High Regenerative Potential Research Articles

Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

Non-developmental dimensions of adult regeneration in Hydra.

An essential dimension of 3D regeneration in adult animals is developmental, with the formation of organizers from somatic tissues. These organizers produce signals that recruit surrounding cells and drive the restoration of the missing structures (organs, appendages, body parts). However, even in animals with a high regenerative potential, this developmental potential is not sufficient to achieve regeneration as homeostatic conditions at the time of injury need to be "pro-regenerative". In Hydra, we identified four distinct homeostatic properties that provide a pro-regenerative framework and we discuss here how these non-developmental properties impact regeneration. First, both the epithelial and the interstitial-derived cells are highly plastic along the animal body, a plasticity that offers several routes to achieve regeneration. Second, the abundant stocks of continuously self-renewing adult stem cells form a constitutive pro-blastema in the central body column, readily activated upon bisection. Third, the autophagy machinery in epithelial cells guarantees a high level of fitness and adaptation to detrimental environmental conditions, as evidenced by the loss of regeneration in animals where autophagy is dysfunctional. Fourth, the extracellular matrix, named mesoglea in Hydra, provides a dynamically-patterned environment where the molecular and mechanical signals induced by injury get translated into a regenerative process. We claim that these homeostatic pro-regenerative features contribute to define the high regenerative potential of adult Hydra.

The role of fish scale derived scaffold and platelet rich plasma in healing of rabbit tibial defect: an experimental study

Fish scale is rich in collagen type I and hydroxyapatite, resembling bone structure. It is readily available, cost effective and can compensate for the limitations of grafting methods such as unavailability, zoonotic disease transmission, and high cost. The aim of this study was to evaluate in vivo the fish scale potential and the possible synergistic effect of platelet rich plasma (PRP) with this scaffold in bone regeneration. Fifteen male white New Zealand rabbits were randomly divided into six groups, each involving 5 limbs. Full thickness bicortical defects were created in the proximal tibia of both pelvic limbs of rabbits. The defect was left untreated in the negative control group. In experimental groups the defect was filled with PRP (group 1), cellular fish scale (group 2), combination of cellular fish scale and PRP (group 3), acellular fish scale (group 4), and a combination of acellular fish scale and PRP (group 5). Fresh fish scales were decellularized to increase biocompatibility and reduce immunity reactions. Decellularization was confirmed by DAPI (4',6-diamidino-2-phenylindole) staining. The microstructure and surface characteristics of fish scales were assessed by scanning electron microscopy (SEM). Histopathological evaluation of bone healing was performed on day 56. Although there was no significant difference in the bone union among experimental groups, the union was superior in all experimental groups compared to control. Spongiosa and cortex formation were superior in the acellular groups. Furthermore, PRP promoted bone marrow formation. We concluded that fish scale is a biocompatible scaffold with a high regenerative potential.

Tissue Extracts for Quantitative Mass Spectrometry of Planarian Proteins Using SILAC.

SILAC (stable isotope labeling by amino acids in cell culture) proteomics enables the relative quantification of proteins in one or more biological samples by mass spectrometry. This technology is based on the metabolic incorporation of heavy isotope-labeled essential amino acids into nascent proteins in vivo. Here, we describe the preparation of SILAC protein samples from planarians, flatworms with high regenerative potential and tissue plasticity. Applications for SILAC proteomics of planarians include the analysis of protein abundances, protein-protein interactions and turnover rates during stem cell-based regeneration and tissue homeostasis.

Micropropagation of Asparagus densiflorus via axillary shoots, indirect organogenesis and somatic embryogenesis

AbstractThe present study has described a simple protocol for efficient plant regeneration of Asparagus densiflorus ‘Sprengeri’ and ‘Myriocladus’ using single-node spear explants, and indirect organogenesis via callogenesis induced on internode explants. The results showed that the genotypes ‘Sprengeri’ and ‘Myriocladus’ regenerated to complete plants via nodal cultures and callus tissue, but the plant regeneration response was higher in secondary explants on MS medium with NAA + kinetin (1+1 mg dm-3) after transfer onto a multiplication medium with IAA+BAP (1+4 mg dm-3), and then onto a rooting medium supplemented with IBA (10 mg dm-3) or NAA + kinetin (1+1 mg dm-3). Primary explants of both cultivars showed high regenerative potential (via the callus stage) on MS medium with IAA+BAP. The cultivar Sprengeri also regenerated via somatic embryogenesis. Both kinds of ‘Meyeri’ explants have a morphogenetic potential for the formation of shoots, which, however, were not capable of rooting. This confirms that the explant, genotype and culture medium are determining factors in the in vitro plant regeneration system.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

