Cell therapy with human MSCs isolated from the umbilical cord Wharton jelly associated to a PVA membrane in the treatment of chronic skin wounds.

The healing process of the skin is a dynamic procedure mediated through a complex feedback of growth factors secreted by a variety of cells types. Despite the most recent advances in wound healing management and surgical procedures, these techniques still fail up to 50%, so cellular therapies involving mesenchymal stem cells (MSCs) are nowadays a promising treatment of skin ulcers which are a cause of high morbidity. The MSCs modulate the inflammatory local response and induce cell replacing, by a paracrine mode of action, being an important cell therapy for the impaired wound healing. The local application of human MSCs (hMSCs) isolated from the umbilical cord Wharton's jelly together with a poly(vinyl alcohol) hydrogel (PVA) membrane, was tested to promote wound healing in two dogs that were referred for clinical examination at UPVET Hospital, showing non-healing large skin lesions by the standard treatments. The wounds were infiltrated with 1000 cells/µl hMSCs in a total volume of 100 µl per cm(2) of lesion area. A PVA membrane was applied to completely cover the wound to prevent its dehydration. Both animals after the treatment demonstrated a significant progress in skin regeneration with decreased extent of ulcerated areas confirmed by histological analysis. The use of Wharton's jelly MSCs associated with a PVA membrane showed promising clinical results for future application in the treatment of chronic wounds in companion animals and humans.

Challenges for nerve repair using chitosan-siloxane hybrid porous scaffolds.

The treatment of peripheral nerve injuries remains one of the greatest challenges of neurosurgery, as functional recover is rarely satisfactory in these patients. Recently, biodegradable nerve guides have shown great potential for enhancing nerve regeneration. A major advantage of these nerve guides is that no foreign material remains after the device has fulfilled its task, which spares a second surgical intervention. Recently, we studied peripheral nerve regeneration using chitosan-γ-glycidoxypropyltrimethoxysilane (chitosan-GPTMS) porous hybrid membranes. In our studies, these porous membranes significantly improved nerve fiber regeneration and functional recovery in rat models of axonotmetic and neurotmetic sciatic nerve injuries. In particular, the number of regenerated myelinated nerve fibers and myelin thickness were significantly higher in rat treated with chitosan porous hybrid membranes, whether or not they were used in combination with mesenchymal stem cells isolated from the Wharton's jelly of the umbilical cord. In this review, we describe our findings on the use of chitosan-GPTMS hybrids for nerve regeneration.

Response of human osteoblastic and osteoclastic cells to AH plus and pulp canal sealer containing quaternary ammonium polyethylenimine nanoparticles.

INTRODUCTION: The incorporation of quaternary ammonium polyethylenimine (QPEI) nanoparticles into endodontic sealers induces alterations in their structure and surface properties, which may affect the compatibility with the periapical tissues. This work addressed the behavior of human bone cells exposed to extracts from commercial and QPEI containing AH Plus (DeTrey, Konstanz, Germany) and Pulp Canal Sealer EWT (PCS; Kerr Italia Srl, Salerno, Italy). METHODS: Freshly mixed AH Plus and PCS or containing 2% QPEI (0.3 mL spread over the well bottom of a 24-well plate) were extracted with culture medium (1.5 mL for 24 hours at 37°C) and diluted (1:20-1:5000). Osteoblastic or osteoclastic cells were cultured in the presence of QPEI particles (1%-10%) and were exposed to the extracts from unmodified and QPEI containing sealers. RESULTS: QPEI nanoparticles, at 1% and 2%, did not affect cell behavior. On osteoblastic cells, AH Plus and PCS increased DNA at 1:2500 dilution (levels ≤1:100 were cytotoxic). Alkaline phosphatase activity decreased at dilutions ≤1:500. Comparatively, QPEI containing AH Plus increased DNA at 1:2500 and 1:500 dilutions, and QPEI containing PCS induced ALP activity at 1:2500 and 1:500 dilutions. Regarding osteoclastic cells, DNA increased (AH Plus) or was not affected (PCS) at dilutions up to 1:500 and decreased with more concentrated extracts. Tartrate-resistant acid phosphatase activity decreased with dilutions ≤1:500 for both sealers. QPEI containing sealers presented a similar behavior. The sealers affected some intracellular signaling pathways, and QPEI containing sealers further modulate these mechanisms. CONCLUSIONS: QPEI nanoparticles, at 2%, did not affect cell behavior. However, the incorporation of 2% QPEI particles into AH Plus and PCS modulates the proliferation and differentiation of bone cells, depending on the sealer and the cell type, without increasing the sealers' cytotoxicity.

Hydroxyapatite surface roughness: complex modulation of the osteoclastogenesis of human precursor cells.

