Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury.However,whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear.In the present study,we established a rat model of spinal cord injury based on impact injury from a dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells.We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury,decreased expression of the microglial pyroptosis markers NLRP3,GSDMD,caspase-1,and interleukin-1β,promoted axonal and myelin regeneration,and inhibited the formation of glial scars.In addition,in a lipopolysaccharide-induced BV2 microglia model,conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway.These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway,thereby promoting the recovery of neurological function after spinal cord injury.Therefore,conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Enhancing Erectile Function and Alleviating Andropause Symptoms: Clinical Efficacy of a Human Stem Cell Conditioned Medium Cream

Erectile dysfunction (ED) is increasingly prevalent in Japan, exceeding 30%, and increasing with age. Unhealthy lifestyle habits, obesity, insufficient exercise, and smoking have been implicated in its pathogenesis, along with endothelial dysfunction of the corpora cavernosa and impaired blood flow to the penis considered underlying factors. However, the current treatments are limited to Phosphodiesterase-5 (PDE5) inhibitors. ED is the primary symptom of andropathy. This study reports the clinical efficacy of human stem cell-conditioned medium cream for ED treatment. Ten men without underlying diseases suspected of andropause with ED (mean age 43.2 ± 4.4 y, Hb 15.2 ± 0.6 gm/dL, AST/ALT 30.2/37.9 ± 12.4/14.0, eGFR 82.7 ± 12.4 mL/min/1.73 m2) were targeted. The cream was applied twice daily to the genital and scrotal areas. The erectile hardness score (EHS), International Index of Erectile Function-5 (IIEF-5), and Aging Male Symptoms (AMS) scale were used to evaluate the participants before and 30 days after use, and the results were compared using paired t-tests. The post-use qualitative opinions were collected through interviews. Significant improvements were observed compared to baseline in the IIEF-5 (11.8 ± 4.6→17.2 ± 5.1, P < 0.001), and AMS (46.3 ± 6.7→37.6 ± 5.3, P < 0.001) scores post cream use. EHS did not show a statistically significant difference, but a trend towards improvement was observed. Qualitative feedback included increased morning erection, improved maintenance of erection during intercourse, and reduced post work fatigue. Human stem cell-conditioned medium contains endothelial growth factors that potentially contribute to the improvement of ED and andropause by enhancing corporal endothelial function. Future studies should include control groups to further investigate the efficacy of these treatments.

Extraction, Purification and Identification of Bone Morphogenetic Protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

Endothelial progenitor cells secrete a variety of growth factors that inhibit inflammation, promote angiogenesis and exert neuroprotective effects. Therefore, in this study, we investigated whether endothelial progenitor cell-conditioned medium might have therapeutic effectiveness for the treatment of spinal cord injury using both in vitro and in vivo experiments. After primary culture of bone marrow-derived macrophages, lipopolysaccharide stimulation was used to classically activate macrophages to their proinflammatory phenotype. These cells were then treated with endothelial progenitor cell-conditioned medium or control medium. Polymerase chain reaction was used to determine mR NA expression levels of related inflammatory factors. Afterwards, primary cultures of rat spinal cord neuronal cells were prepared and treated with H2O2and either endothelial progenitor cell-conditioned medium or control medium. Hoechst 33258 and propidium iodide staining were used to calculate the proportion of neurons undergoing apoptosis. Aortic ring assay was performed to assess the effect of endothelial progenitor cell-conditioned medium on angiogenesis. Compared with control medium, endothelial progenitor cell-conditioned medium mitigated the macrophage inflammatory response at the spinal cord injury site, suppressed apoptosis, and promoted angiogenesis. Next, we used a rat model of spinal cord injury to examine the effects of the endothelial progenitor cell-conditioned medium in vivo. The rats were randomly administered intraperitoneal injection of PBS, control medium or endothelial progenitor cell-conditioned medium, once a day, for 6 consecutive weeks. Immunohistochemistry was used to observe neuronal morphology. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay was performed to detect the proportion of apoptotic neurons in the gray matter. The Basso, Beattie and Bresnahan Locomotor Rating Scale was used to evaluate the recovery of motor function of the bilateral hind limbs after spinal cord injury. Compared with the other two groups, the number of axons was increased, cavities in the spinal cord were decreased, the proportion of apoptotic neurons in the gray matter was reduced, and the Basso, Beattie and Bresnahan score was higher in the endothelial progenitor cell-conditioned medium group. Taken together, the in vivo and in vitro results suggest that endothelial progenitor cell-conditioned medium suppresses inflammation, promotes angiogenesis, provides neuroprotection, and promotes functional recovery after spinal cord injury.