Vegetation dynamics in Bishrampur collieries of northern Chhattisgarh, India: eco-restoration and management perspectives.

Phytosociological study in and around reclaimed coal mine site is an essential requirement for judging restoration impact on a disturbed site. Various studies have been aimed towards assessing the impact of different restoration practices on coal mine wastelands. Plantation scheme in a scientific way is the most suitable approach in this context. During the present investigation, an effort have been made to assess the vegetation dynamics through structure, composition, diversity, and forest floor biomass analysis in and around Bishrampur collieries, Sarguja division, northern Chhattisgarh, India. We have tried to develop strategies for eco-restoration and habitat management of the concerned study sites. Four sites were randomly selected in different directions of the study area. We classified the vegetation community of the study sites into various strata on the basis of height. Two hundred forty quadrats were laid down in various directions of the study area to quantify vegetation under different strata. During our investigation, we found eight different tree species representing four families in the different study sites. The density of the various tree species ranged between 40 and 160 individuals ha-1. The density of sapling, seedling, shrub, and herb ranged between 740 and 1620; 2000 and 6000; 1200 and 2000; and 484,000 and 612,000 individuals ha-1, respectively, in different directions. The diversity indices of the tree reflected highest Shannon index value of 1.91. Simpsons index ranged between 0.28 and 0.50, species richness ranged between 0.27 and 0.61, equitability up to 1.44, and Beta diversity ranged between 2.00 and 4.00. Total forest floor biomass ranged between 4.20 and 5.65t/ha among the study sites. Highest forest floor biomass occurred in the south direction and lowest at east direction. Total forest floor biomass declined by 6.19% in west, 13.10% in north, and 25.66% in east direction, respectively. The mining activities resulted significant damage to natural vegetation and its dynamics. The study indicated that Acacia mangium, Cassia siamea, and Dalbergia sissoo can be recommended for effective eco-restoration of the concerned sites due to cosmopolitan distribution, high regeneration potential, as well as existence in the form of various girth classes with stable population structure.

Short-term exclusion of degraded pasture in the permafrost zone: aspects of soil microbial community

The properties of alas soils were studied in short-term isolation (4–5 years). According to the speed of the response to the isolation of degraded pastures, components of the cryoarid–alas ecosystem form the following decreasing series: above-ground vegetation—functional range of cultivated soil microbial community—colony-forming units—underground phytomass, total mobile soil carbon, and nitrification activity. The response rates, respectively, are equal to 1, 4, 5, and over 5 years. The biodynamic features of degraded grasslands in the early years of isolation indicate that these ecosystems have a high regenerative potential. The high activity of autotrophic nitrification indicates a continuing imbalance in the circulation of substances in the isolated area.

CARACTERIZAÇÃO DA PRODUÇÃO DE SERAPILHEIRA E DA CHUVA DE SEMENTES EM UMA RESERVA DE FLORESTA ESTACIONAL SEMIDECIDUAL, PARANÁ

