ABSTRACT
This corrects the article 10.3791/64016.
ABSTRACT
The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded ß-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4+ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.
Subject(s)
Macrophage Activation , Osteogenesis , CD4-Positive T-Lymphocytes , Cells, Cultured , Macrophages , T-Lymphocytes , HumansABSTRACT
Bone fractures and critical-size bone defects are significant public health issues, and clinical treatment outcomes are closely related to the intrinsic properties of the utilized implant materials. Zinc (Zn)-based biodegradable metals (BMs) have emerged as promising bioactive materials because of their exceptional biocompatibility, appropriate mechanical properties, and controllable biodegradation. This review summarizes the state of the art in terms of Zn-based metals for bone repair and regeneration, focusing on bridging the gap between biological mechanism and required bioactivity. The molecular mechanism underlying the release of Zn ions from Zn-based BMs in the improvement of bone repair and regeneration is elucidated. By integrating clinical considerations and the specific bioactivity required for implant materials, this review summarizes the current research status of Zn-based internal fixation materials for promoting fracture healing, Zn-based scaffolds for regenerating critical-size bone defects, and Zn-based barrier membranes for reconstituting alveolar bone defects. Considering the significant progress made in the research on Zn-based BMs for potential clinical applications, the challenges and promising research directions are proposed and discussed.
ABSTRACT
Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-ß), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients' sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-ß. However, regulation of TGF-ß target genes, such as CTGF, COL1A1, or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-ß isoforms were expressed with increased levels of TGF-ß1 and TGF-ß3 and a reporter assay confirmed that the expressed TGF-ß was activated. However, Western blots and immunostaining showed decreased canonical TGF-ß signaling in the respective chronic wound tissues, suggesting the presence of a TGF-ß inhibitor. As a potential regulatory mechanism, the TGF-ß proteome profiler array suggested elevated levels of the TGF-ß pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation.
Subject(s)
Transforming Growth Factor beta3 , Transforming Growth Factor beta , Humans , Biological Assay , Blotting, Western , Inflammation , Membrane ProteinsABSTRACT
Autologous bone transplantation is still considered as the gold standard therapeutic option for bone defect repair. The alternative tissue engineering approaches have to combine good hardiness of biomaterials whilst allowing good stem cell functionality. To become more useful for load-bearing applications, mechanical properties of calcium phosphate materials have to be improved. In the present study, we aimed to reduce the brittleness of ß-tricalcium phosphate (ß-TCP). For this purpose, we used three polymers (PDL-02, -02a, -04) for coatings and compared resulting mechanical and degradation properties as well as their impact on seeded periosteal stem cells. Mechanical properties of coated and uncoated ß-TCP scaffolds were analyzed. In addition, degradation kinetics analyses of the polymers employed and of the polymer-coated scaffolds were performed. For bioactivity assessment, the scaffolds were seeded with jaw periosteal cells (JPCs) and cultured under untreated and osteogenic conditions. JPC adhesion/proliferation, gene and protein expression by immunofluorescent staining of embedded scaffolds were analyzed. Raman spectroscopy measurements gave an insight into material properties and cell mineralization. PDL-coated ß-TCP scaffolds showed a significantly higher flexural strength in comparison to that of uncoated scaffolds. Degradation kinetics showed considerable differences in pH and electrical conductivity of the three different polymer types, while the core material ß-TCP was able to stabilize pH and conductivity. Material differences seemed to have an impact on JPC proliferation and differentiation potential, as reflected by the expression of osteogenic marker genes. A homogenous cell colonialization of coated and uncoated scaffolds was detected. Most interesting from a bone engineer's point of view, the PDL-04 coating enabled detection of cell matrix mineralization by Raman spectroscopy. This was not feasible with uncoated scaffolds, due to intercalating effects of the ß-TCP material and the JPC-formed calcium phosphate. In conclusion, the use of PDL-04 coating improved the mechanical properties of the ß-TCP scaffold and promoted cell adhesion and osteogenic differentiation, whilst allowing detection of cell mineralization within the ceramic core material.
