IWP-2 modulates the immunomodulatory properties of human dental pulp stem cells in vitro.

AIM: To investigate the effect of IWP-2, Wnt inhibitor, on human dental pulp stem cells (hDPSCs) responses. METHODOLOGY: hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 µM IWP-2 for 24 h, and subsequently, the gene expression profile was examined using high-throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP-2 was investigated in both normal and LPS-induced hDPSCs (inflamed hDPSCs). CD4+ T cells and CD14+ monocyte-derived macrophages were cultured with conditioned media of IWP-2 treated hDPSCs to observe the immunosuppressive property. RESULTS: RNA sequencing indicated that IWP-2 significantly downregulated several KEGG pathways, including cytokine-cytokine receptor interaction, IL-17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP-2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro-inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP-2 in LPS-induced inflammation. In terms of immune cells, IWP-2-treated-inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP-2-treated inflamed hDPSCs conditioned media. CONCLUSION: IWP-2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP-2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues.

Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 µg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

Site-specific periosteal cells with distinct osteogenic and angiogenic characteristics.

OBJECTIVES: This study aimed to investigate the site-specific characteristics of rat mandible periosteal cells (MPCs) and tibia periosteal cells (TPCs) to assess the potential application of periosteal cells (PCs) in bone tissue engineering (BTE). MATERIALS AND METHODS: MPCs and TPCs were isolated and characterized. The potential of proliferation, migration, osteogenesis and adipogenesis of MPCs and TPCs were evaluated by CCK-8, scratch assay, Transwell assay, alkaline phosphatase staining and activity, Alizarin Red S staining, RT‒qPCR, and Western blot (WB) assays, respectively. Then, these cells were cocultured with human umbilical vein endothelial cells (HUVECs) to investigate their angiogenic capacity, which was assessed by scratch assay, Transwell assay, Matrigel tube formation assay, RT‒qPCR, and WB assays. RESULTS: MPCs exhibited higher osteogenic potential, higher alkaline phosphatase activity, and more mineralized nodule formation, while TPCs showed a greater capability for proliferation, migration, and adipogenesis. MPCs showed higher expression of angiogenic factors, and the conditioned medium of MPCs accelerated the migration of HUVECs, while MPC- conditioned medium induced the formation of more tubular structure in HUVECs in vitro. These data suggest that compared to TPCs, MPCs exert more consequential proangiogenic effects on HUVECs. CONCLUSIONS: PCs possess skeletal site-specific differences in biological characteristics. MPCs exhibit more eminent osteogenic and angiogenic potentials, which highlights the potential application of MPCs for BTE. CLINICAL RELEVANCE: Autologous bone grafting as the main modality for maxillofacial bone defect repair has many limitations. Constituting an important cell type in bone repair and regeneration, MPCs show greater potential for application in BTE, which provides a promising treatment option for maxillofacial bone defect repair.

Potential role of dental pulp stem cells conditioned medium for odontoblastic differentiation.

BACKGROUND: Functional bioengineered tooth regeneration using autologous or allogeneic alternative differentiated cells sources are thought to have a great potential in replacing conventional dentures. This study investigated the potential of dental pulp stem cells (DPSCs) conditioned medium for odontoblastic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs). The DPSCs derived from healthy adult permanent first molars were cultured at high confluence prior to conditioned medium collection. The WJMSCs were cultured in six different treatments, with varying ratios of culture media to DPSCs-conditioned medium. MTT assay was used to measure the rate of proliferation of WJMSCs, while immunocytochemistry staining was utilised to detect the expression of dental matrix protein 1 (DMP-1). The deposited calcium was detected and analysed via Alizarin-Red Staining (ARS). RESULTS: It was found that the proliferation of WJMSCs cultured under the mixture of complete medium and DPSCs conditioned medium showed significantly lower than the control; presumably the cells started to exit proliferative state prior differentiation. In 14 days of induction, the cells in all treatments showed osteoblastic-like morphology, calcium compound deposits were observed at day 7, 10 and 14 of differentiation suggested that DPSCs conditioned medium could lead to osteoblastic/odontoblastic differentiation. However, the DMP-1 protein can be seen only expressed minimally at day 14 of conditioned medium induction. CONCLUSIONS: In conclusion, DPSCs conditioned medium appeared as a potential odontoblastic induction approach for WJMSCs. To further investigate the stimulatory effects by DPSCs conditioned medium, specific signalling pathway need to be elucidated to enhance the differentiation efficiency.