O objetivo do estudo foi analisar a produção de serapilheira e a chuva de sementes em um fragmento de Floresta Estacional Semidecidual do oeste do Paraná com 242 ha, que é conhecido como Reserva Particular de Patrimônio Natural Fazenda Santa Maria. O material foi coletado mensalmente de junho/2011 a maio/2012, em nove parcelas de 20 x 20 m, com o uso quatro coletores de 0,5 x 0,5 m em cada parcela. A serapilheira foi triada e pesada, e a chuva de sementes foi avaliada quanto à riqueza, forma de vida, categoria sucessional e síndrome de dispersão das espécies encontradas. A produção anual de serapilheira foi de 11.886 kg ha-1, sendo agosto e setembro os meses de maiores produções. A fração foliar foi a mais representativa com 58,52% do total. Na chuva de sementes foram coletadas 18.300 sementes, distribuídas em 79 morfoespécies, das quais 51 foram identificadas em nível de espécie, oito em nível de gênero e seis em nível de família. Mikania sp., Cecropia pachystachya, Pisonia aculeata, Gouania ulmifolia e Dendropanax cuneatus foram as espécies com maiores densidades relativas. Os meses com maior abundância de sementes foram setembro (19%), outubro (20%), novembro (27%) e março (15%). A forma de vida predominante foi arbórea com 76,27% das espécies, seguida de lianas com 20,34% e herbáceas com apenas 3,39%. Entre as espécies arbóreas, 37% foram representadas pela categoria sucessional de pioneiras, enquanto as categorias secundárias iniciais e tardias representaram 22% cada e as climácicas 20%. A zoocoria predominou entre as síndromes de dispersão (52,54%), enquanto a anemocoria e a autocoria ocorreram em 38,98 e 8,47%, respectivamente. Os resultados foram semelhantes aos encontrados em outros trabalhos realizados em Florestas Estacionais Semideciduais tardias. Assim, por meio deste estudo sobre deposição da serapilheira e chuva de sementes, o fragmento florestal pode ser considerado uma área de floresta tardia e com elevado potencial de regeneração.

P 185 - Low-fluence photodynamic treatment modifies immunogenicity of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) represent one of the most important component of tissue microenvironment. They can be characterized by high proliferative and regenerative potential as well as low immunogenicity that serves MSC application for regenerative therapy. The same properties make them the key players in the pathogenesis of different diseases. Most of these physiological and pathological processes involving MSCs are accompanied by reactive oxygen species (ROS). However, the ROS effects on MSC functional activity are still poorly investigated. In present study we demonstrated that ROS can modify MSC immunogenicity. MSCs were obtained from stromal vascular fraction of human adipose tissue. Intracellular ROS were induced by low fluence (0.25 J/cm2) photodynamic treatment (PDT) with Al-phthalocynine. We demonstrated that low-fluence PDT significantly increase MSC immunogenicity as manifested by decreased MSC viability after co-culture with human peripheral blood mononuclear cells PBMNs. It was accompanied by enhanced FasL expression in MSCs. In addition, it was found out that intracellular ROS induction resulted in dramatic decrease of CCL2 and increase of IL8 but not IL6 production by MSCs. Low-fluence PDT did not affect Fas, MICA/B, Nectin-2, HLA-ABC expression in MSC as well as co-cultivation with PDT-MSC did not influence the expression of DNAM-1, NKG2D and CD69 in NK.

Distraction osteogenesis on microvascular bone flaps: histological analysis

Despite the high osteoinductive potential of vascularized autograft, being used to close significant jaw defects, the processes of osteoresorpton during remodeling in the oral cavity also affect them. The possibilities of distraction osteogenesis allow to restore the necessary volume of bone's tissue at one stage with minimal traumaticity and predictable result, however it's use on vascularized autografts causes a lot of controversy regarding its effectiveness. There has been held a clinical-experimental study of 60 people with jaw defects with various etiologic factors, which were divided into 2 groups: 1 group with defects without previously made osseous plastic, 2 group with defects partially restored by microvascular autografts. Both groups of patients underwent the method of distraction osteogenesis as pre-implantation preparation. At the stage of dental implantation, histological biopsies were taken from the area of the obtained regenerate, to study and visualize the processes during distraction osteogenesis of micro-vascular autografts. There have been received convincing results about the consistency of the distraction regenerate of both groups and have been revealed the high regenerative potential of microvascular autografts.

Molecular Determinants of Cephalopod Muscles and Their Implication in Muscle Regeneration.

