Positive Staining For Collagen Type Research Articles

CARGEL Bioscaffold improves cartilage repair tissue after bone marrow stimulation in a minipig model

PurposeTo gain knowledge of the repair tissue in critically sized cartilage defects using bone marrow stimulation combined with CARGEL Bioscaffold (CB) compared with bone marrow stimulation (BMS) alone in a validated animal model.MethodsSix adult Göttingen minipigs received two chondral defects in each knee. The knees were randomized to either BMS combined with CB or BMS alone. The animals were euthanized after 6 months. Follow-up consisted of histomorphometry, immunohistochemistry, semiquantitative scoring of the repair tissue (ICRS II), and μCT of the trabecular bone beneath the defect.ResultsThere was significantly more fibrocartilage (80% vs 64%, p = 0.04) and a trend towards less fibrous tissue (15% vs 30%, p = 0.05) in the defects treated with CB. Hyaline cartilage was only seen in one defect treated with CB and none treated with BMS alone.For histological semiquantitative score (ICRS II), defects treated with CB scored lower on subchondral bone (69 vs. 44, p = 0.04). No significant differences were seen on the other parameters of the ICRS II. Immunohistochemistry revealed a trend towards more positive staining for collagen type II in the CB group (p = 0.08). μCT demonstrated thicker trabeculae (p = 0.029) and a higher bone material density (p = 0.028) in defects treated with CB.ConclusionTreatment of cartilage injuries with CARGEL Bioscaffold seems to lead to an improved repair tissue and a more pronounced subchondral bone response compared with bone marrow stimulation alone. However, the CARGEL Bioscaffold treatment did not lead to formation of hyaline cartilage.

Effect of BMP-2 with Microfracture on Osteochondral Defect in a Rabbits Talus

Category:Ankle, Basic Sciences/BiologicsIntroduction/Purpose:Microfracture is a technically safe, simple and cost-effective treatment option for osteochondral defect of the talus. However, fibrous tissue and fibrocartilage represent the predominant repairing tissues derived from these procedures and these tissues types do not exhibit the mechanical properties of native hyaline cartilage, long time for cartilage reconstruction and long-term failure results. Bone morphogenetic protein-2 (BMP-2) has been shown to stimulate matrix synthesis and increase in synthesis of cartilage-specific collagen type IIB thereby enhance regeneration of articular cartilage in vitro. The purpose of this study is to evaluate the treatment ability of combining BMP-2 with microfracture on osteochondral defect in rabbit talus.Methods:Full-thickness chondral defects (3 x 3 x 2 mm) was created in the center talus dome. Twenty four male white New Zealand rabbits were divided into four groups of 6 animals into each group dependent on treatment method; Group I (control, no treatment), II (microfracture only), III (10 µL rhBMP-2 treatment), IV (microfracture combine with 10 µL rhBMP-2 treatment). The animals were sacrificed at 2, 4, 6 weeks post operatively. The macroscopic assessment of the repaired tissue was evaluated using the International Cartilage Repair Society (ICRS) Macroscopic Score, which considers the degree of defect repair, the integration to the border zone, and the macroscopic appearance. Subchondral bone regeneration on defect was evaluated by micro-CT scan and the histological findings were scored by using a modified version of the established scoring system for osteochondral repair described by Wakitani, immunohistochemical staining was used for analysis of collagen type I and II.Results:: Excessive bone formation that originated from the subchondral bone region was not observed in any sample. Micro-CT shows higher subchondral bone regeneration on defect in group III, IV than group I, II at 4 and 6 weeks, respectively. According to macroscopic and histological results, group V shows higher quality of cartilage and faster cartilage regeneration compare with another group. In immunohistochemical analysis, group IV shows stronger positive staining for collagen type II in the area of the defect.Conclusion:These findings indicate that the combination BMP-2 with microfracture is effective at accelerating and improving quality of full-thickness cartilage defect repair in a rabbit talus model.

Autologous bioscaffolds based on different concentrations of platelet rich plasma and synovial fluid as a vehicle for mesenchymal stem cells.

