The proximal medial sural nerve biopsy model: a standardised and reproducible baseline clinical model for the translational evaluation of bioengineered nerve guides.

Autologous nerve transplantation (ANT) is the clinical gold standard for the reconstruction of peripheral nerve defects. A large number of bioengineered nerve guides have been tested under laboratory conditions as an alternative to the ANT. The step from experimental studies to the implementation of the device in the clinical setting is often substantial and the outcome is unpredictable. This is mainly linked to the heterogeneity of clinical peripheral nerve injuries, which is very different from standardized animal studies. In search of a reproducible human model for the implantation of bioengineered nerve guides, we propose the reconstruction of sural nerve defects after routine nerve biopsy as a first or baseline study. Our concept uses the medial sural nerve of patients undergoing diagnostic nerve biopsy (≥ 2 cm). The biopsy-induced nerve gap was immediately reconstructed by implantation of the novel microstructured nerve guide, Neuromaix, as part of an ongoing first-in-human study. Here we present (i) a detailed list of inclusion and exclusion criteria, (ii) a detailed description of the surgical procedure, and (iii) a follow-up concept with multimodal sensory evaluation techniques. The proximal medial sural nerve biopsy model can serve as a preliminary nature of the injuries or baseline nerve lesion model. In a subsequent step, newly developed nerve guides could be tested in more unpredictable and challenging clinical peripheral nerve lesions (e.g., following trauma) which have reduced comparability due to the different nature of the injuries (e.g., site of injury and length of nerve gap).

Transplantation of Schwann cells in a collagen tube for the repair of large, segmental peripheral nerve defects in rats.

OBJECT: Segmental nerve defects pose a daunting clinical challenge, as peripheral nerve injury studies have established that there is a critical nerve gap length for which the distance cannot be successfully bridged with current techniques. Construction of a neural prosthesis filled with Schwann cells (SCs) could provide an alternative treatment to successfully repair these long segmental gaps in the peripheral nervous system. The object of this study was to evaluate the ability of autologous SCs to increase the length at which segmental nerve defects can be bridged using a collagen tube. METHODS: The authors studied the use of absorbable collagen conduits in combination with autologous SCs (200,000 cells/µl) to promote axonal growth across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. Control groups were treated with serum only-filled conduits of reversed sciatic nerve autografts. Animals were assessed for survival of the transplanted SCs as well as the quantity of myelinated axons in the proximal, middle, and distal portions of the channel. RESULTS: Schwann cell survival was confirmed at 4 and 16 weeks postsurgery by the presence of prelabeled green fluorescent protein-positive SCs within the regenerated cable. The addition of SCs to the nerve guide significantly enhanced the regeneration of myelinated axons from the nerve stump into the proximal (p < 0.001) and middle points (p < 0.01) of the tube at 4 weeks. The regeneration of myelinated axons at 16 weeks was significantly enhanced throughout the entire length of the nerve guide (p < 0.001) as compared with their number in a serum-only filled tube and was similar in number compared with the reversed autograft. Autotomy scores were significantly lower in the animals whose sciatic nerve was repaired with a collagen conduit either without (p < 0.01) or with SCs (p < 0.001) when compared with a reversed autograft. CONCLUSIONS: The technique of adding SCs to a guidance channel significantly enhanced the gap distance that can be repaired after peripheral nerve injury with long segmental defects and holds promise in humans. Most importantly, this study represents some of the first essential steps in bringing autologous SC-based therapies to the domain of peripheral nerve injuries with long segmental defects.

Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells.

To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.

Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study.

Periodontal-ligament-derived cells (PDL cells) have stem-cell-like properties and, when implanted into periodontal defects in vivo, can induce periodontal regeneration including the formation of new bone, cementum, and periodontal ligament. We have previously demonstrated that PDL cell sheets, harvested from temperature-responsive cell culture dishes, have a great potential for periodontal regeneration. The purpose of this study has been to validate the safety and efficacy of human PDL (hPDL) cell sheets for use in clinical trials. hPDL tissues from three donors were enzymatically digested, and the obtained cells were cultured with media containing autologous serum in a cell-processing center (CPC). The safety and efficacy of hPDL cell sheets were evaluated both in vitro and in vivo. In vitro studies showed that the hPDL cell sheets had high alkaline phosphatase activity and periostin expression (known PDL markers) and no contamination with microorganisms. In vivo studies revealed that hPDL cell sheets, implanted with dentin blocks, induced the formation of cementum and PDL-like tissue in immunodeficient mice. The hPDL cells presented no evidence of malignant transformation. Thus, hPDL cell sheets created in CPCs are safe products and possess the potential to regenerate periodontal tissues.

Ultrasound-stimulated peripheral nerve regeneration within asymmetrically porous PLGA/Pluronic F127 nerve guide conduit.

