RESUMO
Trauma arising from landmines and improvised explosive devices promotes heterotopic ossification, the formation of extra-skeletal bone in non-osseous tissue. To date, experimental platforms that can replicate the loading parameter space relevant to improvised explosive device and landmine blast wave exposure have not been available to study the effects of such non-physiological mechanical loading on cells. Here, we present the design and calibration of three distinct in vitro experimental loading platforms that allow us to replicate the spectrum of loading conditions recorded in near-field blast wave exposure. We subjected cells in suspension or in a three-dimensional hydrogel to strain rates up to 6000 s-1 and pressure levels up to 45 MPa. Our results highlight that cellular activation is regulated in a non-linear fashion-not by a single mechanical parameter, it is the combined action of the applied mechanical pressure, rate of loading and loading impulse, along with the extracellular environment used to convey the pressure waves. Finally, our research indicates that PO MSCs are finely tuned to respond to mechanical stimuli that fall within defined ranges of loading.
Assuntos
Materiais Biocompatíveis/química , Explosões , Técnicas In Vitro/métodos , Pressão , Substâncias ExplosivasRESUMO
OBJECTIVES: It has recently been shown that enamel matrix derivative (EMD) components (Fraction C, containing <6 kDa peptides (mainly a 5.3 kDa tyrosine-rich amelogenin peptide (TRAP)), and Fraction A, containing a mixture of >6 kDa peptides (including a leucine-rich amelogenin peptide (LRAP))) differentially regulate osteogenic differentiation of periodontal ligament (PDL) cells. The present study examined whether EMD and the EMD Fractions (i) bind and internalize into PDL cells and (ii) precipitate and form insoluble complexes on PDL cells. MATERIALS AND METHODS: Biotin-labelled EMD/EMD Fractions were incubated with PDL cells under various different culture conditions and confocal and electron microscopies were carried out to examine the binding and intracellular trafficking of these proteins. RESULTS: The results reported here show, for the first time, that at least some components in Fraction A and the TRAP peptide in Fraction C can bind and be internalized by human PDL cells via receptor-mediated endocytosis. In addition, Fraction A was found to form insoluble aggregate-like structures on PDL cells, whereas Fraction C was soluble in culture media. CONCLUSION: Soluble amelogenin isoform TRAP appears to be internalizing into a subset of PDL cells. Moreover, TRAP uptake is most likely controlled by receptor-mediated endocytosis. CLINICAL RELEVANCE: Information on interaction between PDL cells and EMD/TRAP might prove useful in designing targeted interventions (i.e. use of chemically prepared soluble amelogenin peptides) to repair/regenerate periodontal tissues. Such interventions can also (i) avoid the use of rather crude animal-derived enamel matrix protein (EMP)/EMD preparation and (ii) preparation of cost-effective and more controlled chemically synthesized amelogenin peptides for the clinical use.
Assuntos
Proteínas do Esmalte Dentário/farmacologia , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Coloração e RotulagemRESUMO
The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile(148)Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.
Assuntos
Ácidos Graxos/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Triglicerídeos/metabolismo , Linhagem Celular , Estresse do Retículo Endoplasmático , Células Hep G2 , Humanos , Insulina/metabolismo , Lipase/genética , Proteínas de Membrana/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Tissue-engineering strategies for the treatment of osteoarthritis would benefit from the ability to induce chondrogenesis in precursor cells. One such cell source is bone marrow-derived stromal cells (BMSCs). Here, we examined the effects of moderate-strength static magnetic fields (SMFs) on chondrogenic differentiation in human BMSCs in vitro. Cells were cultured in pellet form and exposed to several strengths of SMFs for various durations. mRNA transcript levels of the early chondrogenic transcription factor SOX9 and the late marker genes ACAN and COL2A1 were determined by reverse transcription-polymerase chain reaction, and production of the cartilage-specific macromolecules sGAG, collage type 2 (Col2), and proteoglycans was determined both biochemically and histologically. The role of the transforming growth factor (TGF)-ß signaling pathway was also examined. Results showed that a 0.4 T magnetic field applied for 14 days elicited a strong chondrogenic differentiation response in cultured BMSCs, so long as TGF-ß3 was also present, that is, a synergistic response of a SMF and TGF-ß3 on BMSC chondrogenic differentiation was observed. Further, SMF alone caused TGF-ß secretion in culture, and the effects of SMF could be abrogated by the TGF-ß receptor blocker SB-431542. These data show that moderate-strength magnetic fields can induce chondrogenesis in BMSCs through a TGF-ß-dependent pathway. This finding has potentially important applications in cartilage tissue-engineering strategies.
