Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.

A collagen prolyl 4-hydroxylase inhibitor reduces adhesions after tendon injury

Collagen synthesis inhibition potentially can reduce adhesion formation after tendon injury but also may affect cutaneous wound healing. We hypothesized that a novel orally administered collagen synthesis inhibitor (CPHI-I) would substantially reduce flexor tendon adhesions after injury, without any clinically important effect on cutaneous wound healing. The experiments were performed in a rat model with an in-continuity crush injury model in the rat hindfoot flexor tendon to provoke adhesion formation. Assays of dermal collagen production and the rate of healing of an excised wound were performed to assess cutaneous wound healing. Animals in the treatment groups received CPHI-I for 1, 2, or 6 weeks and were assessed at either 2 or 6 weeks. The work of flexion in the injured digit was reduced in the CPHI-I-treated animals compared with control animals, (0.188 J versus 0.0307 J at 2 weeks, and 0.0231 J versus 0.0331 J at 6 weeks) The cutaneous wound healing rate was similar in all animals, but dermal collagen synthesis was reduced in the treated animals. The CPHI-I seems to reduce tendon adhesion, and although collagen synthesis was reduced in cutaneous wounds, CPHI-I did not retard wound healing.

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

The effect of amphiphilic siloxane oligomers on fibroblast and keratinocyte proliferation and apoptosis

The formation of hypertrophic scars is a frequent medical outcome of wound repair and often requires further therapy with treatments such as Silicone Gel Sheets (SGS) or apoptosis-inducing agents, including bleomycin. Although widely used, knowledge regarding SGS and their mode of action is limited. Preliminary research has shown that small amounts of amphiphilic silicone present in SGS have the ability to move into skin during treatment. We demonstrate herein that a commercially available analogue of these amphiphilic siloxane species, the rake copolymer GP226, decreases collagen synthesis upon exposure to cultures of fibroblasts derived from hypertrophic scars (HSF). By size exclusion chromatography, GP226 was found to be a mixture of siloxane species, containing five fractions of different molecular weight. By studies of collagen production, cell viability and proliferation, it was revealed that a low molecular weight fraction (fraction IV) was the most active, reducing the number of viable cells present following treatment and thereby reducing collagen production as a result. Upon exposure of fraction IV to human keratinocytes, viability and proliferation was also significantly affected. HSF undergoing apoptosis after application of fraction IV were also detected via real-time microscopy and by using the TUNEL assay. Taken together, these data suggests that these amphiphilic siloxanes could be potential non-invasive substitutes to apoptotic-inducing chemical agents that are currently used as scar treatments.

Expansion and chondrogenic potential of human articular chondrocytes on macroporous microcarriers

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro. High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed. Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.

