Ana Colette Maurício

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.

Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats

In rat gait kinematics, the method most frequently used for measuring hindlimb movement involves placing markers on the skin surface overlying the joints being analyzed. Soft tissue movement around the knee joint has been considered the principle source of error when estimating hindlimb joint kinematics in rodents. However, the motion of knee marker was never quantified, nor the different variations in joint angle associated with this gait analysis system. The purpose of this study was two-fold. The first purpose was to expand upon the limited pool of information describing the effect of soft tissue movement over the knee upon the angular positions of the hip, knee and ankle of rats during treadmill locomotion. Secondly, it was a goal of this study to document the magnitude of the skin displacement when using markers that were attached superficially to the knee joint. This was examined by comparing the hindlimb kinematics in sagittal plane during treadmill locomotion determined from the marker attached to the knee and when the knee position was determined indirectly by computer analysis. Results showed that there is a considerable skin movement artefact which propagates to knee joint position and hindlimb kinematics estimates. It was concluded that these large errors can decrease data reliability in the research of rat gait analysis.

A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats

The convenience of the motor-driven treadmill makes it an attractive instrument for investigating rat locomotion. However, no data are available to indicate whether hindlimb treadmill kinematic findings may be compared or generalized to overground locomotion. In this investigation, we compared overground and treadmill locomotion for differences in the two-dimensional angular kinematics and temporal and spatial measurements for the hindlimb. Ten female rats were evaluated at the same speed for natural overground and treadmill walking. The walking velocity, swing duration and stride length were statistically indistinguishable between the two testing conditions. Significant differences were found between overground and treadmill locomotion for step cycle duration and stance phase duration parameters. During the stance phase of walking, the angular movement of the hip, knee and ankle joints were significantly different in the two conditions, with greater flexion occurring on the overground. Despite this, the sagittal joint movements of the hindlimb were similar between the two walking conditions, with only three parameters being significantly different in the swing. Hip height and angle-angle cyclograms were also only found to display subtle differences. This study suggests that reliable kinematic measurements can be obtained from the treadmill gait analysis in rats.

Methylprednisolone fails to improve functional and histological outcome following spinal cord injury in rats

Currently, methylprednisolone sodium succinate (MPSS) is the standard treatment following acute spinal cord injury (SCI) as a consequence of the results obtained from the National Acute Spinal Cord Injury Studies. However, many have questioned the efficacy of MPSS because of its marginal effects. Additionally there has been criticism of both study design and statistical interpretation. The functional consequences of experimental SCI have been assessed in many ways. The purpose of this investigation was to determine the effects of MPSS vs. saline solution (SS) following moderate T10 contusion injury in rat. Functional recovery was evaluated using the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale, the inclined plane, the beam walk, footprint analysis and the horizontal ladder. To optimize the precision and accuracy of functional results we examined the locomotion on a treadmill using three-dimensional (3D) analysis. Stereology was used to estimate the amount of damaged tissue. The results of the traditional functional methods showed that administration of the NASCIS dosage of MPSS following acute spinal cord contusion did not lead to any significant differences in the functional recovery of MPSS- vs. SS-treated animals. More importantly, the results of the 3D kinematic showed that the MPSS administration did not affect the flexion/extension of the hip, knee and ankle joints during the step cycle. Finally, stereological results revealed no statistically significant differences between the two experimental groups. Altogether, our results support data previously reported by several authors, suggesting that MPSS does not lead to improved functional outcome following experimental acute SCI.

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.

Surface engineered surgical tools and medical devices

Surface engineered surgical tools and medical devices / , Surface engineered surgical tools and medical devices / , کتابخانه دیجیتال جندی شاپور اهواز

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.