ABSTRACT
Dexamethasone (dexa) is commonly used to stimulate osteogenic differentiation of mesenchymal stem/stromal cells (MSCs) in vitro. However, it is paradoxical that glucocorticoids (GCs) such as dexa lead to bone loss and increased fracture risk in patients undergoing glucocorticoid therapy, causing glucocorticoid-induced osteoporosis (GIOP). In a recent publication, we demonstrated that osteogenic differentiation of progenitor cells isolated from jaw periosteal tissue (JPCs) does not depend on dexa, if the medium is supplemented with human platelet lysate (hPL) instead of fetal bovine serum (FBS). This allows the in vitro conditions to be much closer to the natural situation in vivo and enables us to compare osteogenic differentiation with and without dexa. In the present study, we demonstrate that the absence of dexa did not reduce mineralization capacity, but instead slightly improved the osteogenic differentiation of jaw periosteal cells. On the other hand, we show that dexa supplementation strongly alters the gene expression, extracellular matrix (ECM), and cellular communication of jaw periosteal cells. The secretome of periosteal cells previously treated with an osteogenic medium with and without dexa was used to investigate the changes in paracrine secretion caused by dexa. Dexa altered the secretion of several cytokines by jaw periosteal cells and strongly induced osteoclast differentiation of peripheral blood mononuclear cells (PBMCs). This study demonstrates how dexa supplementation can influence the outcome of in vitro studies and highlights a possible role of periosteal cells in the pathogenesis of glucocorticoid-induced osteoporosis. The methods used here can serve as a model for studying bone formation, fracture healing, and various pathological conditions such as (glucocorticoid-induced) osteoporosis, osteoarthritis, bone cancer, and others, in which the interactions of osteoblasts with surrounding cells play a key role.
ABSTRACT
Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.
Subject(s)
Cartilage, Articular , Osteoarthritis , Humans , Aged , Microscopy, Atomic Force/methods , ArtifactsABSTRACT
Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.
Subject(s)
Bone Resorption , Osteoporosis , Bone Resorption/diagnostic imaging , Humans , Leukocytes, Mononuclear/metabolism , Osteoclasts , OsteogenesisABSTRACT
Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (ß-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.
Subject(s)
Osteogenesis , Tissue Scaffolds , Calcium Phosphates/pharmacology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Hydrodynamics , Tissue Engineering/methods , Tissue Scaffolds/chemistryABSTRACT
Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.
Subject(s)
Induced Pluripotent Stem Cells , Mesenchymal Stem Cells , Collagen/metabolism , Humans , Hypoxia/metabolism , Induced Pluripotent Stem Cells/metabolism , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Mesenchymal Stem Cells/metabolism , Neovascularization, Pathologic/metabolism , SecretomeABSTRACT
The jaw periosteal tissue is generally recognized as a suitable source for the isolation of mesenchymal stem cells (MSCs). In previous studies we showed evidence that two- and three-dimensionally cultured jaw periosteum-derived MSCs (JPCs) are able to induce a more immature phenotype of dendritic cells (DCs). To further expand our knowledge of JPCs' immunoregulative function, we investigated the effects of JPC secretomes derived from undifferentiated (CO) or osteogenically differentiated cells (treated with or without dexamethasone: OB+/-D) on CD14+ monocyte-derived DCs (MoDCs). We detected a remarkably reduced formation of MoDC homotypic clusters under the influence of secretomes from osteogenically induced JPCs. Further, significantly decreased numbers of CD83+ cells, up-regulated CD209 and down-regulated CD80, CD86 and CD197 expression levels were detected on the surface of MoDCs. Whereas secretomes from JPCs osteogenically stimulated with dexamethasone significantly enhanced FITC-dextran uptake capacity of MoDCs, the increase by secretomes of JPCs treated without dexamethasone did not reach significance. The analysis of mixed lymphocyte reactions revealed that OB+/-D secretomes were able to significantly reduce the numbers of proliferating CD14- peripheral blood mononuclear cells (PBMCs) and of proliferating CD4+ T cells. The OB-D secretome significantly promoted the expansion of regulatory CD25+ T cells. Regarding gene expression of MoDCs, remarkably up-regulated mRNA expression of CD209, HLA-DRA, CSF3, IL10 and IL8 was detected when DCs were cultured in the presence of OB+/-D secretomes. At the same time, secretomes seemed to have an impact in the down-regulation of IFNγ and IL12B gene expression. At protein level, OB+/-D secretomes significantly up-regulated IL-10 and IDO (indoleamine-pyrrole 2,3-dioxygenase) levels whereas IL-12/IL-23p40 levels were down-regulated in supernatants of MoDCs when cultured under the presence of OB+/-D secretomes. Taken together, while secretomes from untreated JPCs had only little effects on the process of maturation of MoDCs, secretomes derived from osteogenically induced JPCs were able to inhibit the phenotypic and functional maturation of MoDCs.
