships between cartilage cells and surrounding cartilage matrix. In the present study, we investigated localization patterns of these macromolecules in human nasal septal cartilage by immunohistochemical methods. Samples of human nasal septal cartilage were obtained from patients with nasal septum deviations who underwent septoplastic operation and were prepared for immunohistochemical examination. Distribution patterns of cartilage matrix macromolecules correlated with those found in other cartilage tissues. Diffuse staining of collagen type II was found in the cartilage matrix, chondroitin 4-sulfate immunostaining was present in the cytoplasm and like a pericellular ring around chondrocytes. Laminin immunostaining was found in the cytoplasm of chondrocytes, and fibronectin was localized in the pericellular matrix and in capsules of human nasal septal cartilage. Moreover, fibronectin was also detected at high levels in the interconnecting segments between adjacent chondrons. In conclusion, similar localisation patterns of the components investigated in human septal cartilage as in other tissues indicate that these macromolecules may play a role in both cell-matrix adhesion and matrix-matrix cohesion in the pericellular microenvironment surrounding nasal septal cartilage chondrocytes as in other cartilage tissues.     

Binding of Haemophilus ducreyi to extracellular matrix proteins

We developed an enzyme-linked immunosorbent assay-based assay to assess Haemophilus ducreyi binding to extracellular matrix (ECM) proteins. H. ducreyi 35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in ftpA or losB bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for H. ducreyi to bind to ECM proteins.     

Changes of glycoprotein and collagen immunolocalization in the uterine artery wall of postmenopausal women with and without pelvic organ prolapse

Pelvic organ prolapse (POP) is accompanied by an altered composition of the extracellular matrix (ECM). However, it is unclear whether the changed ECM is the cause or the consequence of POP, as stretching of the tissue may have an effect on the composition of the ECM. To address this question, we analyzed the connective tissues of the uterine artery wall of postmenopausal women with and without POP. The uterine artery wall is stretched in patients with POP, but this stretching is unlikely to cause the POP. Twenty-one women (13 with POP and 8 without POP) hospitalized for hysterectomy were included in this study. Tissue samples from the uterine artery were analyzed for collagen (types I, III, IV, V and VI) and other ECM proteins (fibronectin, laminin, tenascin, vitronectin and elastin) using immunofluorescence microscopy. Results revealed that uterine artery samples of women with prolapse showed a significantly weaker immunoreactivity to type VI collagen, vitronectin and elastin and a stronger immunostaining for type III collagen and tenascin as compared to control samples.Our results suggest that the ECM may be altered in response to mechanical stretch. Changes in the ECM composition as observed in POP may not necessarily be the reason for the development of pelvic floor relaxation in postmenopausal women.     

Type VI collagen. In situ hybridizations and immunohistochemistry reveal abundant mRNA and protein levels in human neurofibroma, schwannoma and normal peripheral nerve tissues

Cutaneous neurofibromas contain an extensive extracellular matrix composed of collagenous and non-collagenous macromolecules. In this study, the expression of type VI collagen genes in cutaneous neurofibromas was examined by a combination of in situ hybridizations and immunohistochemistry. In situ hybridizations with a 32P-labeled human type VI collagen-specific cDNA revealed that the majority of cells within neurofibromas expressed the gene for alpha 2(VI) collagen chain. The number of cells expressing clearly detectable levels of alpha 2(VI) collagen mRNA was considerably higher than that of cells actively expressing the pro alpha 1(I) or pro alpha 1(III) collagen genes. The presence of type VI collagen epitopes within the neurofibromas was also demonstrated by immunostaining with specific polyclonal antibodies. The expression of type VI collagen genes in neural tissues was further examined by immunostaining of a benign schwannoma tissue specimen consisting of Schwann cells. The results indicated close association of type VI collagen epitopes with the neoplastic Schwann cells. Immunolocalization of type VI collagen epitopes within normal human peripheral nerve revealed pericellular staining of perineurial cells and Schwann cells, suggesting synthesis of type VI collagen by these cell types. These results suggest that the expression of type VI collagen gene is active in nerve-derived tissues, and that type VI collagen may be a major component of the extracellular matrix in neural connective tissues.     

Novel nanofiber-based scaffold for rotator cuff repair and augmentation

The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a profound effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.     

Localization of fibrillin-1 in human endometrium and decidua during the menstrual cycle and pregnancy

We have examined the existence and distribution of fibrillin-1 within the endometrium and decidua to ascertain the effect of decidualization upon its synthesis, and its relationship to other extracellular matrix proteins. Formalin-fixed, paraffin-embedded tissues were stained using monoclonal antibodies to collagen IV, elastin, fibrillin-1 and laminin, by immunocytochemistry. Fibrillin-1 was present throughout the menstrual cycle and appeared to be cell-associated. Similar staining for fibrillin-1 was detected within the Fallopian tube of ectopic pregnancy material. In first trimester, second trimester and term decidua, staining for fibrillin-1 was more intense than that during the menstrual cycle and was detected as a thick layer encapsulating decidual cells, but also in fibrillar form inter-connecting these cells. In comparison with the association of collagen IV and laminin with all basal laminae, fibrillin-1 was absent from the vascular and glandular elements of pregnancy endometrium. Elastin was absent in all tissues examined. Hence, fibrillin-1 exists within the uterus in association with basal lamina components rather than with elastin- containing fibrils, where its presence in the endometrium and decidua could be of functional significance.      

