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Objective: Cartilage defect treatment strategies are dependent on the lesion size and severity. Osteochondral explant models are a platform to test cartilage repair strategies ex vivo. Current models lack in mimicking the variety of clinically relevant defect scenarios. In this controlled laboratory study, an automated device (artificial tissue cutter, ARTcut®) was implemented to reproducibly create cartilage defects with controlled depth. In a pilot study, the effect of cartilage defect depth and oxygen tension on cartilage repair was investigated. Design: Osteochondral explants were isolated from porcine condyles. 4 âmm chondral and full thickness defects were treated with either porcine chondrocytes (CHON) or co-culture of 20% CHON and 80% MSCs (MIX) embedded in collagen hydrogel. Explants were cultured with tissue specific media (without TGF-ß) under normoxia (20% O2) and physiological hypoxia (2% O2). After 28 days, immune-histological stainings (collagen II and X, aggrecan) were scored (modified Bern score, 3 independent scorer) to quantitatively compare treatment outcome. Results: ARTcut® represents a software-controlled device for creation of uniform cartilage defects. Comparing the scoring results of the MIX and the CHON treatment, a positive relation between oxygen tension and defect depth was observed. Low oxygen tension stimulated cartilaginous matrix deposition in MIX group in chondral defects and CHON treatment in full thickness defects. Conclusion: ARTcut® has proved a powerful tool to create cartilage defects and thus opens a wide range of novel applications of the osteochondral model, including the relation between oxygen tension and defect depth on cartilage repair.
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OBJECTIVES: Before application in dental practice, novel dental materials are tested in vitro and in vivo to ensure safety and functionality. However, transferability between preclinical and clinical results is often limited. To increase the predictive power of preclinical testing, a biomimetic in vitro test system that mimics the wound niche after implantation was developed. METHODS: First, predetermined implant materials were treated with human blood plasma, M2 macrophages and bone marrow stromal stem cells. Thereby, the three-dimensional wound niche was simulated. Samples were cultured for 28 days, and subsequently analyzed for metabolic activity and biomineralization. Second test level involved a cell-infiltrated bone substitute material for an osseointegration assay to measure mechanical bonding between dental material and bone. Standard and novel dental materials validated the developed test approach. RESULTS: The developed test system for dental implant materials allowed quantification of biomineralization on implant surface and assessment of the functional stability of mineralized biomaterial-tissue interface. Human blood plasma, M2 macrophages and bone marrow stromal stem cells proved to be crucial components for predictive assessment of implant materials in vitro. Biocompatibility was demonstrated for all tested materials, whereas the degree of deposited mineralized extracellular matrix and mechanical stability differed between the tested materials. Highest amount of functional biomineralization was determined to be on carbon-coated implant surface. SIGNIFICANCE: As an ethical alternative to animal testing, the established in vitro dental test system provides an economic and mid-throughput evaluation of novel dental implant materials or modifications thereof, by applying two successive readout levels: biomineralization and osseointegration.
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Implantes Dentários , Animais , Biomimética , Implantação Dentária Endóssea , Materiais Dentários , Planejamento de Prótese Dentária , Humanos , Técnicas In Vitro , Osseointegração , Propriedades de Superfície , TitânioRESUMO
A key step in the tissue engineering of articular cartilage is the chondrogenic differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells). Chondrogenesis is regulated by transforming growth factor-ß (TGF-ß), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications aim to maximise the yield of differentiated MSCs. Recent experiments involve seeding a hydrogel construct with a layer of MSCs lying below a layer of chondrocytes, stimulating the seeded cells in the construct from above with exogenous TGF-ß and then culturing it in vitro. To investigate the efficacy of this strategy, we develop a mathematical model to describe the interactions between MSCs, chondrocytes and TGF-ß. Using this model, we investigate the effect of varying the initial concentration of TGF-ß, the initial densities of the MSCs and chondrocytes, and the relative depths of the two layers on the long-time composition of the tissue construct.
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An integral approach toward in situ tissue engineering through scaffolds that mimic tissue with regard to both tissue architecture and biochemical composition is presented. Monolithic osteochondral and meniscus scaffolds are prepared with tissue analog layered biochemical composition and perpendicularly oriented continuous micropores by a newly developed cryostructuring technology. These scaffolds enable rapid cell ingrowth and induce zonal-specific matrix synthesis of human multipotent mesenchymal stromal cells solely through their design without the need for supplementation of soluble factors such as growth factors.
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Células-Tronco , Condrócitos , Humanos , Menisco , Células-Tronco Mesenquimais , Mimetismo Molecular , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Millions of people worldwide suffer from trauma- or age-related orthopedic diseases such as osteoarthritis, osteoporosis, or cancer. Tissue Engineering (TE) and Regenerative Medicine are multidisciplinary fields focusing on the development of artificial organs, biomimetic engineered tissues, and cells to restore or maintain tissue and organ function. While allogenic and future autologous transplantations are nowadays the gold standards for both cartilage and bone defect repair, they are both subject to important limitations such as availability of healthy tissue, donor site morbidity, and graft rejection. Tissue engineered bone and cartilage products represent a promising and alternative approach with the potential to overcome these limitations. Since the development of Advanced Therapy Medicinal Products (ATMPs) such as TE products requires the knowledge of diverse regulation and an extensive communication with the national/international authorities, the aim of this review is therefore to summarize the state of the art on the clinical applications of human bone marrow-derived stromal cells for cartilage and bone TE. In addition, this review provides an overview of the European legislation to facilitate the development and commercialization of new ATMPs.
