Paola Brun

Semisynthetic resorbable materials from hyaluronan esterification

In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan consisting of the partial or total esterification of the carboxyl groups of this natural polymer. This review on the properties of the new materials reports some of their biocompatibility and characterization aspects based on findings from studies conducted on the ethyl and benzyl hyaluronan esters, two representative members of this new class of compounds, and is intended to arouse interest in the potential of other, as yet unexplored derivatives. From the results of a number of investigations, the various derivatives appear to possess different physico-chemical properties, especially as far as the degree of hydration and polymer stability are concerned. In addition, the type of esterification and extent of chemical esterification of hyaluronan considerably affects the biological properties of these materials, offering a range of polymers either favouring or, conversely, inhibiting the adhesion of certain types of cell.

Hyaluronan‐based biopolymers as delivery vehicles for bone‐marrow‐derived mesenchymal progenitors

The tolerability and safety of hyaluronan-based three-dimensional scaffolds as a culture vehicle for mesenchymal progenitor cells was investigated in this pilot study. The proliferation patterns and extracellular matrix production of rabbit and human mesenchymal, bone-marrow-derived progenitors first were characterized in vitro. Subsequently rabbit autologous cells were cultured in this hyaluronan-based scaffold and implanted in a full-thickness osteochondral lesion. In vitro histologic findings showed that mesenchymal progenitor cells adhered and proliferated onto the hyaluronan-derived scaffold. Human stem cells were shown to produce the main extracellular matrix molecules, accompanied by an occasional synthesis of mature type II collagen. In vivo data demonstrated that the biomaterial, with or without mesenchymal progenitors, did not elicit any inflammatory response and was completely degraded within 4 months after implantation. With regard to the efficacy of this cell therapy, even among the small number of animals tested there was histologic evidence that lesions filled with the biomaterial, either seeded or unseeded with cells, achieved a faster and better healing compared to empty controls. The present data suggest that the hyaluronan-based scaffolds are well tolerated and safe and may be a valuable delivery vehicle for tissue engineering in the repair of articular cartilage defects.

T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients

There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)‐17 as their effector cytokine under the control of IL‐22 and IL‐23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and IL‐23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age‐matched control subjects. The expression of IL‐17A, IL‐17F, IL‐21, IL‐22, IL‐23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL‐22+ and IL‐23+ immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL‐17A+ and IL‐22+ immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non‐smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL‐22+ cells correlated significantly with the number of both CD4+ and CD8+ cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL‐17A and IL‐22. Increased expression of the Th17‐related cytokines IL‐17A, IL‐22 and IL‐23 in COPD patients may reflect their involvement, and that of specific IL‐17‐producing cells, in driving the chronic inflammation seen in COPD.

Chondrocyte aggregation and reorganization into three-dimensional scaffolds

Articular cartilage has a very limited self-repairing capacity; thus, chondral lesions normally result in chronic degeneration and, eventually, osteoarthritis development. Currently, tissue engineering offers a new tool for the clinical treatment of osteochondral defects. The present investigation aimed to develop an in vitro engineered cartilage using a new class of semisynthetic scaffolds. Two nonwoven meshes of hyaluronan esters (Hyaff(R) derivatives) were seeded with sternal chick embryo chondrocytes cultured for up to 21 days, after which time they were assessed for both the cellular growth profile and histological features. Avian chondrocytes easily adhered and proliferated onto hyaluronan-based scaffolds, demonstrating a significant preference for the fully esterified benzylic form. Histochemical staining revealed the presence of a neosynthesized glycosaminoglycan-rich extracellular matrix, and immunohistochemistry confirmed the deposition of collagen type II. Moreover, ultrastructural observations supported evidence that chondrocytes grown onto a hyaluronan-derived three-dimensional scaffold maintained their unique phenotype and organization in a cartilage-like extracellular matrix. These findings support the further pursuit of a transplantable engineered cartilage using human chondrocytes for the regeneration of chondral lesions.

In vitro engineering of human skin‐like tissue

Coverage of large, full-thickness burns presents a challenge for the surgeon due to the lack of availability of the patients own skin. Currently, tissue engineering offers the possibility of performing a suitable therapeutic wound coverage after early burn excision by using cultured keratinocyte sheets supported by a dermal layer. The aim of this study was to develop and characterize a skin substitute composed of both epidermal and dermal elements. For this purpose we grew keratinocytes and fibroblasts separately for 15 days within two different types of biomaterials. Cells then were co-cultured for an additional period of 15 days, after which samples were taken and processed with either classic or immunohistochemical stainings. Results showed that (1) human fibroblasts and keratinocytes can be cultured on hyaluronic acid-derived biomaterials and that (2) the pattern of expression of particular dermal-epidermal molecules is similar to that found in normal skin. The data from this study suggest that our skin equivalent might be useful in the treatment of both burns and chronic wounds.