Subject(s)
Monocytes , Secretome , Monocytes/metabolism , Leukocytes, Mononuclear , Cells, Cultured , Phenotype , Dendritic Cells/metabolism , Dexamethasone/pharmacologyABSTRACT
Jaw periosteum-derived mesenchymal stem cells (JPCs) represent a promising cell source for bone tissue engineering in oral and maxillofacial surgery due to their high osteogenic potential and good accessibility. Our previous work demonstrated that JPCs are able to regulate THP-1-derived macrophage polarization in a direct coculture model. In the present study, we used an innovative horizontal coculture system in order to understand the underlying paracrine effects of JPCs on macrophage phenotype polarization. Therefore, JPCs and THP-1-derived M1/M2 macrophages were cocultured in parallel chambers under the same conditions. After five days of horizontal coculture, flow cytometric, gene and protein expression analyses revealed inhibitory effects on costimulatory and proinflammatory molecules/factors as well as activating effects on anti-inflammatory factors in M1 macrophages, originating from multiple cytokines/chemokines released by untreated and osteogenically induced JPCs. A flow cytometric assessment of DNA synthesis reflected significantly decreased numbers of proliferating M1/M2 cells when cocultured with JPCs. In this study, we demonstrated that untreated and osteogenically induced JPCs are able to switch macrophage polarization from a classical M1 to an alternative M2-specific phenotype by paracrine secretion, and by inhibition of THP-1-derived M1/M2 macrophage proliferation.
ABSTRACT
Biodegradable zinc (Zn) and Zn-based alloys have been recognized as promising biomaterials for biomedical implants. Sterilization is an essential step in handling Zn-based implants before their use in clinical practice and there are various sterilization methods are available. However, how these treatments influence the Zn-based biomaterials remains unknown and is of critical relevance. In this study, three commonly-applied standard sterilization methods, namely gamma irradiation, hydrogen peroxide gas plasma and steam autoclave, were used on pure Zn and Zn3Cu (wt%) alloy. The treated Zn and ZnCu alloy were investigated to compare the different influences of sterilizations on surface characteristics, transient and long-term degradation behavior and cytotoxicity of Zn and Zn alloy. Our results indicate that autoclaving brought about apparently a formation of inhomogeneous zinc oxide film whereas the other two methods produced no apparent alterations on the material surfaces. Consequently, the samples after autoclaving showed significantly faster degradation rates and more severe localized corrosion, especially for the ZnCu alloy, owing to the incomplete covering and unstable zinc oxide layer. Moreover, the autoclave-treated Zn and ZnCu alloy exhibited apparent cytotoxic effects towards fibroblasts, which may be due to the excessive Zn ion releasing and its local concentration exceeds the cellular tolerance capacity. In contrast, gamma irradiation and hydrogen peroxide gas plasma had no apparent adverse effects on the biodegradability and cytocompatibility of Zn and ZnCu alloy. Our findings may have significant implications regarding the selection of suitable sterilization methods for Zn-based implant materials among others.
Subject(s)
Absorbable Implants , Zinc , Alloys/pharmacology , Biocompatible Materials/pharmacology , Corrosion , Materials Testing , SterilizationABSTRACT
Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties.
Subject(s)
Neovascularization, Physiologic/genetics , Peptides/pharmacology , Tissue Engineering , Vascular Endothelial Growth Factor A/genetics , Angiopoietin-1/genetics , Collagen/chemistry , Collagen/pharmacology , Durapatite/chemistry , Durapatite/pharmacology , Gene Expression Regulation, Developmental/drug effects , Hepatocyte Growth Factor/genetics , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Insulin-Like Growth Factor Binding Protein 1/genetics , Insulin-Like Growth Factor Binding Protein 2/genetics , Interleukin-8/genetics , Matrix Metalloproteinase 1/genetics , Neovascularization, Physiologic/drug effects , Neovascularization, Physiologic/physiology , Proto-Oncogene Proteins c-met/genetics , Tissue Scaffolds/chemistry , Vascular Cell Adhesion Molecule-1/genetics , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.
Subject(s)
Blood Coagulation/drug effects , Calcium Phosphates/pharmacology , Jaw/cytology , Periosteum/cytology , Tissue Scaffolds/chemistry , Blood Cell Count , Blood Platelets/drug effects , Blood Platelets/metabolism , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Complement System Proteins/metabolism , Humans , Osteogenesis/drug effects , Polylactic Acid-Polyglycolic Acid Copolymer/pharmacologyABSTRACT
Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.
