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Multi-omics analyses reveal DjTcf4 critical for proper timing of differentiation in planarian regeneration.

The blastema is key to forming complete tissues in regenerating Dugesia japonica (D. japonica). However, the dynamic changes in cellular compositions and transcription landscapes in blastema during regeneration are understudied. Here, through genome reannotation, 3D spatial transcriptome construction, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses of changes in gene expression and chromatin structures, we delineate key transcription factors regulating the developmental trajectories of major cell clusters in the regenerating head. Importantly, we find that the T cell factor 4 (DjTcf4)-positive cells highly accumulate at wound areas, and its gene network is critical for the proper timing of development during regeneration in multiple progenitor cells. Depletion of DjTcf4 and its target genes leads to singular eye and/or dull tail phenotypes and delays regeneration. Taken together, we build multi-omics atlases in D. japonica and reveal the noncanonical function of the DjTcf4 network in developmental pattern formation, laying a foundation for studies of regeneration in D. japonica.

Animals↗

Isolation and characterization of fibroblasts derived from regenerating human periodontal defects.

In an attempt to understand better the cells responsible for periodontal regeneration, cells from human gingiva, periodontal ligament and regenerating periodontal defects treated with expanded polytetrafluorethylene membranes were isolated, cultured and characterized. Guided tissue regeneration procedures were carried out on three human volunteers around molar teeth destined for extraction. After a 6-week 'healing phase', fibroblast cell cultures were established from explants of the regenerating soft connective tissue (RTF), as well as from the associated periodontal ligament (PLF) and gingiva (GF). Following stimulation with platelet-derived growth factor-beta (PDGF) and insulin-like growth factor-1 (IGF-1), [3H]thymidine-uptake and dye-binding assays were used to assess the rate of DNA synthesis and cell proliferation, respectively. Northern blotting was used to measure the expression of mRNA for the extracellular matrix proteoglycans decorin, biglycan and versican. The results show that the GF and RTF proliferated more quickly than the PLF. PDGF and IGF-1 were mitogenic for all three cell types. Decorin mRNA expression was stronger in the GF than the RTF and PLF, whereas versican mRNA expression was stronger in the GF and PLF than the RTF. Biglycan mRNA expression was strong in the PLF, moderate in the GF and weak in the RTF. The growth factors did not affect the mRNA expression for biglycan, but they upregulated versican and downregulated decorin mRNA. It can be concluded that RTF exhibits properties characteristic of a reparative phenotype. More specifically, it proliferates faster than PLF, from which it is derived, while exhibiting a unique pattern of proteoglycan mRNA expression. Therefore, this study demonstrates that fibroblasts obtained from the regenerating periodontal defects exhibit characteristics consistent with their ability to facilitate periodontal regeneration.

Adult↗

Comparison of pericranium and eggshell as space fillers used in combination with guided bone regeneration: an experimental study.

PURPOSE: The purpose of the study was to determine the potential adjunctive role of 2 space fillers, used in combination with guided tissue regeneration, on bone regeneration in rat skull defects. MATERIALS AND METHODS: The study was conducted on 45 adult Wistar rats. A bilateral 6-mm-wide full-thickness skull defect was created in the parietal region of each animal. The right defect was chosen as the experiment site, and the left defect was left empty as a control. Each experiment site was covered by an inner and outer polytetrafluoroethylene membrane. The 45 rats were divided into 3 groups; no space filler between the 2 membranes was used in group I (n = 15), a free pericranial autograft was used as a space filler between the 2 membranes in group II (n = 15), and purified eggshell powder was used as space filler between the 2 membranes in group III (n = 15). Five animals in each group were killed at 15, 30, and 90 days. The harvested specimens were subjected to contact radiography and standard microscopic examination, and the rates of osteogenesis were assessed by a semiquantitative method. RESULTS: No evidence of bone regeneration was seen in any animals of the three groups at 15 days. At 30 days, bone regeneration only appeared in group I (P > .05). At 90 days, complete bone regeneration was observed in the group I in 3 of 5 animals (P < .05). In group II, the pericranial graft showed no osteogenic properties. In group III, the eggshell powder showed no resorption, but no osteoconduction was noticed. CONCLUSIONS: Although the osteogenic mechanism of guided tissue regeneration is not clear, this study suggests that the physical properties of the membrane are more important than the use of an adjunctive space filler.