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Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Transplante Ósseo , Osso e Ossos/patologia , Cartilagem/patologia , Humanos , Células-Tronco Mesenquimais/patologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/terapia , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose/terapia , Engenharia Tecidual/legislação & jurisprudência , Engenharia Tecidual/normasRESUMO
The differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells), or chondrogenesis, is a key step in the tissue engineering of articular cartilage, where the motility and high proliferation rate of MSCs used as seed cells are exploited. Chondrogenesis is regulated by transforming growth factor-beta (TGF-ß), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications require the complete differentiation of an initial population of MSCs, and two common strategies used to achieve this in vitro are (1) co-culture the MSCs with chondrocytes, which constitutively produce TGF-ß; or (2) add exogenous TGF-ß. To investigate these strategies we develop an ordinary differential equation model of the interactions between TGF-ß, MSCs and chondrocyte. Here the dynamics of TGF-ß are much faster than those of the cell processes; this difference in time-scales is exploited to simplify subsequent model analysis. Using our model we demonstrate that under strategy 1 complete chondrogenesis will be induced if the initial proportion of chondrocytes exceeds a critical value. Similarly, under strategy 2 we find that there is a critical concentration of exogenous TGF-ß above which all MSCs will ultimately differentiate. Finally, we use the model to demonstrate the potential advantages of adopting a hybrid strategy where exogenous TGF-ß is added to a co-culture of MSCs and chondrocytes, as compared to using either strategy 1 or 2 in isolation.
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Condrócitos/citologia , Condrogênese , Técnicas de Cocultura/métodos , Células-Tronco Mesenquimais/citologia , Modelos Teóricos , Fator de Crescimento Transformador beta/farmacologia , Animais , Humanos , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: The most common cause of lower back pain is the pathological degeneration of the nucleus pulposus (NP). Promising NP regeneration strategies involving human mesenchymal stem cells (hMSCs) would require specific markers to confirm successful differentiation into the NP lineage and to distinguish the articular cartilage (AC). OBJECTIVE: We sought specific NP mRNA markers that are upregulated in native NP cells but not in dedifferentiated NP cells, undifferentiated hMSCs or chondrocytes. We also considered the suitability of non-invasive Raman spectroscopy to distinguish among these classes of cells. METHOD: We used quantitative real-time PCR and Raman spectroscopy to analyse undifferentiated hMSCs in monolayers and embedded in hydrogels, and compared the results with dedifferentiated and redifferentiated human NP and AC cells. RESULTS: The redifferentiation of NP cells induced the expression of annexin A3 (ANXA3), collagen type II (COL2) and proteoglycan mRNAs, whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of ANXA3, COL2, paired box 1 (PAX1) and OCT4 mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of OCT4 mRNA, indicating that only ANXA3, COL2 and PAX1 are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status. CONCLUSION: We recommend ANXA3, COL2 and PAX1 as markers to determine the success of hMSC-based differentiation to regenerate NP cells. Raman spectroscopy can be used to determine cell type and differentiation status especially in the context of clinical trials.
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The development of scaffold formulations based on extracellular matrix (ECM)-inspired synthetic materials constitutes an important resource for the advance of cell-based therapies in bone tissue engineering approaches, where both cell and scaffold implantation are often needed. Culturing cells on porous microcarriers (MCs) allows cell expansion in a three-dimensional microenvironment and constitutes a possible solution for minimally invasive cell and scaffold simultaneous delivery, but the reduced pore dimension and pore interconnection diameter of several commercially available MCs limits de facto cell ingrowth, and ultimately their suitability for in vivo cell delivery. In this study we investigated the potential of a new macroporous MC based on a collagen I-based recombinant peptide (Cellnest™) for C2C12 cells and human bone marrow-derived mesenchymal stromal cells (hBMSCs) expansion and we analyzed the influence of dehydrothermal (DHT), hexamethylene diisocyanate (HMDIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking strategies on cell vitality, proliferation, and hBMSCs differentiation. We established a double emulsification protocol for the manufacturing of MCs characterized by external pores of 20-40 µm diameter, 73% porosity, and 20 ± 3 µm pore interconnection diameter supporting cell ingrowth and proliferation into the MC. MCs cross-linked with DHT and HMDIC supported higher cell proliferation comparing to a commercially available equivalent over the course of 7 days and resulted in higher cell yield by day 28. Moreover, while hBMSCs expansion on Cellnest-MCs did not lead to a significant upregulation of the early markers of osteogenic differentiation Col1a1 and Runx2, their differentiation potential into the osteogenic lineage was preserved when cultured in differentiation medium, as confirmed by mineralized ECM deposition. We believe that Cellnest-MCs will help in reaching clinically relevant cell quantities and ultimately help in accelerating the translation of cell-based therapies for bone tissue engineering in the clinical practice.
