RESUMO
Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.
Assuntos
Sobrevivência Celular , Ácido Hialurônico , Hidrogéis , Hidrogéis/química , Camundongos , Animais , Sobrevivência Celular/efeitos dos fármacos , Ácido Hialurônico/química , Viscosidade , Linhagem Celular , Teste de Materiais , Materiais Biocompatíveis/química , Engenharia Tecidual/métodos , Dodecilsulfato de Sódio/química , Gelatina/química , Fibrina/químicaRESUMO
For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.
Assuntos
Osso e Ossos/fisiologia , Custos e Análise de Custo , Técnicas de Cultura de Tecidos/economia , Técnicas de Cultura de Tecidos/métodos , Idoso , Idoso de 80 Anos ou mais , Agrecanas/genética , Agrecanas/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Proliferação de Células , Sobrevivência Celular , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Esclerose , Sobrevivência de Tecidos , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.
Assuntos
Células da Medula Óssea/metabolismo , Transplante Ósseo/métodos , Osso e Ossos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Biossíntese de Proteínas , Engenharia Tecidual/métodos , Adulto , Bioimpressão/métodos , Células da Medula Óssea/citologia , Osso e Ossos/citologia , Diferenciação Celular/genética , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , Impressão Tridimensional , Alicerces Teciduais , Transplante AutólogoRESUMO
Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.
Assuntos
Anel Fibroso/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/genética , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Anel Fibroso/patologia , Proteína Morfogenética Óssea 2/farmacologia , Células Cultivadas , Matriz Extracelular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Deslocamento do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/patologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regeneração/efeitos dos fármacos , Regeneração/genética , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features. RESULTS: Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1ß) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFß. Further investigations identified CD14+ cells as major recipient cell subset of CardAP-EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells. CONCLUSIONS: CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.
Assuntos
Doenças Cardiovasculares/terapia , Vesículas Extracelulares/imunologia , Monócitos/imunologia , Miócitos Cardíacos/imunologia , Doenças Cardiovasculares/imunologia , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Imunomodulação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Monócitos/citologia , Miócitos Cardíacos/citologia , Regeneração , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV-cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter® miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application.
Assuntos
Vasos Sanguíneos/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Mediadores da Inflamação/metabolismo , Miocárdio/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Sinais (Psicologia) , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/citologia , Tetraspanina 30/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Quimiocinas CC/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Engenharia Tecidual , Animais , Cartilagem Articular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Fêmur/citologia , Fêmur/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Osteoartrite/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/administração & dosagem , Suínos , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.
Assuntos
Cartilagem/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Osteoartrite/metabolismo , Transcriptoma , Animais , Cartilagem/metabolismo , Células Cultivadas , Matriz Extracelular , Osteoartrite/genética , Suínos , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)ß3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFß3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.
Assuntos
Anel Fibroso/citologia , Anel Fibroso/metabolismo , Movimento Celular , Quimiocinas CC/metabolismo , Quimiotaxia , Matriz Extracelular/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Proteoglicanas/metabolismo , Fator de Crescimento Transformador beta3/metabolismoRESUMO
A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.
Assuntos
Anel Fibroso/metabolismo , Núcleo Pulposo/metabolismo , Transcriptoma , Adulto , Idoso , Anel Fibroso/citologia , Anel Fibroso/patologia , Biomarcadores/metabolismo , Biópsia/normas , Feminino , Perfilação da Expressão Gênica/normas , Humanos , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/citologia , Núcleo Pulposo/patologiaRESUMO
In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/metabolismo , Animais , Morte Celular/genética , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Osteoartrite/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The negligible self-repair potential of the myocardium has led to cell-based tissue engineering approaches to restore heart function. There is more and more consensus that, in addition to cell development, paracrine effects in particular play a pivotal role in the repair of heart tissue. Here, we present two complementary murine P19 and P19CL6 embryonic carcinoma cell-based in vitro test approaches to study the potential of repair cells and the factors secreted by these cells to induce cardiomyogenesis. P19 cells were 3-dimensionally cultured in hanging drops and P19CL6 cells in a monolayer. Both systems, capable of inducible differentiation towards the cardiomyogenic lineage shown by the appearance of beating cells, the expression of connexin 43 and cardiac troponins T and I, were used to test the cardiomyogenesis-inducing potential of human cardiac-derived adherent proliferating (CardAP) cells, which are candidates for heart repair. CardAP cells in coculture as well as CardAP cell-conditioned medium initiated beating in P19 cells, depending on the cell composition and concentration of the medium. CardAP cell-dependent beating was not observed in P19CL6 cultures, but connexin 43 and cardiac troponin formation as well as expression of GATA-binding protein 4 indicated the dose-dependent stimulatory cardiomyogenic effect of human CardAP cells. In summary, in different ways, P19 and P19CL6 cells have shown their capability to detect paracrine effects of human CardAP cells. In a complementary approach, they could be beneficial for determining the stimulatory cardiomyogenic potential of candidate cardiac-repair cells in vitro.