Wnt5a-Ror2 signaling mediates root resorption.

INTRODUCTION: This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. METHODS: The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In in vivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. RESULTS: The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. CONCLUSIONS: Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

Promotion effect of apical tooth germ cell-conditioned medium on osteoblastic differentiation of periodontal ligament stem cells through regulating miR-146a-5p.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). METHODS AND RESULTS: First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. CONCLUSIONS: In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

Control of the rubber anthracnose fungus Colletotrichum gloeosporioides using culture filtrate extract from Streptomyces deccanensis QY-3.

Colletotrichum gloeosporioides is a main cause of rubber anthracnose, which results in a huge loss for the natural rubber industry. In this study, an actinomycete strain QY-3 was isolated and had good antagonistic activity against C. gloeosporioides with an inhibition rate of 86.6%. Strain QY-3 was identified as Streptomyces deccanensis preliminarily. Millet medium was selected as the optimal fermentation broth for antifungal metabolites production by S. deccanensis QY-3. The culture filtrate extract (CFE) from the millet broth of S. deccanensis QY-3 exhibits broad-spectrum antifungal activity against plant pathogenic fungi, and its EC50 inhibiting the mycelial growth of C. gloeosporioides is 6.3 µg/mL. The CFE has good thermal and pH stabilities, and it can break the hyphae and inhibit the conidial germination of C. gloeosporioides. 100 µg/mL of CFE had an obvious control effect on rubber anthracnose, and the control efficacy was 63.7% on 5 days after inoculation. Two compounds with inhibitory effects on C. gloeosporioides, anthranilic acid and sangivamycin, were isolated from the CFE. The MICs of both compounds against C. gloeosporioides were 29.3 and 23.0 µg/mL respectively. In conclusion, the CFE from S. deccanensis QY-3 has great potential to be a promising fungicide for rubber anthracnose.

Direct Comparison of Therapeutic Effects on Diabetic Polyneuropathy between Transplantation of Dental Pulp Stem Cells and Administration of Dental Pulp Stem Cell-Secreted Factors.

Stem cell transplantation is a potential novel therapy for diabetic polyneuropathy. Dental pulp stem cells (DPSCs) are attractive stem cell sources because DPSCs can be isolated from extracted teeth and cryopreserved while retaining viability. In this study, we directly compared the efficacy of the transplantation of DPSCs and the administration of the secreted factors from DPSCs (DPSC-SFs) on diabetic polyneuropathy. Eight weeks after streptozotocin injection, DPSCs (1.0 × 106 cells/rat) or DPSC-SFs (1.0 mL/rat) were administered into the unilateral hindlimb skeletal muscles of diabetic Sprague-Dawley rats. DPSC transplantation and DPSC-SF administration did not affect blood glucose levels and body weights in the diabetic rats. Both DPSC transplantation and DPSC-SF administration significantly ameliorated sciatic nerve conduction velocity and sciatic nerve blood flow, accompanied by increases in muscle bundle size, vascular density in the skeletal muscles and intraepidermal nerve fiber density in the diabetic rats, while there was no difference between the results for DPSCs and DPSC-SFs. These results suggest that the efficacy of both DPSC transplantation and DPSC-SF administration for diabetic polyneuropathy four weeks after transplantation/administration was mainly due to the multiple secretomes secreted from transplanted DPSCs or directly injected DPSC-SFs in the early phase of transplantation/administration.

