Comparative model of minimal spinal cord injury reveals a rather anti-inflammatory response in the lesion site as well as increased proliferation in the central canal lining in the neonates compared to the adult rats.

Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20 ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration.

Plasma-derived extracellular vesicle surface markers CD45, CD326 and CD56 correlate with the stage of osteoarthritis: a primary study of a novel and promising diagnostic tool of the disease.

Recently, there is a growing interest in the research based on extracellular vesicles (EVs) which represent paracrine factors secreted by almost all cell types. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and compositions. EVs play an important role in intercellular communication, mechanism and tissue repair. Moreover, EVs could help not only in the treatment of diseases but also in their diagnostics. This work focused on the evaluation of the potential of EVs being used as biomarkers for the diagnosis of osteoarthritis (OA) based on a comparison of the composition of EVs separated from platelet-poor plasma (PPP) of healthy donors and OA patients at different stages of OA. OA is established as a complex syndrome with extensive impact on multiple tissues within the synovial joint. It is a chronic disease of musculoskeletal system that mainly affects the elderly. Depending on the use of the Kellgren-Lawrence classification system, there are four grades of OA which have a negative impact on patients' quality of life. It is very difficult to detect OA in its early stages, so it is necessary to find a new diagnostic method for its timely detection. PPP samples were prepared from whole blood. PPP-EVs were separated from 3 groups of donors-healthy control, early stage OA, end-stage OA, and their content was compared and correlated. EVs from PPP were separated by size exclusion chromatography and characterized in terms of their size, yield and purity by NTA, western blotting, ELISA and flow cytometry. Detection of surface markers expression in EVs was performed using MACSPlex approach. Inflammatory and growth factors in EVs were analysed using MAGPix technology. Our study confirmed significant differences between EVs surface markers of patients and healthy controls correlating with the age of donor (CD63, CD31 and ROR1) and stage of OA (CD45, CD326 and CD56), respectively. Circulating EVs have been under extensive investigation for their capability to predict OA pathology diagnosis as potential targets for biomarker discovery. Taken together, obtained results indicated that PPP-EVs surface markers could be used as potential biomarkers in the early diagnosis of OA.

The role of primed and non-primed MSC-derived conditioned media in neuroregeneration.

Introduction: With growing significance in nervous system repair, mesenchymal stem cell-derived conditioned media (MSCCM) have been used in cell-free therapies in regenerative medicine. However, the immunomodulatory and neuroregenerative effects of MSCCM and the influence of priming on these effects are still poorly understood. Methods: In this study, by various methods focused on cell viability, proliferation, neuron-like differentiation, neurite outgrowth, cell migration and regrowth, we demonstrated that MSCCM derived from adipose tissue (AT-MSCCM) and amniotic membrane (AM-MSCCM) had different effects on SH-SY5Y cells. Results and discussion: AT-MSCCM was found to have a higher proliferative capacity and the ability to impact neurite outgrowth during differentiation, while AM-MSCCM showed more pronounced immunomodulatory activity, migration, and re-growth of SH-SY5Y cells in the scratch model. Furthermore, priming of MSC with pro-inflammatory cytokine (IFN-γ) resulted in different proteomic profiles of conditioned media from both sources, which had the highest effect on SH-SY5Y proliferation and neurite outgrowth in terms of the length of neurites (pAT-MSCCM) compared to the control group (DMEM). Altogether, our results highlight the potential of primed and non-primed MSCCM as a therapeutic tool for neurodegenerative diseases, although some differences must be considered.

Two Amnion-Derived Mesenchymal Stem-Cells Injections to Osteoarthritic Elbows in Dogs-Pilot Study.

The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance (p < 0.05) in nine elbows. Correlation analyses proved statistical significance (p < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant (p < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance (p < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance (p < 0.05) between day 14 and the control group.

The Role of Mesenchymal Stromal Cells and Their Products in the Treatment of Injured Spinal Cords.

