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Blood cells and their role in regeneration. II. Effects of putative immunological manipulations on circulating blood cell counts during regeneration.

The participation of leukocytes in regeneration was studied by determining changes in circulating leukocyte counts following putative immunological manipulations. Splenectomy failed to produce leukopenia during regeneration, although a 24-35% reduction in leukocytes occurred in otherwise intact newts. Bovine serum albumin and anti-lymphocyte serum produced initial lympho- and granulocytopenias, but blood counts soon returned to more normal levels. Lymphocytosis followed treatment with cobra venom factor, but marked lymphopenia occurred shortly thereafter. Regeneration occurred in all cases. These data failed to establish a clear correlation between the nature of quantitative changes in circulating leukocyte levels following these treatments and regenerative capacity.

Animals↗

[Comparative study of the radiosensitivity of normal and regenerating tissues. 1. Interphase cell death and degree of aplasia of regenerating and normal bone marrow and spleen tissues in C57Bl mice].

A comparative study of radiosensitivity of cells of normal and regenerating tissues of bone marrow and spleen has demonstrated that single exposure to X-rays produces a lesser damaging effect on regenerating tissues than on normal ones. The data obtained indicate that the increase in radioresistance of the organism during active regeneration of the haemopoietic organs is due not merely to the increase in the dividing cell pool of these organs but also to qualitative changes in their functional state.

Animals↗

Regeneration of bovine pancreatic ribonuclease A. 2. Kinetics of regeneration.

Analysis of the experimental data of the previous paper [Rothwarf, D. M., & Scheraga, H. A. (1993) Biochemistry (first of four papers in this issue)], using the method of Konishi et al. [Konishi, Y., Ooi, T., & Scheraga, H. A. (1981) Biochemistry 20, 3945-3955; Konishi, Y., Ooi, T., & Scheraga H. A. (1982) Biochemistry 21, 4734-4740], and a discussion of the validity of the steady-state kinetic treatment of the data analyzed here are presented. The analysis reveals that RNase A regenerates with oxidized and reduced dithiothreitol (DTTox and DTTred, respectively) through a rearrangement pathway involving one or more three-disulfide species; i.e., multiple pathways could be involved. This pathway is different from that observed when RNase A is regenerated with oxidized and reduced glutathione (GSSG and GSH, respectively). These differences result primarily from the very different characteristics of the oxidation of thiols with DTTox and GSSG, respectively. In addition, the concept of multiple pathways, as applied to the regeneration of RNase A, is developed.

Animals↗

Isolation and regeneration of protoplasts from the yeast and mycelial form of the dimorphic zygomycete Benjaminiella poitrasii: role of chitin metabolism for morphogenesis during regeneration.

Experimental parameters for isolation and regeneration of protoplasts from the mycelial and yeast form cells of the dimorphic zygomycete Benjamininiella poitrasii are reported. Using a chitosanase containing preparation from Streptomyces sp. MCl we obtained protoplasts after 5 h incubation with a yield of 2+/-0.3 x 10(6) ml(-1) and 3+/-0.4 x 10(7) ml(-1) for the mycelial and yeast form, respectively. During regeneration under conditions triggering dimorphism the two morphological forms were observed after 36 h. Initially, for 10-12 h only an irregular mass was formed as a result of deregulated cell wall synthesis. Among the tested inhibitors influencing cell wall metabolism, chitin metabolism inhibitors showed distinctive effects on the regeneration of protoplasts suggesting that the respective enzymes significantly contribute to determining the morphogenesis of the dimorphic fungus B. poitrasii.

Acetylglucosaminidase↗

[Variations in the completeness of skin regeneration during post-traumatic regeneration].

The healing of the full-thickness skin wounds on the abdomen and the back of hedgehogs was investigated. The wound healing on the abdomen proved to result mainly from the wound contraction. In most cases a small regenerated area containing new hair and sebaceous glands was formed in the centre of the wound. Contraction of the wound was practically absent on the back and its healing occurred chiefly by new tissue formation; due to this vast regenerated areas were formed in the wound. Connective tissue frame-work of the regenerated area on the abdomen and the back resembled intact derma by fiber distribution.

Animals↗

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.

Animals↗

Effect of polylactide/glycolide (PLGA) membranes loaded with metronidazole on periodontal regeneration following guided tissue regeneration in dogs.