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Células da Medula Óssea/metabolismo , Calcificação Fisiológica , Colágeno Tipo I/química , Matriz Extracelular/química , Células-Tronco Mesenquimais/metabolismo , Peptídeos/química , Alicerces Teciduais/química , Células da Medula Óssea/citologia , Linhagem Celular , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Porosidade , Proteínas Recombinantes/químicaRESUMO
Recombinant peptides have the power to harness the inherent biocompatibility of natural macromolecules, while maintaining a defined chemistry for use in tissue engineering. Creating scaffolds from peptides requires stabilization via crosslinking, a process known to alter both mechanics and density of adhesion ligands. The chemistry and mechanics of linear scaffolds from a recombinant peptide based on human collagen type I (RCP) was investigated after crosslinking. Three treatments were compared: dehydrothermal treatment (DHT), hexamethylene diisocyanate (HMDIC), and genipin. With crosslinking, mechanical properties were not significantly altered, ranging from 1.9 to 2.7 kPa. However, the chemistry of the scaffolds was changed, affecting properties such as water uptake, and initial adhesion of human mesenchymal stem cells (hMSCs). Genipin crosslinking supported the lowest adhesion, especially during osteoblastic differentiation. While significantly altered, RCP scaffold chemistry did not affect osteoblastic differentiation of hMSCs. After four weeks in vitro, all scaffolds showed excellent cellular infiltration, with up-regulated osteogenic markers (RUNX2, Osteocalcin, Collagen type I) and mineralization, regardless of the crosslinker. Thus, it appears that, without significant changes to mechanical properties, crosslinking chemistry did not regulate hMSC differentiation on scaffolds from recombinant peptides, a growing class of materials with the ability to expand the horizons of regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1856-1866, 2017.
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Calcificação Fisiológica , Colágeno Tipo I/química , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Peptídeos/química , Alicerces Teciduais/química , Idoso , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Proteínas Recombinantes/químicaRESUMO
There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.
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Cartilagem , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , Alternativas aos Testes com Animais , Animais , Osso e Ossos , Cartilagem/citologia , Células Cultivadas , Condrócitos/citologia , Modelos Biológicos , Osteoblastos/citologia , Fatores de TempoRESUMO
For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 µg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of â¼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.
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Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Mesenquimais/citologia , RNA/análise , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Engenharia Tecidual/métodos , Células Cultivadas , Compostos de Cetrimônio/química , HumanosRESUMO
The analysis of live cells and tissue is gaining increasing importance in research and development. A number of demands are thus being made on the cultivation and analysis of cell response as well. For certain research approaches, in vitro three-dimensional (3D) cultivation best mimics natural conditions. Nevertheless, the imaging of such 3D environments is still a problem. To understand cell function in 3D, a number of in vivo imaging methods have been developed. This article introduces this field and proposes some interesting and promising imaging techniques for this approach.
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Técnicas de Cultura de Células/métodos , Imageamento Tridimensional , Bioengenharia , Microscopia de Fluorescência , Imagem Molecular/métodosRESUMO
INTRODUCTION: One common cause of disability in modern society is low back pain. The main reason for this pain is the degeneration of the intervertebral disc (IVD), particularly of the nucleus pulposus (NP). For the early degeneration stage, a cell-based therapy could constitute a minimally invasive method of treatment. Therefore, adequate cells are needed. As the usage of NP cells is limited because of their insufficient amount or vitality, a promising alternative is the application of human mesenchymal stem cells (hMSCs) OBJECTIVE: To investigate the potential of various growth factors to induce the differentiation of hMSCs into NP cells and thereby to obtain an alternative cell source for the treatment of IVD degeneration. METHODS: hMSC-TERT were cultivated three-dimensionally in a hydrogel for 21 days to form NP cells. Cell survival and proliferation were determined using SybrGreen/propidium iodide double staining and the WST-test. To investigate the ability of several growth factors to differentiate hMSCs into NP cells, fluorescence immunostaining of NP-specific marker proteins (e.g., chondroadherin (CHAD) and the recently discovered cytokeratin 19) were performed. RESULTS: Following the procedure described above, cells are able to maintain their viability and proliferation capacity throughout the cultivation time. By using a previously established immunofluorescence protocol, we were able to indicate the ability of three different growth factors for differentiating hMSCs into NP-like cells. CONCLUSION: The expression of several marker proteins in all differentiation experiments indicates the ability of IGF-1, FGF-2 and PDGF-BB to differentiate hMSCs into NP-like cells apart from the usually applied TGF-beta3. Furthermore, our findings preclude the application of Cytokeratin 19 as a specific marker protein for NP cells. Further experiments have to be done to find real specific NP marker proteins to indisputably verify the differentiation of hMSCs into NP cells. If so, application of these three growth factors would possibly be an option to obtain sufficient NP cells for minimally invasive IVD regeneration.