Effects of Olfactory Mucosa Stem/Stromal Cell and Olfactory Ensheating Cells Secretome on Peripheral Nerve Regeneration.

Cell secretome has been explored as a cell-free technique with high scientific and medical interest for Regenerative Medicine. In this work, the secretome produced and collected from Olfactory Mucosa Mesenchymal Stem Cells and Olfactory Ensheating Cells was analyzed and therapeutically applied to promote peripheral nerve regeneration. The analysis of the conditioned medium revealed the production and secretion of several factors with immunomodulatory functions, capable of intervening beneficially in the phases of nerve regeneration. Subsequently, the conditioned medium was applied to sciatic nerves of rats after neurotmesis, using Reaxon® as tube-guides. Over 20 weeks, the animals were subjected to periodic functional assessments, and after this period, the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed to confirm the beneficial effects resulting from the application of this therapeutic combination. The administration of conditioned medium from Olfactory Mucosal Mesenchymal Stem Cells led to the best results in motor performance, sensory recovery, and gait patterns. Stereological and histomorphometric evaluation also revealed the ability of this therapeutic combination to promote nervous and muscular histologic reorganization during the regenerative process. The therapeutic combination discussed in this work shows promising results and should be further explored to clarify irregularities found in the outcomes and to allow establishing the use of cell secretome as a new therapeutic field applied in the treatment of peripheral nerves after injury.

Combined Use of Chitosan and Olfactory Mucosa Mesenchymal Stem/Stromal Cells to Promote Peripheral Nerve Regeneration In Vivo.

Peripheral nerve injury remains a clinical challenge with severe physiological and functional consequences. Despite the existence of multiple possible therapeutic approaches, until now, there is no consensus regarding the advantages of each option or the best methodology in promoting nerve regeneration. Regenerative medicine is a promise to overcome this medical limitation, and in this work, chitosan nerve guide conduits and olfactory mucosa mesenchymal stem/stromal cells were applied in different therapeutic combinations to promote regeneration in sciatic nerves after neurotmesis injury. Over 20 weeks, the intervened animals were subjected to a regular functional assessment (determination of motor performance, nociception, and sciatic indexes), and after this period, they were evaluated kinematically and the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed confirming the beneficial effects of using these therapeutic approaches. The use of chitosan NGCs and cells resulted in better motor performance, better sciatic indexes, and lower gait dysfunction after 20 weeks. The use of only NGGs demonstrated better nociceptive recoveries. The stereological evaluation of the sciatic nerve revealed identical values in the different parameters for all therapeutic groups. In the muscle histomorphometric evaluation, the groups treated with NGCs and cells showed results close to those of the group that received traditional sutures, the one with the best final values. The therapeutic combinations studied show promising outcomes and should be the target of new future works to overcome some irregularities found in the results and establish the combination of nerve guidance conduits and olfactory mucosa mesenchymal stem/stromal cells as viable options in the treatment of peripheral nerves after injury.

The sensitivity of two-dimensional hindlimb joint kinematics analysis in assessing functional recovery in rats after sciatic nerve crush.

Walking analysis in the rat is increasingly used to assess functional recovery after peripheral nerve injury. Here we assess the sensitivity and specificity of hindlimb joint kinematics measures during the rat gait early after sciatic nerve crush injury (DEN), after twelve weeks of recovery (REINN) and in sham-operated controls (Sham) using discriminant analysis. The analysis addressed gait spatiotemporal variables and hip, knee and ankle angle and angular velocity measures during the entire walking cycle. In DEN animals, changes affected all studied joints plus spatiotemporal parameters of gait. Both the spatiotemporal and ankle kinematics parameters recovered to normality within twelve weeks. At this time point, some hip and knee kinematics values were still abnormal when compared to sham controls. Discriminant models based on hip, knee and ankle kinematics displayed maximal sensitivity to identify DEN animals. However, the discriminant models based on spatiotemporal and ankle kinematics data showed a poor performance when assigning animals to the REINN and Sham groups. Models using hip and knee kinematics during walking showed the best sensitivity to recognize the reinnervated animals. The model construed on the basis of hip joint kinematics was the one combining highest sensitivity with robustness and high specificity. It is concluded that ankle joint kinematics fails in detecting minor functional deficits after long term recovery from sciatic nerve crush and extending the kinematic analysis during walking to the hip and knee joints improves the sensitivity of this functional test.

