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Регенерация тканей у позвоночных животных

Журнальные статьи

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Agrali O.B. et al. Evaluation of the Effectiveness of Esterified Hyaluronic Acid Fibers on Bone Regeneration in Rat Calvarial Defects // Biomed Res. Int. 2018. P. 3874131.

Hyaluronic acid (HA) constitutes one of the major components of the extracellular matrix domain in almost all mammals. The aim of this study was to evaluate the regenerative capacity of HA matrix in rat calvarial bone defects and compare with those of different combinations of resorbable collagen membrane (M) and bovine-derived xenograft (G). Twenty-four 3-month-old male Sprague-Dawley rats weighing 200-250 g were included. Control group was created by leaving one defect empty from 2 critical size defects with 5 mm diameter formed in the calvarial bones of 8 rats. In the same rats, the other defect was treated with HA matrix alone. One of the 2 defects formed in other 8 rats was treated with HA + G and the other with HA + M. One of the 2 defects formed in the remaining 8 rats was treated wilh G+M and the other with HA+G+M. The animals were sacrificed at 4 weeks. Histologic, histomorphometric, and immunohistochemical analyses were performed. Both HA matrix alone and its combinalions with G and M supported new bone formation (NBF). However, NBF was significantly greater in G+M and HA+G+M groups compared to control and HA alone (P < 0.00l). Bone morphogenetic protein-2 was expressed with varying degrees in all groups, without any difference among them. Within the limitations of the present study, HA matrix, used alone or in combination with G and M, did not contribute significantly to bone regeneration in rat calvarial bone defects.


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Al Haj Baddar N.W., Chithrala A., Voss S.R. Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration // Dev. Dyn. 2019. Vol. 248, № 2. P. 189–196.

Background Among vertebrates, salamanders are unparalleled in their ability to regenerate appendages throughput life. However, little is known about early signals that initiate regeneration in salamanders. Results Ambystoma mexicanum embryos were administered tail amputations to investigate the timing of reactive oxygen species (ROS) production and the requirement of ROS for regeneration. ROS detected by dihydroethidium increased within minutes of axolotl tail amputation and levels remained high for 24 hr. Pharmacological inhibition of ROS producing enzymes with diphenyleneiodonium chloride (DPI) and VAS2870 reduced ROS levels. Furthermore, DPI treatment reduced cellular proliferation and inhibited tail outgrowth. Conclusions The results show that ROS levels increase in response to injury and are required for tail regeneration. These findings suggest that ROS provide instructive, if not initiating cues, for salamander tail regeneration. Developmental Dynamics 248:189-196, 2019. (c) 2018 Wiley Periodicals, Inc.


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Alibardi L. Microscopic observations show invasion of inflammatory cells in the limb blastema and epidermis in pre-metamorphic frog tadpoles which destroy the Apical Epidermal CAP and impede regeneration // Ann. Anat.-Anat. Anz. 2017. Vol. 210. P. 94–102.

Some limb regeneration in tadpoles of Rana dalmatina occurs at stages 44-48 when small hind-limbs are present while scarring occurs at stages 51-52 when forelimbs have developed and metamorphosis is approaching. Ultrastructural analysis of cells forming the regenerating blastema detects mesenchymal cells and an Apical Epidermal Cap (AEC) in regenerating limb blastema 5-6 days post-amputation at stages 46-48. In contrast, granulocytes and numerous macrophages and lymphocytes prevail over mesenchymal cells in limb blastema at stages 51-52, which are destined to form scars. An increase in inflammatory cells in limb blastema prior to metamorphosis suggests a negative influence of immune cells on limb regeneration. Inflammatory cells invade the apical wound epidermis where stem keratinocytes are likely destroyed, impeding the formation of an AEC, the microregion which leads to limb regeneration. The invasion of immune cells, however, may also represent a physiological consequence of the death of cell populations in the tadpoles occurring with approaching metamorphosis. The passage from an aquatic to a terrestrial life in this frog elicits the typical amniote scarring reaction after wounding, and the limb cannot regenerate. The present observations sustain the hypothesis that the evolution of the adaptive immunity in tetrapods while efficiently preserving adult self-condition, determined the loss of tissue regeneration since the embryonic antigens evocated in blastema cells are removed by immune cells of the adult. (C) 2016 Elsevier GmbH. All rights reserved.


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Azzouz A. et al. Iron prevents demyelination of frog sciatic nerves // Environ. Toxicol. Pharmacol. 2017. Vol. 55. P. 51–54.

Metal ions are of particular importance in nervous system function, notably iron. However, very little has been done to investigate its physiological role in frog peripheral nervous system. The present research aim to evaluate i) the time-effect of sciatic nerve ligation and/or ii) iron sulphate (1.50 mg/kg, in lymphatic sac) on frog myelin sheaths. Histological sections following ligation shows degeneration of some fibres with axonal and myelin breakdown associated to a decrease of Schwann cells number following 2 h (45.00 +/- 0.30, p < 0.0001), 24 h (28.00 +/- 0.020, p < 0.0001). Interestingly, iron administration reduces the degeneration of myelin sheaths classically observed in frog ligated sciatic nerve associated with an increase of Schwann cells number (139.00 +/- 0.50, p < 0.0001). Thus, iron could prevent degeneration or promote regeneration induced by ligation in frog sciatic nerve.


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Chen W. et al. Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co-culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs, human induced pluripotent stem cell-derived MSCs and human embryonic stem cell-derived MSCs // J. Tissue Eng. Regen. Med. 2018. Vol. 12, № 1. P. 191–203.

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co-culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co-cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC-MSCs) and embryonic stem cells (hESC-MSCs). Constructs were implanted in 8mm cranial defects of rats for 12weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary-like structures were successfully formed on CPC in vitro in all four co-cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co-cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co-cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co-cultured with hUCMSCs, hiPSC-MSCs and hESC-MSCs achieved new bone and vessel density similar to hUVECs co-cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC-MSCs and hESC-MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co-cultures of hUVECs with hUCMSCs, hiPSC-MSCs, hESC-MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co-culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright (C) 2017 John Wiley & Sons, Ltd.


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Chien K.-H. et al. Promoting Induced Pluripotent Stem Cell-driven Biomineralization and Periodontal Regeneration in Rats with Maxillary-Molar Defects using Injectable BMP-6 Hydrogel // Sci Rep. 2018. Vol. 8. P. 114.

Periodontal disease may cause considerable destruction of alveolar bone, periodontal ligaments (PDLs) and cementum and even lead to progressive oral dysfunction. Periodontal tissue regeneration is the ultimate goal of periodontal disease treatment to reconstruct both structures and functions. However, the regenerative efficiency is low, possibly due to the lack of a proper periodontal microenvironment. In this study, we applied an injectable and thermosensitive chitosan/gelatin/glycerol phosphate hydrogel to provide a 3D environment for transplanted stem cells and to enhance stem cell delivery and engraftment. The iPSCs-BMP-6-hydrogel complex promoted osteogenesis and the differentiation of new connective tissue and PDL formation. In animal models of maxillary-molar defects, the iPSCs-BMP-6-hydrogel-treated group showed significant mineralization with increased bone volume, trabecular number and trabecular thickness. Synergistic effects of iPSCs and BMP-6 increased both bone and cementum formation. IPSCs-BMP-6-hydrogel-treated animals showed new bone synthesis (increased ALP- and TRAP-positive cells), new PDL regeneration (shown through Masson's trichrome staining and a qualification assay), and reduced levels of inflammatory cytokines. These findings suggest that hydrogel-encapsulated iPSCs combined with BMP-6 provide a new strategy to enhance periodontal regeneration. This combination not only promoted stem cell-derived graft engraftment but also minimized the progress of inflammation, which resulted in highly possible periodontal regeneration.


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Cho G. et al. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane // Biochim. Biophys. Acta-Gen. Subj. 2015. Vol. 1850, № 4. P. 784–793.

Background: The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Methods: Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Results: Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient When AQP5 was present in the nuclear membrane, nuclear size decreased. Conclusion: The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. General significance: AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. (C) 2015 Elsevier B.V. All rights reserved.


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Dawson N.J., Storey K.B. Passive regeneration of glutathione: glutathione reductase regulation in the freeze-tolerant North American wood frog, Rana sylvatica // J. Exp. Biol. 2017. Vol. 220, № 17. P. 3162–3171.

Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity (K-m) for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l(-1)) and a increase (157%) when measured in the presence of levels of urea (75 mmol l(-1)) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the K-m for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing.


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Droz S.T., McLaughlin K.A. Use of Xenopus Frogs to Study Renal Development/Repair // Kidney Development and Disease / ed. Miller R.K. Cham: Springer International Publishing Ag, 2017. Vol. 60. P. 77–107.

The Xenopus genus includes several members of aquatic frogs native to Africa but is perhaps best known for the species Xenopus laevis and Xenopus tropicalis. These species were popularized as model organisms from as early as the 1800s and have been instrumental in expanding several biological fields including cell biology, environmental toxicology, regenerative biology, and developmental biology. In fact, much of what we know about the formation and maturation of the vertebrate renal system has been acquired by examining the intricate genetic and morphological patterns that epitomize nephrogenesis in Xenopus. From these numerous reports, we have learned that the process of kidney development is as unique among organs as it is conserved among vertebrates. While development of most organs involves increases in size at a single location, development of the kidney occurs through a series of three increasingly complex nephric structures that are temporally distinct from one another and which occupy discrete spatial locales within the body. These three renal systems all serve to provide homeostatic, osmoregulatory, and excretory functions in animals. Importantly, the kidneys in amphibians, such as Xenopus, are less complex and more easily accessed than those in mammals, and thus tadpoles and frogs provide useful models for understanding our own kidney development. Several descriptive and mechanistic studies conducted with the Xenopus model system have allowed us to elucidate the cellular and molecular mediators of renal patterning and have also laid the foundation for our current understanding of kidney repair mechanisms in vertebrates. While some species-specific responses to renal injury have been observed, we still recognize the advantage of the Xenopus system due to its distinctive similarity to mammalian wound healing, reparative, and regenerative responses. In addition, the first evidence of renal regeneration in an amphibian system was recently demonstrated in Xenopus laevis. As genetic and molecular tools continue to advance, our appreciation for and utilization of this amphibian model organism can only intensify and will certainly provide ample opportunities to further our understanding of renal development and repair.


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Edwards-Faret G. et al. Cellular composition and organization of the spinal cord central canal during metamorphosis of the frog Xenopus laevis // J. Comp. Neurol. 2018. Vol. 526, № 10. P. 1712–1732.

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.


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Elliott K.L., Houston D.W., Fritzsch B. Sensory afferent segregation in three-eared frogs resemble the dominance columns observed in three-eyed frogs // Sci Rep. 2015. Vol. 5. P. UNSP 8338.

The formation of proper sensory afferent connections during development is essential for brain function. Activity-based competition is believed to drive ocular dominance columns (ODC) in mammals and in experimentally-generated three-eyed frogs. ODC formation is thus a compromise of activity differences between two eyes and similar molecular cues. To gauge the generality of graphical map formation in the brain, we investigated the inner ear projection, known for its well-defined and early segregation of afferents from vestibular and auditory endorgans. In analogy to three eyed-frogs, we generated three-eared frogs to assess to what extent vestibular afferents from two adjacent ears could segregate. Donor ears were transplanted either in the native orientation or rotated by 90 degrees. These manipulations should result in either similar or different induced activity between both ears, respectively. Three-eared frogs with normal orientation showed normal swimming whereas those with a rotated third ear showed aberrant behaviors. Projection studies revealed that only afferents from the rotated ears segregated from those from the native ear within the vestibular nucleus, resembling the ocular dominance columns formed in three-eyed frogs. Vestibular segregation suggests that mechanisms comparable to those operating in the ODC formation of the visual system may act on vestibular projection refinements.


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Fei J.-F. et al. Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration // Proc. Natl. Acad. Sci. U. S. A. 2017. Vol. 114, № 47. P. 12501–12506.

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (similar to 5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.


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Garcia-Perez M.M. et al. A modified chemical protocol of decellularization of rat sciatic nerve and its recellularization with mesenchymal differentiated Schwann-Like cells: Morphological and functional assessments // Histol. Histopath. 2017. Vol. 32, № 8. P. 779–792.

The functional reconstruction of large neural defects usually requires the use of peripheral nerve autografts, though these have certain limitations. As a result, interest in new alternatives for autograft development has risen. The acellular peripheral nerve graft is an alternative for peripheral nerve injury repair, but to date there is not a standardized chemical decellularization method widely accepted. The objective of this study was to propose a modified chemical protocol of decellularization of rat sciatic nerve and its recellularization in vitro with mesenchymal differentiated Schwann-like cells. After the transplantation, an evaluation of its regeneration was performed using morphological and functional tests. The study consisted of two phases; in phase 1, different concentrations and times of exposure of rat sciatic nerves to detergents were tested, to establish a modified chemical protocol for nerve decellularization. The chemical treatment with 3% triton X-100 and 4% sodium deoxycholate for 15 days allowed a complete decellularization whilst conserving the extracellular matrix of the harvested nerve. In phase 2, the decellularized and recellularized alografts were compared against autografts. The morphological analysis showed a higher positivity to specific myelin antibodies in the recellularized group compared to the autograft. There were no differences in this parameter between the control limb and the experimental limb (recellularized group). The functional analysis showed no statistical differences at week 15 in the Sciatic Function Index in the autograft group vs the other groups. This study sets the morphological and functional bases for posterior studies about nerve defects regeneration in humans.


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Gerber T. et al. Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration // Science. 2018. Vol. 362, № 6413. P. 421–+.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.


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Gozeneli O. et al. Effects of thymoquinone and curcumin on the regeneration of rat livers subject to 70% hepatectomy // Acta Cir. Bras. 2018. Vol. 33, № 2. P. 110–116.

