Collagen transport and related pathways in Osteogenesis Imperfecta.

Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.

An immunohistochemical prostate cell identification key indicates that aging shifts procollagen 1A1 production from myofibroblasts to fibroblasts in dogs prone to prostate-related urinary dysfunction.

BACKGROUND: The identity and spatial distribution of prostatic cell types has been determined in humans but not in dogs, even though aging- and prostate-related voiding disorders are common in both species and mechanistic factors, such as prostatic collagen accumulation, appear to be shared between species. In this publication we characterize the regional distribution of prostatic cell types in the young intact dog to enable comparisons with human and mice and we examine how the cellular source of procollagen 1A1 changes with age in intact male dogs. METHODS: A multichotomous decision tree involving sequential immunohistochemical stains was validated for use in dog and used to identify specific prostatic cell types and determine their distribution in the capsule, peripheral, periurethral and urethral regions of the young intact canine prostate. Prostatic cells identified using this technique include perivascular smooth muscle cells, pericytes, endothelial cells, luminal, intermediate, and basal epithelial cells, neuroendocrine cells, myofibroblasts, fibroblasts, fibrocytes, and other hematolymphoid cells. To enhance rigor and transparency, all high resolution images (representative images shown in the figures and biological replicates) are available through the GUDMAP database at https://doi.org/10.25548/16-WMM4. RESULTS: The prostatic peripheral region harbors the largest proportion of epithelial cells. Aging does not change the density of hematolymphoid cells, fibroblasts, and myofibroblasts in the peripheral region or in the fibromuscular capsule, regions where we previously observed aging- and androgen-mediated increases in prostatic collagen abundance Instead, we observed aging-related changes the procollagen 1A1 positive prostatic cell identity from a myofibroblast to a fibroblast. CONCLUSIONS: Hematolymphoid cells and myofibroblasts are often identified as sources of collagen in tissues prone to aging-related fibrosis. We show that these are not the likely sources of pathological collagen synthesis in older intact male dogs. Instead, we identify an aging-related shift in the prostatic cell type producing procollagen 1A1 that will help direct development of cell type and prostate appropriate therapeutics for collagen accumulation.

Efficacy and safety of spironolactone in the heart failure with mid-range ejection fraction and heart failure with preserved ejection fraction: A meta-analysis of randomized clinical trials.

BACKGROUND: Recent studies have shown the efficacy for using spironolactone to treat heart failure with reduced ejection fraction (HFrEF), but the efficacy of spironolactone for heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF) is unclear. This meta-analysis investigated the efficacy and safety of spironolactone in patients with HFmrEF and HFpEF. METHODS AND RESULTS: We searched several databases including PubMed and the Cochrane Collaboration, for randomized controlled trials (RCTs) that assessed spironolactone treatment in HFmrEF and HFpEF. Eleven RCTs including 4539 patients were included. Spironolactone reduced hospitalizations (odds ratio [OR], 0.84; 95% confidence interval [CI], 0.73-0.95; P = .006), improved New York Heart Association functional classifications (NYHA-FC) (OR, 0.35; 95% CI, 0.19-0.66; P = .001), decreased the levels of brain natriuretic peptide (BNP) (mean difference [MD], - 44.80 pg/mL; 95% CI, -73.44--16.17; P = .002), procollagen type I C-terminal propeptide (PICP) (MD, -27.04 ng/mL; 95% CI, -40.77--13.32, P < .001) in HFmrEF and HFpEF. Besides, it improved 6-minute walking distances (6-MWD) (standard weighted mean difference [SMD], 0.45 m; 95% CI, 0.27-0.64; P < .001), decreased amino-terminal peptide of procollagen type-III (PIIINP) (SMD, -0.37 µg/L; 95% CI, -0.59--0.15; P = .001) in HFpEF only. The risks of hyperkalemia (P<.001) and gynecomastia (P<.001) were increased. CONCLUSION: Patients with HFmrEF and HFpEF could benefit from spironolactone treatment, with reduced hospitalizations, BNP levels, improved NYHA-FC, alleviated myocardial fibrosis by decreasing serum PICP in HFmrEF and HFpEF, decreased PIIINP levels and increased 6-MWD only in HFpEF. The risks of hyperkalemia and gynecomastia were significantly increased with the spironolactone treatment.

The medicinal mushroom Auricularia auricula-judae (Bull.) extract has antioxidant activity and promotes procollagen biosynthesis in HaCaT cells.

