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The development of an effective amphotericin B (AmB) formulation to replace actual treatments available for leishmaniasis, which present serious drawbacks, is a challenge. Here we report the development of hyaluronic acid–amphotericin B self-assembled nanocomplexes (HA–AmB), processed by freeze-drying (FD) or nano spray-drying (SD), using a simple process that favors the non-covalent drug–polysaccharide association in an amorphous state. These water-soluble formulations, which presented a nanometric size (300–600 nm), high colloidal stability (zeta potential around −39 mV) and an AmB loading (15–18%) in aggregated and super aggregated states, demonstrated less in vitrocytotoxic and hemolytic effects compared to the free-drug. A significant decrease in the number of intramacrophagic L. infantumamastigotes upon treatment (IC50of 0.026 and 0.030 μM for HA–AmB FD and HA–AmB SD, respectively) was also observed, and the best selectivity index (SI) was observed for the HA–AmB SD nanocomplex (SI of 651). Intravenous administration of the HA–AmB SD nanocomplex for 3 alternate days showed an effective parasite reduction in the spleen and liver of C57BL/6 mice without signs of toxicity commonly observed upon free-AmB treatment. Although lower than that achieved with AmBisome® in the liver, the observed parasite reduction for the nanocomplex was of a similar order of magnitude. The efficacy, stability, safety and low cost of the HA–AmB SD nanocomplex highlight its potential as an alternative treatment for leishmaniasis.

Development of synthetic bone substitutes has arisen as a major research interest in the need to find an alternative to autologous bone grafts. Using an ovine model, the present pre-clinical study presents a synthetic bone graft (Bonelike®) in combination with a cellular system as an alternative for the regeneration of non-critical defects. The association of biomaterials and cell-based therapies is a promising strategy for bone tissue engineering. Mesenchymal stem cells (MSCs) from human dental pulp have demonstrated both in vitroand in vivoto interact with diverse biomaterial systems and promote mineral deposition, aiming at the reconstruction of osseous defects. Moreover, these cells can be found and isolated from many species. Non-critical bone defects were treated with Bonelike®with or without MSCs obtained from the human dental pulp. Results showed that Bonelike®and MSCs treated defects showed improved bone regeneration compared with the defects treated with Bonelike®alone. Also, it was observed that the biomaterial matrix was reabsorbed and gradually replaced by new bone during the healing process. We therefore propose this combination as an efficient binomial strategy that promotes bone growth and vascularization in non-critical bone defects.

Peripheral nerve regeneration following severe events is still a challenging topic in the regenerative medicine field, especially when nerve tissue is lost and direct suturing is not feasible. Given the limited success observed in currently available techniques, researchers have been putting efforts towards the development and optimization of these techniques, aiming at the best recovery chances for affected patients. The present work explores the combination of two methods of synthetic biomaterial tubes functionalization: the effect of electroconductive biomaterials and its association to an active cellular system. A tube-guide comprised of polyvinyl alcohol (PVA) loaded with COOH-functionalized multiwall carbon nanotubes (CNTs) was produced and studied alone or in combination with a cellular system of mesenchymal stem cells (MSCs) isolated from the umbilical cord Wharton's jelly (WJ). Tube-guides were assessed for in vitrocytocompatibility to the WJ MSCs and tested for in vivoperformance in a neurotmesis rodent model. Animals were assigned to either PVA-CNTs, PVA-CNTs-MSCs, graft or end-to-end reconstruction groups and assessed after 20 weeks of regeneration. Structural analysis revealed overall more evident recovery in the PVA-CNTs-MSCs group, with significant differences to the cell-free group and similar to End-to-End repaired and Grafted groups. Surprisingly, PVA-CNTs-MSCs did no benefit neurogenic muscle atrophy recovery. Overall, the electrofucntionalized tube-guides post an interesting option for nerve reconstruction alone or in combination to MSCs cellular systems.

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca2+]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivotesting was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL).

The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.

A 6-year-old Lusitano stallion was referred to our centre due to an enlarged left testicle. Anamnesis indicated that the stallion had a chronic hypertrophy of the left testicle, with no apparent ill effect on work (dressage training) or semen production. Prolonged use of anti-inflammatory drugs (NSAIDs) and antibiotics were probable. Upon examination of the animal, it was found that clinical signs were compatible with chronic testicular degeneration or fibrosis. Ultrasound scanning did not evidence the exuberant macroscopic lesions seen upon hemicastration of the left testicle, but it showed in the left spermatic cord a conspicuous absence of the typical hypoechogenic areas representing the pampiniform plexus. Swabbing of the penis, prepuce, and distal urethra resulted in the isolation of Rhodococcus equi and Corynebacterium spp. However, histopathological examination did not support infectious orchitis as cause of the lesions and no bacterial growth was obtained from swabbing of the parenchyma in the excised testicle. Histopathological findings were compatible with chronic orchitis with fibrosis and necrosis, probably secondary to ischemia of the testicular parenchyma. After hemi-castration, the stallion resumed semen production at acceptable levels.