Purpose: To investigate thymoquinone, curcumin and a combination of these two drugs were effective or not at the growth of liver. Methods: Forty female Wistar-Albino rats distributed into five groups of eight rats each, control, thymoquinone, curcumin, and thymoquinone/curcumin groups. Pathological specimens were studied using the Ki-67 Proliferation Index(PI); and arginase(Arg), tissue plasminogen activator(tPA), ceruloplasmin(Cer) and nitric oxide(NO) were studied in biochemical analysis. Results: Our results showed that Ki-67 proliferation index was low in Groups 1. The proliferation coefficient was significantly higher in the Group 2 and Group 4 than in the Group 1 and Group 3.(P < 0.001 between Groups 1 and 2, 1 and 4, and 3 and 4). There was no difference between Groups 2 and 4 (P = 1). The results of the biochemical Arg, tPA and Cer test showed statistically between the Group 1 and Group 2. NO showed significant differences Group 1 and 3. Conclusions: Thymoquinone and curcumin both have known positive effects on the organism. Histological and biochemical tests showed that thymoquinone is more effective than curcumin.


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Grigoryan E.N., Markitantova Y.V. Cellular and Molecular Preconditions for Retinal Pigment Epithelium (RPE) Natural Reprogramming during Retinal Regeneration in Urodela // Biomedicines. 2016. Vol. 4, № 4. P. UNSP 28.

Many regeneration processes in animals are based on the phenomenon of cell reprogramming followed by proliferation and differentiation in a different specialization direction. An insight into what makes natural (in vivo) cell reprogramming possible can help to solve a number of biomedical problems. In particular, the first problem is to reveal the intrinsic properties of the cells that are necessary and sufficient for reprogramming; the second, to evaluate these properties and, on this basis, to reveal potential endogenous sources for cell substitution in damaged tissues; and the third, to use the acquired data for developing approaches to in vitro cell reprogramming in order to obtain a cell reserve for damaged tissue repair. Normal cells of the retinal pigment epithelium (RPE) in newts (Urodela) can change their specialization and transform into retinal neurons and ganglion cells (i.e., actualize their retinogenic potential). Therefore, they can serve as a model that provides the possibility to identify factors of the initial competence of vertebrate cells for reprogramming in vivo. This review deals mainly with the endogenous properties of native newt RPE cells themselves and, to a lesser extent, with exogenous mechanisms regulating the process of reprogramming, which are actively discussed.


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Guo Q. et al. Chitosan conduits filled with simvastatin/Pluronic F-127 hydrogel promote peripheral nerve regeneration in rats // J. Biomed. Mater. Res. Part B. 2018. Vol. 106, № 2. P. 787–799.

Treating peripheral nerve defects represents a clinical challenge, and nerve conduits lacking an internal scaffold lead to limited large nerve gap regeneration. Here, we bridged 10-mm sciatic nerve defects in rats with a chitosan conduit filled with 0, 0.5, or 1.0 mg of simvastatin in Pluronic F-127 hydrogel. We assessed subsequent nerve regeneration using the sciatic functional index (SFI), electrophysiological assessments, Fluoro-Gold (FG) retrograde tracing, gastrocnemius muscle mass measurements, and histological and immunohistochemical assessments of nerve regeneration. Ten weeks after implantation, the chitosan conduit filled with simvastatin/Pluronic F-127 hydrogel promoted nerve regeneration; there were significant increases in the SFI, compound muscle action potential peak amplitude, motor nerve conduction velocity, FG-labeled neuron number in the dorsal root ganglia, myelin sheath thickness, axon diameter, gastrocnemius wet weight, and muscle fiber area percentage in the gastrocnemius muscle (all p<0.05). The expression levels of neurotrophic factors, such as pleiotrophin, hepatocyte growth factor, vascular endothelial growth factor, and glial cell line-derived neurotrophic factor, were also found to be increased. The results suggest that chitosan conduits filled with simvastatin/Pluronic F-127 hydrogel improved peripheral nerve regeneration and functional recovery in rats, which may have been related to the increased expression of several endogenous neurotrophic factors. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 787-799, 2018.


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Hirashima S. et al. Effects of an in Vitro Reconstructed Three-dimensional Hematopoietic Microenvironment on Bone Regeneration in a Rat Calvarial Defect Model // J. Hard Tissue Biol. 2018. Vol. 27, № 3. P. 185–194.

The regeneration of large bone defects is limited by reduced angiogenesis and cell migration from the remaining bone and tissue. Although scaffolds are required as autogenous bone grafts or artificial substrates for efficient bone tissue engineering, cellular implants can also facilitate bone regeneration, and bone marrow is frequently applied to treat such defects. Here, we attempted to reconstruct a three-dimensional hematopoietic environment on a newly developed porous polyimide membrane using a modified Dexter culture method and found that the membrane could maintain CD34-, CD45-, and TER-119-positive cells. We further applied membrane-enclosed, hematopoietic-lineage cells to a rat calvarial bone defect model to evaluate the effects of a multicellular environment on bone regeneration. Our results suggested that the cultured hematopoietic environment on the porous membrane facilitated new bone formation. The bone volume and bone mineral content values of the coculture group at 8 weeks post-surgery were significantly higher than those in samples where only bone marrow stromal cells were used. Thus, coculture with multiple cell types accelerated bone formation, and culturing diverse cells on a membrane may facilitate cell transplantation in bone tissue engineering.


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Jeong J.H. et al. The Effect of Biocomposite Screws on Bone Regeneration in a Rat Osteoporosis Model // World Neurosurg. 2017. Vol. 106. P. 964–972.

OBJECTIVE: To evaluate the efficacy of biocomposite screws used in ovariectomy (OVX)-induced osteoporotic rats. METHODS: Twenty-four female Wistar rats (250-300 g, 12 weeks old) were divided into 4 groups: sham group (control), OVX-induced osteoporosis group (OVX), OVX and biodegradable poly(lactic-co-glycolic acid) (PLGA) without tricalcium phosphate (beta-TCP) screw insertion group (OVX/BSR), and OVX and biocomposite (PLGA with b-TCP) screw insertion group (OVX/CSR). Three groups underwent bilateral OVX, and of these, 2 groups had 2 different types of screw inserted at the proximal tibia. At 25 weeks after OVX, the bilateral tibias were extracted. The extracted tibiae were scanned by ex vivo micro-computed tomography (CT) and stained with hematoxylin and eosin (H&E) and with Masson's trichrome stain for pathological assessment. RESULTS: Compared with the ovariectomized groups, the control group had the highest values for bone mineral density (BMD), bone volume (BV)/total volume (TV), and trabecular number (Tb.N) and the lowest values for trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). In the pairwise comparison among ovariectomized groups, the OVX/CSR group had significantly higher BMD, BV/TV, and Tb.N values than the other 2 groups (OVX and OVX/BSR) and significantly lower Tb.Sp. Micro-CT scans showed clear evidence of new trabecular formation near the screw insertion site in the OVX/CSR group only. Analyses of H&E- and Masson's trichrome-stained sections showed more and thicker trabecular bone around the implant in the OVX/CSR group compared with the OVX and OVX/BSR groups. CONCLUSIONS: Biocomposite screws can improve local bone quality and facilitate osteoconductivity in an osteoporotic rat model.


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Ji J. et al. Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation // Cytotherapy. 2018. Vol. 20, № 1. P. 74–86.

Background aims. Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation. Methods. In this study, decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was manufactured using a chemical agent method (0.5% ammonium hydroxideTriton + 20 mmol/L NH4OH) in combination with a biological agent method (DNase solution), and the resulting DMA were evaluated by scanning electron microscopy (SEM) and immunocytochemistry. The effect of DMA on RPC proliferation and differentiation was evaluated by quantitative polymerase chain reaction, Western blot and immunocytochemistry analysis. Results. DMA was successfully fabricated, as demonstrated by SEM and immunocytochemistry. Compared with tissue culture plates, DMA may effectively enhance the proliferation of RPCs by activating Akt and Erk phosphorylation; when the two pathways were blocked, the promoting effect was reversed. Moreover, DMA promoted the differentiation of RPCs toward retinal neurons, especially rhodopsin-and recoverin-positive photoreceptors, which is the most interesting class of cells for retinal degeneration treatment. Conclusions. These results indicate that DMA has important roles in governing RPC proliferation and differentiation and may contribute to the application of RPCs in treating retinal degenerative diseases.


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Kim J.P. et al. The effect of full dose composite tissue allotransplantation immunosuppression on allograft motor nerve regeneration in a rat sciatic nerve model // Microsurgery. 2018. Vol. 38, № 1. P. 66–75.

Background: The purpose of this study was to identify which triple immunosuppressive protocols, currently used for vascularized composite allotransplantation in the clinic, will have the best effect on motor function recovery following nerve allograft reconstruction. Methods: Eighty-eight Lewis rats underwent a 1-cm sciatic nerve allograft transplantation and skin graft from 44 Brown-Norway rats. Group I received 0.9% isotonic saline (control); Group II, 2 mg/kg FK506; Group III, 1 mg/kg FK506 with 15 mg/kg mycophenolate mofetil (MMF); and Group IV, 2 mg/kg FK506 with 30 mg/kg MMF and prednisone. Each group consisted of 11 rats. After 12 weeks, motor function recovery was evaluated with isometric tetanic force, muscle mass, ankle contracture angle, electrophysiology, and nerve histomorphometry. Adequacy of immunosuppression was monitored with the transplanted skin graft. All data are expressed as a percentage of the contralateral side. Results: Isometric tetanic force showed significantly better functional recovery in all groups treated with immunosuppression compared to control. Within the immunosuppression groups no significant difference was found: 42.1 +/- 6.4% (Group I), 56.1 +/- 12.4% (Group II), 58.4 +/- 10.7% (Group III), and 61.3 +/- 11.2% (Group IV). Group IV was superior to all other groups regarding ankle contracture (P<. 05) and electrophysiology (P<. 001). Skin graft rejection occurred in 41 and 0% (Groups III and IV, respectively). Conclusions: FK506 significantly enhanced motor recovery after allograft reconstruction. This effect was comparable between combination treatment (low-dose FK506 and MMF) and triple therapy (high-dose FK506 and MMF plus prednisolone). However, triple therapy was more effective in suppressing skin rejection.


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Kwon J.-W. et al. Engraftment and regenerative effects of bone marrow stromal cell transplantation on damaged rat olfactory mucosa // Eur. Arch. Oto-Rhino-Laryn. 2016. Vol. 273, № 9. P. 2585–2590.

To develop a new therapeutic method to treat olfactory deficits, we investigated the engraftment and regenerative effects of transplanted bone marrow stromal cells (BMSCs) on damaged rat olfactory mucosa. To induce olfactory nerve degeneration, one side of the olfactory mucosa of Sprague-Dawley rats was damaged via Triton X-100 irrigation. Phosphate-buffered saline containing syngeneic BMSCs was injected into the olfactory mucosa for transplantation. PKH fluorescent cell dye labeling of BMSCs was used to monitor the transplanted cells. After transplantation of BMSCs, the thickness and regeneration of olfactory mucosa were analyzed using hematoxylin-eosin (H&E) staining. S100 immunohistochemical staining was used to measure nerve sheath regeneration. The increase in NGF (nerve growth factor) level in the olfactory mucosa was measured by Western blot analysis. Transplanted bone marrow stromal cells were engrafted to the lamia propria of damaged mucosa. The mean time for normalization of thickness and morphological recovery of the olfactory mucosa was 4 weeks in the therapeutic group and 9 weeks in the control group. S100 immunoreactivity was higher on the BMSC-treated side than on the control side. During regeneration, the expression of NGF increased in the olfactory mucosa of the experimental group. Based on these results, BMSC transplantation accelerated regeneration of olfactory mucosa damaged by Triton X-100, and NGF may be essential to this regenerative process.


23. PDF
Li M. et al. Tanshinone IIA attenuates nerve transection injury associated with nerve regeneration promotion in rats // Neurosci. Lett. 2017. Vol. 659. P. 18–25.

Tanshinone IIA (Tan IIA) is the major pharmacological constituent of Salvia miltiorrhiza Bunge (Danshen) for the therapeutic purpose of preventing ischemic injury and treating cerebrovascular disease. The aim of the present study was to explore the potential neuroprotective effects of Tan IIA in sciatic nerve transection injury. We investigated the possible beneficial effects of Tan IIA in promoting nerve regeneration after nerve transection injury in rats. Nerve transection injury was induced in male Sprague-Dawley rats by left sciatic nerve transection. After neuroanastomosis, the rats were intraperitoneally (IP) injected with 6 mg/kg, 15 mg/kg, or 40 mg/ kg Tan IIA once daily for 12 weeks; the vehicle and positive control groups were injected with normal saline and mecobalamin (MeCbl, 100 mu g/kg), respectively. Axonal regeneration and functional recovery were evaluated by a range of morphological and functional measures 12 weeks after neuroanastomosis. The administration of 15 mg/kg and 40 mg/kg Tan IIA and MeCbl achieved better axonal regeneration with significant restoration of motor function as well as a marked decrease in Fluoro-Gold (FG)-labeled neurons and increased nerve regeneration. At 12 weeks post-surgery, 40 mg/kg Tan IIA showed a better neuroprotective effect than 15 mg/kg Tan IIA and MeCbl. There were no statistical differences between the 15 mg/kg Tan IIA and MeCbl groups or the control and 6 mg/kg Tan IIA groups. Our findings demonstrate that Tan IIA can alleviate nerve injury and promote nerve regeneration in a sciatic nerve transection model in rats, providing supportive evidence for Tan IIA as an effective potential therapeutic remedy for peripheral nerve injury.


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Li X. et al. OM-LV20, a novel peptide from odorous frog skin, accelerates wound healing in vitro and in vivo // Chem. Biol. Drug Des. 2018. Vol. 91, № 1. P. 126–136.

