Ana Lúcia Luís

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.

MicroRNA profile: a promising ancillary tool for accurate renal cell tumour diagnosis

Background:Renal cell tumours (RCTs) are clinically, morphologically and genetically heterogeneous. Accurate identification of renal cell carcinomas (RCCs) and its discrimination from normal tissue and benign tumours is mandatory. We, thus, aimed to define a panel of microRNAs that might aid in the diagnostic workup of RCTs.Methods:Fresh-frozen tissues from 120 RCTs (clear-cell RCC, papillary RCC, chromophobe RCC (chRCC) and oncocytomas: 30 cases each), 10 normal renal tissues and 60 cases of ex-vivo fine-needle aspiration biopsies from RCTs (15 of each subtype validation set) were collected. Expression levels of miR-21, miR-141, miR-155, miR-183 and miR-200b were assessed by quantitative reverse transcription–PCR. Receiver operator characteristic curves were constructed and the areas under the curve were calculated to assess diagnostic performance. Disease-specific survival curves and a Cox regression model comprising all significant variables were computed.Results:Renal cell tumours displayed significantly lower expression levels of miR-21, miR-141 and miR-200b compared with that of normal tissues, and expression levels of all miRs differed significantly between malignant and benign RCTs. Expression analysis of miR-141 or miR-200b accurately distinguished RCTs from normal renal tissues, oncocytoma from RCC and chRCC from oncocytoma. The diagnostic performance was confirmed in the validation set. Interestingly, miR-21, miR-141 and miR-155 expression levels showed prognostic significance in a univariate analysis.Conclusion:The miR-141 or miR-200b panel accurately distinguishes RCC from normal kidney and oncocytoma in tissue samples, discriminating from normal kidney and oncocytoma, whereas miR-21, miR-141 and miR-155 convey prognostic information. This approach is feasible in fine-needle aspiration biopsies and might provide an ancillary tool for routine diagnosis.

Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair

Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

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.

Studies on the biocompatibility of bacterial cellulose

Bacterial cellulose was functionalized with a chimeric protein containing a cellulose-binding module and the adhesion peptide Arg-Gly-Asp. Small-diameter bacterial cellulose membranes were produced and subcutaneously implanted in sheep for 1–32 weeks. The implants triggered a biological response similar to other high surface-to-volume implants. There were no significant differences in the inflammation degree between the bacterial cellulose coated with the recombinant protein Arg-Gly-Asp–cellulose-binding module and the native bacterial cellulose. The implants were considered to be mildly irritating to the tissue compared to the negative control sample (expanded polytetrafluoroethylene). The analysis of the fluorescence microscopy revealed that, apart from increasing cell adhesion, the presence of Arg-Gly-Asp stimulated an even cell distribution, while the cells on the untreated bacterial cellulose seemed to form aggregates. Furthermore, the cells on the Arg-Gly-Asp–treated bacterial cellulose presented a more elongated morphology. Mechanical tests indicated that the small-diameter bacterial cellulose tubes were more elastic than the human arteries and veins.

Biocompatibility and hemocompatibility of polyvinyl alcohol hydrogel used for vascular grafting--In vitro and in vivo studies.

Polyvinyl alcohol hydrogel (PVA) is a synthetic polymer with an increasing application in the biomedical field that can potentially be used for vascular grafting. However, the tissue and blood-material interactions of such gels and membranes are unknown in detail. The objectives of this study were to: (a) assess the biocompatibility and (b) hemocompatibility of PVA-based membranes in order to get some insight into its potential use as a vascular graft. PVA was evaluated isolated or in copolymerization with dextran (DX), a biopolymer with known effects in blood coagulation homeostasis. The effects of the mesenchymal stem cells (MSCs) isolated from the umbilical cord Whartons jelly in the improvement of PVA biocompatibility and in the vascular regeneration were also assessed. The biocompatibility of PVA was evaluated by the implantation of membranes in subcutaneous tissue using an animal model (sheep). Histological samples were assessed and the biological response parameters such as polymorphonuclear neutrophilic leucocytes and macrophage scoring evaluated in the implant/tissue interface by International Standards Office (ISO) Standard 10993-6 (annex E). According to the scoring system based on those parameters, a total value was obtained for each animal and for each experimental group. The in vitro hemocompatibility studies included the classic hemolysis assay and both human and sheep bloods were used. Relatively to biocompatibility results, PVA was slightly irritant to the surrounding tissues; PVA-DX or PVA plus MSCs groups presented the lowest score according to ISO Standard 10993-6. Also, PVA was considered a nonhemolytic biomaterial, presenting the lowest values for hemolysis when associated to DX.

Perspectives of employing mesenchymal stem cells from the Wharton's jelly of the umbilical cord for peripheral nerve repair.

Mesenchymal stem cells (MSCs) from Whartons jelly present high plasticity and low immunogenicity, turning them into a desirable form of cell therapy for the injured nervous system. Their isolation, expansion, and characterization have been performed from cryopreserved umbilical cord tissue. Great concern has been dedicated to the collection, preservation, and transport protocols of the umbilical cord after the parturition to the laboratory in order to obtain samples with higher number of viable MSCs without microbiological contamination. Different biomaterials like chitosan-silicate hybrid, collagen, PLGA90:10, poly(DL-lactide-ɛ-caprolactone), and poly(vinyl alcohol) loaded with electrical conductive materials, associated to MSCs have also been tested in the rat sciatic nerve in axonotmesis and neurotmesis lesions. The in vitro studies of the scaffolds included citocompatibility evaluation of the biomaterials used and cell characterization by imunocytochemistry, karyotype analysis, differentiation capacity into neuroglial-like cells, and flow cytometry. The regeneration process follow-up has been performed by functional analysis and the repaired nerves processed for stereological studies permitted the morphologic regeneration evaluation. The MSCs from Whartons jelly delivered through tested biomaterials should be regarded a potentially valuable tool to improve clinical outcome especially after trauma to sensory nerves. In addition, these cells represent a noncontroversial source of primitive mesenchymal progenitor cells, which can be harvested after birth, cryogenically stored, thawed, and expanded for therapeutic uses. The importance of a longitudinal study concerning tissue engineering of the peripheral nerve, which includes a multidisciplinary team able to develop biomaterials associated to cell therapies, to perform preclinical trials concerning animal welfare and the appropriate animal model is here enhanced.

MSCs CONDITIONED MEDIA AND UMBILICAL CORD BLOOD PLASMA METABOLOMICS AND COMPOSITION

Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.

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 Whartons 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 cm2 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 Whartons 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.