Conditioned Medium from the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid-binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid-binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS.

Growth factors and experimental arterial grafts.

BACKGROUND: The production of growth factors from several experimental arterial conduits was determined. METHODS: We implanted 105 experimental arterial grafts that were 1 cm long in the abdominal aorta of Lewis rats (average weight, 250 g). Five different types of grafts were analyzed: arterial isografts, vein grafts, arterial allografts, and polytetrafluoroethylene (PTFE) grafts with normal or decreased compliance. Animals were killed humanely 4 weeks after surgery and the production of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-ß, tumor necrosis factor-α, and interleukin-1 was analyzed. RESULTS: Myointimal hyperplasia (MH) was evident in vein grafts, arterial allografts, and PTFE grafts, but not in arterial isografts. Growth factor production was increased for grafts prone to develop MH like vein, PTFE grafts, and arterial allografts. PDGF and bFGF were increased significantly for PTFE and vein grafts, but not for arterial allografts. The importance of bFGF and PGDF was confirmed by the capability of antibody to PDGF and to bFGF to reduce the mitogenic activity of smooth muscle cells, in vivo and in vitro, for PTFE and vein grafts, but not for arterial allografts, in which a predominant role was played by interleukin-1 and tumor necrosis factor-α. CONCLUSIONS: Agents able to neutralize this increased production of growth factors, either directly or by competition with their receptors, can prevent MH formation.

The effect of nitrogen containing bisphosphonates, zoledronate and alendronate, on the production of pro-angiogenic factors by osteoblastic cells.

Bisphosphonates (BPs) have been shown to influence angiogenesis. This may contribute to BP-associated side-effects such as osteonecrosis of the jaw (ONJ) or atypical femoral fractures (AFF). The effect of BPs on the production of angiogenic factors by osteoblasts is unclear. The aims were to investigate the effect of (1) alendronate on circulating angiogenic factors; vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) in vivo and (2) zoledronate and alendronate on the production of VEGF and ANG-1 by osteoblasts in vitro. We studied 18 post-menopausal women with T score⩽-2 randomized to calcium/vitamin D only (control arm, n=8) or calcium/vitamin D and alendronate 70mg weekly (treatment arm, n=10). Circulating concentrations of VEGF and ANG-1 were measured at baseline, 3, 6 and 12months. Two human osteoblastic cell lines (MG-63 and HCC1) and a murine osteocytic cell line (MLO-Y4) were treated with zoledronate or alendronate at concentrations of 10(-12)-10(-6)M. VEGF and ANG-1 were measured in the cell culture supernatant. We observed a trend towards a decline in VEGF and ANG-1 at 6 and 12months following treatment with alendronate (p=0.08). Production of VEGF and ANG-1 by the MG-63 and HCC1 cells decreased significantly by 34-39% (p<0.01) following treatment with zoledronate (10(-9)-10(-6)M). Treatment of the MG-63 cells with alendronate (10(-7) and 10(-6)) led to a smaller decrease (25-28%) in VEGF (p<0.05). Zoledronate (10(-10)-10(-)(6)M) suppressed the production of ANG-1 by MG-63 cells with a decrease of 43-49% (p<0.01). Co-treatment with calcitriol (10(-8)M) partially reversed this zoledronate-induced inhibition. BPs suppress osteoblastic production of angiogenic factors. This may explain, in part, the pathogenesis of the BP-associated side-effects.

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue.

Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

Nemotic human dental pulp fibroblasts promote human dental pulp stem cells migration.