BACKGROUND: Bioabsorbable membranes have been successfully used for guided tissue regeneration (GTR) and local delivery systems because they are biocompatible and do not require second surgery for removal. Several studies have demonstrated that metronidazole, when applied topically, produced immediate effects on microbiological and clinical parameters, most notably a reduction in probing depth and loss of attachment. The purpose of this study was to evaluate the regenerative potential of a metronidazole-loaded biodegradable (polylactide/glycolide) (PLGA) GTR membrane in dogs. METHODS: Six male adult dogs with 36 created osseous defects were enrolled. Bilateral dehiscence type defects in 5 mm diameter were created at buccal aspect of the alveolar bone in maxillary premolar teeth. After full thickness flap elevation, exposed root surfaces were thoroughly planed. In the experimental sites, PLGA membranes with or without metronidazole were fitted and placed over the defects. On the control defects only root planing was performed. Gingival flaps were replaced slightly coronal to the cemento-enamel junction. Animals were sacrificed at 60 days. The histometric analysis was evaluated with the following parameters: defect height (DH), apical extension of junctional epithelium (AEJE), new cementum height (NCH), new bone height (NBH), and new gingival connective tissue height (NCTH). RESULTS: Postoperative clinical healing was similar in the 3 groups. There were no statistically significant differences between the 2 experimental groups in any parameters. Statistically significant differences were observed for AEJE, NCH, NBH, and NCTH in experimental groups when compared with the controls. Statistically significantly greater NCH, NBH, and NCTH were seen in the experimental groups than the controls and control defects showed longer AEJE than the experimental defects. CONCLUSIONS: These results suggest that PLGA membranes with and without metronidazole may have a beneficial effect on periodontal regeneration.

Absorbable Implants↗

Using absorbable collagen membranes for guided tissue regeneration, guided bone regeneration, and to treat gingival recession.

This article reviews the role of barrier membranes in guided tissue regeneration (GTR) and guided bone regeneration (GBR), including the advantages of using absorbable barrier membranes in GTR and GBR and the unique properties of collagen membranes. The indications and contraindications for using collagen membranes for these procedures are examined, and successful cases are presented. Finally, the role of collagen membranes in the future of regenerative therapy is considered.

Absorbable Implants↗

Scanning electron microscopy of the normal and denervated limb regenerate in the newt, Notophthalmus, including observations on embryonic amphibia limb-bud mesenchyme and blastemas of fish-fin regenerates.

The present work deals with the morphology of normal and denervated early limb regenerates in the newt and compares them with early limb-bud mesenchyme and blastemas of fish-fin regenerates using the three-dimensional properties of the SEM. It shows that blastemal cells are polymorphic, in contrast to those of the stump-cell population, which are fairly homogeneous and from which blastema cells are said to arise by "dedifferentiation." The appearance of cells had not changed by two days following denervation even though previous biochemical studies have detected many changes in metabolism. Longterm denervation (7 days) did result in morphological changes. A comparison between well-known cell types-myoblasts, Schwann cells, osteoblasts observed in vitro, limb-bud mesenchyme and mesenchymatous fin blastema-was carried out in order to suggest explanations for the blastema-cell polymorphism.

Amphibians↗

Trajectories of regenerating retinal axons in the goldfish tectum: II. Exploratory branches and growth cones on axons at early regeneration stages.

HRP was applied to small sites in the dorsotemporal or dorsonasal retina in fish at 10-36 days after optic nerve section. The anterogradely labeled axons were visualized in tectal whole mounts. Axons traveled through all regions of the tectum in various abnormal routes. Misrouted axons were also seen to alter their orientation and to direct their course toward their target. At all regeneration stages the majority of dorsotemporal axons coursed and achieved target-related orientations preferentially within the rostral tectal half whereas dorsonasal axons proceeded into the caudal tectum. The growing axons exhibited various morphologies. All axons in the superficial fascicle layer stratum opticum (SO) and some in the synaptic layer stratum fibrosum et griseum superficiale (SFGS) were unbranched and tipped with a leading growth cone. Other axons in the synaptic layer carried one to several growth cones at their ends and often filopodia proximal to the growth cone, or they had sprouted numerous side branches with growth cones and filopodia on the shaft and on branches. Some axons at retinotopic or ectopic sites gave rise to several long branches of several hundred microns in length, with growth cones and filopodia. From 32 days onward axons ending in terminal arbors at retinotopic sites became apparent. Thus, numerous axons at early regeneration stages go through a phase of exploratory growth on their way toward their target sites.

Animals↗

beta-Endorphins (beta-EP) in amphibians: higher beta-EP levels during regenerating stages of anuran life cycle and immunocytochemical localization of beta-EP in regeneration blastemata.

