Immunohistochemical Analysis Of Collagen Type Research Articles

Comparative assessment of the morphology and antigenicity of human osteochondral units using formalin and coagulant fixatives

Fixation is crucial for preserving tissue integrity during processing, and the most commonly used cross-linking fixative in immunohistochemistry is neutral buffered formalin, which requires antigen retrieval as a crucial step. The successful use of newer coagulant fixatives like methacarn and EMA to preserve isotopes and eliminate the need for antigen retrieval has been reported recently, but their role in decalcified osteochondral unit samples remains unknown. Limited information on the use of coagulant fixatives as formalin substitutes makes it important to comparatively evaluate their effects on osteochondral samples and the impact of antigen retrieval on different days. Osteochondral units from a patient with osteoarthritis who underwent total knee-replacement surgery were fixed with three studied fixatives (Formalin, Methacarn, EMA) and divided into four portions, for different time periods (Day 1, 3, 7, 10). Sections were decalcified, stained with Safranin O, Alcian Blue, Toluidine Blue, PicroSirius Red, Hematoxylin and Eosin, and immunohistochemical analysis of Collagen type II and type X with and without antigen retrieval was conducted. Formalin showed better hematoxylin uptake than coagulant-based fixatives, while all fixatives preserved tissue morphology without necrosis or cellular degeneration with comparable staining quality. Methacarn and EMA-fixed tissues showed higher uptake of collagen type II compared to formalin-fixed tissues, with collagen type II uptake occurring only in the cartilage region and collagen type X uptake occurring only in the bone region. The study highlights the effectiveness of methacarn and EMA as efficient alternatives to formalin, preserving tissue morphology and antigen specificity.

An improved method for processing chondroprogenitor pellets following chondrogenic differentiation for histology and immunohistochemical staining using agarose

PurposeIn-vitro models of cartilage regeneration based on pellet cultures have been widely used to evaluate chondrogenic potential of the cell of interest and predict probable in-vivo behavior. However, pellet processing is a major challenge during handling (due to small size and possible damage to structural contour following sectioning and staining). The present study aimed to utilize human articular cartilage derived chondroprogenitors to assess if agarose-encapsulation of pellets prior to paraffin processing enable easier handling without affecting tissue morphology, glycosaminoglycan staining and immunohistochemical analysis of Collagen type II protein. MethodsPassage 3 chondroprogenitors (n = 3) were evaluated for MSC markers using flow cytometry and subjected to chondrogenic differentiation as pellets cultures. Post-differentiation, the pellets were subjected to either: a) paraffin embedding, b) agarose encapsulation followed by paraffin embedding or c) agarose encapsulation followed by cryosectioning. All sections were subjected to histological staining for glycosaminoglycan uptake: Alcian blue, Safranin O (Bern score) and Toluidine blue with immunohistochemical processing for collagen type II protein deposition. ResultsWith respect to staining and structural integrity, comparable uptake was seen in both paraffin sections and agarose embedded sections while the latter exhibited notably uniform pellets with distinct marginal demarcation. Although plain paraffin and agarose encapsulated sections demonstrated equivalent staining as represented by comparable Bern scores, glycosaminoglycan uptake, and Collagen type II deposition, cryosections exhibited significantly poor staining properties. ConclusionAgarose encapsulation of differentiated pellets prior to routine paraffin embedding, eases handling difficulties whilst maintaining structural integrity with optimal staining outcomes.

A comparative study of the epiligament of the medial collateral and the anterior cruciate ligament in the human knee. Immunohistochemical analysis of collagen type I and V and procollagen type III

BackgroundRecent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament. Materials and methodsWe used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory’s trichrome stain and Van Gieson’s stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software. ResultsOur observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament. ConclusionsThe results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing.

Effect of silicon-doped calcium phosphate bone grafting materials on bone regeneration and osteogenic marker expression after implantation in the ovine scapula.

Compared to the currently clinically available bone grafting materials for alveolar ridge augmentation, there is a great demand for bioactive bone substitutes with higher resorbability, which enhance osteogenesis at the same time. This has prompted the development of a silicon-doped rapidly resorbable calcium alkali orthophosphate (Si-CAOP) and silicon-doped β-tricalcium phosphate (Si-TCP). This study evaluated the effect of these two particulate graft materials as compared to the currently clinically used β-TCP on bone formation and osteogenic marker expression after 2 weeks, 1, 3, 6, 12, and 18 months of implantation in critical size defects in the sheep scapula. Immunohistochemical analysis of collagen type I, alkaline phosphatase, and osteocalcin expression was performed on resin embedded sections. The bone and particle area fraction and the bone-biomaterial contact were determined histomorphometrically. After 2 weeks and 1 month defects grafted with Si-CAOP displayed a significantly greater bone area fraction, bone-particle-contact, osteogenic marker expression and significantly lower particle area fraction than defects grafted with Si-TCP and TCP. By 3 and 6 months all materials studied mediated excellent defect regeneration with further bone remodeling at 12 and 18 months. Taken together, Si-CAOP induced the most expeditious bone regeneration of critical size defects in the sheep scapula. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 594-614, 2019.