The healing of chronic wounds remains a considerable challenge in clinical trials and imposes severe financial and physiological burdens on patients. Many works are being tried to find ideal clinical promoting wound healing biomaterials. Small bioactive peptides with low cost and easy production, store and transfer become excellent candidates. Here, we identified a novel peptide (named OM-LV20) from skin secretions of odorous frog Odorrana margaretae. The peptide had an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, contained an intramolecular disulfide bridge at the C-terminus, and was produced by post-translational processing of a 71-residue prepropeptide. Our results showed that OM-LV20 had no direct microbe-killing effects, hemolytic activity, or acute toxicity, but did exhibit weak antioxidant activity. OM-LV20 promoted wound healing against human keratinocytes (HaCaT) and human skin fibroblasts (HSF) in both time- and dose-dependent manners. In addition, it induced the proliferation of HaCaT but not HSF cells. Of note, OM-LV20 showed strong wound healing-promoting activity in a mice model of full-thickness skin wound. Our research indicates the cellular and animal level wound healing potential of OM-LV20, and thus provides a novel bioactive peptide template for the development of wound healing agents and medicine.


25. PDF
Liang B. et al. Local delivery of a novel PTHrP via mesoporous bioactive glass scaffolds to improve bone regeneration in a rat posterolateral spinal fusion model // RSC Adv. 2018. Vol. 8, № 22. P. 12484–12493.

With the development of tissue engineering, bone defects, such as fractured long bones or cavitary lesions, may be efficiently repaired and reconstructed using bone substitutes. However, high rates of fusion failure remain unavoidable in spinal fusion surgery owing to the lack of appropriate materials for bone regeneration under such challenging conditions. Parathyroid hormone (PTH), a major regulator of bone remodeling, exerts both anabolic and catabolic effects. In this study, we modified PTH(1-34) and designed and synthesized a novel PTH-related peptide, namely PTHrP-1. Further, we fabricated a local PTHrP delivery device from mesoporous bioactive glass (MBG) to address the need for a suitable material in spinal fusion surgery. Using MBG scaffolds as a control, the biological properties of PTHrP-MBG scaffolds were evaluated in terms of attachment, proliferation, and alkaline phosphatase activity, as well as osteogenic gene and angiogenic gene expression in co-cultured rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Furthermore, PTHrP-1-MBG scaffolds were tested in a rat posterolateral spinal fusion model. Our data showed that PTHrP-1-MBG scaffolds possessed good ability to facilitate attachment and stimulation of rBMSC proliferation and differentiation. Importantly, the in vivo results revealed that the PTHrP-1-MBG scaffolds facilitated faster new bone formation and a higher rate and quality of spinal fusion. Therefore, the results suggest that devices consisting of the present novel PTHrP and MBG possess wider potential applications in bone regeneration and should serve as a promising bone substitute for spinal fusion.


26. PDF
Luo L. et al. Exosomal MicroRNA-10a Is Associated with Liver Regeneration in Rats through Downregulation of EphA4 // Chin. Med. J. 2018. Vol. 131, № 4. P. 454–460.

Background: MicroRNAs (miRNAs) have been reported to play vital roles in liver regeneration. Previous studies mainly focused on the functions of intracellular miRNAs, while the functions of circulating exosomal miRNAs in liver regeneration remain largely unknown. The aim of this study was to identify the key exosomal miRNA that played vital roles in liver regeneration. Methods: The Sprague-Dawley male rats were assigned to 70% partially hepatectomized group (n = 6) and sham surgery group (n = 6). The peripheral blood of both groups was collected 24 h after surgery. The exosomal miRNAs were extracted, and microarray was used to find out the key miRNA implicated in liver regeneration. Adenovirus was used to overexpress the key miRNA in rats, and proliferating cell nuclear antigen (PCNA) staining was applied to study the effect of key miRNA overexpression on liver regeneration. Western blotting was used to validate the predicted target of the key miRNA. Results: Exosomal miR-10a was upregulated more than nine times in hepatectomized rats. The level of miR-10a was increased in the early phase of liver regeneration, reached the top at 72 h postsurgery, and decreased to perioperative level 168 h after surgery. Moreover, enforced expression of miR-10a by adenovirus facilitated the process of liver regeneration as evidenced by immunohistochemical staining of PCNA. Erythropoietin-producing hepatocellular receptor A4 (EphA4) has been predicted to be a target of miR-10a. The protein level of EphA4 was decreased in the early phase of liver regeneration, reached the bottom at 72 h postsurgery, and rose to perioperative level 168 h after surgery, which was negatively correlated with miR-10a, confirming that EphA4 served as a downstream target of miR-10a. Moreover, inhibition of EphA4 by rhynchophylline could promote the proliferation of hepatocytes by regulating the cell cycle. Conclusion: Exosomal miR-10a might accelerate liver regeneration through downregulation of EphA4.


27. PDF
Makanae A., Mitogawa K., Satoh A. Cooperative inputs of Bmp and Fgf signaling induce tail regeneration in urodele amphibians // Dev. Biol. 2016. Vol. 410, № 1. P. 45–55.

Urodele amphibians have remarkable organ regeneration ability. They can regenerate not only limbs but also a tail throughout their life. It has been demonstrated that the regeneration of some organs are governed by the presence of neural tissues. For instance, limb regeneration cannot be induced without nerves. Thus, identifying the nerve factors has been the primary focus in amphibian organ regeneration research. Recently, substitute molecules for nerves in limb regeneration, Bmp and Fgfs, were identified. Cooperative inputs of Bmp and Fgfs can induce limb regeneration in the absence of nerves. In the present study, we investigated whether similar or same regeneration mechanisms control another neural tissue governed organ regeneration, i.e., tail regeneration, in Ambystoma mexicanum. Neural tissues in a tail, which is the spinal cord, could transform wound healing responses into organ regeneration responses, similar to nerves in limb regeneration. Furthermore, the identified regeneration inducer Fgf2+Fgf8+Bmp7 showed similar inductive effects. However, further analysis revealed that the blastema cells induced by Fgf2+Fgf8+Bmp7 could participate in the regeneration of several tissues, but could not organize a patterned tail. Regeneration inductive ability of Fgf2+Fgf8+Bmp7 was confirmed in another urodele, Pleurodeles waltl. These results suggest that the organ regeneration ability in urodele amphibians is controlled by a common mechanism. (C) 2015 The Authors. Published by Elsevier Inc.


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Mendez-Olivos E.E., Munoz R., Larrain J. Spinal Cord Cells from Pre-metamorphic Stages Differentiate into Neurons and Promote Axon Growth and Regeneration after Transplantation into the Injured Spinal Cord of Non-regenerative Xenopus laevis Froglets // Front. Cell. Neurosci. 2017. Vol. 11. P. 398.

Mammals are unable to regenerate its spinal cord after a lesion, meanwhile, anuran amphibians are capable of spinal cord regeneration only as larvae, and during metamorphosis, this capability is lost. Sox2/3(+) cells present in the spinal cord of regenerative larvae are required for spinal cord regeneration. Here we evaluate the effect of the transplantation of spinal cord cells from regenerative larvae into the resected spinal cord of non-regenerative stages (NR-stage). Donor cells were able to survive up to 60 days after transplantation in the injury zone. During the first 3-weeks, transplanted cells organize in neural tube-like structures formed by Sox2/3(+) cells. This was not observed when donor cells come from non-regenerative froglets. Mature neurons expressing NeuN and Neurofilament-H were detected in the grafted tissue 4 weeks after transplantation concomitantly with the appearance of axons derived from the donor cells growing into the host spinal cord, suggesting that Sox2/3(+) cells behave as neural stem progenitor cells. We also found that cells from regenerative animals provide a permissive environment that promotes growth and regeneration of axons coming from the host. These results suggest that Sox2/3 cells present in the spinal cord of regenerative stage (R-stage) larvae are most probably neural stem progenitor cells that are able to survive, proliferate, self-organize and differentiate into neurons in the environment of the non-regenerative host. In addition, we have established an experimental paradigm to study the biology of neural stem progenitor cells in spinal cord regeneration.


29. PDF
Mizuno M. et al. Parathyroid Hormone (1-34) Enhances Bone Regeneration in Rats with Cranial Bone Defects // J. Hard Tissue Biol. 2018. Vol. 27, № 4. P. 303–308.

Intermittent administration of parathyroid hormone (PTH) is known to increase bone mass for the treatment of osteoporosis. The aim of this study was to evaluate the benefits of local intermittent administration of PTH for bone regeneration in rats with cranial bone defects. Cranial bone defects were induced under anesthesia using a trephine bur (diameter, 4.3 mm) in 8-week-old male Wistar rats, which were then divided into four groups for further treatment. In the PTH-3 and PTH-1 groups, animals received PTH at 14.1 mu g/kg in an absorbable collagen sponge placed near the bone defect. Animals in the collagen group received saline at 0.05 ml using the same method, while control animals received only sham surgery. Following surgery, PTH-3 animals received two subcutaneous injections of PTH (14.1 mu g/kg) at the experimental site, while the PTH-1, collagen. and control animals each received saline (0.05 ml). All animals were sacrificed at 21 days after surgery. The ratio of new bone mineral content to total defect volume (BMC/TV) at the experimental sites was evaluated using micro-computed tomography. Tissue sections were analyzed by hematoxylin and eosin staining and immunohistochemistry with anti-alkaline phosphatase (ALP), anti-dentin matrix protein 1 (DMP1) and anti-Osterix antibodies. The BMC/TV ratio was significantly higher in the PTH-3 and PTH-1 groups than in the collagen group. The ratio of new bone to defect area (N/D%) was significantly higher in PTH-3 animals than in controls. ALP-positivity was more widely distributed in new bone in PTH-3 animals than in the other groups, and regions of DMP1-positivity and Osterix-positivity were also more widespread in these animals. These findings suggest that local intermittent administration of PTH enhances bone regeneration in rats with cranial bone defects.


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Mohamadi F. et al. Use new poly (epsilon-aprolactone/collagen/NBG) nerve conduits along with NGF for promoting peripheral (sciatic) nerve regeneration in a rat // Artif. Cell. Nanomed. Biotechnol. 2018. Vol. 46. P. 34–45.

Regeneration of peripheral nerve defects remained a remarkable clinical challenge. Engineered nerve conduits represent a promising strategy to improve functional recovery in peripheral nerve injury repair. However, nerve conduits require additional factors such as neurotrophic factors to create a more conducive microenvironment for nerve regeneration. Neurotrophic factors have well-demonstrated abilities to improve neurite outgrowth, making them great candidates for repairing of defected nerves. To this end, we examined the beneficial effects of repairing the transected rat sciatic nerve by loading of nerve growth factor (NGF) in nerve conduits. The PCL/Collagen/NBG conduits were interposed into the 10mm right sciatic nerve defects. Twenty-four rats were randomly allocated into four groups: 1- nerve autograft group, 2- a nongrafted group with gap 10-mm, 3- conduit group and 4- the conduits loaded with NGF. Motor and sensory functional recovery, the evoked muscle action potential, and motor distal latency showed significant improvement in rats treated with NGF. The histology and immunohistochemistry studies revealed less fibrosis and a high level of expression of CD31 and NF-200 protein at the crush site in the Conduit+NGF group. In conclusion, the PCL/Collagen/NBG conduit loaded with NGF, which exhibited nanometer-scale features, neurotrophic activity, favorable mechanical properties and biocompatibility could improve sciatic nerve regeneration in rats.


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Molchanov A.Y., Burlakova O.V., Golichenkov V.A. Regeneration of the skin pigment system during larval development of the clawed frog // Russ. J. Dev. Biol. 2017. Vol. 48, № 1. P. 75–80.

We demonstrate regeneration capability of the skin pigment system of clawed frog larvae after local damage to melanophores without skin rupture. The contribution to recovery of pigmentation of the injured area of de novo differentiation of melanophores is compared to contribution of mitotic division of undamaged melanophores localized on the boundaries of the injured area. The regeneration process is observed during various stages of pigment system development of larvae. We establish that, compared to ontogenetic dynamics, pigmentation development in animals is more intense during the regeneration.


32. PDF
Nakamura R. et al. Heparin cross-linked collagen sponge scaffolds improve functional regeneration of rat tracheal epithelium // J. Tissue Eng. Regen. Med. 2017. Vol. 11, № 11. P. 3027–3037.

Tracheal epithelial cells maintain airway homeostasis by mediating mucociliary clearance. Following tracheal reconstruction, timely epithelial regeneration is required to prevent respiratory compromise and infectious diseases. To achieve rapid tracheal epithelial regeneration, a heparin cross-linked collagen sponge containing fibroblast growth factor-2 (FGF-2) was prepared as a graft for tracheal reconstruction. The heparin cross-linked sponge exhibited a high FGF-2 retaining capacity, and tracheal epithelial and mesenchymal cells cultured in this sponge containing FGF-2 showed high proliferative capacities. Subsequently, heparin-free collagen sponge scaffolds (C/F scaffold) and collagen sponge scaffolds cross-linked with 10 mu g/ml heparin retained FGF-2 (C/H10/F scaffold), and were transplanted into rats with tracheal defects. Invasion of both epithelial and non-epithelial cells was greater in rats treated with the C/H10/F scaffold at 1 week post-transplantation than in rats treated with the C/F scaffold. Moreover, at 2 weeks after transplantation, improved cilia formation was observed in the C/H10/F scaffold group, with higher motility and more potent posterior-anterior flow generation than in the C/F scaffold group. These results suggest that heparin improves functional regeneration of tracheal epithelium. Copyright (c) 2017 John Wiley & Sons, Ltd.


33. PDF
Nakayama E. et al. Transplantation of dedifferentiation fat cells promotes intervertebral disc regeneration in a rat intervertebral disc degeneration model // Biochem. Biophys. Res. Commun. 2017. Vol. 493, № 2. P. 1004–1009.