Plasma beta-endorphin (beta-EP) levels were measured at different stages of metamorphosis in anuran species, namely Rana catesbeiana, R. pipiens, and Xenopus laevis. The mean levels of beta-EP in tadpoles and postmetamorphic juveniles were significantly higher than adult levels in all three species examined. The pattern of high tadpole levels and low adult levels corresponds with the limb regeneration ability of the tadpole and restriction in the adult. We have also reported plasma beta-EP levels in two urodele species, Ambystoma tigrinum and A. jeffersonianum. Localization of beta-EP by immunocytochemistry shows bright fluorescence to beta-EP antiserum in the wound epithelium of the regeneration blastema. The mesoderm and nuclei of all blastema cells remain nonreactive. It is suggested that receptors to beta-EP reside in the epidermis.

Ambystoma↗

Regenerating liver-specific transacting factors of H2B histone gene are newly synthesized during liver regeneration.

We have investigated DNA synthesis and levels of H2B histone mRNA, and the binding pattern of nuclear proteins to various elements in the rat H2B histone gene upstream region with DNase I footprinting assay. Both DNA synthesis and H2B histone mRNA level were increased with maximal stimulation reached at 24 hrs and 36 hrs after partial hepatectomy, respectively. In DNase I footprinting analysis, the nuclear factors interacting with the three elements, TATA at -19 bp (site AR), site B at -29 bp, and CAAT at -69 bp (site C) were required during maximal increase of H2B histone mRNA level after partial hepatectomy. The DNase I protection pattern by nuclear extract of the cycloheximide-treated regenerating liver showed the same results with normal liver. These results suggest that transcriptional regulation of H2B histone gene during liver regeneration may be mediated by nuclear factors that are newly induced by partial hepatectomy.

Animals↗

Plant regeneration from callus of apomictic and sexual lines of Paspalum simplex and RFLP analysis of regenerated plants.

Culture conditions have been established for the induction of callus from different explants of Paspalum simplex. Fast-growing calli were obtained from hypocotyls and roots excised from 5-day-old seedlings on culture medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Rapid plant regeneration from both apomictic and sexual lines was achieved when the medium was supplemented with alpha-naphthaleneacetic acid and benzylaminopurine. Restriction fragment length polymorphism analysis of the apomixis-controlling region of the regenerated plants showed an absence of restriction site variation for the loci analysed, whereas various degrees of variation were detected for the DNA methylation sites of the same loci.

Adenine↗

Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions.

To examine the question of whether or not prevention of axonal regrowth after injury affects the molecular responses of neurons to axotomy, Northern blotting and in situ hybridization were used to study changes in the mRNA levels of neurofilament (NF) proteins and tubulins in rat dorsal root ganglion (DRG) cells. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration-permissive condition) or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration-prohibitive condition). At 14 days post-injury, the relative levels of the low (NF-L) and middle (NF-M) molecular weight NF protein mRNAs, as well as those of beta II- and beta III-tubulin, were examined in the L4 and L5 DRG. The data showed that the levels of NF-L and NF-M mRNAs decreased while beta II- and beta III-tubulin mRNA levels increased in the DRG after either crush axotomy or cut/ligation axotomy of the sciatic nerve, suggesting that the elicitation of these molecular changes by axon disconnection is independent of the ultimate success or failure of the axonal regrowth process. However, cut/ligation axotomy had a more pronounced effect than did crush injury on the mRNA changes. This result suggests that feedback mechanisms from regrowing axons are important in regulating the extent of the cytoskeletal mRNA changes in injured neurons.

Animals↗

Dental nerve regeneration in rats. II. Autoradiographic studies of nerve regeneration to molar pulp and dentin.