Subject(s)
Cell Differentiation , Immunomodulation , Jaw/cytology , Macrophage Activation , Macrophages/physiology , Mesenchymal Stem Cells/cytology , Periosteum/cytology , Adolescent , Adult , Cytokines/metabolism , Female , Humans , Macrophages/immunology , Male , THP-1 Cells , Young AdultABSTRACT
Appropriately adapted comprehensive mechanical properties, degradation behavior and biocompatibility are prerequisites for the application of Zn-based biodegradable implants. In this study, hot-extruded Zn-0.5Cu-xFe (x = 0.1, 0.2 and 0.4 wt%) alloys were fabricated as candidates for biodegradable materials for guided bone regeneration (GBR) membranes. The hot-extrusion process and Cu alloying were expected mostly to enhance the mechanical properties, and the Fe alloying was added mainly for regulating the degradation. The microstructure, mechanical properties and in vitro degradation behavior were systematically investigated. The ZnCuFe alloys were composed of a Zn matrix and FeZn13 phase. With increasing Fe content, a higher FeZn13 phase precipitation with larger particles was observed. Since elongation declined significantly until fracture with increasing Fe content up to 0.4 wt%, the ZnCuFe (0.2 wt%) alloy achieved a good balance between mechanical strength and ductility, with an ultimate tensile strength of 202.3 MPa and elongation at fracture of 41.2%. Moreover, the addition of Fe successfully accelerated the degradation of ZnCuFe alloys. The ZnCuFe (0.2 wt%) alloy showed relatively uniform corrosion in the long-term degradation test. Furthermore, extracts of the ZnCuFe (0.2 wt%) alloy showed no apparent cytotoxic effects against L929 fibroblasts, Saos-2 osteoblasts or TAg periosteal cells. The ZnCuFe (0.2 wt%) alloy exhibited the potential to inhibit bacterial adhesion of Streptococcus gordonii and mixed oral bacteria. Our study provides evidence that the ZnCuFe (0.2 wt%) alloy can represent a promising material for the application as a suitable GBR membrane.
ABSTRACT
Mesenchymal stem cells (MSCs) have gained attraction not only in the field of regenerative medicine but also in the field of autoimmune disease therapies or organ transplantation due to their immunoregulatory and/or immunosuppressive features. Dendritic cells (DCs) play a crucial role in initiating and regulating immune reactions by promoting antigen-specific T cell activation. In this study, we investigated the effect of human jaw periosteal progenitor cells (JPCs) seeded in beta-tricalcium phosphate (ß-TCP) scaffolds on monocyte-derived DC differentiation. Significantly lower numbers of differentiated DCs were observed in the presence of normal (Co) and osteogenically induced (Ob) JPCs-seeded ß-TCP constructs. Gene expression analysis revealed significantly lower interleukin-12 subunit p35 (IL-12p35) and interleukin-12 receptor beta 2 (IL-12Rß2) and pro-inflammatory cytokine interferon-gamma (IFN-γ) levels in DCs under Ob conditions, while interleukin-8 (IL-8) gene levels were significantly increased. Furthermore, in the presence of JPCs-seeded ß-TCP constructs, interleukin-10 (IL-10) gene expression was significantly induced in DCs, particularly under Ob conditions. Analysis of DC protein levels shows that granulocyte-colony stimulating factor (G-CSF) was significantly upregulated in coculture groups. Our results indicate that undifferentiated and osteogenically induced JPCs-seeded ß-TCP constructs have an overall inhibitory effect on monocyte-derived DC maturation.
Subject(s)
Calcium Phosphates/pharmacology , Coculture Techniques , Dendritic Cells/drug effects , Jaw/cytology , Cells, Cultured , Dendritic Cells/metabolism , Humans , Jaw/metabolismABSTRACT
Extensive efforts were undertaken to develop suitable biomaterials for tissue engineering (TE) applications. To facilitate clinical approval processes and ensure the success of TE applications, bioinspired concepts are currently focused on. Working on bone tissue engineering, we describe in the present study a method for biofunctionalization of collagen/hydroxyapatite composites with BMP-2 mimetic peptides. This approach is expected to be fundamentally transferable to other tissue engineering fields. A modified BMP-2 mimetic peptide containing a negatively charged poly-glutamic acid residue (E7 BMP-2 peptide) was used to bind positively charged hydroxyapatite (HA) particles by electrostatic attraction. Binding efficiency was biochemically detected to be on average 85% compared to 30% of BMP-2 peptide without E7 residue. By quartz crystal microbalance (QCM) analysis, we could demonstrate the time-dependent dissociation of the BMP-2 mimetic peptides and the stable binding of the E7 BMP-2 peptides on HA-coated quartz crystals. As shown by immunofluorescence staining, alkaline phosphatase expression is similar to that detected in jaw periosteal cells (JPCs) stimulated with the whole BMP-2 protein. Mineralization potential of JPCs in the presence of BMP-2 mimetic peptides was also shown to be at least similar or significantly higher when low peptide concentrations were used, as compared to JPCs cultured in the presence of recombinant BMP-2 controls. In the following, collagen/hydroxyapatite composite materials were prepared. By proliferation analysis, we detected a decrease in cell viability with increasing HA ratios. Therefore, we chose a collagen/hydroxyapatite ratio of 1:2, similar to the natural composition of bone. The following inclusion of E7 BMP-2 peptides within the composite material resulted in significantly elevated long-term JPC proliferation under osteogenic conditions. We conclude that our advanced approach for fast and cost-effective scaffold preparation and biofunctionalization is suitable for improved and prolonged JPC proliferation. Further studies should prove the functionality of composite scaffolds in vivo.
ABSTRACT
Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-ß-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.