Animals↗

Unbiased stereological methods used for the quantitative evaluation of guided bone regeneration.

The present study describes the use of unbiased stereological methods for the quantitative evaluation of the amount of regenerated bone. Using the principle of guided bone regeneration the amount of regenerated bone after placement of degradable or non-degradable membranes covering defects in rabbit calvaria was compared. Forty rabbits were divided into 5 groups. A titanium microplate was placed over the defect to prevent collapse of the membrane. The non-degradable expanded polytetrafluoroethylene membrane and the degradable Polyglactin 910 material were both placed unicortically and bicortically. Undecalcified sections were prepared for stereologic evaluation after an observation period of 8 weeks. Complete bone healing of the defects was not observed in any of the specimens. Unbiased stereologic estimates revealed 48% bone regeneration in defects covered by 2 ePTFE membranes, and 12% in defects covered by 2 Polyglactin 910 membranes. Defects covered by 1 ePTFE or 1 Polyglactin 910 membranes revealed 10% or 18% bone regeneration, respectively. The control group regenerated 14%. The major difference of the estimates was caused by real difference between specimens, i.e. biologic variation, whereas only minimal variance was added by the stereologic estimation procedure.

Animals↗

Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans.

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters and it seems to be a prerequisite for normal skeletal mineralization. Also, ALP is the most widely recognized marker of osteoblast phenotypes. By a tissue regenerative technique called Guided Bone Regeneration (GBR), it is possible nowadays to regenerate small bony defects. The aim of the present study was to investigate early events in bone healing and neogenesis by studying histochemically the temporal and local appearance of the marker Alkaline Phosphatase (ALP) in a GBR model system. Nine healthy volunteers (5 males, 4 females, mean age 31.7 years) participated in the experiment. After raising a mucoperiosteal flap from the mandibular second molar to the retromolar area in each volunteer, a hollow titanium test cylinder was placed into a congruent bony bed and the coronal end of the cylinder was closed with an ePTFE-membrane. Then the flap was adapted and sutured to obtain primary wound closure. After 2, 7 and 12 weeks, the regenerated tissue within the cylinders was harvested. Histologically, ALP activity was observed associated with the osteoid seams in the very basal part of the regenerate where new bone trabeculae were in the process of being formed. More coronally, large round cells seemed to secrete an ALP-positive substance since in the center of such cell clusters strong ALP activity located extracellularly was detected. In the present experiment, ALP seemed to have been an early sign of osteoblast secretion of a matrix which subsequently was determined to become osteoid. ALP activity was never seen isolated within connective tissue and away from bone. This is an indication that its source is linked to existing bone. The present study has documented for the first time the appearance of ALP activity in guided bone regenerations in humans. It has revealed that: 1) Osteogenesis in guided bone regeneration is preceded by localized, marked expression of ALP in an organized connective tissue environment. 2) Bone neogenesis is an early event in this experimental setup and may be detected already 2 weeks after wounding. 3) Expression of ALP and subsequent bone neogenesis is originating from and topographically linked to pre-existing bone structures.

Adult↗

SOME FEATURES OF MITOCHONDRIA AND FLUFFY LAYER IN REGENERATING RAT LIVER.

1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD-isocitrate dehydrogenase and NADP-isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18.4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4.7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD- and NADP-isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP-isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1.198, light mitochondria 1.193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.

Chemistry Techniques, Analytical↗

Retardation of regeneration and division of Blepharisma by ultraviolet radiation and its photoreversal.