Spinal cord injury (SCI) is a destructive condition that results in lasting neurological damage resulting in disruption of the connection between the central nervous system and the rest of the body. Currently, there are several approaches in the treatment of a damaged spinal cord; however, none of the methods allow the patient to return to the original full-featured state of life before the injury. Cell transplantation therapies show great potential in the treatment of damaged spinal cords. The most examined type of cells used in SCI research are mesenchymal stromal cells (MSCs). These cells are at the center of interest of scientists because of their unique properties. MSCs regenerate the injured tissue in two ways: (i) they are able to differentiate into some types of cells and so can replace the cells of injured tissue and (ii) they regenerate tissue through their powerful known paracrine effect. This review presents information about SCI and the treatments usually used, aiming at cell therapy using MSCs and their products, among which active biomolecules and extracellular vesicles predominate.

Characterisation of mesenchymal stem cells conditioned media obtained at different conditioning times: their effect on glial cells in in vitro scratch model.

In this study, the bone marrow mesenchymal stem cells conditioned media (BMMSC-CM) obtained by conditioning for 24(CM24), 48(CM48) and 72(CM72) hours was characterised. In vitro, the impact of BMMSC-CM on the astrocyte migratory response and oligodendrocyte density was evaluated using the scratch model. The proteomic profiles of individual secretomes were analysed by mass spectrometry and the concentrations of four selected neurotrophins (BDNF, NGF, GDNF and VEGF) were determined by ELISA. Our results revealed an increased number of proteins at CM72, many of which are involved in neuroregenerative processes. ELISA documented a gradual increase in the concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro, the different effect of individual BMMSC-CM on astrocyte migration response and oligodendrocyte density was observed, most pronounced with CM72. The outcomes demonstrate that the prolonged conditioning results in increased release of detectable proteins, neurotrophic factors concentration and stronger effect on reparative processes in neural cell cultures.

Mesenchymal Stem Cells in the Treatment of Human Spinal Cord Injury: The Effect on Individual Values of pNF-H, GFAP, S100 Proteins and Selected Growth Factors, Cytokines and Chemokines.

At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers (S100, GFAP, and pNF-H) as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury.

The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis.

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1ß and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs' response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy.

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs' uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.

Effect of Intra-Articular Injection of Platelet-Rich Plasma on the Serum Levels of Osteoarthritic Biomarkers in Patients with Unilateral Knee Osteoarthritis.

BACKGROUND: The aim of this study is to determine the effect of three doses of intra-articular injection of platelet-rich plasma (PRP) into the osteoarthritic (OA) knee joint on the functional status and on the changes in the levels of specific OA biomarkers in blood serum. METHODS: Forty patients with unilateral primary knee osteoarthritis were enrolled in this single center, prospective clinical trial. For each patient, three intra-articular PRP injections were administered one week apart. Clinical and laboratory assessment was performed before the first PRP injection (baseline), and 3 months after the third PRP application (3-month follow up). Pain in the affected knee joint was assessed with the Visual Analog Scale for Pain (VAS). Change in clinical status was evaluated with the Western Ontario and McMaster Universities Arthritis Index Questionnaire (WOMAC). Concentrations of 19 biomarkers (EGF, Eotaxin, FGF-2, GRO, IL-10, IL-1RA, IL-8, IP-10, MCP-1, PDGF-AB/BB, RANTES, MMP-3, MMP-13, Collagen type 2, BMP-2, TIMP-1, TIMP-2, TGF beta 1, and COMP) in the serum of studied patients were quantified. RESULTS: At 3-month follow up, there was a significant decrease in the VAS score and significant improvement in the WOMAC score. There was a significant decrease in the levels of Eotaxin, MCP-1, MMP-1, IL-10, EGF, PDGF-AB/BB, TGF- ß1 compared to baseline levels. A significant increase in markers BMP-2, COMP, Collagen type 2 and GRO was found at the same time point. There was no significant change in the concentrations of other biomarkers (FGF-2, IL-1RA, IL-8, IL-10, MMP-3, RANTES, TIMP-1, TIMP-3). CONCLUSIONS: We found an increase in specific pro-anabolic and anti-inflammatory biomarkers with a concomitant decrease in pro-inflammatory biomarkers at 3 months after three intra-articular applications of PRP. Significant improvement in VAS and WOMAC scores was observed. Treatment with PRP may be an effective therapeutic option with anti-inflammatory and regenerative potential in patients with primary knee OA.