Our group has reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multi-lineage differentiation potential similar to that observed in mesenchymal stem cells. In the present study, we examined whether DFAT cell transplantation could contribute to intervertebral disc regeneration using a rat intervertebral disc degeneration (IDD) model. The IDD was created in Sprague-Dawley rats by puncturing at level of caudal intervertebral disc under fluoroscopy. One week after injury, rat DFAT cells (5 x 10(4), DFAT group, n = 13) or phosphate-buffered saline (PBS, control group, n = 13) were injected into the intervertebral disc. Percent disc height index (%DHI) was measured every week and histology of injured disc was evaluated at 8 weeks after transplantation. Radiographic analysis revealed that the %DHI in the DFAT group significantly higher than that in the control group at 2-3 weeks after transplantation. Histological analysis revealed that ectopic formation of nucleus pulposus (NP)-like tissue at the outer layer of annulus fibrosus was frequently observed in the DFAT group but not in the control group. Transplantation experiments using green fluorescent protein (GFP)-labeled DFAT cells revealed that the ectopic NP-like tissue was positive for GFP, suggesting direct differentiation of DFAT cells into NP-like cells. In conclusion, DFAT cell transplantation promoted the regeneration of intervertebral disc and improved intervertebral disc height in the rat IDD model. Because adipose tissue is abundant and easily accessible, DFAT cell transplantation may be an attractive therapeutic strategy against IDD. (C) 2017 The Authors. Published by Elsevier Inc.


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Nguyen M. et al. Retinoic acid receptor regulation of epimorphic and homeostatic regeneration in the axolotl // Development. 2017. Vol. 144, № 4. P. 601–611.

Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we target specific retinoic acid receptors (RARs) to either PD duplicate (RA treatment or RAR. agonist) or truncate (RAR beta antagonist) regenerating limbs. RARE-EGFP reporter axolotls showed divergent reporter activity in limbs undergoing PD duplication versus truncation, suggesting differences in patterning and skeletal regeneration. Transcriptomics identified expression patterns that explain PD duplication, including upregulation of proximal homeobox gene expression and silencing of distal-associated genes, whereas limb truncation was associated with disrupted skeletal differentiation. RAR beta antagonism in uninjured limbs induced a loss of skeletal integrity leading to long bone regression and loss of skeletal turnover. Overall, mechanisms were identified that regulate the multifaceted roles of RARs in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.


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Ni C. et al. Stem Leydig cell regeneration in the adult rat testis is inhibited after a short-term triphenyltin exposure // Toxicol. Lett. 2019. Vol. 306. P. 80–89.

Triphenyltin (TPT) is an organotin compound and may be an endocrine disruptor, impairing the male reproductive system. However, the effect of short-term TPT exposure on stem Leydig cell regeneration later on remains unknown. Here, we show that TPT affects stem Leydig cell regeneration in the adult rat testis. Adult male Sprague Dawley rats were gavaged with TPT (0, 0.5, 1.0, 2.0 mg/kg body weight/day) for 10 days, followed by a single intraperitoneal injection of ethane dimethane sulfonate (EDS, 75 mg/kg body weight) to eliminate Leydig cells. Testis parameters and hormone levels were investigated on post-EDS days 21, 35, and 56. TPT significantly reduced serum testosterone levels, decreased Leydig cell number and cell size, and down-regulated its specific gene and protein expression at 1.0 and 2.0 mg/kg even 56 days after cession of treatment. TPT lowered PCNA-labeling index of progenitor Leydig cells on post-EDS day 21. TPT also lowered AKT1 and AKT2, and ERK1/2 phosphorylation on post-EDS day 56. This study reveals that a short-term exposure to TPT blocks stem Leydig cell regeneration in the long term thus delaying spermatogenesis.


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Otsuka-Yamaguchi R. et al. Cells From Subcutaneous Tissues Contribute to Scarless Skin Regeneration in Xenopus laevis Froglets // Dev. Dyn. 2017. Vol. 246, № 8. P. 585–597.

Background: Mammals cannot regenerate the dermis and other skin structures after an injury and instead form a scar. However, a Xenopus laevis froglet can regenerate scarless skin, including the dermis and secretion glands, on the limbs and trunk after skin excision. Subcutaneous tissues in the limbs and trunk consist mostly of muscles. Although subcutaneous tissues beneath a skin injury appear disorganized, the cellular contribution of these underlying tissues to skin regeneration remains unclear. Results: We crossed the inbred J strain with a green fluorescent protein (GFP)-labeled transgenic Xenopus line to obtain chimeric froglets that have GFP-negative skin and GFP-labeled subcutaneous tissues and are not affected by immune rejection after metamorphosis. We found that GFP-positive cells from subcutaneous tissues contributed to regenerating the skin, especially the dermis, after an excision injury. We also showed that the skin on the head, which is over bone rather than muscle, can also completely regenerate skin structures. Conclusions: Cells derived from subcutaneous tissues, at least in the trunk region, contribute to and may be essential for skin regeneration. Characterizing the subcutaneous tissue-derived cells that contribute to skin regeneration in amphibians may lead to the induction of cells that can regenerate complete skin structures without scarring in mammals. (C) 2017 Wiley Periodicals, Inc.


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Pascual-Gil S. et al. Cytokine-loaded PLGA and PEG-PLGA microparticles showed similar heart regeneration in a rat myocardial infarction model // Int. J. Pharm. 2017. Vol. 523, № 2. P. 531–533.

Neuregulin (NRG1) and fibroblast growth factor (FGF1) are well known growth factors implicated in cardiomyocyte proliferation and survival, as well as in angiogenesis, the development of adult heart and the maintenance of cardiac function. NRG1 and FGF1 have become promising therapeutic agents to treat myocardial infarction (MI) disorder. Unfortunately, clinical trials performed so far reported negative efficacy results, because growth factors are rapidly degraded and eliminated from the biological tissues once administered. In order to increase their bioavailability and favour their therapeutic effects, they have been combined with poly(lactic-co-glycolic acid) and polyethylene glycol microparticles (PLGA MPs and PEG-PLGA MPs). Here we compare both types of microparticles loaded with NRG1 or FGF1 in terms of efficacy in a rat MI model. Our results showed that intramyocardial injection of NRG1 or FGF1-loaded PLGA and PEG-PLGA MPs brought about similar improvements in the ejection fraction, angiogenesis and arteriogenesis after administration into the infarcted hearts. PEG coating did not add any effect regarding MP efficacy. Both PLGA and PEG-PLGA MPs were equally phagocyted in the heart. To our knowledge, this is the first study analysing the opsonisation process in heart tissue. The results allow us to conclude that the opsonisation process is different in heart tissue compared to blood. (C) 2016 Elsevier B.V. All rights reserved.


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Peloso A. et al. Creation and implantation of acellular rat renal ECM-based scaffolds // Organogenesis. 2015. Vol. 11, № 2. P. 58–74.

Kidney transplantation is the only potentially curative treatment for patient facing end-stage renal disease, and it is now routinely used. Its use is mainly limited by the supply of transplantable donor organs, which far exceeds the demand. Regenerative medicine and tissue engineering offer promising means for overcoming this shortage. In the present study, we developed and validated a protocol for producing acellular rat renal scaffolds. Left kidneys were removed from 26 male Lewis rats (weights: 250-350 g) and decellularized by means of aortic anterograde perfusion with ionic and anionic detergents (Triton X-100 1% and SDS 1%, respectively). 19 scaffolds thus obtained (and contralateral native kidneys as controls) were deeply characterized in order to evaluate the decellularization quality, the preservation of extracellular matrix components and resultant micro-angioarchitecture structure. The other 7 were transplanted into 7 recipient rats that had undergone unilateral nephrectomy. Recipients were sacrificed on post-transplantation day 7 and the scaffolds subjected to histologic studies. The dual-detergent protocol showed, with only 5 h of perfusion per organ, to obtain thoroughly decellularized renal scaffolds consisting almost exclusively of extracellular matrix. Finally the macro-and the microarchitecture of the renal parenchyma were well preserved, and the grafts were implanted with ease. Seven days after transplant, the scaffolds were morphologically intact although all vascular structures were obstructed with thrombi. Production and implantation of acellular rat renal scaffolds is a suitable platform for further studies on regenerative medicine and tissue engineering.


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Saberi K. et al. Melatonin preconditioning of bone marrow-derived mesenchymal stem cells promotes their engraftment and improves renal regeneration in a rat model of chronic kidney disease // J. Mol. Histol. 2019. Vol. 50, № 2. P. 129–140.

Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has shown to be effective in treating chronic kidney disease. However, the effectiveness of this strategy is constrained by low homing and survival rate of transplanted cells in the injured organs. Therefore, developing strategies to improve homing and cell survival rate and therapeutic potential in cell-based therapies seems necessary. The purpose of this study is to evaluate the effect of pretreating BMMSCs with melatonin (MT) on the prosurvival and renoprotective of transplanted cells into the irreversible model of unilateral ureteral obstruction. Adult male Sprague-Dawley rats were randomized into four groups: Sham, UUO, UUO+BMMSCs, and UUO+BMMSCs+MT. The results of our study demonstrated that preconditioning with MT enhanced homing of BMMSCs into the injured kidney. MT reduced the number of TUNEL positive transplanted cells in the UUO+BMMSCs+MT group. The UUO+BMMSCs+MT group showed lower expressions of TGF-1, -SMA and TNF- at both gene and protein levels but higher expression of E-cadherin compared with the UUO+BMMSCs group. In addition, MT preconditioned BMMSCs ameliorated basement membrane disruption and histological status of injured renal tubules and also reduced fibrosis in damaged kidneys. In conclusion, our results show that stem cells pretreated by MT may represent a feasible approach for improving the beneficial effects of stem cell therapy and significantly enhance their survival after transplantation to the injured kidney.


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Sabin K.Z. et al. Ap-1(cFos/JunB)/miR-200a regulate the pro-regenerative glial cell response during axolotl spinal cord regeneration // Commun. Biol. 2019. Vol. 2. P. 91.

Salamanders have the remarkable ability to functionally regenerate after spinal cord transection. In response to injury, GFAP+ glial cells in the axolotl spinal cord proliferate and migrate to replace the missing neural tube and create a permissive environment for axon regeneration. Molecular pathways that regulate the pro-regenerative axolotl glial cell response are poorly understood. Here we show axolotl glial cells up-regulate AP-1cFos/JunB after injury, which promotes a pro-regenerative glial cell response. Injury induced upregulation of miR-200a in glial cells supresses c-Jun expression in these cells. Inhibition of miR-200a during regeneration causes defects in axonal regrowth and transcriptomic analysis revealed that miR-200a inhibition leads to differential regulation of genes involved with reactive gliosis, the glial scar, extracellular matrix remodeling and axon guidance. This work identifies a unique role for miR-200a in inhibiting reactive gliosis in axolotl glial cells during spinal cord regeneration.


41. PDF
Sasaki R.T. et al. Effect of Laser Photobiomodulation with Gradual or Constant Doses in the Regeneration of Rats’ Mental Nerve After Lesion by Compression // Photomed. Laser Surg. 2017. Vol. 35, № 8. P. 408–414.

Objective: Assess morphologically the efficacy of constant dose (CD) or gradual dose (GD) in photobiomodulation therapy (PBMT) during the regeneration process of rats' mental nerve after compression lesion. Materials and methods: Forty-eight male Wistar rats were used and divided into four groups (n = 12): negative control (NC): lesion by compression; positive control (PC): no lesion; GD: lesion by compression and PBMT with GD; and CD: lesion by compression and PBMT with CD. One day after the surgery, the groups GD and CD underwent PBMT daily in three equidistant points around the incision area. The parameters were wavelength of 808 nm, 100 mW, CD received treatment with 120 J/cm(2), while GD underwent the protocol of application: 1st and 4th sessions: 80 J/cm(2); 5th to 8th sessions: 90 J/cm(2); 9th to 12th sessions: 100 J/cm(2); 13th to 16th sessions: 110 J/cm(2); and 17th to 20th sessions: 120 J/cm(2). Euthanasias were performed at 3, 7, 14, and 21 days. Qualitative and quantitative analysis of the mental nerves were performed with ANOVA (analysis of variance) and Tukey tests (p <= 0.05). Results: It was observed that PBMT was able to accelerate the process of nerve regeneration presenting an increase in the number of myelinated fibers starting at 14 days of treatment for groups CD and GD, and at 21 days they were similar to PC. It was observed a better lamellar organization of myelin sheath at 7 days for GD and at 14 days for CD, similar to PC. Both GD and CD presented significant differences compared to NC and PC for thickness of the myelin sheath, outer perimeter, internal area, and number of myelin fibers. Conclusions: PBMT presented positive effect on the regeneration of nerve starting at 14 days, and after 21 days there was no difference between GD and CD.


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Satoh A. et al. Reactivation of larval keratin gene (krt62.L) in blastema epithelium during Xenopus froglet limb regeneration // Dev. Biol. 2017. Vol. 432, № 2. P. 265–272.

Limb regeneration is considered a form of limb redevelopment because of the molecular and morphological similarities. Forming a regeneration blastema is, in essence, creating a developing limb bud in an adult body. This reactivation of a developmental process in a mature body is worth studying. Xenopus laevis has a biphasic life cycle that involves distinct larval and adult stages. These distinct developmental stages are useful for investigating the reactivation of developmental processes in post -metamorphic frogs (froglets). In this study, we focused on the re-expression of a larval gene (krt62.L) during Xenopus froglet limb regeneration. Recently renamed krt62.L, this gene was known as the larval keratin (xlk) gene, which is specific to larval-tadpole stages. During limb regeneration in a froglet, krt62.L was re-expressed in a basal layer of blastema epithelium, where adult-specific keratin (Krt12.6.S) expression was also observable. Nerves produce important regulatory factors for amphibian limb regeneration, and also play a role in blastema formation and maintenance. The effect of nerve function on krt62.L expression could be seen in the maintenance of krt62.L expression, but not in its induction. When an epidermis-stripped limb bud was grafted in a froglet blastema, the grafted limb bud could reach the digit-forming stage. This suggests that krt62.L-positive froglet blastema epithelium is able to support the limb development process. These findings imply that the developmental process is locally reactivated in an postmetamorphic body during limb regeneration.