In this study, Auricularia auricula-judae (Bull.) extract (AAE) had potent antioxidant activity in vitro and promoted the biosynthesis of procollagen, a precursor of collagen in HaCaT cells. In addition, the expression of HAS-3 (hyaluronic acid synthase), which is a moisturizing factor, was increased in HaCaT cells in response to AAE. Therefore, this work suggests that AAE has the potential to exhibit antioxidant activity and promote procollagen biosynthesis in HaCaT cells.

Wound Healing Properties of Selected Natural Products.

Wound healing is a complex process of recovering the forms and functions of injured tissues. The process is tightly regulated by multiple growth factors and cytokines released at the wound site. Any alterations that disrupt the healing processes would worsen the tissue damage and prolong repair process. Various conditions may contribute to impaired wound healing, including infections, underlying diseases and medications. Numerous studies on the potential of natural products with anti-inflammatory, antioxidant, antibacterial and pro-collagen synthesis properties as wound healing agents have been performed. Their medicinal properties can be contributed by the content of bioactive phytochemical constituents such as alkaloids, essential oils, flavonoids, tannins, saponins, and phenolic compounds in the natural products. This review highlights the in vitro, in vivo and clinical studies on wound healing promotions by the selected natural products and the mechanisms involved.

Beneficial impact of zinc supplementation on the collagen in the bone tissue of cadmium-exposed rats.

Cadmium (Cd) is a toxic metal that damages bone tissue by affecting its mineral and organic components. The organic matrix is mainly (90%) composed of collagen, which determines the biomechanical strength of bone. The aim of this study was to evaluate the effect of zinc (Zn) supplementation (30 or 60 mg l-1 ) under moderate and relatively high exposure to Cd (5 and 50 mg l-1 ) on collagen in the rat tibia proximal epiphysis and diaphysis (regions abundant in trabecular and cortical bone, respectively). Significant decrease in collagen type I biosynthesis was found in both regions of the tibia in Cd-treated rats, whereas the supplementation with Zn provided significant protection against this effect. Western blot confirmed the presence of the major type I collagen in the tibia epiphysis and diaphysis, but collagen type II was revealed only in the epiphysis. Acetic acid- and pepsin-soluble collagen concentration in the tibia epiphysis and diaphysis was significantly increased due to the exposure to Cd, whereas the supplementation with Zn protected, partially or totally, from these effects, depending on the used concentration. The supplementation with Zn also provided protection from unfavorable Cd impact on the maturation of the bone collagen, as the ratio of cross-links to monomers was higher compared to the Cd-treated group. This report confirms our previous findings on the preventive action of Zn against harmful effects of Cd on bone, but additionally, and to the best of our knowledge for the first time, explains the possible mechanism of the beneficial influence of this bioelement.

Orobanche cernua Loefling Attenuates Ultraviolet B-mediated Photoaging in Human Dermal Fibroblasts.

UV radiation is the primary cause of skin photoaging, which results in an increase in matrix metalloproteinases and degradation of collagen. Developing new natural antioxidant as photoprotective agents has become a popular area of research. Orobanche cernua Loefling is a parasitic plant that is rich in phenylethanoid glycosides (PhGs). This study investigated the photoprotective effects of the ethanolic extract of Orobanche cernua Loefling (OC) and its principal component acteoside on UVB-induced photoaging as well as their underlying molecular mechanisms in normal human dermal fibroblasts (NHDFs). Biological testing demonstrated that OC and acteoside possessed significant photoprotective activities, reducing MMP and IL-6 levels while improving type-I procollagen synthesis in UVB-irradiated NHDFs. Further study showed that the protective mechanisms were the improvement of transcription factor Nrf2-mediated antioxidant defensive system, suppression of MAPK/AP-1 and activation of the TGF-ß/Smad pathway. Together, our results suggested that OC might be a promising antiphotoaging agent against UV radiation-induced skin damage.

Severe disruption and disorganization of dermal collagen fibrils in early striae gravidarum.