Pre-clinical evaluation of amphiphilic silicone oligomers for scar remediation

The formation of hypertrophic scars is a frequent outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS; Perkins et al., 1983). Although widely used, knowledge regarding SGS and their mechanism of action on hypertrophic scars is limited. Furthermore, SGS require consistent application for at least twelve hours a day for up to twelve consecutive months, beginning as soon as wound reepithelialisation has occurred. Preliminary research at QUT has shown that some species of silicone present in SGS have the ability to permeate into collagen gel skin mimetics upon exposure. An analogue of these species, GP226, was found to decrease both collagen synthesis and the total amount of collagen present following exposure to cultures of cells derived from hypertrophic scars. This silicone of interest was a crude mixture of silicone species, which resolved into five fractions of different molecular weight. These five fractions were found to have differing effects on collagen synthesis and cell viability following exposure to fibroblasts derived from hypertrophic scars (HSF), keloid scars (KF) and normal skin (nHSF and nKF). The research performed herein continues to further assess the potential of GP226 and its fractions for scar remediation by determining in more detail its effects on HSF, KF, nHSF, nKF and human keratinocytes (HK) in terms of cell viability and proliferation at various time points. Through these studies it was revealed that Fraction IV was the most active fraction as it induced a reduction in cell viability and proliferation most similar to that observed with GP226. Cells undergoing apoptosis were also detected in HSF cultures exposed to GP226 and Fraction IV using the Tunel assay (Roche). These investigations were difficult to pursue further as the fractionation process used for GP226 was labour-intensive and time inefficient. Therefore a number of silicones with similar structure to Fraction IV were synthesised and screened for their effect following application to HSF and nHSF. PDMS7-g-PEG7, a silicone-PEG copolymer of low molecular weight and low hydrophilic-lipophilic balance factor, was found to be the most effective at reducing cell proliferation and inducing apoptosis in cultures of HSF, nHSF and HK. Further studies investigated gene expression through microarray and superarray techniques and demonstrated that many genes are differentially expressed in HSF following treatment with GP226, Fraction IV and PDMS7-g-PEG7. In brief, it was demonstrated that genes for TGFβ1 and TNF are not differentially regulated while genes for AIFM2, IL8, NSMAF, SMAD7, TRAF3 and IGF2R show increased expression (>1.8 fold change) following treatment with PDMS7-g-PEG7. In addition, genes for αSMA, TRAF2, COL1A1 and COL3A1 have decreased expression (>-1.8 fold change) following treatment with GP226, Fraction IV and PDMS7-g-PEG7. The data obtained suggest that many different pathways related to apoptosis and collagen synthesis are affected in HSF following exposure to PDMS7-g-PEG7. The significance is that silicone-PEG copolymers, such as GP226, Fraction IV and PDMS7-g-PEG7, could potentially be a non-invasive substitute to apoptosis-inducing chemical agents that are currently used as scar treatments. It is anticipated that these findings will ultimately contribute to the development of a novel scar therapy with faster action and improved outcomes for patients suffering from hypertrophic scars.

Functional and mechanistic investigation of Shikonin in scarring

Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.

Collagen induced MMP-2 activation in human breast cancer

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen(TM): Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP- 2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

A new low temperature synthesis route of fraipontite (Zn,Al)3(Si,Al)2O5(OH)4

A low temperature synthesis method based on the decomposition of urea at 90°C in water has been developed to synthesise fraipontite. This material is characterised by a basal reflection 001 at 7.44 Å. The trioctahedral nature of the fraipontite is shown by the presence of a 06l band around 1.54 Å, while a minor band around 1.51 Å indicates some cation ordering between Zn and Al resulting in Al-rich areas with a more dioctahedral nature. TEM and IR indicate that no separate kaolinite phase is present. An increase in the Al content however, did result in the formation of some SiO2 in the form of quartz. Minor impurities of carbonate salts were observed during the synthesis caused by to the formation of CO32- during the decomposition of urea.

Synthesis and characterization of K2Ca5(SO4)6· H2O, the equivalent of görgeyite, a rare evaporite mineral

Görgeyite, K2Ca5(SO4)6··H2O, is a very rare monoclinic double salt found in evaporites related to the slightly more common mineral syngenite. At 1 atmosphere with increasing external temperature from 25 to 150 °C, the following succession of minerals was formed: first gypsum and K2O, followed at 100 °C by görgeyite. Changes in concentration at 150 °C due to evaporation resulted in the formation of syngenite and finally arcanite. Under hydrothermal conditions, the succession is syngenite at 50 °C, followed by görgyeite at 100 and 150 °C. Increasing the synthesis time at 100 °C and 1 atmosphere showed that initially gypsum was formed, later being replaced by görgeyite. Finally görgeyite was replaced by syngenite, indicating that görgeyite is a metastable phase under these conditions. Under hydrothermal conditions, syngenite plus a small amount of gypsum was formed, after two days being replaced by görgeyite. No further changes were observed with increasing time. Pure görgeyite showed elongated crystals approximately 500 to 1000 µ m in length. The infrared and Raman spectra are mainly showing the vibrational modes of the sulfate groups and the crystal water (structural water). Water is characterized by OH-stretching modes at 3526 and 3577 cm–1 , OH-bending modes at 1615 and 1647 cm–1 , and an OH-libration mode at 876 cm–1 . The sulfate 1 mode is weak in the infrared but showed strong bands at 1005 and 1013 cm–1 in the Raman spectrum. The 2 mode also showed strong bands in the Raman spectrum at 433, 440, 457, and 480 cm–1 . The 3 mode is characterized by a complex set of bands in both infrared and Raman spectra around 1150 cm–1 , whereas 4 is found at 650 cm–1.