New therapeutic approaches of mesenchymal stem cells-derived exosomes.

Mesenchymal stem cells (MSCs) have been demonstrated to have a great potential in the treatment of several diseases due to their differentiation and immunomodulatory capabilities and their ability to be easily cultured and manipulated. Recent investigations revealed that their therapeutic effect is largely mediated by the secretion of paracrine factors including exosomes. Exosomes reflect biophysical features of MSCs and are considered more effective than MSCs themselves. Alternative approaches based on MSC-derived exosomes can offer appreciable promise in overcoming the limitations and practical challenges observed in cell-based therapy. Furthermore, MSC-derived exosomes may provide a potent therapeutic strategy for various diseases and are promising candidates for cell-based and cell-free regenerative medicine. This review briefly summarizes the development of MSCs as a treatment for human diseases as well as describes our current knowledge about exosomes: their biogenesis and molecular composition, and how they exert their effects on target cells. Particularly, the therapeutic potential of MSC-derived exosomes in experimental models and recent clinical trials to evaluate their safety and efficacy are summarized in this study. Overall, this paper provides a current overview of exosomes as a new cell-free therapeutic agent.

Polyhydroxybutyrate/Chitosan 3D Scaffolds Promote In Vitro and In Vivo Chondrogenesis.

The articular cartilage is an avascular and aneural tissue and its injuries result mostly in osteoarthritic changes and formation of fibrous tissue. Efforts of scientists worldwide are focused on restoration of cartilage with increase in life quality of patients. Novel polymeric polyhydroxybutyrate/chitosan (PCH) porous 3D scaffolds were developed and characterized. The rat mesenchymal stem cells (MSCs) were seeded in vitro on PCH scaffolds by a simple filtration of MSCs suspension over scaffolds using syringe. The chondrogenesis of cell-scaffold constructs was carried out in supplemented chondrogenic cultivation medium. After 2 and 4 weeks of in vitro culturing cell-scaffold constructs in chondrogenic differentiation medium, the cartilage extracellular matrix components like glycosaminoglycans and collagens were identified in scaffolds by biochemical assays and histological and immunohistochemical staining. Preliminary in vivo experiments with acellular scaffolds, which filled the artificially created cartilage defect in sheep knee were done and evaluated. Cells released from the bone marrow cavity have penetrated into acellular PCH scaffold in cartilage defect and induced tissue formation similar to hyaline cartilage. The results demonstrated that PCH scaffolds supported chondrogenic differentiation of MSCs in vitro. Acellular PCH scaffolds were successfully utilized in vivo for reparation of artificially created knee cartilage defects in sheep and supported wound healing and formation of hyaline cartilage-like tissue.

In vitro predegeneration of peripheral nerve; the effect of predegeneration period on rat Schwann cell cultures.

Peripheral nerve predegeneration has been used as a tool to improve the in vitro cultivation of Schwann cells. The process of predegeneration may be accomplished either in vivo or in vitro. In previously published studies, various predegeneration periods were used, ranging from a few days until up to 5 weeks. The present study systematically evaluated the effect of various durations of in vitro predegeneration on the efficacy of Schwann cell cultivation. The sciatic nerves of adult Wistar rats were harvested and the explanted nerve pieces were maintained in the predegeneration medium for different predegeneration periods. In group A, the dissociation was performed immediately after harvesting. In groups B, C and D, the predegeneration periods were 2, 4 and 6 weeks, respectively. During the predegeneration period, the tissue pieces were repeatedly transferred into new dishes. Afterwards, the nerve tissue was enzymatically dissociated and the cells were seeded onto a six-well culture plate at a defined density. After 3-4 days of incubation, the cultures were passaged by means of the cold jet technique and the cell cultivation was continued for another 21 days. It was observed that the cell cultures in groups A and B were rapidly overgrown by fibroblasts. In group C, numerous wells contained a highly enriched Schwann cell population that had formed a typical monolayer, but in a fraction of the dishes, cultures were debased by fibroblast overgrowth. In group D, all of the cultures had enriched Schwann cell populations. In the experiments of the present study, the positive effect of predegeneration was observed only when the predegeneration periods lasted for 4 weeks or longer. It was concluded that the longer predegeneration periods activated Schwann cells and/or depleted the fibroblast proliferation capacity.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation.

