Daniele Vigo

Adipose-Derived Stem Cell Therapy for Intervertebral Disc Regeneration: An In Vitro Reconstructed Tissue in Alginate Capsules

The degenerative pathologies of the intervertebral disc have a remarkable social impact in the industrialized countries and can provide serious disabilities in the population. The current treatment consists of conservative treatments (such as symptomatic pharmacological therapies and physiokinetic therapy) and surgical treatments (intervertebral fusion, total disc replacement, nucleus pulposus (NP) replacement, or surgical exeresis). Recent advances in cell therapy foresee the possibility of regenerating the damaged disc; the autologous disc tissue can be withdrawn, in vitro regenerated, and re-implanted. The aim of this work was to verify whether autologous adipose-derived adult stem cells can improve the quality of an in vitro reconstructed nucleus pulposus tissue. A three-dimensional (3D) co-culture of NP cells and adipose tissue non-adipocyte fraction cells (nAFs) was assessed in a previously developed alginate 3D culture system following the good manufacturing practice guidelines to ensure patient safety for clinical studies. Morphological investigation of cultured and co-cultured cells was performed using transmission electron microscopy and immunofluorescence for collagen type I, aggrecan, CD90, CD34, and vimentin. Results indicate that co-culture of NP and nAFs improves the quality of the in vitro reconstructed tissue in term of extracellular matrix production and 3D cell organization. Technological resources are available for NP cell encapsulation intended for regenerating the intervertebral disc.

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 °C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities.

Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFNγ, while no effects were observed on TNFα and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use.

Boar semen controlled delivery system: storage and in vitro spermatozoa release

Swine spermatozoa were encapsulated in barium alginate and protamine-barium alginate membranes to lengthen their preservation time and to provide a means of controlling their release. Precocious acrosome reactions and secondary anomalies were measured as indices of semen quality. These characteristics were observed for two forms of encapsulated spermatozoa when stored at 18 and 38 degrees C for 24 h and for semen diluted in a classical extender at both temperatures. The results indicate that encapsulation enhances semen preservation, providing protection against membrane damage upon dilution. The effect is even more evident at the higher temperature (38 degrees C), where cell metabolism is higher. An in vitro release test of spermatozoa showed a massive cell delivery from barium alginate capsules within 6 h, and a slow release from protamine-barium alginate capsules. The properties of spermatozoa 24 h after release did not differ from the semen stored at the same temperature in capsules, indicating that the release process does not impair semen quality.

Boar semen controlled-delivery system: morphological investigation and in vitro fertilization test

A technology for encapsulation of swine semen in barium alginate and protamine alginate has recently been proposed for the controlled release of the spermatozoa, thus reducing the number of instrumental inseminations required. Controlled-release capsules containing swine spermatozoa were prepared by adding saturated BaCl2 solution to ejaculate and dropping the resulting suspension into a sodium alginate solution, leading to the formation of barium alginate capsules. A second type of capsule was obtained by cross-linking the barium alginate with protamine sulfate. Two types of membrane were thus obtained: barium alginate gel and a protamine cross-linked alginate membrane. Morphological (scanning electron microscopy and transmission electron microscopy), functional (motility, membrane integrity and in vitro fertilization test) and technological (capsule structure and weight) approaches were used to characterize the encapsulated spermatozoa and the controlled-delivery system. No differences in terms of morphological and functional characteristics (acrosome integrity and spermatozoa motility) between free and encapsulated semen were found. The technological process did not compromise in vitro fertilization potency of the spermatazoa, although seasonal variability was found. The capsule weight was related to either the pH of the semen or the season. This study represents the starting point for the development of further investigations into the storage and release kinetics of cells from the capsules and for the development of an in vivo fertilization protocol.

Semen controlled-release capsules allow a single artificial insemination in sows

Controlled-release capsules containing boar spermatozoa were developed to extend the preservation time of spermatozoa and maximize the efficiency of a single artificial insemination. A large trial (4245 sows) was performed with these capsules using double/triple conventional artificial insemination as a control. The effect of treatment on pregnancy diagnosis, delivery, and born piglets was investigated, with allowance being made for considering season, spermatozoa amount, and the weaning-to-estrus interval as confounding variables. The same pregnancy rate and prolificacy were obtained by two insemination techniques, and a higher parturition frequency was reached with capsules. The reproductive performance in pigs has therefore been optimized by a single instrumental insemination with controlled-release capsules.

Statistical approach in alginate membrane formulation for cell encapsulation in a GMP-based cell factory

A cell encapsulation technology in alginate has been developed with the aim of obtaining cell controlled release or three-dimensional cultures. The aim of this work is to verify the predictability of alginate capsules for large-scale production by Good Manufacturing Practice (GMP) standardized procedures in a cell factory. A cell-free capsule model was performed following the GMP guidelines: an opaque agent suspension in a bivalent cation solution (Ca(2+), Ba(2+), Sr(2+)) was dropped in a sodium alginate solution, obtaining capsules presenting a liquid core surrounded by a gel alginate membrane. The concentration of the ion, and the treatment with protamine, can considerably vary the characteristics of the capsules (weight, whole diameter, core diameter, gel capsule thickness, capsule strength). It is therefore possible to optimize the performance of the capsules, relating the molecular structure and size of the polymeric membrane to the desired functional properties. Technological resources are available for large-scale cell encapsulation intended for advanced therapies (gene therapy, somatic cell therapy and tissue engineering) in a cell factory, following GMP guidelines.

Barium alginate cell-delivery systems: correlation between technological parameters

The most recent trends for the development of several in vitro cell cultures have been oriented towards the cell immobilisation in 3-dimensional scaffolds and cell encapsulation. In fact, an important requirement of cell survival is self-assembly in functional communities, in the presence of an artificial extracellular matrix. In our research, a previously described technique for spermatozoa encapsulation was applied to obtain capsules loaded with an opaque agent as a model, and to perform a formulative study. A process variable, barium ion concentration, was correlated to some capsule properties, such as weight, gel thickness, total and core diameter. Ion concentration can be modified to obtain capsules with predictable characteristics.

A dry powder formulation from silk fibroin microspheres as a topical auto-gelling device

Abstract With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.

Two-dimensional polyacrylamide gel electrophoresis map of bovine ovarian fluid proteins.

Molecular mechanisms underlying the cystic degeneration of ovarian follicles in the dairy cow have not been clarified yet. A useful approach for complementing endocrinological and clinical studies could be represented by the systematic analysis of the protein patterns in follicular and cystic fluid. With this aim, a two‐dimensional polyacrylamide gel electrophoresis map of proteins contained in fluid from bovine ovarian follicles at different stages of development and from bovine ovarian cyst has been obtained. About 200 spots were detected after silver staining. Polypeptides from nine spots or series of spots have been identified by N‐terminal sequencing, and further analysis of the map has been performed by gel comparison. Alpha‐1‐antitrypsin, albumin, serotransferrin and apolipoprotein A‐I and A‐IV were located on the map. Comparison between protein patterns revealed the differential expression of some spots among follicles of smaller diameter, follicles of larger diameter, and cysts. This could represent the first step toward the identification of proteins differentially expressed and associated with ovarian cyst development.