An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

Morphogenesis in wing imaginal discs: its relationship to changes in the extracellular matrix

An extracellular matrix (ECM) lies between the upper and lower epithelial layers of the wing imaginal discs of moths. Organization and composition of this extracellular matrix, as revealed by staining with ruthenium red, tannic acid, and alcian blue, changes in concert with levels of hormones in the haemolymph. The ECM of the wing imaginal disc is an environment for cellular movements. Reorganization of the matrix and increase in ecdysteroid level is coupled with the proximal----distal migration of tracheal cells as well as the distal----proximal outgrowth of sensory neurons.     

Expression of lumican related to CD34 and VEGF in the articular disc of the human temporomandibular joint

Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP) gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ) disc is made up of fibrocartilage with an extracellular matrix (ECM) composed of collagen and proteoglycans. The existence and behaviour of lumican have not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF) and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy). In both normal and deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblast-like cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells.Key words: TMJ disc, lumican, CD34, VEGF, immunohistochemistry, metachromasia.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

Interaction of rabbit sperm and egg

Summary The localization of proteoglycans in the predentin of the rat incisor was investigated by ultrastructural histochemistry. Ruthenium red stained the cell coat of the odontoblasts as well as intracellular vesicles. There was also a staining of the extracellular matrix, but not of collagen fibers in the predentin. Treatment with the enzyme hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles and the extracellular matrix but not that of the cell coat. Bismuth nitrate and phosphotungstic acid gave similar staining of odontoblast vesicles and extracellular matrix. It is likely that the stained structures contain proteoglycans. The importance of these proteoglycans and their ultrastructural localization are discussed in relation to intracellular transport and the calcification process.     

Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss

Alterations in the composition of intervertebral disc extracellular matrix, mainly collagen and proteoglycans, may cause changes in mechanical properties of the disc, leading to dysfunction, nerve root compression, and herniation with severe clinical manifestations. Matrix metalloproteinases may be involved in degradation by hydrolysing extracellular matrix components. Inhibitors of matrix metalloproteinases, in contrast, function in the maintenance of degradation control. In this study, we investigated: (i) whether the level of matrix degradation correlated with the duration of the symptomatic disease, (ii) roles of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) in intervertebral disc degeneration. Nucleus pulposus of intervertebral discs were obtained from 22 patients and analysed for collagen and proteoglycan contents, and pro-MMP-2, TIMP-2 levels. Collagen content was determined as hydroxyproline and proteoglycan content was measured as glycosaminoglycans. The loss in matrix components did not correlate with the duration of the degenerative disc disease. Pro-MMP-2 levels were higher at early stages of the degenerative disc disease (r = -0.495, P < 0.05). TIMP-2 levels were similar in all samples. Pro-MMP-2 and TIMP-2 levels negatively correlated in herniated discs samples (r = -0.855, P < 0.01). Pro- MMP-2 levels negatively correlated with the collagen content in herniated disc material. Our findings may suggest a silent period of active disease prior to symptomatic outcome during which irreversible matrix loss occurs. Involvement of other proteolytic enzymes at different stages of the disease should also be investigated to help to control the degradation cascade at relatively early stages of disc degeneration before the clinical onset of disease.     

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.     

Comparison of verdeluz orange G and modified Gallego stains

Tumors of the oral cavity include combinations of hard and soft tissues that may be difficult to identify using routine hematoxylin and eosin (H & E) staining. Although combination stains can demonstrate hard and soft tissues, trichrome stains, such as VanGieson and Masson, cannot differentiate dental hard tissues, such as dentin, cementum and osteoid. Modified Gallegos (MGS) and verdeluz orange G-acid fuchsin (VOF) stains can differentiate components of teeth. We used 10 tissue sections of decalcified bone and 10 pathologic tissue sections that contained different calcified tissues including peripheral ossifying fibroma, odontoma, central ossifying fibroma and cemento-ossifying fibroma. Sections were stained with H & E, VOF or MGS. H and E stained both hard tissues pink. VOF stained bone purple-red, cementum red and collagen blue. MGS stained bone green-blue, cementum red and collagen blue. VOF staining intensity and differentiation was better than MGS staining. VOF staining demonstrated hard tissue components distinctly and exhibited good contrast with the surrounding connective tissue. VOF also is a simple, single step, rapid staining procedure.     