Objectives In this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation. Methods Primary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis. Results Immunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact. Conclusion The results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

Axonal outgrowth stimulation after alginate/mesenchymal stem cell therapy in injured rat spinal cord.

Despite strong efforts in the field, spinal cord trauma still belongs among the untreatable neurological conditions at present. Given the complexity of the nervous system, an effective therapy leading to complete recovery has still not been found. One of the potential tools for supporting tissue regeneration may be found in mesenchymal stem cells, which possess anti­inflammatory and trophic factor­producing properties. In the context of transplantations, application of degradable biomaterials which could form a supportive environment and scaffold to bridge the lesion area represents another attractive strategy. In the present study, through a combination of these two approaches we applied both alginate hydrogel biomaterial alone or allogenic transplants of MSCs isolated from bone marrow seeded in alginate biomaterial into injured rat spinal cord at three weeks after spinal cord compression performed at Th8­9 level. Following three­week survival, using immunohistochemistry we studied axonal growth (GAP­43 expression) and both microglia (Iba­1) and astrocyte (GFAP) reactions at the lesion site and in the segments below and above the lesion. To detect functional improvement, during whole survival period we performed behavioral analyses of locomotor abilities using a classical open field test (BBB score) and a Catwalk automated gait analyzing device (Noldus). We found that despite the absence of locomotor improvement, application of both alginate and MSCs caused significant increase in the number of GAP­43 positive axons.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Comparison of dynamic behavior and maturation of neural multipotent cells derived from different spinal cord developmental stages: an in vitro study.

Neural progenitor cells (NPCs) are characterized as undifferentiated cells with the ability of self-renewal and multipotency to give rise to other cells of the nervous system. In our in vitro study we demonstrate the proliferative and differentiative potential of NPCs isolated from the spinal cord at different developmental stages (embryonal, early postnatal, adult), maintained and expanded within neurospheres (NSs). Using the NSs culture system, we examined the size, number of NSs and their fate when exposed to differentiation conditions. Based on immunocytochemical analyses for cell markers (MAP 2, GFAP, RIP) we evaluated the occurrence of various cell types: neurons, astrocytes and oligodendrocytes. The results show that NSs increased in size during cultivation time via NPC proliferation, but proliferation potential decreased Turing maturation stages. In addition, NPCs derived from spinal cord developmentally different stages gave rise to a consistent ratio of glial and neuronal progeny (3:1), and adult tissues represent a comparable source of NPCs compared to embryonal and early postnatal tissues. These data provide useful information for large-scale in vitro expansion of NPCs required for potential cell therapy after spinal cord injury.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds.

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study.

In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties.

Oral administration of inosine promotes recovery after experimental spinal cord injury in rat.

Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to extend axons. We, herein, evaluated the effect of oral inosine treatment on spinal cord injury (SCI) recovery by means of locomotor and bladder function, quantification of neurons and spinal cord tissue sparing. Rats after compression SCI were divided into groups-SCI-Aqua and SCI-Inosine (daily application of aqua for injection or inosine)-locomotion of hind limbs (BBB score) and urinary bladder function were evaluated from day 1 to 28 after SCI. The neuronal profile was determined by immunohistochemistry with NeuN antibodies and tissue sparing by Luxol fast blue staining method. SCI affected the functional movement of hind limbs in both groups with gradual improvement (increased BBB score) during survival. However, we found a significant difference in BBB score and recovery of bladder function between SCI-Aqua and SCI-Inosine groups during the second week of survival following SCI. In addition, the number of NeuN positive cells and percentage of tissue sparing was also significantly higher in SCI-Inosine group when compared with the SCI-Aqua group. Daily oral administration of inosine after SCI throughout the survival was beneficial for locomotion and micturition, neuronal survival and tissue sparing. This indicates that inosine may represent one of the co-stimulatory factors for treatment strategies to promote neuronal plasticity after SCI.