Regeneration of Blepharisma undulans variety japonicus from which the hypostome has been removed is retarded by dosages of 3000 to 4600 ergs/mm.(2) at wavelength 2654A most strongly when the fragment is exposed soon after cutting. Dosages greater than 4600 ergs/mm.(2) prevent regeneration. Regeneration is also retarded strongly when the Blepharisma are cut soon after irradiation. Starvation retards regeneration and potentiates the effect of ultraviolet radiations. Division after regeneration of Blepharisma is also retarded by ultraviolet radiations about equally, regardless of when the Blepharisma are cut indicating a more lasting effect of the radiations upon the cells. Blepharisma cut after irradiation usually recover from the effects of the radiations sooner than uncut individuals given the same dosage. Retardation of division by ultraviolet radiation is subject to photoreversal by visible light, especially in a nitrogen atmosphere, provided the ultraviolet dose is not excessive. Visible light alone if prolonged, retards regeneration or may even kill the cut fragments of Blepharisma.

Ciliophora↗

Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel.

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

Absorbable Implants↗

Conditions for success in guided bone regeneration: retrospective study on 376 implant sites.

BACKGROUND: Used alone or in association with bone, guided bone regeneration is a clinically accepted method to increase the volume of bone during implant placement. Concerns about appropriate surgical techniques and the predictability of the results questions still exist. The aim of this study was to assess the conditions for success. METHODS: Three-hundred-seventy-six implants were placed in association with 214 non-resorbable membranes. In 109 implants, the membrane was secured on a blood clot; for 213 implants, membrane was placed with autogenous bone; and for 54 implants it was placed with allogenic bone. The surgical procedure, the primary closure, the timing of re-entry, and the density of regenerated tissue were studied. RESULTS: In 19 membranes, barrier exposure occurred before 3 months in these instances, the regenerated tissue was removed; another 7 membranes were exposed between 3 and 6 months. The 188 non-exposed membranes were removed between 6 and 12 months. In cases of exposure before 3 months, the regenerated tissue was soft and then removed. After closure for 6 months or more, the regenerated tissue was dense and resistant to probe pressure. The best results were obtained when the membranes were placed with autogenous bone. CONCLUSIONS: If all the clinical steps are appropriately followed, guided bone regeneration using an autogenous bone graft and implant placement is a predictable technique for increasing bone volume.

Adult↗

Participation of periodontal ligament cells with regeneration of alveolar bone.

BACKGROUND: It is important to clarify the participation of periodontal ligament (PDL) cells in the regeneration of alveolar bone to establish a reliable approach for obtaining periodontal regeneration. The aim of this study was to determine whether PDL cells play an important role in alveolar bone repair during the course of periodontal regeneration. METHODS: In an in vitro study, the expression of the osteoblast phenotype, such as alkaline phosphatase activity and parathyroid hormone-dependent 3',5'-cyclic adenosine monophosphate accumulation, was investigated in dog PDL cells (DPLC) and dog bone cells isolated from mandibles (DBC). In a related study, the roots of mandibular third premolars extracted from aged dogs were divided into a PDL(+) group, in which the PDL was preserved, and a PDL(-) group, in which the PDL was removed. These roots were respectively transplanted into surgically created bone cavities with buccal and interproximal bone defects in an edentulous area, prepared in advance by extraction of mandibular fourth premolars. These bone defects with the transplanted roots were completely covered with submerged physical barrier membranes. New bone formation and new connective tissue attachment, which require new cementum and insertion of functionally oriented new collagen fibers of periodontal ligament, were histomorphometrically assessed, and were compared between the PDL(+) and PDL(-) groups 6 weeks after transplantation. RESULTS: Both cultured DPLC and DBC exhibited the osteoblast phenotype. New connective tissue attachment was observed only in the PDL(+) group. However, alveolar bone was almost completely regenerated to the original bone height in both the PDL(+) and PDL(-) groups, and the amount of newly formed bone was not significantly different between the 2 groups. CONCLUSIONS: DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.

Alkaline Phosphatase↗

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

The role of Schwann cells in paradoxical regeneration in the axolotl.

