Daniel Hartmann

The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.

Clinical, biological, and skin histopathologic effects of ionic macrocyclic and nonionic linear gadolinium chelates in a rat model of nephrogenic systemic fibrosis.

Objective:The purpose of this study was to compare the clinical, pathologic, and biochemical effects of repeated administrations of ionic macrocyclic or nonionic linear gadolinium chelates (GC) in rats with impaired renal function. Material and Methods:Rats submitted to subtotal nephrectomy were allocated to single injections of 2.5 mmol/kg of gadodiamide (nonionic linear chelate), nonformulated gadodiamide (ie, without the free ligand caldiamide), gadoterate (ionic macrocyclic chelate), or saline for 5 consecutive days. Blinded semi-quantitative histopathologic and immunohistochemical examinations of the skin were performed, as well as clinical, hematological, and biochemical follow-up. Rats were killed at day 11. Long-term (up to day 32) follow-up of rats was also performed in an auxiliary study. Results:Epidermal lesions (ulcerations and scabs) were found in 4 of the 10 rats treated with nonformulated gadodiamide. Two rats survived the study period. Inflammatory signs were observed in this group. No clinical, hematological, or biochemical signs were observed in the saline and gadoterate- or gadodiamide-treated groups. Plasma fibroblast growth factor-23 levels were significantly higher in the gadodiamide group than in the gadoterate group (day 11). Decreased plasma transferrin-bound iron levels were measured in the nonformulated gadodiamide group. Histologic lesions were in the range: nonformulated gadodiamide (superficial epidermal lesions, inflammation, necrosis, and increased cellularity in papillary dermis) > gadodiamide (small superficial epidermal lesions and signs of degradation of collagen fibers in the dermis) > gadoterate (very few pathologic lesions, similar to control rats). Conclusions:Repeated administration of the nonionic linear GC gadodiamide to renally impaired rats is associated with more severe histologic lesions and higher FGF-23 plasma levels than the macrocyclic GC gadoterate.

Long-term remodeling of a bilayered living human skin equivalent (Apligraf®) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell‐synthesized tissue. Apligraf®, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species‐specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species‐specific immunohistochemistry tools is thus very useful for studying long‐term tissue remodeling of bioengineered living tissues. (WOUND REP REG 2003;11:35–45)

Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study

OBJECTIVE The aim of this study was to investigate the clinical value of serum measurement of C-telopeptide of type II collagen (CTX-II). In correlation with late stages of osteoarthritis (OA) evaluated with histological assessment, the evolution of serum CTX-II concentration was followed during a 20-week longitudinal study in rabbit anterior cruciate ligament transection (ACLT) OA model in adult and growing animals. METHODS OA was induced in five adult and nine growing rabbits. Four adult and four young rabbits were unoperated. Serum sampling was made at week 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16 and 20 after the surgery in all rabbits. Animals were euthanized 20 weeks after the surgery. Serum CTX-II levels were analyzed with a recently available enzyme-linked immunosorbent assay (ELISA) kit, the protocol of which has been modified to increase the sensitivity of the test. RESULTS Significant differences for the CTX-II levels at W3, W6, W8, W10, W12, W14, W16 and W20 were observed between the adult ACLT and the control groups. A negative correlation between CTX-II levels and cartilage thickness of the medial compartment of the knee at W8, W10, W12 and a positive correlation between the CTX-II levels and the histomorphological score of the medial compartment of the knee at W3, W6, W8, W10, W12 were noted in adult animals. In young animals, operated or not, we observed high CTX-II levels at the beginning of the study, which decreased until the end. CONCLUSION Our results suggest the interest of the serum CTX-II monitoring for the OA progression and the relevance of the multiple time point analysis of this biomarker. Moreover, they address the question of the importance of correctly choosing the age of the animals used in the pre-clinical studies of OA.

Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity

The aim of this study was to compare the affinity values obtained for a monoclonal antibody/antigen complex using two different techniques, surface plasmon resonance (SPR) and an enzyme linked immunosorbent assay (ELISA) approach recently described by Bobrovnik S.A. and by Stevens F.J. These two techniques can be used in particular to determine the equilibrium dissociation constant, K(D), of the complex in solution or on a surface. Bobrovniks method gives two K(D) values that differ by a factor of 100, demonstrating that two populations of complexes are present in solution. In an initial step, one protein binds relatively weakly to the other (high K(D)) and this is followed by a conformational change in the most flexible portion of the antigen, which increases the affinity (low K(D)). Only the higher of the two K(D) values can be detected when complex formation in solution is investigated using SPR, because the interaction measured concerns the fibronectin/antibody complexes of lowest affinity. In contrast, when measuring association at the sensor surface, SPR gives an average result between the two K(D) values because complexes corresponding to both affinities can form in this situation. The constants that characterise the kinetics of the fibronectin-antibody interaction obtained by SPR and ELISA are therefore different, because the methods do not allow the same phenomena to be observed. However they are consistent and complementary.