BACKGROUND: Striae gravidarum (SG), or stretch marks of pregnancy, begin as erythematous streaks and mature into hypopigmented atrophic bands. OBJECTIVES: In order to investigate molecular alterations that may promote atrophy of SG, we investigated dermal type I collagen fibrils, which provide human skin with support. METHODS: We obtained skin samples of recently developed, erythematous abdominal SG from pregnant women. To examine the organization of collagen fibrils, second-harmonic generation imaging was performed using multiphoton microscopy. Immunostaining was used to determine protein expression and localization of type I procollagen, the precursor of type I collagen fibrils. Real-time polymerase chain reaction was used to determine gene expression levels. RESULTS: In control (hip) and stretched normal-appearing perilesional abdominal skin, dermal collagen fibrils were organized as tightly packed, interwoven bundles. In SG, collagen bundles appeared markedly separated, especially in the mid-to-deep dermis. In the spaces separating these bundles, loosely packed wavy collagen fibrils lacking organization as bundles were present. These disorganized fibrils persisted into the postpartum period and failed to form densely packed bundles. Numerous large fibroblasts displaying type I procollagen expression were in close proximity to the disorganized fibrils, suggesting that the fibrils are newly synthesized. Supporting this possibility, immunostaining and gene expression of type I procollagen were increased throughout the dermis of SG. CONCLUSIONS: Early SG display marked separation of collagen bundles and emergence of disorganized collagen fibrils that fail to form bundles. These alterations may reflect ineffective repair of collagen bundles disrupted by intense skin stretching. Persistent disruption of the collagenous extracellular matrix likely promotes formation and atrophy of SG.

Preparation, Characterization, and Biological Activities of Topical Anti-Aging Ingredients in a Citrus junos Callus Extract.

In this study, we prepared and characterized a callus extract from Citrus junos and assessed its utility as a source of topical anti-aging ingredients. Callus extract was produced by aqueous extraction from Citrus junos grown on Murashige and Skoog medium with picloram as a growth regulator. After measuring the total phenolic and flavonoid contents, the major phenolic compound in calli was identified as p-hydroxycinnamoylmalic acid (1) by spectroscopic analysis. The total phenol content in the extract was determined to be 24.50 ± 0.43 mg/g of gallic acid equivalents; however, the total flavonoid content of the extract was not determined. The biological activities of the callus extract, in terms of skin anti-aging, were assessed by measuring the anti-tyrosinase activity in, and melanogenesis by, melanoma cells; and proliferation of, and procollagen synthesis by, human fibroblasts. The callus extract was incorporated into nanoliposomes (NLs) to improve its percutaneous absorption. Addition of the callus extract resulted in a 1.85-fold decrease in the melanin content of melanocytes compared with that with arbutin. The extract (500 µg/mL) significantly promoted the proliferation of, and procollagen synthesis by, fibroblasts (by 154% and 176%, respectively). In addition, the flux through the human epidermis of Citrus junos callus extract incorporated into NLs was 17.67-fold higher than that of the callus extract alone. These findings suggest that Citrus junos callus extract-loaded NLs have promise as an anti-aging cosmetic, as well as having a skin-lightening effect.

Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification.

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1 ng/mL and a measurable range of 1-640 ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.

Photodynamic therapy in dermatology beyond non-melanoma cancer: An update.

Photodynamic therapy (PDT) employs a photosensitizer (PS) and visible light in the presence of oxygen, leading to production of cytotoxic reactive oxygen species, which can damage the cellular organelles and cause cell death. In dermatology, PDT has usually taken the form of topical application of a precursor in the heme biosynthesis pathway, called 5-aminolevulinic acid (or its methyl ester), so that an active PS, protoporphyrin IX accumulates in the skin. As PDT enhances dermal remodeling and resolves chronic inflamation, it has been used to treat cutaneous disorders include actinic keratoses, acne, viral warts, skin rejuvenation, psoriasis, localized scleroderma, some non-melanoma skin cancers and port-wine stains. Efforts are still needed to mitigate the side effects (principally pain) and improve the overall procedure.

ROS Scavenging and Anti-Wrinkle Effects of Clitocybin A Isolated from the Mycelium of the Mushroom Clitocybe aurantiaca.

Clitocybin A, an isoindolinone from Clitocybe aurantiaca, was investigated to assess its anti-wrinkle properties, through reactive oxygen species (ROS)-scavenging and elastase inhibitory activities, procollagen synthesis, and matrix metalloproteinase-1 (MMP-1) expression, in human primary dermal fibroblast-neonatal (HDF-N) cells. Clitocybin A exhibited no significant cytotoxicity up to 10 ppm in HDF-N cells, with cell viability and cell proliferation activity greater than 94.6% and 91.9%, respectively. Strong and concentration-dependent ROS radical scavenging activities of clitocybin A were observed following irradiation with UVB at 30 mJ/cm2. Furthermore, clitocybin A treatment of cells at 0.1, 1, and 10 ppm exhibited decreased elastase activity, in a concentration-dependent manner, by 1.97%, 6.6%, and 8.31%, respectively, versus the control group. The effects of clitocybin A on procollagen synthesis and MMP-1 expression were investigated. Clitocybin A treatment of cells at 1, 5, and 10 ppm increased procollagen synthesis, by 67.9%, 74.4%, and 112.9%, respectively, versus the control group. At these concentrations, MMP-1 expression decreased significantly following UV irradiation. Together, these findings suggest that clitocybin A may be an effective ingredient for use in anti-wrinkle cosmetic products.