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.

Effects of coriaria lactone-activated,astrocyte-conditioned medium on estrogen receptor and progesterone receptor expression in rat cortical and hippocampal neurons

BACKGROUND： Coriaria lactone-activated astrocytes released bioactive substances that eventually caused epilepsy. OBJECTIVE： It has been suggested that activated astrocytes alter the expression of the estrogen receptor and progesterone receptor by releasing bioactive substances during epilepsy, thereby affecting neuronal activity in the brain. This study was designed to observe the expression of the estrogen receptor and the progesterone receptor in rat brain following lateral ventricle injection of coriaria lactone-activated, astrocyte-conditioned medium. DESIGN AND SETTING： This immunohistochemical, randomized, controlled, animal study was conducted at the Department of Pathology, Hospital Affiliated to Binzhou Medical College, China. MATERIAL： Coriaria lactone was provided by Huaxi Pharmaceutical Factory, China. METHODS： Forty adult, healthy, male, Sprague Dawley rats were randomly assigned into two groups. Astrocyte-conditioned medium （10 μ L） was injected into rat lateral ventricle in the control group （n = 8）. Coriaria lactone-activated, astrocyte-conditioned medium （10 μL） was infused into the rat lateral ventricle in the coriaria lactone group （n = 32）. At 2, 4, 8 and 12 hours following injection, rats were sacrificed and subjected to immunohistochemistry. Eight rats were studied at each time point. MAIN OUTCOME MEASURES： Behavioral changes were observed in rats of both groups. Expression of the estrogen receptor and the progesterone receptor in rat cortical and hippocampal neurons was measured using immunohistochemistry. RESULTS： Four hours after injection, estrogen receptor levels in rat cortical and hippocampal neurons were significantly higher in the coriaria lactone group than in the control group （P 〈 0.05）. Progesterone receptor levels were significantly lower in the coriaria lactone group than in the control group （P 〈 0.05）. Seizures were not observed in the control group. In the coriaria lactone group, convulsions appeared 30 minutes after injection; seizures reached grade Ⅲ at 45 minutes rat behavior was nearly normal at 2 hours. CONCLUSION： Activated astrocytes can induce seizures in the rat by enhancing estrogen receptor expression and decreasing progesterone receptor expression in cerebral cortical and hippocampal neurons.

Effect of conditioned medium from neural stem cells on glioma progression and its protein expression profile analysis

BACKGROUND Emerging evidence suggests that the spread of glioma to the subventricular zone(SVZ)is closely related to glioma recurrence and patient survival.Neural stem cells(NSCs)are the main cell type in the SVZ region and exhibit tumor-homing ability.AIM To evaluate the effects of conditioned medium(CM)derived from SVZ NSCs on the cancer-related behaviors of glioma cells.METHODS The characteristics of SVZ hNSCs were identified by immunofluorescence.The normoxic-hNSC-CM and hypoxic-hNSC-CM(3%O2,oxygen-glucose deprived[OGD]culturing)were collected from 80%-90%confluent SVZ NSCs in sterile conditions.The CCK8 and Transwell assays were used to compare and evaluate the effects of normoxic-CM and hypoxic-CM on glioma proliferation and invasion.Then proteins secreted from SVZ NSCs into the CM were investigated by mass spectrometry,and the potential effects of candidate protein NCAN in the regulation of glioma progression were examined by CCK8 and Transwell assays.RESULTS The CM from SVZ NSCs significantly increased the proliferation and invasion of glioma cells,particularly the CM from OGD NSCs induced under hypoxic conditions.Furthermore,the secreted protein neurocan(NCAN)in CM from OGD NSCs was identified by proteomic analysis.NCAN was expressed in glioma cells and played regulatory roles in mediating the progression of glioma cells mainly via the Rho/Rho-associated protein kinase pathway.CONCLUSION Our study identified a potential interactive mechanism between SVZ NSCs and glioma cells,in which SVZ NSCs promote glioma progression via the secreted protein NCAN.These findings suggested that exploring the CM derived from cells could be a novel strategy for optimizing treatments and that NCAN derived from SVZ NSCs may be a potential new target in glioma progression.