The autoradiographic technique was used to analyze the degeneration and regeneration of sensory nerves to rat molars and gingiva following cut or crush injury to the right inferior alveolar nerve. At 2 days after nerve injury there was almost complete denervation of the first molar, partial denervation of the second molar, and minimal effect on the innervation to the third molar and gingiva. The degree of sensory deficit and recovery for these same rats had been previously determined. Reinnervation of the first molar was analyzed in terms of axon number and location, intensity of axon labeling, and type of nerve injury. At 6 days, neither the cut injury nor crush injury rats had any reinnervation of their first molars. By 7 days, 3 of 4 rats had axons reinnervating first molars; in those teeth there was approximately one-fourth of the normal number of axons in the pulp, and very few axons in the dentin. These rats still had as large a molar sensory deficit as the 7 day rat and 6 day rats that had no reinnervation. By 3 weeks there were one-half to three-fourths of the normal axon numbers in the pulp, one-fourth to one-half of the normal axon numbers in dentin; and sensitivity was at least half-recovered. By 6 weeks, numbers of axons in the pulp and dentin were either normal or slightly less than normal; axons had grown back into dentin to the same depth as in normal teeth; and complete recovery of sensitivity had occurred. The regenerating axons had greater than normal labeling intensity at 1 week and 3 weeks in all rats. Those with the crush nerve injury had somewhat greater numbers of reinnervating axons at 1 week and 3 weeks than the cut injury rats. A structure-function comparison for the molars showed that return of sensitivity correlated with reinnervation of both pulp and dentin.

Alveolar Process↗

Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.

We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules.

Amino Acid Sequence↗

Peripheral nerve regeneration across an 80-mm gap bridged by a polyglycolic acid (PGA)-collagen tube filled with laminin-coated collagen fibers: a histological and electrophysiological evaluation of regenerated nerves.

We evaluated peripheral nerve regeneration across an 80-mm gap using a novel artificial nerve conduit. The conduit was made of a polyglycolic acid (PGA)-collagen tube filled with laminin-coated collagen fibers. Twelve beagle dogs underwent implantation of the nerve conduit across an 80-mm gap in the left peroneal nerve. In four other dogs used as negative controls, the nerve was resected and left unconnected. Histological observation showed that numerous unmyelinated and myelinated nerve fibers, all smaller in diameter and with a thinner myelin sheath than normal nerve fibers, regrew through and beyond the gap 12 months after implantation. The distribution of the regenerated axonal diameters was different from that of the normal axonal diameters. Compound muscle action potentials, motor evoked potentials, and somatosensory evoked potentials were recorded in most animals 3 months after implantation. Peak amplitudes and latencies recovered gradually, which indicating the functional establishment of the nerve connection with the target organs. In addition to the ordinary electrophysiological recoveries, potentials with distinct latencies originating from Aalpha, Adelta and C fibers became distinguishable at the 6th lumbar vertebra following stimulation of the peroneal nerve distal to the gap 12 months after implantation. The pattern of walking without load was restored to almost normal 10-12 months after implantation. Neither electrophysiological nor histological restoration was obtained in the controls. Our nerve conduit can guide peripheral nerve elongation and lead to favorable functional recovery across a wider nerve gap than previously reported artificial nerve conduits.

Animals↗

Electrical stimulation accelerates and enhances expression of regeneration-associated genes in regenerating rat femoral motoneurons.

1. In this study we investigated whether electrical stimulation accelerates the upregulation of Talpha1-tubulin and GAP-43 (regeneration-associated genes; RAGs) and the downregulation of the medium-molecular-weight neurofilament (NFM), in concert with stimulation-induced acceleration of BDNF and trkB gene expression and axonal regeneration. 2. Two weeks prior to unilateral femoral nerve transection and suture, fluorogold (Fluorochrome Inc., Denver) or fluororuby (Dextran tetramethylrhodamine, Mol. Probes, D-1817, Eugene, OR) was injected into quadriceps muscles of the left and right hindlimbs to label the femoral motoneuron pools as previously described. Over a period of 7 days, fresh spinal cords were processed for semiquantitation of mRNA by using in situ hybridization. 3. There was an increase in Talpha1-tubulin and GAP-43 mRNA and a decline in the NFM mRNA at 7 days after nerve suture and sham stimulation but not in intact nerves. In contrast, 1-h stimulation of sutured but not intact nerves dramatically accelerated the changes in gene expression: mRNA levels of Talpha1-tubulin and GAP-43 were significantly elevated above control levels by 2 days while NFM mRNA was significantly reduced by 2 days in the sutured nerves. Thereby, the neurofilament/tubulin expression ratio was reduced at 2 days after suture and stimulation, possibly allowing more tubulin to be transported faster into the growing axons to accelerate the elongation rate following stimulation. Importantly, the changes in RAGs and NFM gene expression were delayed relative to the accelerated upregulation of BDNF and trkB mRNA by electrical stimulation. 4. The temporal sequence of upregulation of BDNF and trkB, altered gene expression of RAGs and NFM, and accelerated axonal outgrowth from the proximal nerve stump are consistent with a key role of BDNF and trkB in mediating the altered expression of RAGs and, in turn, the promotion of axonal outgrowth after electrical stimulation.

Animals↗