43. PDF
Seo D.K. et al. Enhanced axonal regeneration by transplanted Wnt3a-secreting human mesenchymal stem cells in a rat model of spinal cord injury // Acta Neurochir. 2017. Vol. 159, № 5. P. 947–957.

While pure mesenchymal stem cell (MSC) treatment for spinal cord injury (SCI) is known to be safe, its efficacy is insufficient. Therefore, gene-modified stem cells are being developed to enhance the effect of pure MSCs. We investigated the effect of stem cell therapy through the transfection of a Wnt3a-producing gene that stimulates axonal regeneration. MSCs obtained from the human umbilical cord blood (hMSCs) were multiplied, cultivated, and transfected with the pLenti-Wnt3a-GFP viral vector to produce Wnt3a-secreting hMSCs. A total of 50 rats were injured with an Infinite Horizon impactor at the level of the T7-8 vertebrae. Rats were divided into five groups according to the transplanted material: (1) phosphate-buffered saline injection group (sham group, n = 10); (Pertz et al. Proc Natl Acad Sci USA 105:1931-1936, 39) Wnt3a protein injection group (Wnt3a protein group, n = 10); (3) hMSC transplantation group (MSC group, n = 10); (4) hMSCs transfected with the pLenti vector transplantation group (pLenti-MSC group, n = 10); (5) hMSCs transfected with the pLenti+Wnt3a vector transplantation group (Wnt3a-MSC group, n = 10). Behavioral tests were performed daily for the first 3 days after injury and then weekly for 8 weeks. The injured spinal cords were extracted, and axonal regeneration markers including choline acetyltransferase (ChAT), growth-associated protein 43 (GAP43), and microtubule-associated protein 2 (MAP2) were investigated by immunofluorescence, RT-PCR, and western blotting. Seven weeks after the transplantation (8 weeks after SCI), rats in the Wnt3a-MSC group achieved significantly higher average scores in the motor behavior tests than those in the other groups (p < 0.05). Immunofluorescent stains showed greater immunoreactivity of ChAT, GAP43, and MAP2 in the Wnt3a-MSC group than in the other groups. RT-PCR and western blots revealed greater expression of these proteins in the Wnt3a-MSC group than in the other groups (p < 0.05). Wnt3a-secreting hMSC transplantation considerably improved neurological recovery and axonal regeneration in a rat SCI model.


44. PDF
Seo N. et al. Low-frequency pulsed electromagnetic field pretreated bone marrow-derived mesenchymal stem cells promote the regeneration of crush-injured rat mental nerve // Neural Regen. Res. 2018. Vol. 13, № 1. P. 145–153.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to promote the regeneration of injured peripheral nerves. Pulsed electromagnetic field (PEMF) reportedly promotes the proliferation and neuronal differentiation of BMSCs. Low-frequency PEMF can induce the neuronal differentiation of BMSCs in the absence of nerve growth factors. This study was designed to investigate the effects of low-frequency PEMF pretreatment on the proliferation and function of BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the regeneration of injured peripheral nerve using in vitro and in vivo experiments. In in vitro experiments, quantitative DNA analysis was performed to determine the proliferation of BMSCs, and reverse transcription-polymerase chain reaction was performed to detect S100 (Schwann cell marker), glial fibrillary acidic protein (astrocyte marker), and brain-derived neurotrophic factor and nerve growth factor (neurotrophic factors) mRNA expression. In the in vivo experiments, rat models of crush-injured mental nerve established using clamp method were randomly injected with low-frequency PEMF pretreated BMSCs, unpretreated BMSCs or PBS at the injury site (1 x 10(6) cells). DiI-labeled BMSCs injected at the injury site were counted under the fluorescence microscope to determine cell survival. One or two weeks after cell injection, functional recovery of the injured nerve was assessed using the sensory test with von Frey filaments. Two weeks after cell injection, axonal regeneration was evaluated using histomorphometric analysis and retrograde labeling of trigeminal ganglion neurons. In vitro experiment results revealed that low-frequency PEMF pretreated BMSCs proliferated faster and had greater mRNA expression of growth factors than unpretreated BMSCs. In vivo experiment results revealed that compared with injection of unpretreated BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher myelinated axon count and axon density and more DiI-labeled neurons in the trigeminal ganglia, contributing to rapider functional recovery of injured mental nerve. These findings suggest that low-frequency PEMF pretreatment is a promising approach to enhance the efficacy of cell therapy for peripheral nerve injury repair.


45. PDF
Shu Y. et al. Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between adult female and male Chinese concave-eared frogs (Odorrana tormota) // Gene. 2018. Vol. 678. P. 241–251.

The Chinese concave-eared torrent frog (Odorrana tormota) is the first known non-mammalian vertebrate that can communicate using ultrasound. In this species, females are approximately four times as large as males, in which the female growth rate is obviously higher than that of male. Until now, the molecular mechanisms underlying muscle growth development differences between male and female frogs have not been reported. Here, we integrated mRNA and miRNA expression profiles to reveal growth differences in the hindlimb muscles of 2-year-old frogs. Among 569 differentially expressed genes (DEGs), 69 were associated with muscle growth and regeneration. Fifty-one up-regulated genes in females were potentially involved in promoting muscle growth and regeneration, whereas 18 up-regulated genes in males may lead to muscle growth inhibition and fast-twitch muscle fiber contraction. 244 DEGs were enriched in mTOR and other protein synthesis signaling pathways, and protein degradation pathways, including lysosomal protease, calpain, caspase, and ubiquitin proteasome system pathways. It may interpret why female muscles grow faster than males. Based on expression differences of genes involved in glycolysis and oxidative metabolism, we speculated that the proportion of slow muscle fiber was higher and that of fast muscle fiber was lower in female compared with male muscle. Additionally, 767 miRNAs were identified, including 217 new miRNAs, and 6248 miRNA-negatively regulated mRNAs were predicted. The miRNA target genes were enriched in pathways related to muscle growth, protein synthesis, and degradation. Thus, in addition to the identified mRNA differential expressions, miRNAs may play other important roles in the differential regulation of hindlimb muscle growth between female and male O. tormota.


46. PDF
Sugiura T. et al. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration // Nature. 2016. Vol. 531, № 7593. P. 237–+.

Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed(1-3). In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals(4,5). A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain-and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals.


47. PDF
Tazawa I., Yaoita Y. Vitamin A induced homeotic hindlimb formation on dorsal and ventral sides of regenerating tissue of amputated tails of Japanese brown frog tadpoles // Dev. Growth Diff. 2017. Vol. 59, № 9. P. 688–700.

When anuran tadpoles are treated with vitamin A after tail amputation, hindlimb-like structures can be generated instead of the lost tail part at the amputation site. This homeotic transformation was initially expected to be a key to understanding the body plan of vertebrates. Unfortunately, homeotic limb formation has been reproduced in only some Indian frog species and a European species, but not in experimental anurans such as Xenopus laevis or Rana catesbeiana. Consequently, this fascinating phenomenon has not been well analyzed, especially at the molecular level. In addition, the initial processes of ectopic limb development are also unclear because morphological changes in the early phases have not been analyzed in detail. In this study, we report the induction of homeotic transformation using Japanese brown frogs and present a detailed morphological analysis. Unexpectedly, the ectopic limbs developed not only at the ventral sites, but also at the dorsal sites of the tail regenerates of vitamin A-treated tadpoles. The relationship between position and axial orientation of ectopic limbs suggested the double duplication of positional value order along the rostral-caudal axis and the dorsal-ventral axis of the tail regenerates.


48. PDF
Thygesen M.M. et al. A clinically relevant blunt spinal cord injury model in the regeneration competent axolotl (Ambystoma mexicanum) tail // Exp. Ther. Med. 2019. Vol. 17, № 3. P. 2322–2328.

A randomized controlled and blinded animal trial was conducted in the axolotl (Ambystoma mexicanum), which has the ability to regenerate from transectional spinal cord injury (SCI). The objective of the present study was to investigate the axolotl's ability to regenerate from a blunt spinal cord trauma in a clinical setting. Axolotls were block-randomized to the intervention (n=6) or sham group (n=6). A laminectomy of two vertebrae at the level caudal to the hind limbs was performed. To induce a blunt SCI, a 25 g rod was released on the exposed spinal cord. Multiple modalities were applied at baseline (pre-surgery), and subsequently every third week for a total of 9 weeks. Gradient echo magnetic resonance imaging (MRI) was applied to assess anatomical regeneration. To support this non-invasive modality, regeneration was assessed by histology, and functional regeneration was investigated using swimming tests and functional neurological examinations. MRI suggested regeneration within 6 to 9 weeks. Histological analysis at 9 weeks confirmed regeneration; however, this regeneration was not complete. By the experimental end, all animals exhibited restored full neurological function. The present study demonstrated that the axolotl is capable of regenerating a contusion SCI; however, the duration of complete regeneration required further investigation.


49. PDF
Tinajero Aroni M.A. et al. Loading deproteinized bovine bone with strontium enhances bone regeneration in rat calvarial critical size defects // Clin. Oral Investig. 2019. Vol. 23, № 4. P. 1605–1614.

ObjectiveTo evaluate the effect of grafting with strontium (Sr)-loaded deproteinized bovine bone (DBB) on bone healing in calvarial critical size defects (CSD) in rats.Material and methodsTwo circular bone defects (5mm in diameter) were created in the calvaria of 42 rats. One of the defects, randomly chosen, was grafted with (a) DBB, (b) DBB loaded with 19.6g/g of Sr (DBB/Sr1), or (c) DBB loaded with 98.1g/g of Sr (DBB/Sr2). The other defect was left empty as negative control. Groups of seven animals from each of the groups were euthanized 15 and 60days post-op. Bone healing in the CSD was evaluated by micro-CT and histology/histomorphometry and immunohistochemistry.ResultsDBB/Sr2-grafted sites showed statistically significantly shorter radiographic residual defect length compared with DBB/Sr1- and DBB-grafted sites, and with empty controls at 60days. Further, the amount of new bone formation in the DBB/Sr1- and DBB/Sr2-grafted sites was significantly higher compared with that in the DBB-grafted sites at 60days. A larger number of DBB/Sr1- and DBB/Sr2-grafted sites presented with no- or only limited to mild inflammation, compared with the DBB-grafted sites, especially at 60days. Higher expression of osteocalcin was observed in DBB/Sr1- and DBB/Sr2-grafted sites as compared to DBB-grafted sites.ConclusionGrafting with Sr-loaded DBB enhanced bone formation in CSD in rats, when compared with grafting with non-loaded DBB.Clinical relevanceGrafting with Sr-loaded DBB may enhance bone formation in bone defects.


50. PDF
van Neerven S.G.A. et al. Two-component collagen nerve guides support axonal regeneration in the rat peripheral nerve injury model // J. Tissue Eng. Regen. Med. 2017. Vol. 11, № 12. P. 3349–3361.

Progress in material development has enabled the production of nerve guides that increasingly resemble the characteristics of an autologous nerve graft. In the present study, 20 mm adult rat sciatic nerve defects were bridged with the collagen-based, two-component nerve guide Neuromaix', the commercially available NeuraGen (R) nerve tube or an autologous nerve graft. Neuromaix was able to support structural as well as functional regeneration across this gap. The majority of the axons grew across the scaffold into the distal nerve segment and retrograde tracing confirmed that these axons were of somatosensory and motor origin. Histomorphology revealed that axons regenerating through Neuromaix exhibited reduced myelin sheath thickness, whereas axon diameter and axon density were comparable to those of the autograft. Neuromaix implantation resulted in reinnervation of the gastrocnemius muscle to a level that was not significantly different from that supported by the autograft, as demonstrated by electrophysiology. Our findings show that the use of the Neuromaix scaffold not only allowed axonal regeneration across large nerve gaps, but that the regenerating axons were also able to functionally reinnervate the muscles. These data provide a promising perspective for the first in human application of the materials. Copyright (c) 2016 John Wiley & Sons, Ltd.


51. PDF
Wanner R. et al. Three-Dimensional In vivo Magnetic Resonance Imaging (MRI) of Mouse Facial Nerve Regeneration // Front. Neurol. 2019. Vol. 10. P. 310.

MRI (magnetic resonance imaging) is an indispensable tool in the diagnosis of centrals nervous system (CNS) disorders such as spinal cord injury and multiple sclerosis (MS). In contrast, diagnosis of peripheral nerve injuries largely depends on clinical and electrophysiological parameters. Thus, currently MRI is not regularly used which in part is due to small nerve calibers and isointensity with surrounding tissue such as muscles. In this study we performed translational MRI research in mice to establish a novel MRI protocol visualizing intact and injured peripheral nerves in a non-invasive manner without contrast agents. With this protocol we were able to image even very small nerves and nerve branches such as the mouse facial nerve (diameter 100-300 mu m) at highest spatial resolution. Analysis was performed in the same animal in a longitudinal study spanning 3 weeks after injury. Nerve injury caused hyperintense signal in T-2-weighted images and an increase in nerve size of the proximal and distal nerve stumps were observed. Further hyperintense signal was observed in a bulb-like structure in the lesion site, which correlated histologically with the production of fibrotic tissue and immune cell infiltration. The longitudinal MR representation of the facial nerve lesions correlated well with physiological recovery of nerve function by quantifying whisker movement. In summary, we provide a novel protocol in rodents allowing for non-invasive, non-contrast agent enhanced, high-resolution MR imaging of small peripheral nerves longitudinally over several weeks. This protocol might further help to establish MRI as an important diagnostic and post-surgery follow-up tool to monitor peripheral nerve injuries in humans.


