Marta Cecilia Tosca

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.

GMP-compliant culture of human hair follicle cells for encapsulation and transplantation.

Human hair follicle cells, both bulge and dermal papilla cells, were isolated and cultured in a GMP cell factory, in order to obtain an in vitro hair follicle source for encapsulation end transplantation in alopecia regenerative cell therapy. An in vitro model, constituted by organotypic cultures of human skin sample, was set up to simulate the dermal–epidermal interaction between bulge cells and dermal papilla cells, evaluating the possible new follicles formation and the regenerative potentiality of these hair follicle cells. Both the bulge and dermal papilla cells show an excellent cellular proliferation as well as an abundant extracellular matrix production. The immunofluorescence investigation revealed the positivity of both cell lines to CK15 and CD200, whereas both cell lines were negative to CD71 and Oct-4. The pool of cultured bulge and dermal papilla cells was injected into the deep dermis; at day 28 of culture, some organized areas with a higher cell density can be observed: the cells self-organize into papilla-like lengthened aggregates. In samples in which the follicular cells have been seeded on the dermis surface, an epidermis-like homogeneous monolayer on the dermis surface can be seen, therefore showing a potentiality of these cells for epidermis regeneration. These data show the efficacy of a cellular isolation and amplification approach to obtain an in vitro human hair follicle regenerative source on industrial scale in a GMP cell factory. The results also proved an intrinsic potentiality of follicular cells to in vitro recreate the epidermis for tissue engineering purposes. Thus, it is feasible to produce bioengineered hair follicles in a GMP cell factory, for encapsulation and transplantation in alopecic patients.

Local biological effects of adipose stromal vascular fraction delivery systems after subcutaneous implantation in a murine model

The aim of this study was to test alginate beads and silk fibroin non-woven mats as stromal vascular fraction delivery systems to support cell implantation for tissue repair and regeneration, through trophic and immunomodulant paracrine signaling. Furthermore, in vivo scaffold biocompatibility was histologically analyzed in a murine model at different time endpoints, with particular focus on construct-induced vascularization and neoangiogenesis. The fibroin mat induced a typical foreign body reaction, recruiting macrophages and giant cells and concurrently promoted neovascularization of the implanted construct. Conversely, alginate beads triggered a more circumscribed, chronic inflammatory reaction, which decreased over time. The combined in vivo implantation of alginate beads and fibroin mat with stromal vascular fraction promoted vascularization and integration of scaffolds into the surrounding subcutaneous environment. The new blood vessel ingrowth should, hopefully, support engineered cell viability and functionality, as well as the transport of soluble bioactive molecules. Due to their neovascularization properties, stromal vascular fraction administration, using alginate or fibroin scaffolds, is a new, promising, cost-effective tissue engineering approach.

Stromal Vascular Fraction Loaded Silk Fibroin Mats Effectively Support the Survival of Diabetic Mice after Pancreatic Islet Transplantation

The aim of this study is to assess whether stromal vascular fraction (SVF)-soaked silk fibroin nonwoven mats (silk-SVF) can preserve the functionality of encapsulated pancreatic endocrine cells (alginate-PECs) after transplantation in the subcutaneous tissue of diabetic mice. Silk scaffolds are selected to create an effective 3D microenvironment for SVF delivery in the subcutaneous tissue before diabetes induction: silk-SVF is subcutaneously implanted in the dorsal area of five healthy animals; after 15 d, mice are treated with streptozotocin to induce diabetes and then alginate-PECs are implanted on the silk-SVF. All animals appear in good health, increasing weight during time, and among them, one presents euglycemia until the end of experiments. On the contrary, when PECs are simultaneously implanted with SVF after diabetes induction, mice are euthanized due to suffering. This work clearly demonstrates that silk-SVF creates a functional niche in subcutaneous tissue and preserves endocrine cell survival and engraftment.

Sustained islet allograft function after peritransplant treatment using exenatide with and without everolimus.

Islet transplantation represents now a feasible therapeutical option for selected patients with brittle type 1 diabetes mellitus. Islet graft function has progressively improved over time with a success rate in some centers similar to that of whole pancreas transplantation. The first phases after transplantation appear to be critical. Transplanted islets are exposed to an inflammatory reaction that, together with glucotoxicity in the case of suboptimal glycemic control results in significant reduction of cellular vitality. For these reasons, different protocols of recipient treatment were proposed, such as anti-inflammatory drugs or long-acting glucagon-like peptide 1 analogues to improve insulin secretion and overall metabolic balance. The mammalian target of rapamycin inhibitors in particular have a significant antiinflammatory effect; in comparison to sirolimus, everolimus proved to have a most potent effect, higher bioavailability, and a shorter half-life. Ten patients affected by type 1 diabetes mellitus (7 with brittle diabetes and 3 already under immunosuppression therapy for a previous kidney transplantation) were treated for 1 month after intrahepatic islet allotransplantation with low-dose exenatide 5 μg twice/day and 6 of them with everolimus 3 mg 12 hours before and 12 to 18 hours after the first islet transplantation, too. The immunosuppression therapy included induction with polyclonal ALG (or Basiliximab in

Islet Transplantation in Pediatric Patients: Current Indications and Future Perspectives.

The first islet transplantation in diabetes mellitus was performed more than 20 years ago. Since then, clinical results have progressively improved. Nowadays, islet transplantation can be considered a real therapeutic option after pancreatectomy for painful chronic pancreatitis (autotransplantation) and in selected adult patients affected by type 1 diabetes mellitus (allotransplantation). Better results are mainly due to the advances in the standardization of islet isolation and purification procedures as well as in the pharmacological treatment of recipients. Anti-inflammatory treatments facilitate islet engraftment and prevent metabolic exhaustion and functional β-cell apoptosis; new strategies better control islet graft rejection. As a consequence, islet transplantation activities are no longer confined to few centers only, rather thousands of transplants are now performed all over the world. Many attempts are actually undertaken to find solutions to current problems of islets transplantation, from toxicity of immunosuppressive therapy to the limited engraftment, function and duration. There is general hope that these procedures will offer a safe and feasible therapeutic option for an increasing number of patients suffering from diabetes mellitus, including pediatric patients.

Human Engineered Cartilage and Decellularized Matrix as an Alternative to Animal Osteoarthritis Model

(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening; (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1β to promote cartilage degradation; (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion; furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity; (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.

Human adipose-derived stromal cells as a feeder layer to improve keratinocyte expansion for clinical applications

The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

Long-term Effect of Islet Transplantation on Glycemic Variability:

Islet transplantation has been reported to restore normoglycemia and the overall metabolic control in type 1 diabetes mellitus (DM). In the most experienced centers, islet transplantation clinical outcome is similar to that of the whole pancreas transplantation. Long-term islet transplantation function remains a very interesting matter worth discussing. A progressive islet function decrease was reported, probably due to islet exhaustion. In 5 islet-transplanted patients with at least 3-yr follow-up and still insulin independent, their glycemic control was characterized by a blinded retrospective continuous glucose monitoring system (CGMS). Islet transplantation restored glycemic control and glucose variability. Data were compared with patients in the waiting list. All the parameters of glycemic variability tested had improved significantly in patients who had islet transplantation compared with those patients who were on the waiting list. In conclusion, islet transplantation is able to maintain a proper glucose control and normalize glycemic variability in selected patients. A blinded retrospective CGMS is a useful method to characterize glucose homeostasis deeply in vivo in islet-transplanted patients.