Toll-Like Receptor 2 Regulates Intestinal Inflammation by Controlling Integrity of the Enteric Nervous System

BACKGROUND & AIMS In the intestines, Toll-like receptor 2 (TLR2) mediates immune responses to pathogens and regulates epithelial barrier function; polymorphisms in TLR2 have been associated with inflammatory bowel disease phenotype. We assessed the effects of TLR2 signaling on the enteric nervous system (ENS) in mice. METHODS TLR2 distribution and function in the ileal neuromuscular layer of mice were determined by immunofluorescence, cytofluorimetric analysis, immunoprecipitation, and immunoblot analyses. We assessed morphology and function of the ENS in Tlr2(-/-) mice and in mice with wild-type Tlr2 (wild-type mice) depleted of intestinal microbiota, using immunofluorescence, immunoblot, and gastrointestinal motility assays. Levels and signaling of glial cell line-derived neurotrophic factor (GDNF) were determined using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and immunoprecipitation analyses. Colitis was induced by administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid to Tlr2(-/-) mice after termination of GDNF administration. RESULTS TLR2 was expressed in enteric neurons, glia, and smooth muscle cells of the intestinal wall. Tlr2(-/-) mice had alterations in ENS architecture and neurochemical profile, intestinal dysmotility, abnormal mucosal secretion, reduced levels of GDNF in smooth muscle cells, and impaired signaling via Ret-GFRα1. ENS structural and functional anomalies were completely corrected by administration of GDNF to Tlr2(-/-) mice. Wild-type mice depleted of intestinal microbiota had ENS defects and GDNF deficiency, similar to Tlr2(-/-) mice; these defects were partially restored by administration of a TLR2 agonist. Tlr2(-/-) mice developed more severe colitis than wild-type mice after administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid; colitis was not more severe if Tlr2(-/-) mice were given GDNF before dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid. CONCLUSIONS In mice, TLR2 signaling regulates intestinal inflammation by controlling ENS structure and neurochemical coding, along with intestinal neuromuscular function. These findings provide information as to how defective TLR2 signaling in the ENS affects inflammatory bowel disease phenotype in humans.

Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds

The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.

In vitro reconstructed dermis implanted in human wounds: degradation studies of the HA-based supporting scaffold

The objective of the present study was to demonstrate the safety and efficacy of a dermal replacement for cutaneous wounds of diverse origin. Autologous fibroblasts were cultured in fleece scaffolds made from benzyl esters of hyaluronic acid and applied onto cutaneous lesions. The cases presented are (1) skin removal for multiple epithelioma and (2) chronic deep decubitus ulcer. Dermal-like tissue applied by the surgeon elicited no adverse reactions, and was fully integrated and well-vascularized by 1-3 weeks. In Case 1, the material was fully integrated after 1 week, and after 3 weeks an epidermal autograft was overlaid which showed good take with excellent integration observed after 4 weeks. At 12 months, skin demonstrated visual normo-elastic properties and no signs of excessive scarring. In Case 2, 2-3 weeks after the dermal implant was applied, the wound was invaded with granulation tissue and healing occurred by secondary intention. The ulcer was healed by 8 weeks, with the biomaterial completely resorbed and a complete re-epithelialization over the dermal-like tissue. These results suggest that autologous fibroblast culture in hyaluronan-derived scaffolds may be successfully grafted in diverse cutaneous pathologies and constitute a suitable bed for further epidermal implantation.

MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells

We previously demonstrated that MG-2477 (3-cyclopropylmethyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one) inhibits the growth of several cancer cell lines in vitro. Here we show that MG-2477 inhibited tubulin polymerization and caused cells to arrest in metaphase. The detailed mechanism of action of MG-2477 was investigated in a non-small cell lung carcinoma cell line (A549). Treatment of A549 cells with MG-2477 caused the cells to arrest in the G2/M phase of the cell cycle, with a concomitant accumulation of cyclin B. Moreover, the compound induced autophagy, which was followed at later times by apoptotic cell death. Autophagy was detected as early as 12h by the conversion of microtubule associated protein 1 light chain 3 (LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. After 48h of MG-2477 exposure, phosphatidylserine externalization on the cell membrane, caspase-3 activation, and PARP cleavage occurred, revealing that apoptotic cell death had begun. Pharmacological inhibition of autophagy with 3-methyladenine or bafilomycin A1 increased apoptotic cell death, suggesting that the autophagy caused by MG-2477 played a protective role and delayed apoptotic cell death. Additional studies revealed that MG-2477 inhibited survival signaling by blocking activation of Akt and its downstream targets, including mTOR, and FHKR. Treatment with MG-2477 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase and 4E-BP1. Overexpression of Akt by transfection with a Myr-Akt vector decreased MG-2477 induced autophagy, indicating that Akt is involved. Taken together, these results indicated that the autophagy induced by MG-2477 delayed apoptosis by exerting an adaptive response following microtubule damage.

Decreased T lymphocyte infiltration in bronchial biopsies of subjects with severe chronic obstructive pulmonary disease

Background Studies on the inflammatory process in the large airways of patients with mild/moderate COPD have shown a prevalent T lymphocyte and macrophage infiltration of the bronchial mucosa. However, bronchial inflammation in more severe disease has not been extensively studied.