The ability to regenerate whole-body structures has been studied for many decades and is of particular interest for stem cell research due to its therapeutic potential. Several vertebrate and invertebrate species have been used as model systems to study pathways involved in regeneration in the past. Among invertebrates, cephalopods are considered as highly evolved organisms, which exhibit elaborate behavioral characteristics when compared to other mollusks including active predation, extraordinary manipulation, and learning abilities. These are enabled by a complex nervous system and a number of adaptations of their body plan, which were acquired over evolutionary time. Some of these novel features show similarities to structures present in vertebrates and seem to have evolved through a convergent evolutionary process. Octopus vulgaris (the common octopus) is a representative of modern cephalopods and is characterized by a sophisticated motor and sensory system as well as highly developed cognitive capabilities. Due to its phylogenetic position and its high regenerative power the octopus has become of increasing interest for studies on regenerative processes. In this paper we provide an overview over the current knowledge of cephalopod muscle types and structures and present a possible link between these characteristics and their high regenerative potential. This may help identify conserved molecular pathways underlying regeneration in invertebrate and vertebrate animal species as well as discover new leads for targeted tissue treatments in humans.

Human cardiac progenitor cells with regenerative potential can be isolated and characterized from 3D-electro-anatomic guided endomyocardial biopsies

AimsIn the present study, we aimed to develop a percutaneous approach and a reproducible methodology for the isolation and expansion of Cardiac Progenitor Cells (CPCs) from EndoMyocardial Biopsies (EMB) in vivo. Moreover, in an animal model of non-ischemic heart failure (HF), we would like to test whether CPCs obtained by this methodology may engraft the myocardium and differentiate. Methods and resultsEMB were obtained using a preformed sheath and a disposable bioptome, advanced via right femoral vein in 12 healthy mini pigs, to the right ventricle. EMB were enzymatically dissociated, cells were expanded and sorted for c-kit. We used 3D-Electro-Anatomic Mapping (3D-EAM) to obtain CPCs from 32 patients affected by non-ischemic cardiomyopathy. The in vivo regenerative potential of CPCs was tested in a rodent model of drug-induced non-ischemic cardiomyopathy. c-kit positive CPCs replicative capacity was assessed in 30 patients. Telomere length averaged 7.4±0.4kbp and telomerase activity was present in all preparations (1.7×105 copies). The in situ hybridization experiments showed that injected human CPCs may acquire a neonatal myocyte phenotype given the expression of the alpha-sarcomeric actin together with the presence of the Alu probe, suggesting a beneficial impact on LV performance. ConclusionsThe success in obtaining CPCs characterized by high regenerative potential, in vitro and in vivo, from EMB indicates that harvesting without thoracotomy in patients affected by either ischemic or non-ischemic cardiomyopathy is feasible. These initial results may potentially expand the future application of CPCs to all patients affected by HF not undergoing surgical procedures.

Bone Morphogenetic Proteins and myostatin pathways: key mediator of human sarcopenia

BackgroundSarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis that the balance between BMPs and myostatin pathways regulates the age-related muscle degeneration in OP and OA patients. To this end, we investigated the relationship among the expression of BMP-2/4-7, myostatin and phosphorylated Smads1-5-8 and the muscle quality, evaluated in term of fibers atrophy and satellite cells activity.MethodsIn this retrospective study, we collected 123 biopsies of vastus lateralis: 48 biopsies from patients who underwent hip arthroplasty for subcapital fractures of the femur (OP), 55 biopsies from patients who underwent hip arthroplasty for osteoarthritis (OA) and 20 biopsies from patients who underwent hip arthroplasty for high-energy hip fractures (CTRL). Muscle biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Serial sections were used for morphometrical and immunohistochemical analysis (BMP/2/4-7, myostatin, Smads1-5-8, Pax7 and myogenin). In addition, 1 mm3 of muscle tissue of each patient was embedded in epon for ultrastructural study.ResultsMorphometric data indicated an increase of the number of atrophic fibers in OP patients compare to OA. In line with these data, we found an high regenerative potential in muscle tissues of OA patients due to the significant amount of both Pax7 and myogenin positive satellite cells detected in OA group. In addition, our data showed the decrease of BMP2/4 and -7 expression in OP patients compared to both OA group and CTRL. Conversely, OP patients were characterized by high levels of myostatin expression. A different expression profile was also found for phosphorylated Smad1-5-8 between OP and OA patients. In particular, OP patients showed a low number of positive phosphorylated Smad1-5-8 nuclei.ConclusionThe identification of molecular pathways involved in the pathogenesis of sarcopenia open new prospective for the development of drugs able to prevent/treat the muscle impairment that occur in elderly. Results here reported, highlighting the role of BMPs and myostatin pathways in physio-pathogenesis of human sarcopenia, allow us to propose human recombinant BMP-2/7 and anti-myostatin antibodies as a possible therapeutic option for the sarcopenia.