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 chitosan scaffolds for repairing rat sciatic nerve defects.

Neurotmesis must be surgically treated by direct end-to-end suture of the two nerve stumps or by a nerve graft harvested from elsewhere in the body in case of tissue loss. To avoid secondary damage due to harvesting of the nerve graft, a tube-guide can be used to bridge the nerve gap. Previously, our group developed and tested hybrid chitosan membranes for peripheral nerve tubulization and showed that freeze-dried chitosan type III membranes were particularly effective for improving peripheral nerve functional recovery after axonotmesis. Chitosan type III membranes have about 110 microm pores and about 90% of porosity, due to the employment of freeze-drying technique. The present study aimed to verify if chitosan type III membranes can be successfully used also for improving peripheral nerve functional recovery after neurotmesis of the rat sciatic nerve. Sasco Sprague-Dawley adult rats were divided into 6 groups: Group 1: end-to-end neurorrhaphy enwrapped by chitosan membrane type III (End-to-EndChitll); Group 2: 10mm-nerve gap bridged by an autologous nerve graft enwrapped by chitosan membrane type III (Graf180degreeChitIII); Group 3: 10 mm-nerve gap bridged by chitosan type III tube-guides (GapChitIII); These 3 experimental groups were compared with 3 control groups, respectively: Group 4: 10 mm-nerve gap bridged by an autologous nerve graft (Graft180degree); Group 5: 10 mm-nerve gap bridged by PLGA 90:10 tube-guides (PLGA); Group 6: end-to-end neurorrhaphy alone (End-to-End). Motor and sensory functional recovery were evaluated throughout a healing period of 20 weeks using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. Regenerated nerves withdrawn at the end of the experiment were analysed histologically. Results showed that nerve regeneration was successful in all experimental and control groups and that chitosan type III tubulization induced a significantly better nerve regeneration and functional recovery in comparison to PLGA tubulization control. Further investigation is needed to explore the mechanisms at the basis of the positive effects of chitosan type III on axonal regeneration.

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.

PLGA 90/10 and caprolactone biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve: 1) one made of PLGA in a novel proportion (90:10) of the two polymers poly(L-lactide):poly(glycolide); 2) another made of (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, by comparing its healing efficacy with that of the more traditional methods of end-to-end nerve suture and autologous graft. Motor and sensory functional recovery were assessed throughout the healing period of 20 weeks, and the repaired nerves were processed for morphological and histomorphometrical analysis. Both motor and sensory functions improved significantly in all experimental nerve repaired groups. At the end of the 20-week follow-up, the end-to-end group showed better recovery of motor function when compared with the groups treated with guiding tubes. However, at this time point, the level of motor function in the Neurolac(R) and PLGA groups was similar to the one of the graft group. Nociception function also recovered faster in the end-to-end group compared with the Neurolac(R) and PLGA groups, and in this case, recovery was also delayed in the graft group. At the end of follow-up, nociception was similar in all experimental groups. Morphological and histomorphometrical analysis showed that axon regeneration occurred in both PLGA and Neurolac(R) experimental groups, with no significant differences in the total number of regenerated fibers, but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterials are a good substrate for preparing tubular nerve guides, and their different pattern of degradation does not seem to influence the degree of nerve regeneration.

Pediatric surgeons and gastroesophageal reflux.