In the field of tissue engineering, diverse types of bioscaffolds are being developed currently for osteochondral defect applications. In this work, a novel scaffold based on platelet rich plasma (PRP) and hyaluronic acid with mesenchymal stem cells (MSCs) has been evaluated to observe its effect on immobilized cells. The bioscaffolds were prepared by mixing different volumes of synovial fluid (SF) with PRP from patients obtaining three formulations at PRP-SF ratios of 3:1, 1:1 and 1:3 (v/v). The live/dead staining revealed that although the cell number of each type of bioscaffold was different, these this constructs provide cells with a suitable environment for their viability and proliferation. Moreover, immobilized MSCs showed their ability to secrete fibrinolytic enzymes, which vary depending on the fibrin amount of the scaffold. Immunohistochemical analysis revealed the positive staining for collagen type II in all cases, proving the biologic action of SF derived MSCs together with the suitable characteristics of the bioscaffold for chondrogenic differentiation. Considering all these aspects, this study demonstrates that these cells-based constructs represent an attractive method for cell immobilization, achieving completely autologous and biocompatible scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 377-385, 2018.

Rescued Chondrogenesis of Mesenchymal Stem Cells under Interleukin 1 Challenge by Foamyviral Interleukin 1 Receptor Antagonist Gene Transfer.

Background: Mesenchymal stem cells (MSCs) and their chondrogenic differentiation have been extensively investigated in vitro as MSCs provide an attractive source besides chondrocytes for cartilage repair therapies. Here we established prototype foamyviral vectors (FVV) that are derived from apathogenic parent viruses and are characterized by a broad host range and a favorable integration pattern into the cellular genome. As the inflammatory cytokine interleukin 1 beta (IL1β) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1β.Materials and Methods: We generated different recombinant FVVs encoding enhanced green fluorescent protein (EGFP) or IL1RA and examined their transduction efficiencies and transgene expression profiles using different cell lines and human primary MSCs derived from bone marrow-aspirates. Transgene expression was evaluated by fluorescence microscopy (EGFP), flow cytometry (EGFP), and ELISA (IL1RA). For evaluation of the functionality of the IL1RA transgene to block the inhibitory effects of IL1β on chondrogenesis of primary MSCs and an immortalized MSC cell line (TERT4 cells), the cells were maintained following transduction as aggregate cultures in standard chondrogenic media in the presence or absence of IL1β. After 3 weeks of culture, pellets were harvested and analyzed by histology and immunohistochemistry for chondrogenic phenotypes.Results: The different FVV efficiently transduced cell lines as well as primary MSCs, thereby reaching high transgene expression levels in 6-well plates with levels of around 100 ng/ml IL1RA. MSC aggregate cultures which were maintained in chondrogenic media without IL1β supplementation revealed a chondrogenic phenotype by means of strong positive staining for collagen type II and matrix proteoglycan (Alcian blue). Addition of IL1β was inhibitory to chondrogenesis in untreated control pellets. In contrast, foamyviral mediated IL1RA expression rescued the chondrogenesis in pellets cultured in the presence of IL1β. Transduced MSC pellets reached thereby very high IL1RA transgene expression levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21, while IL1RA concentrations of controls were permanently below 200 pg/ml.Conclusion: Our results indicate that FVV are capable of efficient gene transfer to MSCs, while reaching IL1RA transgene expression levels, that were able to efficiently block the impacts of IL1β in vitro. FVV merit further investigation as a means to study the potential as a gene transfer tool for MSC based therapies for cartilage repair.

Effects of Mesenchymal Stem Cell and Growth Factor Delivery on Cartilage Repair in a Mini-Pig Model.