Cartilage production by rabbit articular chondrocytes on polyglycolic acid scaffolds in a closed bioreactor system

Rabbit articular chondrocytes were seeded onto three-dimensional polyglycolic acid (PGA) scaffolds and placed into a closed bioreactor system. After 4 weeks of growth, meshes were examined for cartilage formation. Gross examination revealed solid, glistening material that had the appearance of cartilaginous tissue. Histologic examination revealed cell growth and deposition of extracellular matrix throughout the mesh with a less dense central core. Alcian blue and Safranin 0 staining showed deposition of glycosaminoglycans (GAGs). Immunostaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen. Biochemical analysis showed collagen and GAG values to be 15% and 25% dry weight, respectively. Our results indicate that this type of system compares well with those previously described and should be useful for producing cartilage for evaluation in a clinical setting. (c) 1995 John Wiley & Sons, Inc.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

The use of Histochoice™® for histological examination of articular and growth plate cartilages,intervertebral disc and meniscus

Histochoice?® is a proprietary nontoxic, non-cross-linking fixative designed by the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sections of immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice?® fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.     

Metabolic effects of vitamin D active metabolites in monolayer and micromass cultures of nucleus pulposus and annulus fibrosus cells isolated from human intervertebral disc

Intragenic polymorphisms in the vitamin D receptor gene are linked to disc degeneration features, suggesting that alterations in the vitamer-mediated signalling could be involved in the pathophysiology of the disc and that interaction of disc cells with vitamin D metabolites may be critical for disc health. The vitamer-mediated modulation of disc cells proliferation, metabolic activity, extracellular matrix (ECM) genes expression and proteins production was investigated. It was stated that disc cells express vitamin D receptor and are very sensitive to metabolic stimuli. In monolayer cultures, 1,25(OH)(2)D(3), but not 24,25(OH)(2)D(3), determined an inhibition of the proliferation and regulated also the ECM genes expression in nucleus pulposus and annulus fibrosus cells. Micromass cultures induced a more physiologic expression pattern of extracellular matrix genes. Cells Treatment with vitamin D metabolites did not result in relevant modifications of glycosaminoglycans production, except for annulus cells, whose production was reduced after 1,25(OH)(2)D(3) treatment. Moreover, a reduced glycosaminoglycans staining in both cell types and a significant reduced aggrecan gene expression in annulus cells treated with 1,25(OH)(2)D(3) were observed. A reduction of collagen I and II staining in annulus cells 1,25(OH)(2)D(3) treated, in accordance with a downregulation of collagen genes expression, was also registered. Finally, the vitamin D receptor gene expression did not show significant metabolite-mediated modification in monolayer or micromass cultures. These findings could enhance new insights on the biochemical mechanisms regulated by vitamin D in disc cartilage and possibly involved in the development of physiological/pathological modifications of the disc.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Alterations in the dynamics of inflammation, proliferation and apoptosis in subcutaneous implants of lupus-prone mice

Wound repair is a complex process that involves inflammation, proliferation, extracellular matrix deposition/remodeling and apoptosis. Autoimmune diseases profoundly affect the healing process. We have used histological parameters to characterize the recruitment of mast cells and the proliferative activity and apoptosis in the fibrovascular tissue induced by subcutaneous polyether-polyurethane sponge implants in lupus-prone New Zealand White (NZW) and in control Balb/c mouse strains at days 10 and 21 post implantation. Fibrovascular tissue infiltration (hematoxylin and eosin staining), mast cell number (Dominici staining) and cellular proliferation (AgNOR staining) peaked early (day 10) but collagen deposition (picrosirius red staining) and apoptosis remained high in implants of NZW mice during the experimental period. In contrast, implants of Balb/c animals showed a progressive increase in mast cell recruitment and cellular proliferation but apoptosis fell from day 10 to 21 post-implantation. This divergent response early mast cells recruitment, excessive collagen deposition and disturbed removal of apoptotic cells from the site of injury in NZW mice implies that the genotype trait of NZW mice is a determining factor in abnormal healing response.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration

 Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0–86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-spec ific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (<2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (>70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration. Accepted: 24 April 1997