52. PDF
Wu C.-H. et al. Long-Duration Muscle Dedifferentiation during Limb Regeneration in Axolotls // PLoS One. 2015. Vol. 10, № 2. P. e0116068.

Although still debated, limb regeneration in salamanders is thought to depend on the dedifferentiation of remnant tissue occurring early after amputation and generating the progenitor cells that initiate regeneration. This dedifferentiation has been demonstrated previously by showing the fragmentation of muscle fibers into mononucleated cells and by revealing the contribution of mature muscle fibers to the regenerates by using lineage-tracing studies. Here, we provide additional evidence of dedifferentiation by showing that Pax7 (paired-box protein-7) transcripts are expressed at the ends of remnant muscle fibers in axolotls by using in situ hybridization and by demonstrating the presence of Pax7(+) muscle-fiber nuclei in the early bud and mid-bud stages by means of immunohistochemical staining. During the course of regeneration, the remnant muscles did not progress; instead, muscle progenitors migrated out from the remnants and proliferated and differentiated in the new tissues at an early stage of differentiation. The regenerating muscles and remnant muscles were largely disconnected, and this left a gap between them until extremely late in the late stage of differentiation, at which point the new and old muscles connected together. Notably, Pax7 transcripts were detected in the regions of muscles that faced these gaps; thus, Pax7 expression might indicate dedifferentiation in the remnant-muscle ends and partial differentiation in the regenerating muscles. The roles of this long-duration dedifferentiation in the remnants remain unknown. However, the results presented here could support the hypothesis that long-duration muscle dedifferentiation facilitates the connection and fusion between the new and old muscles that are both in an immature state; this is because immature Pax7(+) myoblasts readily fuse during developmental myogenesis.


53. PDF
Wu J. et al. A frog cathelicidin peptide effectively promotes cutaneous wound healing in mice // Biochem. J. 2018. Vol. 475. P. 2785–2799.

Although cathelicidins in mammals have been well characterized, little is known about the function of cathelicidin in amphibians. In the present study, a novel 24-residue peptide (cathelicidin-NV, ARGKKECKDDRCRLLMKRGSFSYV) belonging to the cathelicidin family was identified from the skin of the plateau frog Nanorana ventripunctata. Cathelicidin-NV showed strong wound healing-promoting activity in a murine model with a full-thickness dermal wound. It directly enhanced the proliferation of keratinocyte cells, resulting in accelerated re-epithelialization of the wound site. Cathelicidin-NV also promoted the proliferation of fibroblasts, the differentiation of fibroblasts to myofibroblasts and collagen production in fibroblasts, which are implicated in wound contraction and repair processes. Furthermore, cathelicidin-NV promoted the release of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, vascular endothelial growth factor and transforming growth factor-beta 1 in vivo and in vitro, which are essential in the wound-healing processes such as migration, proliferation and differentiation. The MAPK (ERK, JNK and p38) signaling pathways were involved in the wound healing-promoting effect. Additionally, unlike other cathelicidins, cathelicidin-NV did not have any direct effect on microbes and showed no cytotoxicity and hemolytic activity toward mammalian cells at concentrations up to 200 mu g/ml. This current study may facilitate the understanding of the cellular and molecular events that underlie quick wound healing in N. ventripunctata. In addition, the combination of these properties makes cathelicidin-NV an excellent candidate for skin wound therapeutics.


54. PDF
Xu K. et al. Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses // Sci Rep. 2016. Vol. 6. P. 19811.

Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells.


55. PDF
Yang H. et al. Vitamin C plus hydrogel facilitates bone marrow stromal cell-mediated endometrium regeneration in rats // Stem Cell Res. Ther. 2017. Vol. 8. P. 267.

Background: Intrauterine adhesion (IUA) is a common uterine cavity disease which can be caused by mechanical damage that may eventually lead to infertility and pregnancy abnormalities. Since the effect of therapeutic drugs appears disappointing, cell therapy has emerged as an alternative choice for endometrium regeneration. The aim of this study is to investigate whether the combination of hydrogel Pluronic F-127 (PF-127), Vitamin C (Vc), and a bone marrow stromal cell (BMSC) mixture could be a feasible strategy to improve the endometrial regeneration in a mechanical damage model of IUA in rats. Methods: Firstly, PF-127 cytotoxicity and the effect of Vc was tested in vitro using the Annexin V/propidium iodide (PI) apoptosis test, cell count kit (CCK) growth test, and enzyme-linked immunosorbent assay (ELISA). For the establishment of the rat IUA model, a 2-mm transverse incision in the uterus was prepared at the upper end, and 1.5 to 2.0 cm endometrial damage was scraped. Rats were randomly assigned to five groups to investigate the combined strategy on IUA uterine regeneration: a sham group, an IUA control group, an IUA BMSC encapsulated in PF-127 plus Vc group, an IUA BMSC plus Vc group, and an IUA PF-127 plus Vc group. A cell mixture was injected into the uterine horn while making the IUA model. Eight weeks after cell transplantation, the rats were sacrificed and the uterine was dissected for analysis. Endometrial thickness, gland number, fibrosis area, and the expression of marker proteins for endometrial membrane were examined by hematoxylin and eosin staining, Masson's staining, and immunohistochemistry. Results: Vc promoted the survival and health of PF-127-encapsulated BMSCs in vitro. When this combination was transplanted in vivo, the endometrium showed better restoration as the endometrium membrane became thicker and had more glands and less fibrosis areas. The expression of cytokeratin, von Willebrand Factor (vWF), was also restored. The proinflammatory cytokine interleukin-1 beta (IL-1 beta) was significantly lower compared with the control group. Conclusions: Vc alleviates the cytotoxic effect of PF-127 and promotes cell survival and growth in rat BMSC encapsulation. Thus, a cell therapy strategy containing biomaterial scaffold, BMSCs and the modulatory factor Vc promotes the restoration of damaged IUA endometrium.


56. PDF
Zhao C. et al. Preparation of decellularized biphasic hierarchical myotendinous junction extracellular matrix for muscle regeneration // Acta Biomaterialia. 2018. Vol. 68. P. 15–28.

Muscle injury and defect affect people's quality of life, and effective treatment is lacking. Herein, we generated a scaffold to obtain decellularized porcine Achilles tendon myotendinous junction (D-MTJ) extra- cellular matrix (ECM) with well-preserved native biphasic hierarchical structure, biological composition, and excellent mechanical properties for muscle regeneration. The combined use of potassium chloride, potassium iodide, Triton-X 100, and sodium-dodecyl sulfate (SDS) can completely remove the main immunogenicity, while maintaining the major biological components and microstructure. The specific biomechanics of D-MTJ is comparable to the native muscle-tendon physiological conditions. Additionally, the D-MTJ ECM scaffold induced minimal immunological reaction (histology analysis) through rat subcutaneous implantation. Moreover, in vitro, muscle satellite cells adhered, proliferated, and infiltrated into the D-MTJ scaffold, and myofiber-like cell differentiation was observed as shown by increased expression of myogenesis-related genes during culture. In vivo, newly formed myofibers were observed in a muscle defect model with D-MTJ orthotopic transplantation, while the control group presented mostly with fibrous tissue deposition. Additionally, the number of Myod and MyHC-positive cells in the ECM scaffold group was higher at day 30. We preliminary explored the mechanisms underlying D-MTJ-mediated muscle regeneration, which may be attributed to its specific biphasic hierarchical structure, bio-components, and attractiveness for myogenesis cells. In conclusion, our findings suggest the D-MTJ ECM scaffold prepared in this study is a promising choice for muscle regeneration. Statement of Significance This study is the first to use decellularization technology obtaining the specifically decellularized myotendinous junction (D-MTJ) with well-preserved biphasic hierarchical structure and constituents, excellent mechanical properties and good biocompatibility. The D-MTJ was further proved to be efficient for muscle regeneration in vitro and in vivo, and the underlying mechanisms may be attributed to its specifically structure and constituents, improved myogenesis and good preservation of repair-related factors. Our study may provide basis for the decellularization of other biphasic hierarchical tissues and a platform for further studies on muscle fiber and tendon integrations in vitro. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


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Zhu J. et al. Effect of decellularized spinal scaffolds on spinal axon regeneration in rats // J. Biomed. Mater. Res. Part A. 2018. Vol. 106, № 3. P. 698–705.

A series of complex influencing factors lead to failure of neural regeneration after spinal cord injury (SCI). Up to now, there is no robust treatment that can restore the loss of function caused by injury. Because damaged spinal axons do not spontaneously regenerate in their naturally inhibitory microenvironments, biomaterials that induce neural regeneration to appear as attractive treatments to improve the microenvironmental conditions after SCI. In this study, we report the novel use of decellularized (DC) scaffolds to provide contact guidance for axonal regrowth in vivo. The idea is that the scaffolds comprise some cytokines and a physical compartment that may facilitate regeneration. To evaluate the efficacy of scaffolds in supporting neural regeneration after SCI, the scaffold was implanted into an injured spinal cord of the rat. The injured spinal scaffolds showed a significant increase of the expression of GAP43, NF200, and Nestin in the scaffold implant groups compared with controls without the scaffold. In addition, the motor function has a better recovery. Together, these results demonstrate that spinal acellular scaffold is capable of promoting axonal regeneration after SCI and may serve as a potential tool in the treatment of SCI. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 698-705, 2018.


58. 040991
Басок Ю.Б., Севастьянов В.И. ТЕХНОЛОГИИ ТКАНЕВОЙ ИНЖЕНЕРИИ И РЕГЕНЕРАТИВНОЙ МЕДИЦИНЫ В ЛЕЧЕНИИ ДЕФЕКТОВ ХРЯЩЕВОЙ ТКАНИ СУСТАВОВ // Вестник трансплантологии и искусственных органов. 2016. Т. 18. № 4. С. 102-122.

Важнейшими проблемами здравоохранения в индустриальном обществе являются повреждение и дегенерация суставного хряща, что связано с ограниченными возможностями ткани к регенерации. В обзоре подробно описаны существующие и разрабатываемые технологии восстановления и замещения поврежденных хрящевых тканей суставов. Дан анализ полученных результатов по двум основным направлениям: стимулирование регенерации поврежденной хрящевой ткани и выращивание элементов хрящевой ткани в биореакторах.


59. 042126
Ваза А.Ю., Макаров М.С., Сластинин В.В., Боровкова Н.В., Клюквин И.Ю., Похитонов Д.Ю., Пономарев И.Н. ЭФФЕКТИВНОСТЬ КОМБИНАЦИИ АЛЛОГЕННОЙ БОГАТОЙ ТРОМБОЦИТАМИ ПЛАЗМЫ С КОЛЛАГЕНОМ ПРИ ЛЕЧЕНИИ ДЕФЕКТОВ БЕДРЕННОЙ КОСТИ У КРЫС // Трансплантология. 2016. № 2. С. 36-44.

Проведен эксперимент по исследованию действия богатой тромбоцитами плазмы (БоТП) в сочетании с коллагеном на процесс регенерации кости в дистальном отделе бедра крыс. Используемая доза крысиной БоТП содержала 130-135 пг/мл тромбоцитарного фактора роста (PDGF). Установлено, что при таком уровне PDGF комбинация БоТП и коллагена значительно ускоряла рост костных трабекул в области дефекта, стимулировала ангиогенез и снижала интенсивность воспалительной реакции. У всех обследованных животных использование аллогенной БоТП позволило в 2 раза сократить сроки репарации дефекта бедренной кости.


60. 000617

Оценивали влияние гиперлипидемии на системную продукцию цитокинов в плазме крови крыс после реконструктивного моделирования передней брюшной стенки эндопротезами из полипропилена ("Эсфил") и политетрафторэтилена ("Экофлон"). В группу сравнения входили животных без гиперлипидемии, которым также проводили пластику передней брюшной стенки данными эндопротезами. Контролем служили здоровые крысы без гиперлипидемии и оперативного вмешательства. В динамике после оперативной реконструкции передней брюшной стенки (с 1-х по 10-е сутки) у животных регистрировалась гиперцитокинемия. При аллопластике политетрафторэтиленом в плазме крови крыс определялась более выраженная продукция про- и противовоспалительных цитокинов, что характеризует преимущественную реактогенность имплантата. При гиперлипидемии изменялась реактивность иммунокомпетентных клеток на эндопротез. К 10-м суткам после эндопротезирования брюшной стенки в условиях гиперлипидемии наблюдалось прогрессивное снижение уровня провоспалительных цитокинов (ИЛ-1b и ФНО-a), более выраженное при использовании политетрафторэтилена. У крыс с гиперлипидемией в 1-е сутки после пластики полипропиленом в плазме крови определялось значительное увеличение содержания про- и противовоспалительных цитокинов (ИЛ-1b, ФНО-a, ИФН-g и ИЛ-10), а к 10-м суткам наблюдалось повышение уровня ИФН-g в плазме крови, что отражает прогрессивную активацию Тh1-лимфоцитов.


61. 005108

Более высокие потенции к регенерации у хвостатых амфибий, в сравнении с возможностями восстановления органов и тканей у других позвоночных, являются предметом многолетних интенсивных исследований. Накопленная информация раскрывает клеточные и молекулярные основы процессов регенерации у Urodela, но не объясняет причины поддержания регенераторных способностей у половозрелых животных. Суммированная в обзоре информация позволяет сделать предположение о том, что педоморфоз, присущий этому семейству животных, обусловливает сохранение ювенильности на всех уровнях организации – от организменного до молекулярно-генетического, что в свою очередь существенно облегчает и допускает инициацию и развитие регенеративных ответов на травму вплоть до эпиморфной регенерации целых органов. В качестве примера прослежены связанные с педоморфозом клеточные и молекулярно-генетические особенности тканей глаза и мозга Urodela, которые играют предположительно пермиссивную роль в их регенерации.