Beneficial Regulation of Fibrillar Collagens, Heat Shock Protein-47, Elastin Fiber Components, Transforming Growth Factor-β1, Vascular Endothelial Growth Factor and Oxidative Stress Effects by Copper in Dermal Fibroblasts

Skin aging is associated with the loss of the structural collagens and the elastin fiber components that form the extracellular matrix (ECM). It is associated with reduced transforming growth factor-β (TGF-β), angiogenesis and increased oxidative stress. Copper has been incorporated into cosmetics for anti-skin aging. This research investigated the mechanism for the anti-skin aging effect copper ions, from cuprous oxide powders. Dermal fibroblasts were exposed to copper and examined for expression (protein and/or promoter levels) of types I, III, V collagen, heat shock protein-47 (HSP-47), elastin, fibrillin-1, and fibrillin-2, TGF-β1, vascular endothelial growth factor (VEGF), and in addition for membrane damage and lipid peroxidation. The direct antioxidant activity of copper was also determined. The research indicates that copper's anti-skin aging and skin regeneration potential is through its stimulation of ECM proteins, TGF-β1, VEGF, and inhibition of oxidative stress effects at physiological concentrations; and supports its use in cosmetics. Dr. Gadi Borkow is the chief medical scientist of Cupron Scientific.     

Effect of Selective Estrogen Receptor Modulator/Raloxifene Analogue on Proliferation and Collagen Metabolism of Tendon Fibroblast

The selective estrogen receptor modulator raloxifene is therapeutically beneficial for postmenopausal connective tissue degradation, such as osteoporosis, vascular sclerosis, and dermal degradation; however, the effects of raloxifene on postmenopausal tendon metabolism have not been clarified. In this study, we investigated the effects of raloxifene analogue (LY117018) on cell proliferation and collagen metabolism using cultured rat Achilles tendon fibroblasts. 17β-Estradiol (E₂; 10^?11–10^?9 M) and LY117018 (10^?9–10^?7 M) had no significant effects on tendon fibroblast proliferation, based on a BrdU (5-bromo-2′-deoxyuridine) incorporation assay (24 hr) and a WST-8 colorimetric assay (2 or 6 days). Neither E₂ nor LY117018 significantly altered the expression of type I collagen, which is a main component of the tendon extracellular matrix (ECM), whereas both E₂ and LY117018 significantly increased the expression of matrix metalloproteinase (MMP)-13, which is responsible for tendon collagen degradation in rat. Also, both E₂ and LY117018 increased the expression of type III collagen and elastin, which are minor components of tendon ECM, but are considered to govern the elastic properties of tendons. These changes in collagen and MMP induced by either E₂ or LY117018 were attenuated by the estrogen receptor alpha blocker ICI 182,780. The results of this study suggest that postmenopausal estrogen deficiency might downregulate tendon collagen turnover and decrease tendon elasticity. Further, raloxifene treatment might restore these changes to premenopausal levels.     

Fluorescence spectroscopy and birefringence of molecular changes in maturing rat tail tendon

Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes--laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-mum histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI(max)), which includes collagen and elastin, increases with animal weight (R(2)=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R(2)=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI(max) at R(2)=0.60).     

Autologous tenocyte therapy using porcine-derived bioscaffolds for massive rotator cuff defect in rabbits

Large and retracted rotator cuff tendon tears fail to repair or retear after surgical intervention. This study attempted to develop novel tissue-engineering approaches using tenocyte-seeded bioscaffolds for tendon reconstruction of massive rotator cuff tendon defect in rabbits. Porcine small intestine submucosa (Restore) and type I/III collagen bioscaffold (ACI-Maix) were chosen as bioscaffold carriers for autologous tenocytes. Biological characterization of autologous tenocytes was conducted before the implantation. The tenocyte-seeded bioscaffolds were implanted as interposition grafts to reconstruct massive rotator cuff tendon defects in rabbits. In situ reimplantation of the autologous rotator cuff tendon, excised during defect creation, served as a positive control. Histological outcomes were analyzed and semi-quantitatively graded at 4 and 8 weeks after surgery. At 4 weeks, both tenocyte-seeded bioscaffolds displayed inflammatory reaction similar to bioscaffold-only cuff reconstruction, and the histological grading were inferior to control repair. However, at 8 weeks, inflammatory reaction of both tenocyte-seeded bioscaffolds were dramatically less than with bioscaffold alone. In addition, bioscaffolds seeded with tenocytes generated a histological appearance similar to that of the positive control. The implantation of autologous tenocytes on collagen-based bioscaffolds results in better rotator cuff tendon healing and remodeling than with the implantation of bioscaffold alone.