Regeneration of the Corneal Epithelium after Debridement of its Central Region: An Autoradiographic Study on Rabbits

Purpose: To investigate the proliferative behavior of the corneal and limbal epithelia after debridement on the central region of the rabbit cornea. Methods: After scraping a circular epithelial area, 5 mm in diameter, in the center of the cornea, (3)H-thymidine ((3)H-TdR) was injected intravitreally, and the rabbits killed from 1 to 49 days afterward. The cornea, together with the adjacent conjunctiva, was processed for autoradiography. Results: The regenerating epithelium at the center of the cornea exhibited high frequencies of labeled nuclei when compared to controls. The mitotic indexes for the limbus were comparable in experimental and control eyes. The unique basal stratum of the limbal epithelium exhibited quick proliferation and vertical migration in all eyes. Cells that remained labeled for four weeks or more were observed throughout the corneal epithelium, including its basal stratum, and this did not depend on epithelial damage. Conclusion: Corneal epithelium wounds are healed by sliding and proliferation of cells surrounding the epithelial gap without any evidence for the participation of the limbal epithelium. Daughter cells labeled with (3)H-TdR were visualized in all layers of the corneal epithelium up to 7 weeks after the DNA precursor injection. However, at this long interval, the only labeled cells in the limbus were in the suprabasal layers.

Reconstruction of mandibular segmental defects using the guided-bone regeneration technique with polylactide membranes and/or autogenous bone graft: A preliminary study on the influence of membrane permeability

Purpose: Bone maintenance after mandibular reconstruction with autogenous iliac crest may be disappointing due to extensive resorption in the long term. The potential of the guided-bone regeneration (GBR) technique to enhance the healing process in segmental defects lacks comprehensive scientific documentation. This study aimed to investigate the influence of polylactide membrane permeability on the fate of iliac bone graft (BG) used to treat mandibular segmental defects. Materials and Methods: Unilateral 10-mm-wide segmental defects were created through the mandibles of 34 mongrel dogs. All defects were mechanically stabilized, and the animals were divided into 6 treatment groups: control, BG alone, microporous membrane (poly L/DL-lactide 80/20%) (Mi); Mi plus BG; microporous laser-perforated (15 cm(2) ratio) membrane (Mip), and Mip plus BG. Calcein fluorochrome was injected intravenously at 3 months, and animal euthanasia was carried out at 6 months postoperatively. Results: Histomorphometry showed that BG protected by Mip was consistently related to larger amounts of bone compared with other groups (P <= .0001). No difference was found between defects treated with Mip alone and BG alone. Mi alone rendered the least bone area and reduced the amount of grafted bone to control levels. Data from bone labeling indicated that the bone formation process was incipient in the BG group at 3 months postoperatively regardless of whether or not it was covered by membrane. In contrast, GBR with Mip tended to enhance bone formation activity at 3 months. Conclusions: The use of Mip alone could be a useful alternative to BG. The combination of Mip membrane and BG efficiently delivered increased bone amounts in segmental defects compared with other treatment modalities. (C) 2008 American Association of Oral and Maxillofacial Surgeons.

Microscopic Evidences That Bone Marrow Mononuclear Cell Treatment Improves Sciatic Nerve Regeneration After Neurorrhaphy

Cell therapy constitutes a possibility for improving nerve regeneration, increasing the success of nerve repair. We evaluate the use of mononuclear cells in the regeneration of the sciatic nerve after axotomy followed by end-to-end neurorrhaphy. Forty adult male Wistar rats (250300 g) were divided into four groups: (1) sham, (2) neurorrhaphy: the sciatic nerve was sectioned and repaired using epineural sutures, (3) culture medium: after the suture, received an injection of 10 mu L of culture medium into the nerve, and (4) mononuclear cell: after the suture, a concentration of 3 X 10(6) of mononuclear cell was injected in epineurium region. Mononuclear cells were obtained from the bone marrow aspirates and separated by Ficoll-Hypaque method. The histological analyses were performed at the 4th postoperative day. The sciatic functional index, histological, and morphometric analyzes were used to evaluate nerve regeneration at the 6th postoperative week. Six rats were used for immunohistochemical analysis on the 4th postoperative day. In the group 4, on the fourth day, the histological analysis demonstrated a more accelerated degenerative process and an increase of the neurotrophic factors was observed. In the 6th week, all the morphometric results of the group 4 were statistically better compared with groups 2 and 3. There was a statistically significant improvement in the sciatic functional index for group 4 compared with groups 2 and 3. Mononuclear cells stimulated nerve regeneration, most probably by speeding up the Wallerian degeneration process as well as stimulating the synthesis of neurotrophic factors. Microsc. Res. Tech. 74:355-363, 2011. (C) 2010 Wiley-Liss, Inc.