ObjectiveWe have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor–β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone.DesignTrochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen.ResultsTreatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P > 0.05). Neither condition had an impact on other histological semiquantitative scores (P > 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together (P > 0.05).ConclusionsAt this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

The formation of human auricular cartilage from microtic tissue: An in vivo study

ObjectivesThis study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold. Study designCell culture experiment with the use of microtic chondrocytes. DesignCell culture experiment with the use of microtic chondrocytes. MethodsAfter ear reconstructive surgery at the Universiti Kebangsaan Malaysia Medical Center, chondrocytes were isolated from microtic cartilage. Chondrocytes isolated from the tissue were cultured expanded until passage 4 (P4). Upon confluency at P4, chondrocytes were harvested and tissue engineered constructs were made with human plasma polymerized to fibrin. Constructs formed later is implanted at the dorsal part of nude mice for 8 weeks, followed by post-implantation evaluation with histology staining (Hematoxylin and Eosin (H&E) and Safranin O), immunohistochemistry and RT-PCR for chondrogenic associated genes expression level. ResultsUnder gross assessment, the construct after 8 weeks of implantation showed similar physical characteristics that of cartilage. Histological staining showed abundant lacunae cells embedded in extracellular matrix similar to that of native cartilage. Safranin O staining showed positive staining which indicates the presence of proteoglycan-rich matrix. Immunohistochemistry analysis showed the strong positive staining for collagen type II, the specific collagen type in the cartilage. Gene expression quantification showed no significant differences in the expression of chondrogenic gene used which is collagen type I, collagen type II, aggrecan core protein (ACP), elastin and sox9 genes when compared to construct formed from normal auricular tissue. ConclusionChondrocytes isolated from microtia cartilage has the potential to be used as an alternative cell source for external ear reconstruction in future clinical application.

Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a ‘cap’ on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

A chondromimetic microsphere for in situ spatially controlled chondrogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have been identified as a viable cell source for cartilage tissue engineering. However, to undergo chondrogenic differentiation hMSCs require growth factors, in particular members of the transforming growth factor beta (TGF-β) family. While in vitro differentiation is feasible through continuous supplementation of TGF-β3, mechanisms to control and drive hMSCs down the chondrogenic lineage in their native microenvironment remain a significant challenge. The release of TGF-β3 from an injectable microsphere composed of the cartilage-associated extracellular matrix molecule hyaluronan represents a readily translatable approach for in situ differentiation of hMSCs for cartilage repair. In this study, chondromimetic hyaluronan microspheres were used as a growth factor delivery source for hMSC chondrogenesis. Cellular compatibility of the microspheres (1.2 and 14.1μm) with hMSCs was shown and release of TGF-β3 from the most promising 14.1μm microspheres to control differentiation of hMSCs was evaluated. Enhanced accumulation of cartilage-associated glycosaminoglycans by hMSCs incubated with TGF-β3-loaded microspheres was seen and positive staining for collagen type II and proteoglycan confirmed successful in vitro chondrogenesis. Gene expression analysis showed significantly increased expression of the chondrocyte-associated genes, collagen type II and aggrecan. This delivery platform resulted in significantly less collagen type X expression, suggesting the generation of a more stable cartilage phenotype. When evaluated in an ex vivo osteoarthritic cartilage model, implanted hMSCs with TGF-β3-loaded HA microspheres were detected within cartilage fibrillations and increased proteoglycan staining was seen in the tissue. In summary, data presented here demonstrate that TGF-β3-bound hyaluronan microspheres provide a suitable delivery system for induction of hMSC chondrogenesis and their use may represent a clinically feasible tissue engineering approach for the treatment of articular cartilage defects.

The naïve effector cells of collagen type I during acute experimental pancreatitis are acinar cells and not pancreatic stellate cells