The experiments described here examine further the conditions under which paradoxical regeneration occurs and provide support for the hypothesis that a proximal migration of Schwann cells is responsible for the phenomenon. When only the hand is shielded from irradiation and the limb is denervated, amputation through the forearm or upper arm sometimes results in regeneration. The effects of variation in the time interval between denervation and amputation, the level of amputation and the method and number of denervations on the incidence of regeneration were investigated. The presence or absecce of viable Schwann cells at the amputation plane was deduced from the remyelination of nerves under conditions which do or do not permit paradoxical regeneration. The nerves of totally irradiated and denervated limbs remain unmyelinated following regrowth of axons and such limbs do not regenerate after amputation. When only the hand was shielded from irradiation before the limbs were denervated, the new axons became completely remyelinated and some of these limbs regenerated when amputated. It is suggested that under these conditions Schwann cells can migrate proximally and can then proliferate further to form a blastema, since they would be the only unirradiated tissue present at the amputation plane.

Ambystoma↗

[The characteristics of the effect of space flight factors on regeneration processes in mammals and Urodela].

Comparative analysis of experimental studies on regeneration of bone and muscle tissue under the influence of space flight factors (SFF) during restoration of experimental fracture in rats and epimorphic limb regeneration in adult newt is presented SFF-induced retardation of regeneration in mammals and its acceleration in tailed amphibians results from changed calcium metabolism in conditions of microgravity. Bidirectional effect of SFF on bone regeneration is due to specific control of calcium homeostasis in these taxa. Upon termination of the space flight (SF) the Wistar SPF rats proved to have increased functional activity of parathyrocytes and blocked calcitonin secretion to the blood by the C-cells of thyroid gland. Hence, inhibition of posttraumatic bone regeneration in rats is due to decreased formation rate and enhanced resorption of bony tissue. Sharp decrease of the calcitonincytes population following the initial hypertrophy of ultimobranchial gland (UBG) is observed in adult newt Pleurodeles waltlii Michah during SF. Progressing demineralization of skeletal elements including the amputated limb stump accelerates the first stages of its regeneration in conditions of SF.

Adaptation, Physiological↗

3H-GABA administration during tail regeneration of lizards and autoradiographical localization.

Thirteen lizards (Anolis carolinensis and Scincella lateralis) were injected with 3H-GABA (tritiated gamma-aminobutyric acid) in order to study its localization inside the regenerating tail spinal cord (SC) by means of light and electron microscopic autoradiography. One and three hours after intraperitoneal injection, the radioactivity was essentially localized in regenerating nerves and pale cells, mostly contacting the central canal of the SC as typical Cerebrospinal-Fluid-Contacting-Neurons (CSFCNs). A smaller number of pale cells did not take up 3H-GABA and some of them appeared to be degenerating. Weaker radioactivity in regenerating SC was observed 6 hours post injection. Among non neural tissues, cells of the regenerating blastema, of the meninges and capillaries surrounding the regenerating SC, showed lower 3H-GABA uptake. This indicates that a certain amount of injected 3H-GABA has been converted to other metabolites. No 3H-GABA uptake was detected 1 or 6 hours post injection in normal SC and brain both in grey and white matter. This research indicates that CSFCNs of the regenerating tail in lizards uptake the aminoacid GABA or derived aminoacids, suggesting these cells subserve a high metabolic activity and may be GABA-ergic

Animals↗

Periodontal regeneration of intrabony defects: an evidence-based treatment approach.

A task force of periodontists established clinical and histologic outcomes (goals) for the treatment of intrabony defects and researched the literature for techniques that would most predictably achieve these goals. The group also identified factors that could influence predictability. The treatment outcomes selected by the task force included regeneration of a true attachment apparatus; gain in bone and probing attachment levels; reduction in probing pocket depth; minimal gingival recession; increased patient comfort; esthetic appearance and state of wellness; and maintenance of health, comfort, and function over time. Based on evidence, it was concluded that guided tissue regeneration, guided tissue regeneration combined with the use of decalcified freeze dried bone allografts and freeze-dried bone allografts alone are the most predictable regenerative procedures for achieving selected treatment outcomes. Various factors, such as patient characteristics, the morphology of the defect, and the surgical technique can influence the healing response of intrabony defects. Patient factors, such as plaque control, compliance, and cigarette smoking, can directly affect predictability of periodontal regeneration. Defect selection is critical, and deep and narrow defects are the most predictable response to regenerative procedures. The number of remaining bony walls is important in grafting procedures, but their influence is questionable in guided tissue regeneration. Various technical procedures, such as flap design, defect debridement, and wound protection, may influence the predictability of regeneration.