Dental stem cell-conditioned medium for tissue regeneration: Optimization of production and storage

BACKGROUND Mesenchymal stem cells(MSC)effects on tissue regeneration are mainly mediated by their secreted substances(secretome),inducing their paracrine activity.This Conditioned medium(CM),including soluble factors(proteins,nucleic acids,lipids)and extracellular vesicles is emerging as a potential alternative to cell therapy.However,the manufacturing of CM suffers from variable procedures and protocols leading to varying results between studies.Besides,there is no welldefined optimized procedure targeting specific applications in regenerative medicine.AIM To focus on conditioned medium produced from dental MSC(DMSC-CM),we reviewed the current parameters and manufacturing protocols,in order to propose a standardization and optimization of these manufacturing procedures.METHODS We have selected all publications investigating the effects of dental MSC secretome in in vitro and in vivo models of tissue regeneration,in accordance with the PRISMA guidelines.RESULTS A total of 351 results were identified.And based on the inclusion criteria described above,118 unique articles were included in the systematic review.DMSC-CM production was considered at three stages:before CM recovery(cell sources for CM),during CM production(culture conditions)and after production(CM treatment).CONCLUSION No clear consensus could be recovered as evidence-based methods,but we were able to describe the most commonly used protocols:donors under 30 years of age,dental pulp stem cells and exfoliated deciduous tooth stem cells with cell passage between 1 and 5,at a confluence of 70%to 80%.CM were often collected during 48 h,and stored at-80°C.It is important to point out that the preconditioning environment had a significant impact on DMSCCM content and efficiency.

Intranasal Administration of Conditioned Medium from Cultured Mesenchymal Stem Cells Improves Cognitive Impairment in Olfactory Bulbectomized Mice

Alzheimer’s disease (AD) is the common cause of dementia which shows the neuro-pathologies like an accumulation of amyloid-β (Aβ) and degeneration of cholinergic neuron. Olfactory bulbectomized (OBX) mice show some of AD features, so they have been used to research as AD model. Mesenchymal stem cells (MSCs) can differentiate into many kinds of cells, including neuronal cells. In this study, we intranasally administrated the conditioned medium derived from cultured umbilical cord (UC) MSCs. The intranasal administration of the MSCs medium restored the cognitive impairment observed in OBX mice. In addition, the decreased number of choline acetyltransferase-positive cells in the medial septum was restored by the conditioned medium administration. In conclusion, MSCs-derived conditioned medium may have protective effects of cholinergic neurons in the medial septum, thereby rescuing the cognitive impairment of OBX.

Cycloheximide prevents production of arresting,a fraction of 30-50 kDa obtained from seminiferous tubule conditioned medium

Aim: To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermato-genesis and sperm count in male rats. Methods: The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined. Results: The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control. Conclusion: The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.

Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SHSY5Y cells induced by Aβ_(25-35)

A progressive neurodegenerative disease,Alzheimer’s disease(AD).Studies suggest that highly expressed protein isoaspartate methyltransferase 1(PCMT1)in brain tissue.In the current study,we explored the effects of neural stem cell-conditioned medium(NSC-CDM)on the PCMT1/MST1 pathway to alleviate Aβ_(25-35)-induced damage in SH-SY5Y cells.Our data suggested that Aβ_(25-35) markedly inhibited cell viability.NSC-CDM or Neural stem cell-complete medium(NSC-CPM)had a suppression effect on toxicity when treatment with Aβ_(25-35),with a greater effect observed with NSC-CDM.Aβ_(25-35)+NSC-CDM group exhibited an increase in PCMT1 expression.sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression.Overexpression of PCMT1 reversed the Aβ_(25-35)-induced decrease in cell proliferation and apoptosis.In summary,our findings suggest that NSC-CDM corrects the Aβ_(25-35)-induced damage to cells by improving PCMT1 expressions,which in turn reduces phosphorylation of MST1.

CONDITIONED MEDIUM OF HUMAN NASOPHARYNGEAL CARCINOMA EPITHELIOID CELL LINE CNE_(1) CONTAINED THE ACTIVITIES OF TRANSFORMED GROWTH-INHIBITING FACTORS

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE1) was assayed by both the 3H-thymidine incorporation test and the soft agar test. It was found that the SFCM stimulated the growth of long-term serum-free cultured CNE4 cells in ac-cordence with the fact that the growth rate of long-term serum-free cultured CNE1 cells was directly proportional to the plating density. Alternatively 5% SFCM inhibited the growth of short-term serum-free cultured CNE4 cells by 51% in which the indicator cell remained the responsiveness state of growing in the serum-supplemented medium to the effector of interest. Furthermore, SFCM resulted in the inhibition of anchorage-independent growth of CNE4 cells and A431 cells. Also in soft agar test. SFCM reduced the colony formation of NRK(?),9F cells in the presence of EGF or EGF plus TGF-β. These finding suggested that CNE4 secreted autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum-free medium, the latter strongly reverse malignant phenotypes of CNE4 and A431 cells in serum-supplemented surrounding.

The Skin Whitening Effect of Co-Cultured Conditioned Medium: Involvement of Synergy between Stem Cells and Immune Cells

Many researchers have described that mesenchymal stem cells conditioned medium and immune cells conditioned medium have a clear whitening effect when they are used as cosmetic ingredients. In this study, we confirmed the whitening efficacy of various concentrations of immune cells and stem cell conditioned media. The author tried to study a conditioned medium that has a strong whitening effect even with a composition of less than 20% (the most used concentration in cosmetics). Because of the fact that the conditioned medium contains various cytokines and growth factors secreted by stem cells or immune cells, it is known to have effects such as wound healing, antioxidant, and whitening effect. Recently, stem cells have been used not only in the development of cosmetic raw materials but also in skincare procedures, and there are reports being released of cosmetics using immune cells conditioned medium. The concentration-dependent whitening effect equivalently increased as the concentration of the mono-cultured conditioned medium was obtained through the stem cells or immune cells culture. In the case of co-culture, whitening results are like the effect of positive control such as arbutin in the medium carrying only 10% of the co-cultured conditioned medium. It is possible that enhanced whitening efficiency in co-cultured conditioned medium leads to a major innovation in the global cosmetic market.

Immobilization of hUC-MSCs conditioned medium on 3D PLLA collagen-coated matrix enhances diabetic wound healing progression

Conditioned medium(CM)derived from human umbilical cord-mesenchymal stem cells(hUC-MSCs)which con-tains numerous amounts of growth factors,has demonstrated potential in treatment of diabetic wounds.However,for practical application,a biodegradable supporting material is needed to hold the CM and fill in the injury site,where deep cavity wounds are often present in diabetic patients.Poly-l-lactic acid matrix coated with collagen(PLLA/CC)is a suitable carrier due to its biodegradability and biocompatibility.Thus,we present a method to immobilize the hUC-MSCs CM on PLLA/CC through freeze-drying process(PLLA/CC CM FD).When seeded on PLLA/CC CM FD,fibroblasts had an increased cellular function in producing collagen;although no enhancement in cell viability was observed.Moreover,implantation of PLLA/CC CM FD on the wound of diabetic rats showed improvement in wound closure and collagen deposition in the wound area.Altogether,this study exhibits the potential of PLLA/CC CM FD as a therapy for diabetic deep cavity wound.

Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells

Background Although the introduction of serum prostate-specific antigen （PSA） measurements into clinical practice has revolutionized the care of patients with prostate cancer, there are well-recognized limitations of PSA, and there is a critical need to identify additional prostate cancer biomarkers to assist in early detection and prognosis. In this regard, high resolution proteomic technology has the unexceptionable superiority to find those high abundance biomarkers. The purpose of this study was to search new tumor markers by proteomic technology. Methods The proteins in conditioned medium （CM） of BPH-1 and LNCaP cells were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry （MALDI-TOF-MS）. The corresponding mRNA levels of some identified proteins were analyzed by RT-PCR. Results Totally 11 differentially expressed proteins （6 up-regulated including creatine kinase, brain （CKB）, triosephosphate isomerase 1 （TPI1）, isocitrate dehydrogenase 2 （IDH2） and 5 down-regulated including glutathione S-transferase pi （GST-pi）） in the CM were identified using MALDI-TOF-MS and database search. The expression pattern between mRNA and CM protein levels of CKB, IDH2, TPI1 and GST-pi in BPH-1 and LNCaP was similar. Conclusion We proved a feasible and effective way to search new tumor markers by a proteomics-based strategy and identified 11 potentially useful proteins in CM of BPH-1 and LNCaP cells to distinguish prostate cancer from benign prostatic hypertrophy.

Conditioned medium-electrospun fiber biomaterials for skin regeneration

Conditioned medium(CM)contains variety of factors secreted by cells,which directly regulate cellular processes,showing tremendous potential in regenerative medicine.Here,for the first time,we proposed a novel regenerative therapy mediated by biodegradable micro-nano electrospun fibers loaded with highly active conditioned medium of adipose-derived stem cells(ADSC-CM).ADSC-CM was successfully loaded into the nanofibers with biological protection and controllable sustained-release properties by emulsion electrospinning and protein freeze-drying technologies.In vitro,ADSC-CM released by the fibers accelerated the migration rate of fibroblasts;inhibited the over proliferation of fibroblasts by inducing apoptosis and damaging cell membrane;in addition,ADSC-CM inhibited the transformation of fibroblasts into myofibroblasts and suppressed excessive production of extracellular matrix(ECM).In vivo,the application of CM-biomaterials significantly accelerated wound closure and improved regeneration outcome,showing superior pro-regenerative performance.This study pioneered the application of CM-biomaterials in regenerative medicine,and confirmed the practicability and significant biological effects of this innovative biomaterials.

Ginsenoside-Rg1 combined with a conditioned medium from induced neuron-like hUCMSCs alleviated the apoptosis in a cell model of ALS through regulating the NF-κB/Bcl-2 pathway

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord.One important aspect of ALS pathogenesis is superoxide dismutase 1(SOD1)mutant-mediated mitochondrial toxicity,leading to apoptosis in neurons.This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1(G-Rg1)and conditioned medium(CM)on a mutational SOD1 cell model,and to explore the underlying mechanisms.We found that the contents of nerve growth factor,glial cell line-derived neurotrophic factor,and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells(hUCMSCs)were exposed to neuron differentiation reagents for seven days.CM or G-Rg1 decreased the apoptotic rate of SOD1^(G93A)-NSC34 cell to a certain extent,but their combination brought about the least apoptosis,compared with CM or G-Rg1 alone.Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups.Proteins associated with mitochondrial apoptotic pathways,such as Bax,caspase 9(Cas-9),and cytochrome c(Cyt c),were downregulated.Furthermore,CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B(NF-κB)signaling pathway by reducing the phosphorylation of p65 and IκBa.CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid(BetA),an agonist of the NF-κB signaling pathway.In summary,the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^(G93A)-NSC34 cells through suppresing the NF-κB/Bcl-2 sgaling pathway(Fig.1 is a graphcal representation of the abstract).