ObjectiveThe purpose of this study was to investigate the expression of collagen type I and the mRNA level of its regulatory factor, TGF-β1, in tissue samples of acute pancreatitis and to determine the significance of collagen type I in predisposition to pancreatic fibrosis during acute pancreatitis. MethodsSprague–Dawley rats were divided into an experimental group (30 rats) and a control group (12 rats). The rats in the experimental group were intraperitoneally injected with cerulein to induce acute pancreatitis. The distribution and expression of collagen type I in the pancreatic tissues were examined by immunohistochemical staining. The mRNA level of TGF-β1 was determined by real-time polymerase chain reaction (PCR). Results(1) Collagen type I was localized in the cytoplasm of pancreatic acinar cells. With pancreatitis progressed, strong positive staining for collagen type I covered whole pancreatic lobules, whereas, the islet tissue, interlobular area, and pancreatic necrotic area were negative for collagen type I. (2) The level of TGF-β1 mRNA in rats from the experimental group increased gradually the establishment of acute pancreatitis, and was significantly higher than that in the control group at every time point. Conclusions(1) During acute pancreatitis, pancreatic acinar cells, not pancreatic stellate cells as traditionally believed, were the naïve effector cells of collagen type I. (2) TGF-β1 played a key role in regulating collagen I expression during acute pancreatitis.

Histological Analysis of Tissue Ingrowth and Organization Following Implantation of a Novel Synthetic Acellular Meniscal Scaffold for the Treatment of Irreparable Partial Meniscus Tears and/or Partial (SS-27A)

Introduction Histological analysis of biopsy tissue to assess tissue ingrowth and organization as it populates a novel, biodegradable, polyurethane meniscal scaffold designed for the treatment of painful irreparable partial meniscus tears and/or partial meniscal tissue loss. Methods This was a prospective, single-arm, multicentre clinical investigation in patients with an irreparable partial medial or lateral meniscal tear or partial meniscus loss. The scaffold is designed to support the body's own physiological pathways of tissue repair by providing a temporary three-dimensional matrix for cellular attachment and vascular ingrowth. A biopsy was obtained at 12 months (±1 month) after implantation of the Actifit scaffold in the 44 patients who had agreed to a relook arthroscopy with a biopsy. All biopsies were taken with an arthroscopic punch from the center of the inner free edge of the implanted scaffold meniscus according to a standardized protocol and fixed in 4%-buffered formol for at least 24 hours. Histochemical staining of the biopsy and cellular markers were used to enable visualization of specific cellular and extracellular structures. Results A total of, 52 patients (34 medial: 18 lateral) were treated with the polyurethane meniscal scaffold. The population recruited into the study were young (mean age, 30.8±9.4 years), and the majority (88%) had undergone 1 or 2 prior surgeries on the involved meniscus. The mean longitudinal length of the meniscal defect was 47.1±10.0 mm. All 44 biopsies showed fully vital material, with no signs of necrosis or cell death, illustrating biocompatibility and successful tissue ingrowth into the scaffold meniscus within the knee joint. Notably, in addition to a fibrous capsule, three distinctly different layers were observed: Layer 1: a superficial fibrotic vascularized, tissue layer, mainly consisting of fibroblasts and some fusiform fibrochondroblast-like cells. Layer 2: an avascular "transition" layer situated between the superficial "layer 1" and the deep "layer 3." Layer 2 consists of a mixture of cell types, of which fusiform cells with a morphology resembling fibrochondroblasts are most prominent. In some biopsies cells with a morphology resembling fibrochondroblasts of the polygonal subtype are prominent. The extracellular matrix is loosely organized with positive staining for collagen type I. Layer 3: a deep, avascular hypocellular layer consisting of cells with a morphology resembling fibrochondroblasts of the polygonal subtype with a matrix rich in fibrin. Conclusion No signs of tissue necrosis or cell death were observed and all biopsies showed complete re-population by cells and tissue that can be regarded as vital structures, illustrating the biocompatibility of the polyurethane scaffold. Importantly, the layered organization of biopsied scaffold meniscus, each with its own histological characteristics, is illustrative of an ongoing process of regeneration, maturation, and remodeling. Moreover, the above-described structures and their organization are similar to that of the native human meniscus. (Actifit Study Group: R. Verdonk, P. Beaufils, J. Bellemans, P. Colombet, R. Cugat, P. Djian, H. Laprell, P. Neyret, H. Paessler.)

