Alessia Di Giancamillo

Animal models for meniscus repair and regeneration.

The meniscus plays an important role in knee function and mechanics. Meniscal lesions, however, are common phenomena and this tissue is not able to achieve spontaneous successful repair, particularly in the inner avascular zone. Several animal models have been studied and proposed for testing different reparative approaches, as well as for studying regenerative methods aiming to restore the original shape and function of this structure. This review summarizes the gross anatomy, function, ultrastructure and biochemical composition of the knee meniscus in several animal models in comparison with the human meniscus. The relevance of the models is discussed from the point of view of basic research as well as of clinical translation for meniscal repair, substitution and regeneration. Finally, the advantages and disadvantages of each model for various research directions are critically discussed. Copyright

Multidifferentiation potential of human mesenchymal stem cells from adipose tissue and hamstring tendons for musculoskeletal cell-based therapy

AIM Adipose-derived stem cells (ASCs) have been deeply characterized for their usefulness in musculoskeletal tissue regeneration; recently, other mesenchymal stem cell (MSC) sources have also been proposed. This study compares for the first time human tendon stem/progenitor cells isolated from hamstring tendons with human ASCs. MATERIALS & METHODS Human TSPCs and ASCs were isolated from hamstring tendon portions and adipose tissue of healthy donors undergoing ACL reconstruction or liposuction, respectively (n = 7). Clonogenic ability, immunophenotype and multi-differentiation potential were assessed and compared. RESULTS Both populations showed similar proliferation and clonogenic ability and expressed embryonic stem cell genes and MSC surface markers. Tendon stem/progenitor cells showed lower adipogenic and osteogenic ability, but after the chondrogenic differentiation, they produced more abundant glycosaminoglycans and expressed higher levels of aggrecan with regards to ASCs. The tenogenic induction with BMP-12 upregulated SCX and DCN gene expression in both populations. CONCLUSION Our results demonstrate that waste hamstring tendon fragments could represent a convenient MSC source for musculoskeletal regenerative medicine.

Evaluation of oxidative stress biomarkers in Zosterisessor ophiocephalus from the Venice Lagoon, Italy.

Several studies carried out in the last years have demonstrated the presence of a wide range of contaminants in some areas of the Venice Lagoon. Many of these contaminants are able to drive free radical reactions, which lead to oxidative stress and can potentially affect fish health. In the present study, oxidative stress biomarkers were examined in three different sites (Porto Marghera, Val di Brenta and Caroman) of the Venice Lagoon and their levels monitored in Zosterisessor ophiocephalus, one of the most common fish species present in the lagoon. Schmorls staining revealed the presence of melanomacrophage centres in spleen and head kidney, and the highest number of melanomacrophage centres was observed in the animals sampled at the Porto Marghera (Porto Marghera vs Val di brenta and Caroman: p<0.01). The cellular localization of HNE and NT, investigated through an immunohistochemical approach, showed that immunopositivity was mainly localized in melanomacrophage centres of spleen and kidney. It is relevant that the animals of the detoxified control group did not exhibit any immunoreactivity. By Western blot, the antibodies against HNE and NT recognized in the liver polypeptides damaged by oxidative stress with molecular weights under 66kDa. Comparing the relative densities, animals from the Val di Brenta site exhibited the lowest levels of HNE adducts (p<0.05), whereas animals from the Porto Marghera site exhibited the highest levels of NT adducts (p<0.05). MDA levels, measured spectrophotometrically by TBARS assay did not exhibit any statistical difference among sites.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Repair of osteochondral defects in the minipig model by OPF hydrogel loaded with adipose-derived mesenchymal stem cells.

AIM Critical knee osteochondral defects in seven adult minipigs were treated with oligo(polyethylene glycol)fumarate (OPF) hydrogel combined with autologous or human adipose-derived stem cells (ASCs), and evaluated after 6 months. METHODS Four defects were made on the peripheral part of right trochleas (n = 28), and treated with OPF scaffold alone or pre-seeded with ASCs. RESULTS A better quality cartilage tissue characterized by improved biomechanical properties and higher collagen type II expression was observed in the defects treated by autologous or human ASC-loaded OPF; similarly this approach induced the regeneration of more mature bone with upregulation of collagen type I expression. CONCLUSION This study provides the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel allow improving osteochondral defect regeneration in a minipig model.

Fabrication of multi‐well chips for spheroid cultures and implantable constructs through rapid prototyping techniques

Three‐dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi‐well chips, which were validated using articular chondrocytes (ACs). Multi‐well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type‐II collagen deposition. Moreover, the use of our multi‐well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical‐grade fibrin to generate implantable multi‐well constructs allowing for the precise distribution of multiple cell types. Multi‐well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi‐well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi‐well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre‐implantation samples. In conclusion, multi‐well chips for the generation and culture of multiple cell spheroids can be fabricated by low‐cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment. Biotechnol. Bioeng. 2015;112: 1457–1471.

Meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth

The meniscus plays important roles in knee function and mechanics and is characterized by a heterogeneous matrix composition. The changes in meniscus vascularization observed during growth suggest that the tissue‐specific composition may be the result of a maturation process. This study has the aim to characterize the structural and biochemical variations that occur in the swine meniscus with age. To this purpose, menisci were collected from young and adult pigs and divided into different zones. In study 1, both lateral and medial menisci were divided into the anterior horn, the body and the posterior horn for the evaluation of glycosaminoglycans (GAGs), collagen 1 and 2 content. In study 2, the menisci were sectioned into the inner, the intermediate and the outer zones to determine the variations in the cell phenotype along with the inner–outer direction, through gene expression analysis. According to the results, the swine meniscus is characterized by an increasing enrichment in the cartilaginous component with age, with an increasing deposition in the anterior horn (GAGs and collagen 2; P < 0.01 both); moreover, this cartilaginous matrix strongly increases in the inner avascular and intermediate zone, as a consequence of a specific differentiation of meniscal cells towards a cartilaginous phenotype (collagen 2, P < 0.01). The obtained data add new information on the changes that accompany meniscus maturation, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint.

Dietary Conjugated Linoleic Acids Decrease Leptin in Porcine Adipose Tissue

We investigated the effects of dietary conjugated linoleic acids (CLA) on white adipose tissue (WAT) in heavy pigs. Twelve pigs were assigned to 1 of 2 groups supplemented with either 0 or 0.75% of a CLA preparation (isomeric mixture) and were slaughtered at 159 +/- 2.3 kg live weight. Their subcutaneous WAT was analyzed by both chemical and microanatomical methods. The WAT of CLA-fed pigs tended to have a higher protein content (P = 0.064) and smaller adipocytes (P = 0.053) than that of control (CTR) pigs. The number of proliferating preadipocytes tended to be greater (P = 0.076) in pigs fed CLA, whereas the number of apoptotic adipocytes was greater (P < 0.01) than in CTR pig. Immunohistochemistry revealed that leptin (Ob) expression was lower (P = 0.048) in adipocytes from treated pigs and Western blot quantification of Ob revealed lower levels (P < 0.05) in CLA-fed pigs. The Ob receptor was not affected by dietary CLA supplementation. Tyrosine hydroxylase activity was higher (P < 0.001) in WAT of CLA fed-pigs than in CTR. It is conceivable that the increased noradrenergic activity due to dietary CLA decreases the Ob expression, although it does not diminish the lipid content of WAT, at least in heavy pigs. This article describes the interaction between CLA and Ob in the WAT of heavy pigs and we hypothesize that there is an increased noradrenergic stimulation of lipolysis directly in the target tissue.

Burial of piglet carcasses in cement: a study of macroscopic and microscopic alterations on an animal model.

AbstractScarce experimental data exist describing postmortem effects of burial in cement. The scanty literature presents several case reports, but no experimental study. To perform a pilot study, the following experimental system was designed: 4 piglet corpses, who died of natural causes, were encased in concrete. After 1, 2, 3, and 6 months, a block was opened, and autopsy and microscopic analyses were performed. At the first month, initial putrefaction had started, and hindlegs were partly skeletonized. At the second month, both forelegs and hindlegs were partly skeletonized, and the abdomen and back showed advanced putrefaction. At the third month, the samples showed areas of mummification at the abdomen within a general context of initial putrefaction. At the sixth month, the sample showed wide adipocere formation. Histological findings revealed in some analyzed tissues (epithelium, dermis, adipose, and subcutaneous muscular tissues) a well-defined histological pattern even at 3 months after encasement in concrete: this means that microscopic changes may be delayed in concrete and that it may be worth performing histological analyses even in such kind of decomposed material.

Sex Impact on Tau-Aggregation and Postsynaptic Protein Levels in the P301L Mouse Model of Tauopathy

P301L transgenic (tg) mice well mimic features of human tauopathies and provide a good model for investigating the role of tau in neurodegenerative events. We here analyzed the possible interactions among phosphorylation of tau (p-tau), spine injury, neuronal death, and sex in the P301L mouse model of tauopathy. When compared to control mice (ctr), P301L transgenic mice (tg) presented a lower body weight, reduced survival rate, hyperphosphorylated tau, spine injury, and neuronal loss in both cerebral cortex and hippocampus at 15 months of age. Importantly, we found that pathological features were more pronounced in female than male tg mice. Recent reports underline that tau may be localized within both pre- and post-synaptic compartments, suggesting that it may possibly induce or contribute to synaptic dysfunction. Therefore, we focused our attention on tau localization at dendritic spines. We detected high levels of both tau and p-tau in dendritic spine of P301L transgenic mice. In addition, p-tau correlated with a significant reduction of post-synaptic markers, such as GluN2A, GluN2B, GluA1, GluA2, Drebrin, and PSD-95, in P301L mice. The p-tau levels are higher in female than in male mice, and the increased p-tau was consistent with a proportional decrease in the post-synaptic marker levels analyzed. The P301L-tg females showed a more severe synaptopathy compared to males. Future investigations on the postsynaptic role of p-tau will be necessary to understand its toxic effects and provide insights into new therapeutic targets for maintaining spine integrity, highlighting the importance of tau toxicity as well as the impact of sex on tau-pathology.