Early alveolar bone regeneration in rats after topical administration of simvastatin

Objectives. The aim of this study was to ultrastructurally examine the influence of simvastatin on bone healing in surgically created defects in rat mandibles. Study design. Bone defects 0.8 mm in diameter were created in the buccal aspect of first mandibular molar roots and filled with 2.5% simvastatin gel, while the controls were allowed to heal spontaneously. The rats were humanely killed 7, 9, 11, or 14 days postoperatively, and the specimens were processed for scanning and transmission electron microscopy, as well as for colloidal gold immunolabeling of osteopontin. Results. The regenerated alveolar bone in the simvastatin-treated defects presented smaller marrow spaces, and the collagen fibrils were regularly packed exhibiting a lamellar bone aspect. Osteopontin was present through the bone matrix during the wound healing and alveolar bone regeneration. Conclusion. The present study provides evidence that a single topical application of 2.5% simvastatin gel improves the quality of the new bone and decreases bone resorption. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112: 170-179)

Expression of matrix metalloproteinases-2 and-9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Acellular dermal matrix as a barrier in guided bone regeneration: a clinical, radiographic and histomorphometric study in dogs

Objectives The purpose of this study was to evaluate the effectiveness of the acellular dermal matrix (ADM) as a membrane for guided bone regeneration (GBR), in comparison with a bioabsorbable membrane. Material and methods In seven dogs, the mandibular pre-molars were extracted. After 8 weeks, one bone defect was surgically created bilaterally and the GBR was performed. Each side was randomly assigned to the control group (CG: bioabsorbable membrane made of glycolide and lactide copolymer) or the test group (TG: ADM as a membrane). Immediately following GBR, standardized digital X-ray radiographs were taken, and were repeated at 8 and 16 weeks post-operatively. Before the GBR and euthanasia, clinical measurements of the width and thickness of the keratinized tissue (WKT and TKT, respectively) were performed. One animal was excluded from the study due to complications in the TG during wound healing; therefore, six dogs remained in the sample. The dogs were sacrificed 16 weeks following GBR, and a histomorphometric analysis was performed. Area measurements of new tissue and new bone, and linear measurements of bone height were performed. Results Post-operative healing of the CG was uneventful. In the TG membrane was exposed in two animals, and one of them was excluded from the sample. There were no statistically significant differences between the groups for any histomorphometric measurement. Clinically, both groups showed an increase in the TKT and a reduction in the WKT. Radiographically, an image suggestive of new bone formation could be observed in both groups at 8 and 16 weeks following GBR. Conclusion ADM acted as a barrier in GBR, with clinical, radiographic and histomorphometric results similar to those obtained with the bioabsorbable membrane. To cite this article:Borges GJ, Novaes AB Jr, de Moraes Grisi MF, Palioto DB, Taba M Jr, de Souza SLS. Acellular dermal matrix as a barrier in guided bone regeneration: a clinical, radiographic and histomorphometric study in dogs.Clin. Oral Impl. Res. 20, 2009; 1105-1115.

Synthesis of chitosan/hydroxyapatite membranes coated with hydroxycarbonate apatite for guided tissue regeneration purposes

Chitosan, which is a non-toxic, biodegradable and biocompatible biopolymer, has been widely researched for several applications in the field of biomaterials. Calcium phosphate ceramics stand out among the so-called bioceramics for their absence of local or systemic toxicity, their non-response to foreign bodies or inflammations, and their apparent ability to bond to the host tissue. Hydroxyapatite (HA) is one of the most important bioceramics because it is the main component of the mineral phase of bone. The aim of this work was to produce chitosan membranes coated with hydroxyapatite using the modified biomimetic method. Membranes were synthesized from a solution containing 2% of chitosan in acetic acid (weight/volume) via the solvent evaporation method. Specimens were immersed in a sodium silicate solution and then in a 1.5 SBF (simulated body fluid) solution. The crystallinity of the HA formed over the membranes was correlated to the use of the nucleation agent (the sodium silicate solution itself). Coated membranes were characterized by means of scanning electron microscopy - SEM, X-ray diffraction - XRD, and Fourier transform infrared spectroscopy - FTIR. The results indicate a homogeneous coating covering the entire surface of the membrane and the production of a semi-crystalline hydroxyapatite layer similar to the mineral phase of human bone. (C) 2010 Elsevier B.V. All rights reserved.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Population biology and regeneration of forbs and shrubs after fire in Brazilian Campos grasslands