62. 000513

Современные достижения биологии в понимании процессов регенерации тканей, выявлении эндогенных источников регенерации, а также развитие методологии индукции и дифференцировки плюрипотентных стволовых клеток открывают широкие перспективы для регенерационной медицины. Однако использование получаемой информации в прикладных биомедицинских исследованиях затруднено недостаточным знанием молекулярных факторов и их композиций, способных регулировать регенерационные ответы клеток-источников регенерации. В обзоре приводятся собственные и литературные данные о категориях клеток, являющихся эндогенными источниками регенерации тканей глаза у низших и высших позвоночных. Рассматриваются особенности профиля экспрессии генов, позволяющего этим клеткам сохранять «молодой» фенотип. Приводятся транскрипционные факторы и сигнальные пути, обеспечивающие это состояние, а также репрограммирование и вход в пролиферативную фазу клеток-источников. Обсуждается роль в данных процессах факторов крови, иммунной системы, гормонов, продуктов окислительного стресса и факторов так называемого «клеточного омоложения» при их взаимодействии с локальными факторами клеточного окружения. Анализируются условия и молекулярные факторы - индукторы репрограммирования и пролиферации клеток, являющихся потенциальными источниками регенерации тканей глаза in vitro. Уделено внимание эпигенетическим изменениям, в т.ч. зависимым от возраста, в процессе конверсии клеток-источников регенерации.


63. 002190

Проанализированы клетки ретинального пигментного эпителия (РПЭ) взрослого тритона, обладающие уникальной способностью к репрограммированию в клетки сетчатки in vivo. Суммированы собственные и имеющиеся в литературе данные об особенностях биологии этих клеток ? от морфологии до молекулярного профиля, которые могут быть ассоциированы со способностью к изменению фенотипа. Установлено, что в РПЭ взрослого тритона сочетаются молекулярные признаки специализированных и малодифференцированных клеток. Отмечено, что персистентная на низком уровне пролиферация и быстрая смена специфических белков цитоскелета могут также способствовать успеху репрограммирования клеток РПЭ в нейрональном направлении. Каждый из рассмотренных факторов компетенции к репрограммированию может быть обнаружен для РПЭ животных, чьи клетки не способны in vivo к смене фенотипа на нейральный, но их совокупность, подкрепленная пермиссивным для конверсии эпигенетическим состоянием, возможно, является внутренним свойством только РПЭ тритона.


64. 045783
Измайлов А.А., Соколов М.Е., Баширов Ф.В., Фадеев Ф.О., Маркосян В.А., Гарифулин Р.Р., Лисюков А.Н., Кузнецов М.С., Исламов Р.Р. СРАВНИТЕЛЬНЫЙ АНАЛИЗ ЭФФЕКТИВНОСТИ ПРЯМОЙ И КЛЕТОЧНО-ОПОСРЕДОВАННОЙ ГЕННОЙ ТЕРАПИИ КРЫС С КОНТУЗИОННОЙ ТРАВМОЙ СПИННОГО МОЗГА // Гены и Клетки. 2017. Т. 12. № 4. С. 53-59.


65. 039335

Статья посвящена совместному и отдельному влиянию препаратов паракринных факторов мезенхимальных стволовых клеток и пероксиредоксина 6 на течение химического ожога кожи. Рассмотрены эффекты воздействия используемых препаратов на процесс регенерации кожи после аппликаций трихлоруксусной кислотой. Оценку результатов проводили на основе визуального осмотра в течение 7 суток с момента ожога. Помимо этого, через 24 часа после нанесения ожога определялась относительная концентрация маркера клеточной пролиферации Ki-67 и маркера апопто-за Cas 3, а так же уровень цитокинов в тканях. Концентрация Ki-67 была намного выше в группах с лечением. Относительная концентрация Cas 3 в группах с лечением ниже относительно контрольной группы. Была показана высокая эффективность применения пероксиредоксина 6 при лечении химического ожога, что связано с подавлением данным ферментом-антиоксидантом окислительного стресса. Исходя из результатов анализа профиля цитокинов, сделан вывод о том, что в группе с совместным применением пероксиредоксина 6 и паракринных факторов мезенхимальных стволовых клеток активнее всего проходят процессы клеточной пролиферации и заживления ткани, так как общий уровень цитокинов стимулирующих воспаление в данной группе снижен.


66. 00080X

Исследовали 50 самок крыс линии Вистар, разделенных на опытную (n=30) и контрольную (n=20) группы. Крысам опытной группы на уровне 9-го грудного позвонка (ThIX) проводили контузию спинного мозга свободно падающим цилиндрическим грузом диаметром 1,8 мм и массой 10 г с высоты 25 мм. Оценку их локомоторной активности осуществляли в соответствии с графиком эксперимента. Степень функционального восстановления функций тазовых конечностей оценивали по стандартной шкале BBB (от 0 баллов - отсутствие движения в тазовых конечностях до 21 балла - нормальная ходьба). Во время выхода из наркоза неврологический статус всех животных опытной группы соответствовал 0 баллов. Локомоторная активность крыс контрольной группы оценивалась в 21 балл. Первые признаки самопроизвольного восстановления двигательной активности тазовых конечностей животных опытной группы регистрировали на 3-и сут после операции. Функции тазовых конечностей в значительной степени восстанавливались в течение 4 нед, после чего кривая восстановления выходила на плато...


67. 00789X

Выбор клеточных ресурсов для рецеллюляризации биологических каркасов и синтетических матриксов остается актуальной проблемой регенеративной медицины. Выделение стромальных клеток из нативных органов и использование их для рецеллюляризации биологических матриксов представляется перспективной альтернативой применению мезенхимных стромальных клеток. В настоящей работе модифицированы протоколы получения стромальных клеток из гомогенизированных тканей легких и диафрагмы крыс. После типирования культур и оценки жизнеспособности клеток на каркасах выполняли ортотопическую трансплантацию тканеинженерной конструкции диафрагмы крысам с гистологической характеристикой эксплантатов через 24 и 45 сут. Каркасы, рецеллюляризированные стромальными клетками из нативных тканей, при прочих равных свойствах, вызывали менее выраженную воспалительную реакцию, чем каркасы, рецеллюляризированные мезенхимными стромальными клетками (МСК), выраженность фиброза также была ниже, чем при использовании МСК для заселения каркасов.


68. 00789X

Использование рецеллюляризированных матриксов позволяет получать тканеинженерные конструкции, в значительной мере воспроизводящие морфофункциональные особенности нативных органов и тканей. Сомнения в целесообразности предимплантационной рецеллюляризации каркасов, обусловленные гибелью предварительно заселенных на матрикс клеток в условиях in vivo, требуют изучения поведения клеточных культур после рецеллюляризации на пластике и на биологических имплантатах. Децеллюляризированный детергент-энзиматическим методом пищевод крыс был заселен GFP-позитивными клетками, интенсивно флуоресцирующими в зеленом спектре, что позволило проследить состояние исходной клеточной популяции при культивировании на пластике и после проведения трансплантаций, рассчитать жизнеспособность и метаболическую активность клеток, провести флуоресцентную детекцию клеток на каркасе до и после имплантации. Данных об активной пролиферации заселенных на матрикс клеток получено не было, однако наблюдали косвенные признаки наличия метаболической активности и синтеза белка GFP клетками после рецеллюляризации каркаса.


69. 002190
Маркитантова Ю.В., Авдонин П.П., Григорян Э.Н. КОМПОНЕНТЫ FGF2 СИГНАЛЬНОГО ПУТИ В ТКАНЯХ ЗАДНЕГО СЕКТОРА ГЛАЗА ВЗРОСЛОГО ТРИТОНА PLEURODELES WALTL // Известия Российской академии наук. Серия биологическая. 2014. № 4. С. 325.

Впервые выявлены компоненты сигнального пути FGF2 в тканях задней стенки нормального и регенерирующего глаза взрослого тритона Pleurodeles waltl. В сетчатке, пигментном эпителии сетчатки и сосудистой оболочке методом полимеразной цепной реакции (ПЦР) обнаружена экспрессия гена fgf2. Доказана высокая гомология нуклеотидной последовательности мРНК наиболее консервативного участка гена fgf2 тритона P. waltl с ортологами fgf2 других позвоночных. Аминокислотная последовательность белка Fgf2 тритона P. waltl демонстрирует еще бо льшую гомологию с этим ростовым фактором у других позвоночных. В исследуемых тканях глаза с использованием Вестерн-блот-гибридизации обнаружен белок Fgf2 с молекулярной массой 35 кДа. Иммуногистохимически изучена локализация белка Fgf2 и его рецепторов Fgfr в пигментном эпителии, хороиде, центральной и ростовой области сетчатки нативного глаза тритона, соединительной ресничке фоторецепторов...


70. 005108
Молчанов А.Ю., Бурлакова О.В., Голиченков В.А. РЕГЕНЕРАЦИЯ ПИГМЕНТНОЙ СИСТЕМЫ КОЖИ В ЛИЧИНОЧНОМ РАЗВИТИИ ШПОРЦЕВОЙ ЛЯГУШКИ // Онтогенез. 2017. Т. 48. № 1. С. 84-90.

Показана возможность восстановления пигментной системы покровов личинок шпорцевой лягушки после локального повреждения меланофоров без разрушения кожного покрова. Сопоставлен вклад в восстановление пигментации поврежденного участка за счет дифференцировок меланофоров de novo и митотических делений неповрежденных меланофоров, локализованных по границе участка. Прослежен процесс регенерации в разные периоды развития пигментной системы личинок. Установлено более интенсивное развитие пигментации у животных при восстановлении по сравнению с онтогенетической динамикой.


71. 004425
Мухаммадиева Г.Ф., Каримов Д.О., Кутлина Т.Г., Валова Я.В., Хуснутдинова Н.Ю., Репина Э.Ф., Бакиров А.Б. ЭКСПРЕССИЯ ГЕНОВ КОНТРОЛЯ КЛЕТОЧНОГО ЦИКЛА, ОКИСЛИТЕЛЬНОГО СТРЕССА И АПОПТОЗА (CHEK1, HMOX1, CASP7) В ПЕЧЕНИ КРЫС В ОТВЕТ НА ТЕТРАХЛОРМЕТАН // Молекулярная биология. 2019. Т. 53. № 1. С. 84-90.

Тетрахлорметан относится к одному из наиболее хорошо изученных гепатотропных ядов. Отравление экспериментальных животных тетрахлометаном схоже с острыми поражениями печени различной этиологии у человека. Исследована экспрессия генов контроля клеточного цикла, апоптоза и окислительного стресса на модели индуцированного тетрахлорметаном токсического гепатита. В работе использованы белые беспородные крысы-самцы, которым однократно вводили 50%-ный масляный раствор тетрахлорметана в дозе 0.125–4.000 г/кг (экспериментальная группа) или оливковое масло (контрольная группа). Через 24 и 72 ч после введения тетрахлорметана животных декапитировали и, используя ПЦР в режиме реального времени, анализировали в печени экспрессию генов гемоксигеназы-1 (Hmox1), киназы-1 контрольной точки клеточного цикла (Chek1) и каспазы-7 (Casp7). Показано, что в печени животных из экспериментальной группы повышена экспрессия генов Hmox1 и Chek1, возможно, принимающих участие в патологических процессах, происходящих в печени при воздействии окислительного стресса. Можно предположить, что при формировании токсического поражения печени под действием тетрахлометана наибольший вклад в гибель клеток вносит некроз.


72. 041265
Николаев С.М., Разуваева Я.Г., Торопова А.А., Убеева И.П., Верлан Н.В., Аюшеева В.В. ВЛИЯНИЕ КОМПЛЕКСНОГО РАСТИТЕЛЬНОГО СРЕДСТВА НА МОРФОФУНКЦИОНАЛЬНОЕ СОСТОЯНИЕ ПЕЧЕНИ БЕЛЫХ КРЫС ПРИ ЭТАНОЛОВОМ ГЕПАТИТЕ // Бюллетень Восточно-Сибирского научного центра Сибирского отделения Российской академии медицинских наук. 2016. Т. 1. № 6 (112). С. 167-170.

Исследовано влияние комплексного растительного средства (Urtica dioica L., Polygonum aviculare L., Achillea millefolium L., Zingiber officinalis L., Cinnamomum cassia L.) на морфофункциональное состояние печени белых крыс при этаноловом гепатите. Установлено, что исследуемое средство ограничивает микроциркуляторные нарушения,уменьшает дистрофические, некротические процессы в гепатоцитах, снижает интенсивность воспалительной инфильтрации печени и стимулирует регенерацию печеночных клеток при этаноловом гепатите.


73. 005108
Новикова Е.Л., Бакаленко Н.И., Нестеренко А.Ю., Кулакова М.А. НОХ-ГЕНЫ И РЕГЕНЕРАЦИЯ У ЖИВОТНЫХ // Онтогенез. 2016. Т. 47. № 4. С. 209-218.

Понятие регенерации тесно связано с представлениями о позицонной информации, то есть распределении в теле зародыша или взрослого организма разного рода сигналов, которые указывают клеткам их местоположение. Прекрасными кандидатами на роль факторов, создающих позиционную информацию, могут быть Нох-гены. Считается, что основная их функция состоит в эмбриональной регионализации и спецификации передне-задней оси тела билатеральных животных в соответствии с правилами временн?й и пространственной коллинеарности. В то же время, показана постэмбриональная экспрессия Hox-генов и установлено их участие во многих процессах, идущих в дефинитивном теле некоторых животных, в том числе и в процессах регенерации у представителей разных эволюционных ветвей. В восстановительных процессах Нох-гены заняты не только разметкой новообразующихся структур, что является отражанием их эмбриональной функции. Картины распределения транскриптов Нох-генов у некоторых взрослых животных и динамика их экспрессии при повреждении указывает на роль Нох-генов в создании позиционной информации во взрослом организме...