Assuntos
Diferenciação Celular , Condrogênese , Campos Magnéticos , Células-Tronco Mesenquimais/citologia , Adulto , Benzamidas/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Dioxóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Fator de Crescimento Transformador beta3/metabolismoRESUMO
To recapitulate the in vivo environment and create neo-organoids that replace lost or damaged tissue requires the engineering of devices, which provide appropriate biophysical cues. To date, bioreactors for cartilage tissue engineering have focused primarily on biomechanical stimulation. There is a significant need for improved devices for articular cartilage tissue engineering capable of simultaneously applying multiple biophysical (electrokinetic and mechanical) stimuli. We have developed a novel high-throughput magneto-mechanostimulation bioreactor, capable of applying static and time-varying magnetic fields, as well as multiple and independently adjustable mechanical loading regimens. The device consists of an array of 18 individual stations, each of which uses contactless magnetic actuation and has an integrated Hall Effect sensing system, enabling the real-time measurements of applied field, force, and construct thickness, and hence, the indirect measurement of construct mechanical properties. Validation tests showed precise measurements of thickness, within 14 µm of gold standard calliper measurements; further, applied force was measured to be within 0.04 N of desired force over a half hour dynamic loading, which was repeatable over a 3-week test period. Finally, construct material properties measured using the bioreactor were not significantly different (p=0.97) from those measured using a standard materials testing machine. We present a new method for articular cartilage-specific bioreactor design, integrating combinatorial magneto-mechanostimulation, which is very attractive from functional and cost viewpoints.
Assuntos
Cartilagem Articular/fisiologia , Magnetismo/instrumentação , Estresse Mecânico , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/farmacologia , Fenômenos Biomecânicos/efeitos dos fármacos , Reatores Biológicos/economia , Calibragem , Cartilagem Articular/efeitos dos fármacos , Bovinos , Desenho de Equipamento , Teste de Materiais , Reprodutibilidade dos Testes , Engenharia Tecidual/economia , Interface Usuário-ComputadorRESUMO
The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo.
Assuntos
Amelogenina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Tirosina/metabolismo , Fosfatase Ácida/metabolismo , Adolescente , Adulto , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Proteínas do Esmalte Dentário/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Neovascularização Fisiológica/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Fosfatase Ácida Resistente a Tartarato , Adulto Jovem , Fator de von Willebrand/metabolismoRESUMO
The adult periodontal ligament (PDL) is considered to contain progenitor cells that are involved in the healing of periodontal wounds. Treatment with enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs), has been shown to be of some clinical benefit in eliciting periodontal regeneration in vivo. Although there is extensive information available about the effects of EMD on periodontal regeneration, the precise influence of this material on alveolar bone and the formation of blood vessels and proprioceptive sensory nerves, prominent features of functionally active periodontal tissue, remain unclear. The aim of the present study was therefore to examine the effects of EMD on the ability of human periodontal ligament cells (HPCs) to undergo multi-lineage differentiation in vitro. Our results showed that HPCs treated with EMD under non-selective growth conditions did not show any evidence of osteogenic, adipogenic, chondrogenic, neovasculogenic, neurogenic and gliogenic "terminal" differentiation. In contrast, under selective lineage-specific culture conditions, EMD up-regulated osteogenic, chondrogenic and neovasculogenic genes and "terminal" differentiation, but suppressed adipogenesis, neurogenesis and gliogenesis. These findings thus demonstrate for the first time that EMD can differentially modulate the multi-lineage differentiation of HPCs in vitro.
Assuntos
Diferenciação Celular , Proteínas do Esmalte Dentário/metabolismo , Ligamento Periodontal/citologia , Linhagem da Célula , Células Cultivadas , Humanos , Técnicas In Vitro , Ligamento Periodontal/metabolismoRESUMO
Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases.
Assuntos
Amelogenina/farmacologia , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/patologia , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Peptídeos/farmacologia , Cicatrização/efeitos dos fármacos , Adulto , Amelogenina/química , Sequência de Aminoácidos , Biomarcadores/metabolismo , Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Fracionamento Químico , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/farmacologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Ligamento Periodontal/citologia , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismoRESUMO
Stem cells isolated from adult human tissue have received increasing attention because of their potential to repair and/or regenerate damaged tissue. However, identification and characterization of such cell populations have been limited due to the lack of adequate methodology for assessing their multi-lineage potential. In the present study, using adult human ligament tissue as a model, we have developed a combination of methods which together can be used to identify adult stem cell compartments based on their ability to undergo a range of differentiation pathways, including osteogenesis, adipogenesis, chondrogenesis, myogenesis, vasculogenesis, angiogenesis, neurogenesis and gliogenesis in vitro. This was carried out using the conventional reverse transcription polymerase chain reaction technique to assess the expression of selected key lineage-associated marker genes and by using histological, immunological and morphological criteria to assess characteristic features of lineage-specific 'terminal' differentiation in vitro.