Tribological Maturation of Regenerated Cartilage was Inhibited by Using Chondrocyte Aggregates

Cartilage tissue was regenerated using fibroin sponge seeded with chondrocyte aggregates. The aggregates were formed on the sponge by micro-folding culture, where chondrocytes were cultured on a substrate containing an array of pits of 100 μm diameter. The aggregate-derived cartilage tissue showed higher safranin O staining intensity and was positive for collagen type II by immunostaining. However, its friction coefficient was higher than that of the cartilage tissue regenerated using individual cells. Moreover, the surface of this aggregate-derived cartilage tissue showed weak positive staining for collagen type I. Thus, regeneration of the aggregate-derived cartilage tissue has the possibility of forming larger amounts of extracellular matrix from a limited number of chondrocytes. However, such tissue showed relatively poor tribological function possibly because of its immature functional layer structure.

Expression of collagen type I, II and III in loose body of osteoarthritis

The expression of collagen type I, II, and III was investigated to evaluate phenotypic change in chondrocytes in loose bodies related to osteoarthritis. We assessed collagen type I, II, and III production in loose bodies from knee joints of ten osteoarthritic patients, using an immunohistochemical method with monoclonal antibodies. Collagen type III expression was identified in all ten loose bodies and was mainly located in cartilage, including chondrocytes and matrices, as well as in a layer of fibroid tissue on the surface. No positive signal for collagen type III was observed in necrotic osteocytes. There was weakly positive staining for collagen type I in chondrocytes. No positive staining for collagen type II could be seen in the cartilage of loose bodies. Cartilage from the non-osteoarthritic knee joints of four people was negative for the expression of collagen type I and III, and positive for the expression of collagen type II. Collagen type I and III expression suggested the dedifferentiation status of chondrocytes in loose bodies.

Immunohistochemical localization of collagen types I and VI in human skin wounds.

A total of 74 human skin wounds were investigated and collagen types I and VI were localized in the wound area by immunohistochemistry. Collagen type I appeared in the form of ramifying string-like structures after approximately 5-6 days, but positive reactions in the form of a spot-like staining around isolated fibroblasts also occurred in a skin wound aged 4 days. Collagen VI was detectable after a post-infliction interval of at least 3 days showing a strongly positive reacting network associated with fibroblasts in the wound area. Both collagens appeared almost constantly after a wound age of 6-7 days and could also be found in wounds aged a few months. Therefore, although a positive reaction for collagen type I in the form of string-like and ramifying structures around wound fibroblasts indicates a wound age of at least 5-6 days, a spot-like positive staining for collagen type I cannot exclude a wound age of at least 4 days. A positive staining for collagen type VI represents a post-infliction time of 3 days or more. The almost constant appearance of these collagen types suggests that negative results in a sufficient number of specimens indicate a wound age of less than 6-7 days, but cannot completely exclude longer post-infliction intervals. Since collagen type I and VI are also found in the granulation/scar tissue of lesions with advanced wound age, the immunohistochemical analysis of these proteins provides no further information for an age determination of older skin wounds.

Distribution of collagen type IV in soft tissue tumors: An immunohistochemical study

The distribution of collagen type IV, one of the major constituents of basement membrane, was studied immunohistologically in a series of 103 soft tissue tumors including those of peripheral nerve origin, smooth muscle origin, striated muscle origin, fibrous tissue origin, fibrohistiocytic origin, adipose tissue origin, synovial tissue origin, and blood vessel origin, paragangliomas, alveolar soft part sarcomas, granular cell tumors, and epithelioid sarcomas. Intensely positive staining for collagen type IV was observed in neurilemomas, neurofibromas, malignant schwannomas, and blood vessel tumors. Weakly to moderately positive staining was seen in leiomyomas, angiomyomas, and leiomyosarcomas. In contrast, synovial, fibroblastic and fibrohistiocytic tumors, benign or malignant, were negative. In paragangliomas, granular cell tumors, and alveolar soft part sarcomas, positive staining was evident surrounding nests or clusters of tumor cells. In all tumors, staining for collagen type IV clearly illustrated the vascular pattern.