Alveolar Bone Loss↗

Guided bone regeneration of bone defects associated with implants: an evidence-based outcome assessment.

Guided bone regeneration is a new technique that evolved following the guided tissue regeneration procedure for regeneration of lost periodontium. The objective of guided bone regeneration is to promote bone formation in osseous deformities either before or in conjunction with endosseous implant placement. Osseous defects consist mainly of extraction sites, dehiscences or fenestrations, and localized ridge deformities. In addition, bone defects may either provide natural spacemaking or be nonspacemaking. A plethora of publications indicate that the guided bone regeneration technique can be used successfully in all types of defects. Nonspacemaking defects usually require bone graft materials to assist in space maintenance and enhance bone formation. Fixation pins are also used, either with or without graft materials, in this regard. The scientific literature on guided bone regeneration was reviewed by a task force to determine techniques proven to increase predictability relative to successful patient outcomes and to develop specific evidence based diagnostic and treatment decision trees.

Alveolar Bone Loss↗

Polypeptide growth factors for periodontal regeneration.

The regeneration of periodontal attachment apparatus is particularly difficult to achieve, primarily because of the presence of many different kinds of tissue that must be restored to produce a functional unit. Traditional methods aimed at regenerating the periodontium have limited indications, and their results are not predictable. Recently, investigators have begun to understand the cellular processes necessary for repair and regeneration of periodontal tissues. Proteins called growth factors have been identified that coordinate these cellular events. The growth factors that may contribute to periodontal regeneration include platelet-derived growth factor, insulin-like growth factor, transforming growth factor-beta, and bone morphogenetic proteins. In vitro studies have demonstrated the positive effects of these factors on a number of cell types essential for periodontal regeneration. For instance, it has been shown that platelet-derived and insulin-like growth factors promote proliferation of osteoblasts an periodontal ligament cell-derived fibroblasts. Animal models have also been used to verify that growth factors can enhance regeneration in vivo following periodontal disease and as an adjunct to implant placement. In the future, human clinical trials will be required to identify the ideal growth factors, their proper doses, and the most suitable carrier system for them.

Animals↗

Biologic and clinical considerations for autografts and allografts in periodontal regeneration therapy.

Although complete periodontal regeneration is unpredictable with any regenerative therapy currently used, periodontal bone grafts show strong potential. A large body of clinical evidence clearly indicates that grafts consistently lead to better bone fill than nongrafted controls. Regardless of which material is used, the average bone fill is 3 mm, or about 60% defect fill according to reported studies. In addition, histologic analyses now show conclusively that regenerative treatment with bone grafting leads to some degree of regenerated bone, cementum, and periodontal ligament. Although bone grafts and other periodontal treatments have improved clinicians' ability to promote osteogeneration, the outcomes are still somewhat unpredictable. Part of the problem is that it is still unclear how periodontal disease affects the supporting bone's regenerative potential and what specific biologic factors are involved. In recent years, however, clinicians have begun to learn much more about how periodontal regeneration works on a cellular and molecular level. This is a key step to developing strategies and materials that allow clinicians to promote periodontal regeneration predictably. Since its introduction during the last decade, GTR has been an invaluable addition to the armamentarium, as has the recognized importance of early wound stability. As more is learned about the biologic process of periodontal regeneration, new materials and techniques, such as growth factors, absorbable membranes, and new graft materials, are expected to make the task of periodontal regeneration even more predictable. It is likely that some combination of techniques may eventually prove to yield the best results.

Alveolar Process↗