Assuntos
Coração/fisiologia , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Animais , Carcinoma Embrionário , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Meios de Cultivo Condicionados , Humanos , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Chitosan-beta glycerophosphate-hydroxyethyl cellulose (CH-GP-HEC) is a biocompatible and biodegradable scaffold exhibiting a sol-gel transition at 37°C. Chondrogenic factors or mesenchymal stem cells (MSCs) can be included in the CH-GP-HEC, and injected into the site of injury to fill the cartilage tissue defects with minimal invasion and pain. The possible impact of the injectable CH-GP-HEC on the viability of the encapsulated MSCs was assessed by propidium iodide-fluorescein diacetate staining. Proliferation of the human and rat MSCs was also determined by MTS assay on days 0, 7, 14 and 28 after encapsulation. To investigate the potential application of CH-GP-HEC as a drug delivery device, the in vitro release profile of insulin was quantified by QuantiPro-BCA™ protein assay. Chondrogenic differentiation capacity of the encapsulated human MSCs (hMSCs) was also determined after induction of differentiation with transforming growth factor ß3. MSCs have very good survival and proliferative rates within CH-GP-HEC hydrogel during the 28-day investigation. A sustained release of insulin occurred over 8 days. The CH-GP-HEC hydrogel also provided suitable conditions for chondrogenic differentiation of the encapsulated hMSCs. In conclusion, the high potential of CH-GP-HEC as an injectable hydrogel for cartilage tissue engineering is emphasised.
Assuntos
Cartilagem/citologia , Quitosana/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Condrogênese , Humanos , Insulina/metabolismo , Ratos , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
It is generally accepted that after differentiation bone marrow mesenchymal stem cells (MSC) become lineage restricted and unipotent in an irreversible manner. However, current results imply that even terminally differentiated cells transdifferentiate across lineage boundaries and therefore act as a progenitor cells for other lineages. This leads to the questions that whether transdifferentiation occurs via direct cell-to-cell conversion or dedifferentiation to a progenitor cells and subsequent differentiation, and whether MSC potency decreases or increases during differentiation. To address these questions, MSC were differentiated into adipogenic lineage cells, followed by dedifferentiation. The process of dedifferentiation was also confirmed by single cell clonal analysis. Finally the dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated MSC, adipogenic-differentiated and dedifferentiated cells were performed. Interestingly, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes have an association with cell cycle arrest in adipogenic-differentiated cells and perhaps narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these genes have an association with cell cycle progression and maybe motivate dedifferentiation of adipogenic-differentiated cells. We found that dedifferentiated cells have a multilineage potency comparable to MSC, and also observed the associative role of proliferation genes with cell cycle arrest and progression. Concluded, our results indicate that transdifferentiation of adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and correlates with cell cycle arresting and deriving genes. Regarding clinical use, the knowledge of potency and underlying mechanisms are prerequisites.
Assuntos
Tecido Adiposo/citologia , Desdiferenciação Celular , Diferenciação Celular , Transdiferenciação Celular , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/citologia , Pontos de Checagem do Ciclo Celular/genética , Células Cultivadas , Condrogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese/genética , Células-Tronco/citologia , Regulação para CimaRESUMO
Photochemical additive manufacturing technologies can produce complex geometries in short production times and thus have considerable potential as a tool to fabricate medical devices such as individualized patient-specific implants, prosthetics and tissue engineering scaffolds. However, most photopolymer resins degrade only slowly under the mild conditions required for many biomedical applications. Herein we report a novel platform consisting of amino acid-based polyphosphorodiamidate (APdA) monomers with hydrolytically cleavable bonds. The substituent on the α-amino acid can be used as a handle for facile control of hydrolysis rates of the monomers into their endogenous components, namely phosphate and the corresponding amino acid. Furthermore, monomer hydrolysis is considerably accelerated at lower pH values. The monomers underwent thiol-yne photopolymerization and could be 3D structured via multiphoton lithography. Copolymerization with commonly used hydrophobic thiols demonstrates not only their ability to regulate the ambient degradation rate of thiol-yne polyester photopolymer resins, but also desirable surface erosion behavior. Such degradation profiles, in the appropriate time frames, in suitably mild conditions, combined with their low cytotoxicity and 3D printability, render these novel photomonomers of significant interest for a wide range of biomaterial applications.