Effectiveness of mesenchymal stem cellconditioned medium in bone regeneration in animal and human models:a systematic review and meta-analysis

Background:Given the limitations of current therapies for the reconstruction of bone defects,regenerative medicine has arisen as a new therapeutic strategy along with mesenchymal stem cells(MSCs),which,because of their osteogenic potential and immunomodulatory properties,have emerged as a promising alternative for the treatment of bone injuries.In vivo studies have demonstrated that MSCs have a positive effect on regeneration due to their secretion of cytokines and growth factors that,when collected in conditioned medium(MSC-CM)and applied to an injured tissue,can modulate and promote the formation of new tissue.Objective:To evaluate the effectiveness of application of conditioned medium derived from mesenchymal stem cells in bone regeneration in animal and human models.Methods:We conducted a systematic review with a comprehensive search through February of 2018 using several electronic databases(MEDLINE,EMBASE,SCOPUS,CENTRAL(Ovid),and LILACS),and we also used the“snowballing technique”.Articles that met the inclusion criteria were selected through abstract review and subsequent assessment of the full text.We assessed the risk of bias with the SYRCLE and Cochrane tools,and three metaanalyses were performed.Results:We included 21 articles,19 of which used animal models and 2 of which used human models.In animal models,the application of MSC-CM significantly increased the regeneration of bone defects in comparison with control groups.Human studies reported early mineralization in regenerated bones,and no bone resorption,inflammation,nor local or systemic alterations were observed in any case.The meta-analysis showed an overall favorable effect of the application of MSC-CM.Conclusions:The application of MSC-CM to bone defects has a positive and favorable effect on the repair and regeneration of bone tissue,particularly in animal models.It is necessary to perform additional studies to support the application of MSC-CM in clinical practice.

EFFECTS OF HYPOXIC ENDOTHELIAL CELL CONDITIONED MEDIUM ON PROLIFERATION AND COLLAGEN SYNTHESIS OF SMOOTH MUSCLE CELLS AND INHIBITORY EFFECTS OF RADIX SALVIAE MILTIORRHIZAE

The effects of hypoxic endothelial cell conditioned medium (HECCM) on proliferation and collagen synthesis of cultured porcine pulmonary arterial smooth muscle cells (PASMCs) were studied by 3H-thymidine (3H-TdR) and 3H-proline incorporations, image analysis for determination of DNA content and colorimetric assay using MTT, and the inhibitory effects of radix salviae miltiorrhizae (RSM) on them were also investigated. The results showed that HECCM could induce enhancement of the enzymatic activity of mitochondria, increase of the nucleic DNA content and increases of the 3H-TdR and 3H-proline incorporations in PASMCs. The 3H-proline incorporation in PASMCs cultured in HECCM was 1.83 times as much as that cultured in normoxic endothelial cell conditioned medium (NECCM). Compared with the control, Chinese herb medicine RSM could inhibit the proliferation of PASMCs cultured in HECCM and decrease the 3H-prolinc incorporation in PASMCs cultured in both HECCM and NECCM (P< 0.001). However, RSM had no ef fects on the nucleic DNA content and 3H-TdR incorporation into DNA of PASMCs cultured in NECCM. It suggests that hypoxia may stimulate the endothelia to synthesize and secrete some cytokines which can stimulate the proliferation and the synthesis of collagen of PASMCs and RSM can inhibit this process.

miRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells

Our previous study found that rat bone marrow–derived neural crest cells(acting as Schwann cell progenitors)have the potential to promote long-distance nerve repair.Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication.Nevertheless,the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear.To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves,we collected conditioned culture medium from hypoxia-pretreated neural crest cells,and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation.The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells.We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells.Subsequently,to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons,we used a microfluidic axonal dissociation model of sensory neurons in vitro,and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons,which was greatly dependent on loaded miR-21-5p.Finally,we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb,as well as muscle tissue morphology of the hind limbs,were obviously restored.These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p.miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome.This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves,and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.