Recently, we developed a novel method to fabricate a nerve guide conduit (NGC) with asymmetrical pore structure and hydrophilicity using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method. From the animal study using a rat model (sciatic nerve defect of rat), we recognized that the unique PLGA/Pluronic F127 tube provided good environments for nerve regeneration. In this study, we applied low-intensity pulsed ultrasound as a simple and noninvasive stimulus at the PLGA/F127 NGC-implanted site transcutaneously in rats to investigate the feasibility of ultrasound for the enhanced nerve regeneration through the tube. The nerve regeneration behaviors within the ultrasound-stimulated PLGA/Pluronic F127 NGCs were compared with the NGCs without the ultrasound treatment as well as normal nerve by histological and immunohistochemical observations. It was observed that the PLGA/Pluronic F127 tube-implanted group applied with the ultrasound had more rapid nerve regeneration behavior (approximately 0.71 mm/day) than the tube-implanted group without the ultrasound treatment (approximately 0.48 mm/day). The ultrasound-treated tube group also showed greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the ultrasound treatment, indicating better nerve regeneration. The better nerve regeneration behavior in the our NGC/ultrasound system may be caused by the synergistic effect of the asymmetrically porous PLGA/Pluronic F127 tube with unique properties (selective permeability, hydrophilicity, and structural stability, which can provide good environment for nerve regeneration) and physical stimulus (stimulation of the Schwann cells and activation of the neurotrophic factors).

Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes.

OBJECTIVE: The accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single-lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons. METHODS: The simultaneous tracing of the tibial and peroneal nerves with fast blue and diamidino yellow was performed 8 weeks after the repair of a 1-cm nerve gap in the rat sciatic nerve to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with diamidino yellow 1 week before repair and fast blue 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons. RESULTS: In the cases in which there was successful regeneration across single-lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single-lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair. CONCLUSION: Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis for the development of a nerve tube for motor nerve repair.

Regeneración tisular guiada. Bases biológicas y clínicas. Parte II: fase de mantenimiento, fundamentos, establecimiento y protocolo de funcionamiento / Guided tissue regeneration. Biological and clinical bases. Part II: maintenance phase: fundamentals, establishment and functioning protocol

La fase de mantenimiento es una parte integral de toda terapia odontológica. Los pacientes deben ser informados del proceso de la enfermedad, alternativas terapéuticas, complicaciones potenciales, resultados esperados y su responsabilidad en el tratamiento. Su no realización resultará en recurrencia y progresión de enfermedades. Desafortunadamente todas estas recomendaciones no se están implementando en los consultorios, clínicas e IPS odontológicas. El propósito de este artículo es proporcionar los fundamentos para el establecimiento de una fase integral de mantenimiento y su protocolo de funcionamiento.

Regeneración tisular guiada: bases biológicas y clínicas / Guided tissue regeneration: biological and clinical bases

Esta revisión de la literatura sobre un tema complejo para los estudiantes y el odontólogo general porporcionará conocimientos más claros que le permitirán entender, tomar decisiones, orientar a los pacientes y finalmente remitirlos al especialista para la realización de terapias regenerativas. El entendimiento de la regeneración tisular guiada y la incorporación de procedimientos para lograrla en la práctica clínica ha cambiado significativamente. Es así como algunos conceptos que se utilizaron en el pasado son reevaluados actualmente, como el uso de acondicionadores de tejidos. Otros son cuestionados y revisados en la literatura constantemente, con el fin de ofrecerle al clínico opciones adecuadas para implementar en los pacientes y lograr resultados exitosos a largo plazo, clínica e histológicamente. La regeneración de tejidos es un fenómeno complejo que sucede en una deliberada y ordenada secuencia de eventos que resultan en la formación de cemento, ligamento periodontal y hueso. Diferentes señales pueden ser usadas para estimular la producción tisular, incluyendo mensajes mitogénicos y factores de diferenciación en la obtención de tejidos duros y blandos. La cadena de eventos celulares incluye quimiotaxis, proliferacion, diferenciación y angiogénesis que conducen a la posterior formación tisular. Mientras existe una sólida racionalización para el uso de una gran variedad de factores de crecimiento y adhesión en regeneración de tejidos bucales, únicamente un pequeño número están siendo evaluados clínicamente. Muchos tipos de terapias han fallado en evaluaciones preclínicas o han resultado en una limitada capacidad regenerativa.

Enhanced bone regeneration and formation around implants using guided bone regeneration.

This study investigated the use of a prototype expanded polytetrafluoroethylene membrane attached to bone with butyl-cyanoacrylate, in facilitating guided bone regeneration into bone defects and around titanium screws in rabbit femora. Two experimental models were used to assess bone growth. The first model investigated two unicortical defects in each femora. The second was bone growth in a 500-micron engineered space around one transcortical titanium screw. In the first model there was a significant increase in bone formation at 1 and 2 months in the membrane groups (p < 0.01) as compared to the controls. In the second model the percentage of bone in contact with the implant was significant at 1 and 2 months in the defects covered with membrane compared to the uncovered defects. The uncovered defects had fibrous tissue adherent to the implant continuous with the overlying soft tissue. Our study demonstrated three points: this membrane can be used to increase bone regeneration into defects, this technique allows bone to grow directly around an implant, and butyl-cyanoacrylate can be used in deep soft tissue and bone applications without any apparent deleterious effects.