74. 000231

Цель - выявить динамику морфометрических показателей гепатоцитов после перелома костей голени и определить влияние аминокислотной смеси на восстановительные процессы в печени. Материал и методы. Печень изучали у 66 самцов мышей линии CBA в возрасте 2-3 мес, разделенных на 3 экспериментальные (54 особи с переломом костей голени) и 1 контрольную (12 интактных особей) группы. Измеряли объем одноядерных форм гепатоцитов и их ядер, ядерно-цитоплазматический индекс, содержание двуядерных форм гепатоцитов и митотическую активность. Наблюдения проводили на 3-, 7-еи 28-е сутки после перелома костей. Мыши экспериментальных групп в восстановительном периоде получали стандартный рацион питания, обедненный белком рацион или рацион с добавлением аминокислотной смеси (в равном весовом соотношении: лейцин, изолейцин, аргинин, метионин). Результаты. Перелом костей голени сопровождается изменением морфометрических показателей гепатоцитов, опосредованным реакцией органа на травму. Источником репаративной регенерации печени являются периферическая и центральная зоны печеночной дольки. Применение аминокислотной смеси оказывает стимулирующее влияние на развитие восстановительных процессов в печени. Выводы. Печень реагирует на переломы костей изменением морфофункциональных показателей гепатоцитов, которые различаются в отдельных зонах дольки. Динамика восстановительных процессов в печени зависит от характера рациона питания.


75. 000513
Панина С.Б., Гуценко О.И., Милютина Н.П., Корниенко И.В., Ананян А.А., Гвалдин Д.Ю., Плотников А.А., Внуков В.В. SKQ1 КОНТРОЛИРУЕТ ЭКСПРЕССИЮ ГЕНА CASP3 И КАСПАЗА-3-ПОДОБНУЮ АКТИВНОСТЬ В ГОЛОВНОМ МОЗГЕ КРЫС ПРИ ОКИСЛИТЕЛЬНОМ СТРЕССЕ //Биохимия. 2018. Т. 83. № 10. С. 1550-1561.

Исследовали влияние митохондриально-направленного антиоксиданта SkQ1 - катионного производного пластохинона - 10-(6'-пластохинонил)децилтрифенилфосфония - на уровень экспрессии гена CASP3 и активность каспазы-3 в коре больших полушарий и митохондриях мозга крыс в норме и при ГБО-индуцированном окислительном стрессе. Установлено, что в физиологических условиях применение SkQ1 (50 нмоль/кг, 5 дней) не приводит к изменению экспрессии гена CАSP3 и активности каспазы-3 в клетках коры больших полушарий, а также не влияет на активность фермента в митохондриях больших полушарий мозга, но способствует умеренному снижению содержания первичных продуктов перекисного окисления липидов (ПОЛ) и повышению уровня восстановленного глутатиона. При окислительном стрессе, индуцированном гипербарооксигенацией (ГБО), (0,5 МПа, 90 мин) выявлено значительное повышение уровня мРНК гена CASP3 и активности каспазы-3 в коре больших полушарий мозга. Помимо этого, наблюдается значительная активации фермента в митохондриях на фоне снижения уровня восстановленного глутатиона, активности глутатионредуктазы и активации перекисного окисления липидов (ПОЛ)...


76. 000617
Повышева Т.В., Семенов В.Э., Галяметдинова И.В., Резник В.С., Кнни К.С., Колесников П.Е., Челышев Ю.А. НОВЫЕ АНАЛОГИ КСИМЕДОНА ДЛЯ СТИМУЛИРОВАНИЯ ПОСТТРАВМАТИЧЕСКОЙ РЕГЕНЕРАЦИИ СПИННОГО МОЗГА КРЫСЫ // Бюллетень экспериментальной биологии и медицины. 2016. Т. 162. № 8. С. 183-187.

На модели дозированной контузионной травмы спинного мозга крысы исследовано влияние системного введения синтетических производных пиримидина — лекарственного средства ксимедона и соединений 29Д и 34Д. Ксимедон оказывает стимулирующее влияние на восстановление двигательной функции. По показателям функциональных тестов в открытом поле и на установке "Rotarod" соединения 29Д и 34Д оказались эффективнее ксимедона. Соединение 29Д более выражено, чем ксимедон, поддерживает численность популяции Olig2+-олигодендроцитов в кортикоспинальном тракте, а также NG2-клеток во всех исследованных зонах белого вещества. В группе с введением соединения 34Д различия в численности популяции NG2+-клеток зарегистрированы только в передних канатиках, где количество этих глиоцитов в 2 раза больше, чем в группе ксимедона. Результаты указывают на возможность терапевтического действия исследованных аналогов ксимедона — соединений 29Д и 34Д через различные молекулярные и клеточные пути.


77. 040949
Пронина Г.И., Корягина Н.Ю., Ревякин А.О., Капанадзе Г.Д., Степанова О.И., Курищенко Ж.О., Петрова Н.В. ВЛИЯНИЕ ТРАНСПЛАНТАЦИИ СТВОЛОВЫХ КЛЕТОК МЫШЕЙ НА РЕГЕНЕРАЦИЮ КОНЕЧНОСТЕЙ АКСОЛОТЛЕЙ // Биомедицина. 2015. № 4. С. 43-50.

Для изучения регенерации была ампутирована часть передней конечности аксолотлей до локтевого сустава. Выявлено, что ксенотрансплантация экспериментальным аксолотлям стволовых клеток мышей-доноров ускоряет регенерацию удаленных конечностей.


78. 041481
Рыкало Н.А., Гуминская О.Ю., Мнихович М.В., Жакота Д.А., Казанцева Г.П., Соколов Д.А., Филин А.А. ОСОБЕННОСТИ РЕПАРАТИВНОЙ РЕГЕНЕРАЦИИ ПЕЧЕНИ НЕПОЛОВОЗРЕЛЫХ КРЫС НА ФОНЕ ХРОНИЧЕСКОГО МЕДИКАМЕНТОЗНОГО ГЕПАТИТА // Журнал анатомии и гистопатологии. 2016. Т. 5. № 1. С. 83-85.

В статье рассматриваются особенности механизмов репаративной регенераций гепатоцитов неполовозрелых крыс на фоне рифампицин-изониазид индуцированного гепатита. Изучение клеточных и молекулярных механизмов, регулирующих репарацию печени после ее повреждений медикаментозного генеза, позволит более тщательным образом исследовать механизмы компенсации структуры и функции печени для разработки наиболее оптимальных вариантов патогенетической коррекции хронического медикаментозного гепатита.


79. 000617
Скурихин Е.Г., Пахомова А.В., Першина О.В., Ермолаева Л.А., Ермакова Н.Н., Крупин В.А., Пан Э.С., Кудряшова А.И., Рыбалкина О.Ю., Жданов В.В., Гольдберг В.Е., Дыгай А.М. ОЦЕНКА РЕГЕНЕРАТОРНОГО ПОТЕНЦИАЛА СТВОЛОВЫХ И ПРОГЕНИТОРНЫХ КЛЕТОК ИШЕМИЗИРОВАННЫХ СЕМЕННИКОВ МЫШЕЙ ЛИНИИ С57BL/6 В КУЛЬТУРЕ И НА МОДЕЛИ УГНЕТЕНИЯ СПЕРМАТОГЕНЕЗА БУСУЛЬФАНОМ // Бюллетень экспериментальной биологии и медицины. 2016. Т. 162. № 9. С. 388-394.

На модели угнетения сперматогенеза бусульфаном и in vitro изучали регенераторный потенциал стволовых и прогениторных клеток из ишемизированных тестикул мышей линии C57Bl/6. Показано, что сперматогониальные стволовые клетки с фенотипом CD117—CD90+ и CD51—CD24+CD52+ из ишемизированных тестикул демонстрировали 33-кратный и 7-кратный прирост клеточной массы и генерировали колонии in vitro. Эпителиальные (CD45—СD31—Sca-1+CD49f+) и эндотелиальные (CD45—CD31+) прекурсоры обладали меньшим потенциалом к самообновлению. Через 30 сут после введения стволовых и прогениторных клеток из ишемизированных тестикул в зону rete testis ""бусульфановых"" тестикул отмечалось увеличение числа сперматогониальных стволовых клеток CD117—CD90+, общего количества и подвижных форм сперматозоидов в семенниках мышей-реципиентов. Кроме того, в ""бусульфановых"" семенниках наблюдалось увеличение количества Sca-1+-клеток, восстановление эпителиосперматогенного слоя в семенных канальцах, появлялись незрелые клетки Лейдига, повышался уровень тканевого тестостерона и индекс плодовитости.


80. 000617
Скурихин Е.Г., Пахомова А.В., Першина О.В., Ермолаева Л.А., Крупин В.А., Ермакова Н.Н., Пан Э.С., Кудряшова А.И., Рыбалкина О.Ю., Павловская Т.Б., Литвяков Н.В., Гольдберг В.Е., Дыгай А.М. РЕГЕНЕРАТИВНЫЙ ПОТЕНЦИАЛ СПЕРМАТОГОНИАЛЬНЫХ СТВОЛОВЫХ КЛЕТОК, ЭНДОТЕЛИАЛЬНЫХ И ЭПИТЕЛИАЛЬНЫХ ПРЕКУРСОРОВ МЫШЕЙ ЛИНИИ С57BL/6 С МЕТАБОЛИЧЕСКИМИ НАРУШЕНИЯМИ // Бюллетень экспериментальной биологии и медицины. 2017. Т. 163. № 2. С. 204-210.

На модели угнетения сперматогенеза бусульфаном и in vitro исследовали свойства сперматогониальных стволовых клеток, эндотелиальных и эпителиальных прекурсоров семенников мышей линии C57Bl/6 в условиях метаболических нарушений. Показано, что сперматогониальные стволовые клетки (CD117-CD90+) и эпителиальные прекурсоры (CD45-СD31-Sca-1+CD49f+) из тестикул мышей с метаболическими нарушениями демонстрируют 17- и 28-кратный прирост клеточной массы соответственно и генерируют колонии invitro. В то же время потенциал к самообновлению сперматогониальных стволовых клеток с иммунофенотипом CD51-CD24+CD52+ снижается. Сперматогониальные стволовые клетки (CD117-CD90+, CD117+CD90+) и эндотелиальные прекурсоры (CD45-CD31+) из тестикул мышей-доноров с метаболическими нарушениями демонстрируют высокую способность к приживлению в пораженных бусульфаном семенниках мышей линии C57Bl/6.


81. 041710
Студеникин А.В., Стадников А.А., Нузова О.Б., Колосова Н.И. ОСОБЕННОСТИ ТЕЧЕНИЯ РАНЕВОГО ПРОЦЕССА У КРЫС НА ФОНЕ АЛЛОКСАНОВОГО ДИАБЕТА ПРИ РАЗЛИЧНЫХ СПОСОБАХ МЕСТНОГО ЛЕЧЕНИЯ // Пермский медицинский журнал. 2016. Т. 33. № 2. С. 98-103.

Цель. Определить клиническую эффективность местного применения «Милиацила» и КВЧ-терапии в лечении гнойных ран на фоне аллоксанового диабета в экспериментальных условиях. Материалы и методы. Исследования проведены с участием 140 белых беспородных крыс. В основной группе для лечения гнойных ран применяли «Милиацил» и КВЧ-терапию. В I контрольной группе лечение ран не проводилось, у крыс II контрольной группы использовали «Милиацил», у 24 крыс III контрольной группы применяли КВЧ-терапию. Результаты. У экспериментальных животных повреждение островных клеток поджелудочной железы в ранние сроки наблюдений нашло отражение в различного вида структурных изменениях, преимущественно дегенеративного характера. Сравнительные изучения тканевых структур гнойных ран в различных условиях воздействия показали бoльшую лечебную эффективность сочетанного использования «Милиацила» и КВЧ-терапии. Усиливались лейкоцитарные и макрофагальные реакции, ускорялось купирование воспалительного процесса и отделение некротических масс. Выводы. Установлен феномен потенцирования противовоспалительного и регенераторного эффекта «Милиацила» и КВЧ-терапии.


82. 009310
Титова А.А., Мавликеев М.О., Певнев Г.О., Билялов А.И., Абызова М.С., Латышев А.А., Сахапов Д.И., Шафигуллина А.К., Деев Р.В., Киясов А.П. ГИСТОЛОГИЧЕСКАЯ ОЦЕНКА ПАТОЛОГИЧЕСКИХ ИЗМЕНЕНИЙ В ИКРОНОЖНОЙ МЫШЦЕ ПРИ МОДЕЛИРОВАНИИ ИШЕМИИ ЗАДНИХ КОНЕЧНОСТЕЙ У КРЫС // Казанский медицинский журнал. 2017. Т. 98. № 1. С. 73-76.

ЦЕЛЬ. Разработка модели ишемии задних конечностей у крыс для оценки эффективности генной и клеточной терапии. МЕТОДЫ. На первом этапе производили лигирование наружной подвздошной артерии сразу после бифуркации общей подвздошной артерии. Затем бедренную артерию лигировали до её разветвления на нисходящую коленную, подколенную и подкожную артерии, после этого следовало рассечение артерии между лигатурами. Второй этап выполняли через 7 дней после первого: ветви подколенной артерии к икроножной мышце и сформировавшиеся коллатерали лигировали и иссекали. Кровоток в конечности оценивали при помощи лазерной допплеровской флоуметрии. Животных выводили из эксперимента на 14-е, 17-е, 21-е, 28-е, 35-е и 42-е сутки после первого этапа. Парафиновые срезы икроножной мышцы окрашивали по Маллори и с антителами к CD34. РЕЗУЛЬТАТЫ. Патогистологический анализ показал продолжительную ишемию оперированной конечности без восстановления нативной структуры скелетных мышц и значительный интерстициальный фиброз (11,89±5,53% против 2,55±2,13% в интактной конечности к 42-му дню, р <0,05)...


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