Assuntos
Aminoácidos , Alicerces Teciduais , Humanos , Alicerces Teciduais/química , Materiais Biocompatíveis , Engenharia Tecidual , Poliésteres , Materiais Dentários , Compostos de Sulfidrila/químicaRESUMO
BACKGROUND: Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. METHOD: Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. RESULTS: Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). CONCLUSION: The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.
Assuntos
Cartilagem Articular/cirurgia , Condrócitos/transplante , Fêmur/cirurgia , Articulações/cirurgia , Teste de Materiais/métodos , Osteotomia , Adesivos Teciduais/química , Engenharia Tecidual/métodos , Adesividade , Animais , Células Cultivadas , Colágeno/química , Reutilização de Equipamento , Adesivo Tecidual de Fibrina/química , Ácido Láctico/química , Modelos Animais , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Suínos , Resistência à Tração , Fatores de Tempo , Alicerces Teciduais , Sobrevivência de TecidosRESUMO
BACKGROUND: Tissue defects in the annulus fibrosus (AF) due to intervertebral disc (IVD) degeneration or after nucleodiscectomy have little self-healing capacity. To prevent progressive degeneration of the IVD, the AF must be repaired. Biological closure has not yet been achieved and is a challenge for the research community. In this study, a scaffold made of absorbable poly (glycolic acid) (PGA) and hyaluronan (HA) that exhibit excellent biocompatibility and cell colonization properties was used to repair AF defects in an ovine model. METHODS: A partial resection was performed in AF in L3/4 or L4/5 of 10 sheep and PGA-HA scaffolds were implanted on the defects (n = 5), while defects in the control group were left untreated (n = 5). Three months post-operation, the lumbar discs were sectioned and stained with hematoxylin and eosin and safranin-O/fast-green. Histological features including proteoglycan content, annular structure, cellular morphology, blood vessel ingrowth and tear/cleft formation were scored using a modified scoring scheme by 3 investigators and evaluated by a pathologist independently. RESULTS: The treated AF exhibited significantly enhanced repair tissue structure with signs of proteoglycan formation compared to the untreated group. The median scores were 4.3 for the treated and 9.8 for the untreated group. Cystic degeneration, perivascular infiltration, inflammation and necrosis were only present in the untreated group. Blood vessel ingrowth and tear/cleft formation were increased, though not significant, in the untreated group while cell morphology was comparable in both groups. CONCLUSION: PGA-HA scaffolds used for AF closure support repair tissue formation in an ovine lumbar disc defect model.
Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Anel Fibroso/patologia , Ácido Hialurônico , Disco Intervertebral/patologia , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/cirurgia , Proteoglicanas , OvinosRESUMO
Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.
Assuntos
Bioimpressão , Cartilagem Articular , Animais , Bioimpressão/métodos , Colágeno Tipo II/química , Gelatina/química , Ácido Hialurônico/química , Hidrogéis/química , Impressão Tridimensional , Proteoglicanas , Suínos , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
BACKGROUND: Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data. RESULTS: The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein. CONCLUSIONS: Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.
Assuntos
Adipogenia/genética , Células da Medula Óssea/metabolismo , Perfilação da Expressão Gênica/métodos , Células-Tronco Mesenquimais/metabolismo , Células da Medula Óssea/citologia , Análise por Conglomerados , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/genética , TranscriptomaRESUMO
PURPOSE: Augmentation of the maxillary sinus with allogenic or alloplastic materials, as well as autologous bone grafts, has inherent disadvantages. Therefore, the aim of our study was to evaluate the long-term clinical repair effect of autologous periosteal bone grafts on atrophic maxillary bone. PATIENTS AND METHODS: In the present retrospective cohort study, augmentation of the edentulous atrophic posterior maxilla was performed using autologous tissue-engineered periosteal bone grafts based on bioresorbable polymer scaffolds and, in a 1-step procedure, simultaneous insertion of dental implants. The clinical evaluation of 10 patients was performed by radiologic assessment of bone formation, with a follow-up of 5 years. Bone formation was further documented by measuring the bone height and by histologic examination. RESULTS: Excellent clinical and radiologic results were achieved as early as 4 months after transplantation of the periosteal bone grafts. The bone height remained significantly (P < .05) greater (median 14.2 mm) than the preoperative atrophic bone (median 6.9 mm) during the 5-year observation period. Histologically, the bone biopsy specimens of 2 patients obtained after 6 months showed trabecular bone with osteocytes and active osteoblasts. No signs of bone resorption, formation of connective tissue, or necrosis were seen. CONCLUSION: Our results suggest that the transplantation of autologous periosteal bone grafts and implantation of dental implants in a 1-step procedure is a reliable procedure that leads to bone formation in the edentulous posterior maxilla, remaining stable in the long term for a period of at least 5 years.