AAV2/9-mediated gene transfer into murine lacrimal gland leads to a long-term targeted tear film modification.

Corneal blindness is the fourth leading cause of blindness worldwide. Since corneal epithelium is constantly renewed, non-integrative gene transfer cannot be used to treat corneal diseases. In many of these diseases, the tear film is defective. Tears are a complex biological fluid secreted by the lacrimal apparatus. Their composition is modulated according to the context. After a corneal wound, the lacrimal gland secretes reflex tears, which contain growth factors supporting the wound healing process. In various pathological contexts, the tear composition can support neither corneal homeostasis nor wound healing. Here, we propose to use the lacrimal gland as bioreactor to produce and secrete specific factors supporting corneal physiology. In this study, we use an AAV2/9-mediated gene transfer to supplement the tear film. First, we demonstrate that a single injection of AAV2/9 is sufficient to transduce all epithelial cell types of the lacrimal gland efficiently and widely. Second, we detect no adverse effect after AAV2/9-mediated nerve growth factor expression in the lacrimal gland. Only a transitory increase in tear flow is measured. Remarkably, AAV2/9 induces an important and long-lasting secretion of this growth factor in the tear film. Altogether, our findings provide a new clinically applicable approach to tackle corneal blindness.

The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility.

Different industrial processes exist to purify allogenic bone, providing safe and cleaned blocks for bone allografting. However, they often make use of chemical reagents that can be aggressive for the bone matrix. Bone samples were processed with several soaking techniques used in industry: NaHCO3, H2O2, NaOH and H2O2+NaOH combined; the consequences on the bone matrix and cytocompoatibility were evaluated on femoral heads from osteoarthritic patients. Alterations of matrix were searched by histochemistry, atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cytocompatibility was evaluated by coculturing human osteoblast-like cells (SaOS-2) on bone slices. Collagen fibers were dramatically altered at the surface of bone treated with H2O2, NaOH (and their association), but not with NaHCO3. A marked reduction in the number of hydroxyapatite crystals was observed on the trabecular surfaces by TEM and morphological changes were evidenced in SEM and AFM. Argyrophilic proteins of the bone matrix were removed by H2O2 and NaOH (and their association), but not by NaHCO3. As a consequence, attachment, spreading, proliferation and alkaline phosphatase activity of SaOS-2 were reduced by H2O2 and NaOH treatments. Strong oxidizing reagents altered matrix integrity by modifying collagenous and non-collagenous proteins. Whether these changes have clinical consequences on the bone bonding and osseointegration in human necessitate further investigations.

ß-TCP granules mixed with reticulated hyaluronic acid induce an increase in bone apposition.

ß beta-tricalcium phosphate (ß-TCP) granules are suitable for repair of bone defects. They have an osteoconductive effect shortly after implantation. However, dry granules are difficult to handle in the surgical room because of low weight and lack of cohesion. Incorporation of granules in a hydrogel could be a satisfactory solution. We have investigated the use of hyaluronic acid (HyA) as an aqueous binder of the granules. ß-TCP granules were prepared by the polyurethane foam technology. Commercially available linear (LHya) and reticulated hyaluronic acid (RHyA) in aqueous solution were used to prepare a pasty mixture that can be handled more easily than granules alone. Thirteen New Zealand White rabbits (3.5-3.75 kg) were used; a 4 mm hole was drilled in each femoral condyle. After flushing, holes were filled with either LHyA, RHyA, dry ß-TCP granules alone, ß-TCP granules + LHyA and ß-TCP granules + RHyA. Rabbits were allowed to heal for one month, sacrificed and femurs were harvested and analysed by microCT and histomorphometry. The net amount of newly formed bone was derived from measurements done after thresholding the microCT images for the material and for the material+bone. LHyA and RHyA did not result in healing of the grafted area. LHyA was rapidly eluted from the grafted zone but allowed deposition of more granules, although the amount of formed bone was not significantly higher than with ß-TCP granules alone. RHyA permitted the deposition of more granules which induced significantly more bone trabeculae without inducing an inflammatory reaction. RHyA appears to be a good vehicle to implant granules of ß-TCP, since HyA does not interfere with bone remodeling.

Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.

A role for the gastro-intestinal tract in controlling bone remodeling is suspected since serum levels of bone remodeling markers are affected rapidly after a meal. Glucose-dependent insulinotropic polypeptide (GIP) represents a suitable candidate in mediating this effect. The aim of the present study was to investigate the effect of total inhibition of GIP signaling on trabecular bone volume, microarchitecture and quality. We used GIP receptor (GIPR) knockout mice and investigated trabecular bone volume and microarchitecture by microCT and histomorphometry. GIPR-deficient animals at 16 weeks of age presented with a significant (20%) increase in trabecular bone mass accompanied by an increase (17%) in trabecular number. In addition, the number of osteoclasts and bone formation rate was significantly reduced and augmented, respectively in these animals when compared with wild-type littermates. These modifications of trabecular bone microarchitecture are linked to a remodeling in the expression pattern of adipokines in the GIPR-deficient mice. On the other hand, despite significant enhancement in bone volume, intrinsic mechanical properties of the bone matrix was reduced as well as the distribution of bone mineral density and the ratio of mature/immature collagen cross-links. Taken together, these results indicate an increase in trabecular bone volume in GIPR KO animals associated with a reduction in bone quality.

Phenotypic effects of continuous or discontinuous treatment with dexamethasone and/or calcitriol on osteoblasts differentiated from rat bone marrow stromal cells.

Osteoblasts are target cells for glucocorticoids and calcitriol, and their phenotype is greatly modified by these hormones. We investigated the effect of continuous or discontinuous hormonal exposure to osteoblasts derived from rat bone marrow stromal cells in long-term subcultures. Stromal cells were grown in primoculture in presence of dexamethasone (dex), but in following subcultures, dex and/or calcitriol were added just after seeding or after a 7-day hormone-free period. Cell proliferation, alkaline phosphatase (ALP) histochemical staining, and enzymatic bioactivity measurement, osteocalcin (OC), ALP and bone sialoprotein (BSP) mRNA expression were used to study the differential effect on osteoblastic phenotype of various conditions of treatment by dex and calcitriol. In primoculture, the osteoblastic differentiation was confirmed by the formation of calcified nodules and by strong expression of ALP, OC, and BSP mRNAs. In subcultures, proliferation of stromal cells was stimulated by dex and inhibited by calcitriol and by both hormones. Cell proliferation was not modified by hormonal lack during 7 days. Continuous hormonal treatment by dex strongly enhanced OC and BSP mRNAs, but apparently did not modified ALP mRNAs expression. Continuous treatment by calcitriol decreased ALP and the dex-induced BSP expression and stimulated the OC mRNAs level, strongly when associated with dex. The population of ALP+ cells and ALP bioactivity were strongly increased by dex, whereas calcitriol or both hormones decreased them. When the subcultures were undergone without hormonal treatment during 7 days, all osteogenic mRNAs strongly decreased even after hormonal recovery. Dex, calcitriol, and both hormones inhibited ALP mRNAs. OC messengers were only weakly detectable with both hormones. ALP+ cell population and ALP bioactivity were decreased after 14 days of hormonal treatment recovery. These results support that continuous presence of glucocorticoids appears as a major key for the permanent expression of the osteoblastic phenotype that is inhibited by calcitriol, in the rat bone marrow.

Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.