In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices

Poly(lactic acid) is nowadays among the most used bioabsorbable materials for medical devices. To promote bone growth on the material surface and increase the degradation rate of the polymer, research is currently focused on organic-inorganic composites by adding a bioactive mineral to the polymer matrix. The purpose of this study was to investigate the ability of a poly(L,DL-lactide)-Bioglass® (P(L,DL)LA-Bioglass(®) 45S5) composite to be used as a bone fixation device. In vitro cell viability testing of P(l,dl)LA based composites containing different amounts of Bioglass(®) 45S5 particles was investigated. According to the degradation rate of the P(L,DL)LA matrix and the cytocompatibility experiments, the composite with 30 wt % of Bioglass® particles seemed to be the best candidate for further investigation. To study its behavior after immersion in simulated physiological conditions, the degradation of the composite was analyzed by measuring its weight loss and mechanical properties and by proceeding with X-ray tomography. We demonstrated that the presence of the bioactive glass significantly accelerated the in vitro degradation of the polymer. A preliminary in vivo investigation on rabbits shows that the addition of 30 wt % of Bioglass(®) in the P(L,DL)LA matrix seems to trigger bone osseointegration especially during the first month of implantation. This composite has thus strong potential interest for health applications.

Complete Unilateral Ureteral Obstruction in the Fetal Lamb. Part II: Long-Term Outcomes of Renal Tissue Development

PURPOSE We analyzed the dynamics of the renal tissue response to experimental fetal urinary flow impairment concerning renal morphology, extracellular matrix composition, regulators of connective tissue degradation and PAX2 protein expression. MATERIALS AND METHODS A total of 26 fetal lambs underwent surgical unilateral ureteral obstruction at 90 days of gestation and 14 twin matched animals served as controls. Kidneys were harvested 10, 20 and 40 days after the prior procedure in groups 1 to 3, respectively and in 1-month-old lambs (group 4). Morphological analysis was done using light microscopy. Picrosirius red staining was used to evaluate the area occupied by extracellular matrix components. Collagen I, III and IV, alpha-smooth muscle actin, MMP-1, 2 and 9, TIMP-1 and 2 and PAX2 protein were assessed using immunochemistry. RESULTS All obstructed kidneys were hydronephrotic without dysplasia. Hypoplasia resulting from a decreased NGG was observed. The inflammatory response to obstruction was poor in fetal obstructed kidneys. From 10 days after obstruction interstitial fibrosis was noted and confirmed by an increase in picrosirius red staining. In obstructed kidneys immunochemistry showed an increase in collagen deposition beginning from the papillae and extending through the whole parenchyma. Aberrant interstitial collagen IV deposition was observed. The increase in alpha-smooth muscle actin staining was mainly localized in the blastema and interstitial cells in obstructed kidneys. MMP and TIMP immunostaining was mainly present in tubules throughout the whole nephrogenic period and persisted in mature kidneys. Beginning from 20 days after obstruction a progressive increase in MMP and TIMP expression was noted. This was associated with ectopic expression in the medullary tubules. PAX2 protein was highly expressed in the nephrogenic zone, decreasing progressively to being markedly decreased in control lamb kidneys. No difference was found in PAX2 expression during the fetal period when comparing unobstructed and obstructed kidneys, it but remained strongly expressed in the dilated collecting ducts of obstructed lambs. CONCLUSIONS Complete unilateral ureteral obstruction performed in fetal lambs at 90 days of gestation led to pure hydronephrotic transformation, hypoplasia and a marked increase in connective tissue deposition. Inflammatory infiltrates and PAX2 dysregulation were not seen as having a decisive role in these modifications.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins.

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation.

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 μg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.

Crystallization processes at the surface of polylactic acid—bioactive glass composites during immersion in simulated body fluid†

We report on the crystallization processes occurring at the surface of PDLLA-Bioglass® composites immersed in simulated body fluid. Composites manufactured by injection molding and containing different amounts (0, 20, 30, and 50 wt %) of 45S5 Bioglass® particles were tested for durations up to 56 days and compared with Bioglass® particles alone. Crystallization processes were followed by visual inspection, X-ray diffraction (with Rietveld analysis) and scanning electron microscopy. Both calcite and hydroxyapatite were formed at the surface of all materials, but their relative ratio was dependent on the Bioglass® content and immersion time. Hydroxyapatite was always the major phase after sufficient immersion time, insuring bioactivity of such composites especially for Bioglass® content higher than 30 wt %. A scenario of crystallization is proposed. Rapid degradation of the composites with 50 wt % was also observed during immersion. Therefore, composites with 30 wt % of Bioglass® particles seem to exhibit the best balance between bioactivity and stability at least during the first weeks of immersion in contact with body fluids.