Surface geometry of poly(ether imide) boosts mouse pluripotent stem cell spontaneous cardiomyogenesis via modulating the embryoid body formation process.

The permanent loss of cardiomyocytes may lead to the irreversible damage of myocardium in cardiovascular diseases. The induced pluripotent stem cells (iPSCs) with the capacity of differentiation into a variety of cell types including cardiomyocytes showed high potential for efficient heart regeneration. The iPSCs and iPSC-derived embryoid bodies (EBs) as well as the differentiated cardiomyocytes are highly sensitive to the biophysical cues of their microenvironment, and accordingly their behavior and function can be largely modulated by microstructure of the cell culture surface. In this study, we investigated the regulatory effect of microscale roughness on both cardiomyogenesis and secretion of EBs using poly(ether imide) (PEI) cell culture inserts with different levels of bottom roughness (R0: flat surface; R1: rough surface, Rq ∼ 4 μm; R2: rough surface, Rq ∼ 23 μm). The proliferation rate and cardiomyogenesis of EBs increased with the increase of surface roughness. The EB secretome derived from R2 surface remarkably enhanced the in vitro new vessel formation of endothelial cells, as compared to those from R0 and R1. These findings highlight the potential to improve the iPSC/EB-based restoration of cardiovascular function via microstructured biomaterials.

Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians.

Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.

BONE REGENERATION POTENTIAL OF BETA TRI-CALCIUM PHOSPHATE AND PLATELET RICH FIBRIN IN LOCALIZED ALVEOLAR RIDGE DEFECT WITH IMMEDIATELY PLACED DENTAL IMPLANT (AN EXPERIMENTAL STUDY IN DOG)

Background: Augmentation of localized alveolar bone defect is one of major problems in implant dentistry. Beta tri-calcium phosphate (β-TCP) is considered a source of synthetic bone substitute for bone regeneration. If it is combined with platelet rich fibrin (PRF) with immediately placed dental implant it may encounter a promising result for bone formation.Objective: The aim of the present study was to compare the regenerative bone potential around immediately placed dental implant in localized alveolar bony defect by the use of β-TCP alone or in combination with PRF.Materials and Methods: Eight clinically healthy dogs were included in this study. Localized alveolar bone defect was created after extraction of the second mandibular premolar bilaterally. After immediate implants were inserted the defect was augmented with following: Group I: β-TCP. Group II: β-TCP with PRF. Group III: Control group without adding any material. After 12 weeks, the animals were sacrificed and bone regeneration in the created defect was evaluated through histological examination, immunohistochemically and histomorphometric analysis.Results: Groups analysis demonstrated signs of bone regeneration with variable degrees. Histomorphometrically, the control group revealed the lowest mean area percentage. Group I: β-TCP and group II: β-TCP combined with PRF showed the highest mean area percentage and both were significantly higher than control group. However, no significant difference observed between group I and II. The results were supported by both histological and immunohistochemical analysis. The signs of bone maturation in group I & group II were more than control group.Conclusion: Beta tri-calcium phosphate with platelet rich fibrin is a promising osteogenic bone substitute with high bone regenerative potential .It could be used as an alternative method for bone regeneration around dental implant in localized alveolar bone defect.