Immunohistochemical analysis of collagen types I, III, matrix metalloproteinases-1, -9, and gonadal steroid receptors alterations on the pathophysiology of female pelvic organ prolapse and urinary incontinence

Immunohistochemical analysis of collagen types I, III, matrix metalloproteinases-1, -9, and gonadal steroid receptors alterations on the pathophysiology of female pelvic organ prolapse and urinary incontinence

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats.

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.

Performance of β-tricalcium phosphate granules and putty, bone grafting materials after bilateral sinus floor augmentation in humans

Sinus floor augmentation (SFA) using bone grafting materials, and in particular calcium phosphates (CaP), is a well-established pre-implantology procedure. The use of CaP simplifies SFA procedures. β-tricalcium phosphate (β-TCP) is amply used for SFA. This study evaluated the clinical and osteogenic performance of β-TCP granules (TCP-G) and a β-TCP putty (TCP-P) bone graft material. TCP-P consisted of TCP-G in a hyaluronic acid (HyA) carrier. Bone formation, volume stability and osteogenic marker expression after bilateral SFA in patients was assessed. Eight patients were selected for a split-mouth design. Biopsies obtained six months after SFA, were processed for immunohistochemical analysis of collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Histomorphometric analysis determined bone, grafting material and marrow space percentages. Cone-beam computed tomography was used to calculate the graft volume and its stability. Both materials allowed excellent bone regeneration and volume stability. TCP-P displayed better surgical handling properties, greater bone formation, higher expression of Col I, ALP, OC and BSP; as well as significantly lower grafting volume reduction values. HyA had no adverse effect on TCP-P performance. Due to its clinical and osteogenic performance, TCP-P can be regarded as excellent bone grafting material for SFA.

Effects of riboflavin/UVA corneal cross‐linking on keratocytes and collagen fibres in human cornea

To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in human corneas. Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross-linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter. Normal corneas exhibited no TUNEL-positive keratocytes and keratoconic and cross-linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP-ribose) polymerase assessed using Western blot. The Ki-67 staining showed a significant increase in the keratocyte proliferation in cross-linked corneas compared with normal and keratoconus. In cross-linked corneas CD34-positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross-linked corneas compared with control and keratoconus. Corneal cross-linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34-positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.

Surgical Method to Create Vocal Fold Injuries in Mice

The goal of this study was to develop a surgical method for the creation of vocal fold injuries in mice, as a precursor to the use of genetically engineered mouse models in the study of vocal fold wound healing and scar formation. Seven FVB strain mice were used in this study. A laryngoscope and 3 micro-instruments were designed and fabricated to facilitate endoscopic vocal fold visualization and the creation of vocal fold surgical injuries. The larynges were harvested 1 and 7 days after surgery, and the vocal fold injury sites were evaluated by routine hematoxylin and eosin staining. Additional immunohistochemical analysis of collagen type I and elastin distribution in the lamina propria was performed for an uninjured control larynx. Endoscopic visualization and vocal fold stripping resulting in thyroarytenoid muscle exposure were successful in all animals. Histologic and immunohistochemical analyses revealed a simple lamina propria structure with relatively even collagen type I and elastin distribution in the control vocal fold, obliteration of vocal fold mucosa 1 day after surgery, and complete reepithelialization by 7 days. These results demonstrate the feasibility of creating reproducible vocal fold injuries via an endoscopic approach in mice. The observation that the mouse lamina propria may have a relatively simple histologic structure indicates that additional characterization should be performed and caution used in translating findings between this and other model systems.

Immunohistochemical analysis of collagen types I, III, IV and α-actin in the urethra of sexually intact and ovariectomized beagles

Urinary incontinence is a widespread problem in both postmenopausal women and ovariectomized dogs. The objective of this study was to investigate the influence of ovariectomy on the immunoreactivity and the distribution pattern of collagens I, III, IV and alpha-actin in the canine urethra. The immunohistochemical results were evaluated in five sexually intact and five ovariectomized beagles. The immunostaining of both collagens I and III delineated urethral connective tissue fibres and co-localized within in the fibres of both groups. The basement membranes of smooth muscle cells and sinusoids showed marked type IV collagen expression, whereas only faint immunoreactivity was present at the urothelial-stromal interface. No differences could be detected in the expression or distribution of the assessed collagen types and actin between ovariectomized and control animals. In conclusion, ovariectomy does not appear to have an effect on urethral collagens I, III, IV and smooth muscle actin in the dog, as ascertained by immunohistochemistry.

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.