BMP1 and TLL1 Are Required for Maintaining Periodontal Homeostasis.

Mutations in bone morphogenetic protein 1 (BMP1) in humans or deletion of BMP1 and related protease tolloid like 1 (TLL1) in mice lead to osteogenesis imperfecta (OI). Here, we show progressive periodontal defects in mice in which both BMP1 and TLL1 have been conditionally ablated, including malformed periodontal ligament (PDL) (recently shown to play key roles in normal alveolar bone formation), significant loss in alveolar bone mass ( P < 0.01), and a sharp reduction in cellular cementum. Molecular mechanism studies revealed a dramatic increase in the uncleaved precursor of type I collagen (procollagen I) and a reduction in dentin matrix protein 1 (DMP1), which is partially responsible for defects in extracellular matrix (ECM) formation and mineralization. We also showed a marked increase in the expression of matrix metallopeptidase 13 (MMP13) and tartrate-resistant acid phosphatase (TRAP), leading to an acceleration in periodontal breakdown. Finally, we demonstrated that systemic application of antibiotics significantly improved the alveolar bone and PDL damage of the knockdown phenotype, which are thus shown to be partially secondary to pathogen-induced inflammation. Together, identification of the novel roles of BMP1 and TLL1 in maintaining homeostasis of periodontal formation, partly via biosynthetic processing of procollagen I and DMP1, provides novel insights into key contributions of the extracellular matrix environment to periodontal homeostasis and contributes toward understanding of the pathology of periodontitis.

Anti-wrinkle effects of Seungma-Galgeun-Tang as evidenced by the inhibition of matrix metalloproteinase-I production and the promotion of type-1 procollagen synthesis.

BACKGROUND: Seungma-Galgeun-Tang (SMGGT), a traditional herbal medicinal formula, has been used to treat various skin problems such as inflammation and rashes in Korean traditional medicine. In order to clarify the scientific evidence for the biological efficacy of SMGGT on the prevention of skin aging and in particular wrinkle formation, molecular anti-wrinkle parameters were evaluated in cultured human dermal fibroblasts. METHODS: Standard SMGGT was prepared from KFDA-certified herbal medicines and the chemical fingerprint of SMGGT was verified by HPLC-ESI-MS to insure the quality of SMGGT. To evaluate the inhibitory effects of SMGGT on the synthesis of matrix metalloproteinase-1 (MMP-1) and type-1 procollagen, the content of MMP-1 and type-1 procollagen synthesizing enzymes in cultured human dermal fibroblasts were measured using an ELISA kit and Western Blot, respectively. RESULTS: The treatment of SMGGT water extract significantly inhibited the production of MMP-1 and promoted type-1 procollagen synthesis concentration dependently. CONCLUSIONS: These results suggest that SMGGT has the potential to prevent wrinkle formation by down-regulating MMP-1 and up-regulating type-1 procollagen in human dermal fibroblasts.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis.

BACKGROUND: Interferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in SSc before overt skin fibrosis develops. METHODS: The expression of 11 IFN type I inducible genes was tested in whole-blood samples from 30 healthy controls (HCs), 12 subjects with primary Raynaud's phenomenon (RP), 19 patients with EaSSc, 7 patients with definite SSc without cutaneous fibrosis, 21 limited cutaneous SSc and 10 diffuse cutaneous SSc subjects. The correlation between IFN activity in monocytes, B cell activating factor (BAFF) mRNA expression and type III procollagen N-terminal propeptide (PIIINP) serum levels was tested. RESULTS: In all the SSc groups, higher IFN scores were observed compared with HC. An IFN score ≥7.09 discriminated HCs from patients with SSc (sensitivity=0.7, specificity=0.88, area under receiving operating characteristic (AUROC)=0.82); the prevalence of an elevated IFN score was: HC=3.3%; RP=33.3%, EaSSc=78.9%, definite SSc=100%, limited cutaneous SSc=42.9%, diffuse cutaneous SSc=70.0%. In monocytes an IFN score ≥4.12 distinguished HCs from patients with fibrotic SSc (sensitivity=0.62, specificity=0.85, AUROC=0.76). Compared with IFN-negative subjects, IFN-positive subjects had higher monocyte BAFF mRNA levels (19.7±5.2 vs 15.20±4.0, p=2.1×10(-5)) and serum PIIINP levels (median=6.0 (IQR 5.4-8.9) vs median=3.9 (IQR 3.3-4.7), p=0.0004). CONCLUSIONS: An IFN type I signature is observed in patients with SSc from the earliest phases of the disease, even before overt skin fibrosis. The presence of IFN type I signature in monocytes is correlated with BAFF mRNA expression and serum PIIINP levels, supporting a contribution in the pathogenesis and progression of SSc.