Fire is an important factor in several ecosystems, affecting plant population biology. Campos grasslands are under constant influence of disturbance, mostly grazing and fire. However, few studies evaluated the effect of fire on plant population biology of grassland species. Therefore, we aim to analyze the effect of fire on the population biology of four species, from different functional groups and regeneration strategies: Chaptalia runcinata (forb, resprouter, absence of belowground organ), Vernonia flexuosa (forb, resprouter, presence of rhizophore), Eupatorium ligulaefolium (shrub, resprouter, presence of xylopodium) and Heterothalamus psiadioides (shrub, obligate seeder). Seven plots were established in different sites in southern Brazil: frequently burned (FB) and excluded from fire since 6 years (E). All plots were subjected to controlled burns during summer. Before experiments, populations were sampled. Further observations were carried out after 90 and after 360 days of fire experiments. In addition, we counted the number of seedlings and resprouters recruited after fire. Heat shock experiments were conducted with two species (H. psiadioides and V. flexuosa), as well as the study of the bud bank of the following species: E. ligulaefolium and V. flexuosa. The obligate seeder species had all individuals killed by fire and established only after 1 year. Resprouters, however, showed new stems immediately after fire. E. ligulaefolium and V. flexuosa showed only vegetative regeneration from belowground organs and more individuals in excluded sites 1 year after the fire. The bud bank of E. ligulaefolium tended to be larger in excluded sites, whilst V. flexuosa showed an opposite result. High temperatures did not enhance nor kill seeds from both studied species. Vegetative regeneration was the most important strategy for all studied species, except for H. psiadioides, the obligate seeder species. Fire thus, plays an important role on population structure and demography, being also important for plant recruitment.

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

POLYGLYCOLIC ACID TUBE ASSOCIATED WITH GM1 IN REGENERATION OF PERIPHERAL NERVES

Introduction: Nerve allografting is regarded as a treatment of choice in large neural tissue losses preventing repair by primary anastomosis. In these cases, a synthetic polyglycolic acid tube is an alternative for nerve grafting. On the other hand, several studies have emphasized the importance of neurotrophic factors on neural regeneration, including substances with potential to optimize neural regeneration, especially the GM1, an neurotrophic enhancer factor. Objective: to compare, in rats, the neural regeneration degree using histological analysis, regenerated myelinized axons count, and functional analysis with the use of neurotube and GM1. Methods: This assessment was performed by interposing allograft (group A), polyglycolic acid tube (group B) and polyglycolic acid tube associated to GM1 (group C) on 5-mm sciatic nerve defects. Results: Neuroma formation was found only on group A. Groups A and C showed similar histological patterns, except for the regenerated axons on group C, which were shown to be better organized and myelinized than in group A. Conclusion: on functional recovery, no statistically significant difference was found for the three groups, despite of qualitative and quantitative histological differences found.

Effects of hyperbaric oxygen therapy on facial nerve regeneration

Conclusion. Hyperbaric oxygen treatment (HBOT) promoted an increase of the mean axonal diameter in the group evaluated 2 weeks after lesion induction, which suggests a more advanced regeneration process. However, the number of myelin nerve fibers of the facial nerve of the rabbits was similar when compared to the control and treatment groups, in both evaluation periods. Objective. To evaluate the effect of HBOT on the histological pattern of the facial nerve in rabbits exposed to a nerve crush injury. Materials and methods. Twenty rabbits were exposed to facial nerve crush injury. Ten rabbits received HBOT, 10 rabbits comprised the control group. The rabbits were sacrificed 2 and 4 weeks after the trauma. Qualitative morphological analysis, measurement of the external axonal diameters and myelin fiber count were carried out in an area of 185 000 mu m(2). Results. There was an increase in the area of the axons and thicker myelin in the 2 weeks treatment group in comparison with the control group. The mean diameter of the axons was of 2.34 mu m in the control group and of 2.81 mu m in the HBOT group, with statistically significant differences. The 2 week control group had a mean number of myelin fibers of 186 +/- 5.2664, and the HBOT group had a mean number of 2026.3 +/- 302; this was not statistically significant. The 4 week control group presented a mean of 2495.1 +/- 479 fibers and the HBOT group presented a mean of 2359.9 +/- 473; this was not statistically significant.

Pulp tissue from primary teeth: new source of stem cells

SHED (stem cells from human exfoliated deciduous teeth) represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.