It is recognized that the surface roughness affects osteoblastic differentiation, but little information is available regarding its effect on osteoclastogenesis. With this work, the osteoclastogenic behaviour of human peripheral blood mononuclear cells (PBMCs), cultured isolated (1.5×10(6)cellscm(-2)) or co-cultured with human bone marrow cells (hBMCs; 10(3)cellscm(-2)), was assessed on surface-abraded hydroxyapatite disks with three different surface roughnesses (R(a) 0.0437-0.582 µm). Monocultures and co-cultures were performed for 21 days in the absence or presence of recombinant M-CSF and RANKL. Results showed that PBMCs supplemented with M-CSF and RANKL or co-cultured with hBMCs displayed typical osteoclastic features, i.e. multinucleated cells with actin rings, vitronectin and calcitonin receptors, gene expression of TRAP, cathepsin K, carbonic anhydrase 2, c-myc and c-src, TRAP activity and resorbing activity. The osteoclastogenic response increased with surface roughness in PBMCs cultured with M-CSF and RANKL but decreased in PBMCs co-cultured with hBMCs. However, co-cultures supplemented with the osteoclastogenic inducers displayed high and similar levels of osteoclast differentiation in the three tested surfaces. In conclusion, modulation of osteoclast differentiation by surface roughness seemed to be dependent on the mechanisms subjacent to the osteoclastogenic stimulus, i.e. the presence of soluble factors or direct cell-to-cell contacts between osteoblastic and osteoclastic cells.

Synthesis and characterization of chitosan-silicate hydrogel as resorbable vehicle for bonelike-bone graft.

The use of bone grafts is required to restore skeletal integrity and enhance bone healing of large defects in several areas of regenerative medicine, such as: orthopedic and maxillofacial procedures. Some of these bone grafts can be resorbed in a time controlled way, in order to allow the correct process of natural re-construction of the involved bone tissue to occur. The Bonelike graft is a bone substitute that mimics the inorganic composition of bone; this biomaterial was developed and characterized over the last decade. In a granular form, Bonelike has proved its highly bioactive behavior in medical applications, such as; maxillofacial and orthopedics surgery. The clinical applications in maxillary bone defects indicated a good bone bonding between new formed bone and the Bonelike granules. The purpose of this study was to develop a new injectable system for the application of Bonelike using a resorbable vehicle which may be used in minimal invasive surgery. A new hydrogel derived from chitosan and y-glycidoxypropyltrimethoxysilane (GPTMS) was synthesized and characterized. The mixture derived from chitosan and GPTMS existed in sol state at room temperature and formed a hydrogel at 37 degrees C. The degradability of the hydrogel could be controlled by the concentration of chitosan and GPTMS, and the presence the presence of Bonelike did not affect its degradability. The pH changes caused by the degradation of this hydrogel were small, so it is not expected to cause any deleterious effect in vivo conditions.

Use of PLGA 90:10 scaffolds enriched with in vitro-differentiated neural cells for repairing rat sciatic nerve defects.

Poly(lactic-co-glycolic acid) (PLGA) nerve tube guides, made of a novel proportion (90:10) of the two polymers, poly(L-lactide): poly(glycolide) and covered with a neural cell line differentiated in vitro, were tested in vivo for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve. Before in vivo testing, the PLGA 90:10 tubes were tested in vitro for water uptake and mass loss and compared with collagen sheets. The water uptake of the PLGA tubes was lower, and the mass loss was more rapid and higher than those of the collagen sheets when immersed in phosphate-buffered saline (PBS) solution. The pH values of immersing PBS did not change after soaking the collagen sheets and showed to be around 7.4. On the other hand, the pH values of PBS after soaking PLGA tubes decreased gradually during 10 days reaching values around 3.5. For the in vivo testing, 22 Sasco Sprague adult rats were divided into four groups--group 1: gap not reconstructed; group 2: gap reconstructed using an autologous nerve graft; group 3: gap reconstructed with PLGA 90:10 tube guides; group 4: gap reconstructed with PLGA 90:10 tube guides covered with neural cells differentiated in vitro. Motor and sensory functional recovery was evaluated throughout a healing period of 20 weeks using sciatic functional index, static sciatic index, extensor postural thrust, withdrawal reflex latency, and ankle kinematics. Stereological analysis was carried out on regenerated nerve fibers. Both motor and sensory functions improved significantly in the three experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the group where the gap was reconstructed using the autologous graft. The presence of neural cells covering the inside of the PLGA tube guides did not make any difference in the functional recovery. By contrast, morphometric analysis showed that the introduction of N1E-115 cells inside PLGA 90:10 tube guides led to a significant lower number and size of regenerated nerve fibers, suggesting thus that this approach is not adequate for promoting peripheral nerve repair. Further studies are warranted to assess the role of other cellular systems as a foreseeable therapeutic strategy in peripheral nerve regeneration.