The role of stem and progenitor cells in the regeneration of the pancreas and testes in metabolic disorders

The aim of this research was to investigate the regenerative potential of stem and progenitor cells derived from the pancreas and testes in metabolic disorders. Materials and methods. The experiments were performing on C57Bl/6 mice. Metabolic disorders (MD) were modeling by streptozotocin and fat diet. Morphological methods were used to assess morphopathological changes in the pancreas and testicular tissue and fertility. We investigated the insulin expression in the islets of Langerhans, CD16 in testes by immunohistochemical methods. We evaluated the blood lipids, level of glucose, inflammatory mediators, testosterone and a glucose-dependent insulinotropic polypeptide levels in biological samples by biochemical methods and ELISA. We used cytometric methods to study the surface markers of stem and progenitor cells, culture methods and transplantation test to investigate the regenerative potential of stem and progenitor cells. Results. After streptozotocin injection and fatty diet we observed the metabolic imbalance of lipids, testosterone, glucose and insulin resistance in C57BL/6 male mice. The inflammation, type 2 diabetes, asthenoand oligozoospermia were developed and the fertility index was decreased after the metabolic disorders. We observed the increase of oligopotent β-cell precursors (CD45- TER119- CD133+CD49flow) and precursors of hemangiogenesis (CD45- TER119- cKit-1+Flk-1+) count in the pancreas, spermatogonial stem cells (CD117- CD90+ and CD117+CD90+) and precursors of hemangiogenesis (CD45- TER119- cKit-1+Flk-1+) in the testes in MD. Stem and progenitor cells had a high clonal activity and self-renewal capacity, the ability to differentiate into mature cells in vitro, the effective engrafment in injured tissue. Conclusions. In experiments in vitro and in vivo we detected a high regenerative potential of precursors of hemangiogenesis and insulin-producing β-cells, spermatogonial stem cells of C57BL/6 male mice with metabolic disorders. Low rates of regeneration of the microvasculature, insulin-producing β-cells and germ cells in metabolic disorders are associated with the inhibitory effect of diabetic factors and inflammation on stem and progenitor cells.

Electrical stimulation drives chondrogenesis of mesenchymal stem cells in the absence of exogenous growth factors.

Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs) have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by driving Ca2+/ATP oscillations, which are known to be essential for prechondrogenic condensation. In subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on extracellular ATP signaling via P2X4 receptors, and ES induced significant increases in TGF-β1 and BMP2 expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates ES-driven condensation. These findings may contribute to the development of electrotherapeutic strategies for cartilage repair using MSCs.

ИССЛЕДОВАНИЕ МЕТАБОЛИЧЕСКОЙ АКТИВНОСТИ И СИНТЕЗА ПРОТЕОГЛИКАНОВ КЛЕТКАМИ МЕЖПОЗВОНКОВОГО ДИСКА ПОД ВОЗДЕЙСТВИЕМ КОСТНОГО МОРФОГЕНЕТИЧЕСКОГО БЕЛКА2 В ЭКСПЕРИМЕНТЕ

Modern therapeutic strategies for intervertebral disc repair mainly focus on targeting molecular pathways of extracel-lular matrix degeneration. Anabolic strategies for regeneration are aimed to increase production of major extracellular molecules. Members of TGF-b superfamily proteins, particularly the bone morphogenetic proteins (BMP) have a high regenerative potential regarding the mesenchymal cells. The goal of this study is to study production of proteoglycans by the intervertebral disc cells under the influence of bone morphogenetic protein 2.Material and methods. The experiment was carried out on the cell cultures derived from the annulus fibrosis cells and nucleus pulposus cells of the human intervertebral disc. We studied cell livability, metabolic activity and proteoglycan expression. Cell livability was assessed using the trypan blue staining. Alamar blue test was used for the estimation of metabolic activity. Amount of sulfated glycosaminoglycans was assessed using the assay based on the reaction with 1,9-Dimethylmethylene Blue.Results. Cultivation with bone morphogenetic protein 2 in different concentrations did not decrease livability of the cells. Study cell cultures with application of bone morphogenetic protein 2 in different concentrations showed significant increase in metabolic activity and proteoglycan synthesis by the annulus fibrosis cells. Despite the relative increase in the number of the nucleus pulposus cells treated with the bone morphogenetic protein 2, the differences in metabolic and synthetic activity compared with control group was not significant. Conclusion. The bone morphogenetic protein 2 has an anabolic effect towards the intervertebral disc cells, particularly in the production of proteoglycans by the annulus fibrosis cells.