Conditioned medium from human bone marrow-derived mesenchymal stem cells promotes skin moisturization and effacement of wrinkles in UVB-irradiated SKH-1 hairless mice.

BACKGROUND: Mesenchymal stem cells (MSCs) are promising therapeutic agents for various diseases. AIMS: To investigate the effects of conditioned medium from human bone marrow-derived mesenchymal stem cells (MSC-CdM) on pro-collagen production and wrinkle formation, we performed in vitro and in vivo experiments. METHODS: We assessed the effects of MSC-CdM on proliferation and photo-aging in human dermal fibroblasts after UVB exposure using enzyme activity assays for collagen type I secretion and MMP-1. To determine the effect of topically applied MSC-CdM on wrinkle formation, MSC-CdM (1% and 10%) and vehicle (propylene glycol: ethanol, 7 : 3) were applied to the dorsal skin of UVB-irradiated hairless mice for 8 weeks. We examined the effects on wrinkle formation by assessing visual skin grading, replica, tape stripping, transepidermal water loss (TEWL), and skin hydration measurement. We also examined histology of the lesions using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining. RESULTS: MSC-CdM markedly reduced UV-induced matrix metalloproteinase-1 expression and increased pro-collagen synthesis in a dose-dependent manner. Our findings suggest that MSC-CdM induces repair of dermal damage and effacement of wrinkles on UVB-irradiated hairless mice through protective effect of hydration. CONCLUSION: These results support an anti-wrinkle effect of MSC-CdM that involves increased collagen synthesis and suggest that MSC-CdM might be a potential candidate for preventing UV-induced skin damage.

Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Development and characterization of a eukaryotic expression system for human type II procollagen.

BACKGROUND: Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. RESULTS: Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. CONCLUSIONS: Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Acute bone response to whole body vibration in healthy pre-pubertal boys.

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton's response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such "stimulation-testing" in clinical practice.

In malignant cartilagenous tumors, immunohistochemical expression of procollagen PC1CP peptide is higher and that of PC2CP lower than in benign cartilaginous lesions.

Few studies on oncogenesis of chondrosarcoma (CS) are available in the literature. Our previously published experimental evidence suggests that while the C-propeptide of procollagen Iα1 (PC1CP), a component of cartilage, favors tumor progression, the C-propeptide of procollagen IIα1 (PC2CP) exerts antitumor properties. In this study, we analyzed expression of PC1CP and PC2CP by immunohistochemistry in a series of enchondromas and CS. Our retrospective series consisted of 88 cases, including 43 CSs, 34 enchondromas and 11 nontumor samples. Immunohistochemical staining for PC1CP and PC2CP was evaluated in the cytoplasm and in the extracellular matrix (ECM). Diffuse staining for PC1CP in ECM was significantly more frequent in tumor than in nontumor samples (32 % vs. 0 %; p = 0.03), and in CSs than in enchondromas (44 vs. 18 %; p = 0.02). ECM semiquantitative score was higher in tumors than in nontumor samples (p < 0.005) and higher in CSs than in enchondromas (p = 0.05). Staining for PC2CP in ECM was more frequently found in enchondromas than in CSs (59 vs. 33 %; p = 0.02). ECM semiquantitative score was higher in enchondromas than in CSs (p = 0.02). Diffuse staining for PC1CP in combination with absence of staining for PC2CP had 94 % specificity for CS but with a sensitivity of only 35 %. Expression of neither PC1CP nor PC2CP correlated with recurrence-free survival or occurrence of metastases. In conclusion, we show that the expression of PC1CP is higher and that of PC2CP lower in malignant cartilaginous tumors. These results support an oncogenic role of PC1CP and anti-oncogenic property of PC2CP in cartilaginous tumors.