BACKGROUND/AIMS: Better antacid medications and the introduction of laparoscopy destabilize the indications for fundoplication. This study aims at raising a discussion among pediatric surgeons on these indications, modalities, and the results of this operation. MATERIALS AND METHODS: A total of 252 refluxing children operated between 1992 and 2006 were divided into groups according to predominant symptoms (93 digestive, 47 respiratory, and 68 neurologic) or to comorbidities (27 esophageal atresia, 10 diaphragmatic hernia, 5 abdominal wall defects, and 2 caustic stricture), and the indications, complications, mortality, and long-term results were reviewed. Features of open (n = 135) and laparoscopic (n = 117) approaches were compared, and long-term integrity of the wrap was analyzed using the Kaplan-Meier method. RESULTS: Digestive, respiratory, and neurologic patients had more often laparoscopic plications, whereas all others rather had an open approach. The rate of complications was 22%, and they were more frequent in children operated by laparotomy (P < .05). Median follow up was 51.3 months (range, 6-160). Overall wrap integrity was maintained in 89% of the children, and the proportions for digestive, respiratory, and neurologic groups were 95%, 95%, and 87%, respectively. For esophageal atresia, congenital diaphragmatic hernia, abdominal wall defects, and caustic stricture, they were 72%, 77%, 100%, and 0%, respectively. The functional results were fully satisfactory in 83% of patients. There were 17 deaths (6.7%), but only 3 in the first postoperative month and only 1 related to the operation (0.4%). CONCLUSIONS: Fundoplication is a powerful method of reflux control. It is indicated after failure of medical treatment in gastroesophageal reflux disease and in symptomatic refluxers with some particular comorbidities. Surgery should be offered only after diagnosis has been firmly established, and the indications must remain identical for open and laparoscopic procedures. High technical standards and rigorous report of the results are required for keeping a relevant place of pediatric surgery in the treatment of this disease.

Evaluation of two biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides, one of PLGA made of a novel proportion (90:10) of the two polymers, Poly(L-lactide):Poly(glycolide), with (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, in promoting nerve regeneration across a 10 mm-gap of the rat sciatic nerve. Finally, end-to-end coaptation was performed. Motor and sensory functional recovery was assessed throughout the healing period of 20 weeks and the repaired nerves were processed for morphological analysis. Both motor and sensory functions improved significantly in all experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the end-to-end group. No significant differences were detected in the comparison between the two types of tubes. Compatible with results of functional tests, morphological analysis showed that axon regeneration occurred in both PLGA and Neurolac experimental groups but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterial are a good substrate for preparing tubular nerve guides and the different pattern of degradation does not seem to influence the degree of nerve regeneration.

Teratomas of the neck and mediastinum in children.

This retrospective study reviews a series of teratomas of the neck and mediastinum aiming at defining the features of these particular locations. We recorded prenatal diagnosis, perinatal management, clinical and radiologic features, pathology, surgical strategies and results in cervical and mediastinal teratomas treated over the last 10 years. During this period we treated 66 children with teratoma of which 11 (6 male and 5 female) had cervicomediastinal locations. Five babies had cervical teratomas extended into the anterior mediastinum in two cases. Prenatal diagnosis was made in three (two with polyhydramnios). Four babies were born by C-section and only one had a successful EXIT procedure. The diagnosis was confirmed by imaging and increased AFP. Surgical treatment involved total tumor removal and in one case subsequent removal of lymph node metastases. All children survived except one in whom airway could not be cleared at birth. Two children bear mild hypothyroidism. During the same period six patients aged 0-17 years were treated for mediastinal teratoma. Only one was prenatally diagnosed and only two had some dyspnea. Removal was performed either by median sternotomy, thoracotomy, or thoracoscopy. They all survive and are free of disease. Teratomas of the neck may cause fetal disease and unmanageable neonatal airway obstruction. Prenatal diagnosis and planned multidisciplinary management are mandatory at birth. In contrast, only some mediastinal tumors cause respiratory embarrassment. Although benign, these tumors are sometimes immature and may metastasize to regional lymph nodes. Total surgical removal is curative. Thyroid insufficiency may be present at birth in cervical teratomas and may be aggravated by surgery.