Dental pulp inflammation has long been perceived as a negative factor leading to pulp disruption. Previous studies have suggested that the inflammatory reaction might be a prerequisite for the burst of progenitors implicated in pulp repair. To investigate the migration of human dental pulp stem cells (hDPSCs) in response to human dental pulp fibroblasts (HDPFs) nemosis, an in vitro model of nemosis-induced inflammation in three-dimensional culture was used in this study. We observed HDPF spheroid formation and that cell-cell adhesion between HDPFs leads to necrosis. Cell death detection and cell counting kit-8 assays showed reduced live cell numbers and increased levels of cell membrane leakage in HDPF spheroids. HDPFs spheroids expressed cyclooxygenase-2 and released an increasing amount of prostaglandin E2 and interleukin-8, indicating inflammation in response to nemosis. The Transwell assays showed that the conditioned medium from HDPFs spheroids significantly induced hDPSCs migration more than the medium from the monolayer. Taken together, these results indicate that HDPFs spheroids induce nemosis and contribute to the migration of hDPSCs. This model might provide a potential research tool for studying interactions between fibroblasts and stem cells, and studies concerning nemosis-targeted stem cells might help treat pulp inflammation.

Periostin regulates integrin expression in gingival epithelial cells.

OBJECTIVE: Human gingival epithelial cells (HGECs) function as a mechanical barrier against invasion by pathogenic organisms through epithelial cell-cell junction complexes, which are complex components of integrin. Integrins play an important role in the protective functions of HGECs. Human periodontal ligament (HPL) cells regulate periodontal homeostasis. However, periodontitis results in the loss of HPL cells. Therefore, as replenishment, HPL cells or mesenchymal stem cells (MSCs) can be transplanted. Herein, HPL cells and MSCs were used to elucidate the regulatory mechanisms of HGECs, assuming periodontal tissue homeostasis. METHODS: Human gingival fibroblasts (HGFs), HGECs, HPL cells, and MSCs were cultured, and the conditioned medium was collected. With or without silencing periostin mRNA, HGECs were cultured under normal conditions or in a conditioned medium. Integrin and periostin mRNA expression was determined using real-time polymerase chain reaction. Integrin protein expression was analyzed using flow cytometry, and periostin protein expression was determined via western blotting. RESULTS: The conditioned medium affected integrin expression in HGECs. Higher expression of periostin was observed in MSCs and HPL cells than in HGFs. The conditioned medium that contained periostin protein regulated integrin expression in HGECs. After silencing periostin in MSCs and HPL cells, periostin protein was not detected in the conditioned medium, and integrin expression in HGECs remained unaffected. CONCLUSIONS: Integrins in HGECs are regulated by periostin secreted from HPL cells and MSCs. This result suggests that periostin maintains gingival cell adhesion and regulates bacterial invasion/infection. Therefore, the functional regulation of periostin-secreting cells is important in preventing periodontitis.

Degrees of macrophage-facilitated healing in aneurysm occlusion devices.

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm.

Conditioned Medium From Stem Cells of Human Exfoliated Deciduous Teeth Alleviates Mouse Osteoarthritis by Inducing sFRP1-Expressing M2 Macrophages.

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1ß-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.

MSCs' conditioned media cytokine and growth factor profiles and their impact on macrophage polarization.

BACKGROUND: There is a growing body of evidence that multipotent mesenchymal stromal cells' (MSCs') remarkable therapeutic potential is attributed not only to their differentiation and regenerative capacity, but also to the paracrine effect, underlying their immunomodulatory properties. MSCs' secretome (i.e., cytokines, growth factors, and extracellular vesicles) is therefore increasingly discussed in the context of their ability to modulate inflammatory response and promote regeneration. There is evidence that 2D or 3D culturing conditions have an impact on the cells' secretome, and here we aimed to compare the secretion of cytokines and growth factors in human MSCs from different sources cultured in 2D and 3D conditions and assess their effect on human macrophages polarization in vitro. METHODS: MSCs were derived from human adipose tissue, bone marrow, gingiva, placenta, and umbilical cord, cultured as monolayers or as cell spheroids. Their cytokine profiles were analyzed, and data standardization was carried out using a z-score. Human peripheral blood mononuclear cells-derived macrophages were then treated with umbilical cord-derived MSCs' conditioned media and their effect on macrophages polarization was assessed. RESULTS: Our findings suggest that umbilical cord-derived MSCs' conditioned media demonstrated the highest cytokine and growth factor levels and despite mostly pro-inflammatory cytokine profile were able to promote anti-inflammatory macrophage polarization. CONCLUSIONS: Umbilical cord-derived MSCs' conditioned media hold great potential for therapeutic use, demonstrating significant anti-inflammatory effect on human macrophages.

In vitro induction of in vivo-relevant stellate astrocytes in 3D brain-derived, decellularized extracellular matrices.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications.

Dental Pulp Stem Cell-Derived Conditioned Medium Alleviates Subarachnoid Hemorrhage-Induced Microcirculation Impairment by Promoting M2 Microglia Polarization and Reducing Astrocyte Swelling.

Aneurysmal subarachnoid hemorrhage (SAH) can cause severe neurological deficits and high mortality. Early brain edema following SAH contributes to the initiation of microcirculation impairment and may further lead to delayed ischemic neurologic deficit (DIND). This study aimed to investigate whether dental pulp stem cell conditioned medium (DPSC-CM) ameliorates SAH-induced microcirculation impairment and the underlying mechanisms. SAH was induced via intrathecal injection of fresh autologous blood in Wistar male adult rat. DPSC-CM or DPSC-CM + insulin growth factor-1 (IGF-1) antibody was randomly administered by intrathecal route 5 min after SAH induction. To evaluate the underlying mechanisms of DPSC-CM in the treatment of SAH, primary rat astrocyte and microglia co-cultures were challenged with hemolysate or SAH-patient CSF in the presence or absence of DPSC-CM. The results showed that in vivo, DPSC-CM treatment decreased the brain water content, improved microcirculation impairment and enhanced functional recovery at 24 h post-SAH. DPSC-CM treatment also alleviated the expressions of water channel protein aquaporin-4 (AQP4) and pro-inflammatory cytokines, and enhanced the expressions of anti-inflammatory factors in the cortical region. However, all the beneficial effects of DPSC-CM were abrogated after treatment with IGF-1 neutralizing antibody. The in vitro results further showed that DPSC-CM treatment reduced hemolysate/SAH-patient CSF-induced astrocyte swelling and promoted M2 microglia polarization, partially through IGF-1/AKT signaling. The data suggested that DPSC-CM significantly reduced brain edema and rescued microcirculation impairment with concomitant anti-inflammatory benefits after SAH, and may potentially be developed into a novel therapeutic strategy for SAH.

The role of different macrophages-derived conditioned media in dental pulp tissue regeneration.

Macrophages have been reported to play important roles in tissue repair and regeneration. While it is known that macrophages are present in the dental pulp, their role in dental pulp regeneration is not fully understood. In the present study, we investigated the effects of different phenotype macrophages conditioned medium on the cellular behaviors of hDPSCs and their extracellular matrix (cell sheets) in vitro. Moreover, twenty-four root fragments inserted with cell sheets cultured with different conditioned media were placed into the back subcutaneous space of 6-8-week-old male BALB/c nude mouse. The regenerated tissues in the root fragments were assessed via histologic analysis after 8 weeks of transplantation. M2 macrophages could promote the proliferation, migration, and osteogenic differentiation of hDPSCs. Dental pulp-like tissue with an odontoblast-like layer lining the dentinal surface and well-arranged collagen fibers was harvested in root fragment combined with M2 conditioned medium cultured cell sheet, whereas a large amount of calcium salt deposition and disorganization of collagen fibers were observed in root fragments combined with M1 conditioned medium cultured cell sheet. Therefore, promoting the transformation of M1 into M2 macrophage in dental pulp tissue regeneration may be a potential way for dental pulp regeneration via functional healing.