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1.
Acta Biomater ; 54: 81-94, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28242454

RESUMO

Tissue engineering (particularly for the case of load-bearing cardiovascular and connective tissues) requires the ability to promote the production and accumulation of extracellular matrix (ECM) components (e.g., collagen, glycosaminoglycan and elastin). Although different approaches have been attempted in order to enhance ECM accumulation in tissue engineered constructs, studies of underlying signalling mechanisms that influence ECM deposition and degradation during tissue remodelling and regeneration in multi-cellular culture systems have been limited. The current study investigated vascular smooth muscle cell (VSMC)-monocyte co-culture systems using different VSMC:monocyte ratios, within a degradable polyurethane scaffold, to assess their influence on ECM generation and degradation processes, and to elucidate relevant signalling molecules involved in this in vitro vascular tissue engineering system. It was found that a desired release profile of growth factors (e.g. insulin growth factor-1 (IGF-1)) and hydrolytic proteases (e.g. matrix-metalloproteinases 2, 9, 13 and 14 (MMP2, MMP9, MMP13 and MMP14)), could be achieved in co-culture systems, yielding an accumulation of ECM (specifically for 2:1 and 4:1 VSMC:monocyte culture systems). This study has significant implications for the tissue engineering field (including vascular tissue engineering), not only because it identified important cytokines and proteases that control ECM accumulation/degradation within synthetic tissue engineering scaffolds, but also because the established culture systems could be applied to improve the development of different types of tissue constructs. STATEMENT OF SIGNIFICANCE: Sufficient extracellular matrix accumulation within cardiovascular and connective tissue engineered constructs is a prerequisite for their appropriate function in vivo. This study established co-culture systems with tissue specific cells (vascular smooth muscle cells (VSMCs)) and defined ratios of immune cells (monocytes) to investigate extracellular matrix (ECM) generation and degradation processes, revealing important mechanisms underlying ECM turnover during vascular tissue regeneration/remodelling. A specific growth factor (IGF-1), as well as hydrolytic proteases (e.g. MMP2, MMP9, MMP13 and MMP14), were identified as playing important roles in these processes. ECM accumulation was found to be dependent on achieving a desired release profile of these ECM-promoting and ECM-degrading factors within the multi-cellular microenvironment. The findings enhance our understanding of ECM deposition and degradation during in vitro tissue engineering and would be applicable to the repair or regeneration of a variety of tissues.


Assuntos
Colagenases/metabolismo , Citocinas/metabolismo , Matriz Extracelular/química , Monócitos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Engenharia Tecidual , Técnicas de Cocultura , Humanos , Monócitos/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia
2.
J Biomater Sci Polym Ed ; 25(11): 1159-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24898697

RESUMO

This study investigated the interaction of human circulating angiogenic cells (CACs) with a degradable polar hydrophobic ionic polyurethane (D-PHI) which has been previously shown to exhibit anti-inflammatory character and favorable interactions with human endothelial cells (ECs). Given the implication of the CACs in microvessel development it was of intrinsic interest to expand our knowledge of D-PHI biocompatibility with this relevant primary cell involved in angiogenesis. The findings will be compared to a well-established benchmark substrate for CACs, fibronectin-coated tissue culture polystyrene (TCPS). Immunoblotting analysis showed that CACs were a heterogeneous population of cells composed mostly of monocytic cells expressing the CD14 marker. Assessment of the cytokine release profile, using ELISA, showed that D-PHI supported a higher concentration of interleukin-10 (IL-10) when compared to the concentration of tumor necrosis factor alpha, which is indicative of an anti-inflammatory phenotype, and was different from the response with TCPS. It was found that the CACs were attached to D-PHI and remained viable and functional (nitric oxide production) during the seven days of culture. However, there did not appear to be any significant proliferation on D-PHI, contrary to the CAC growth on fibronectin-coated TCPS. It was concluded that D-PHI displayed some of the qualities suitable to enable the retention of CACs onto this substrate, as well as maintaining an anti-inflammatory phenotype, characteristics which have been reported to be important for angiogenesis in vivo.


Assuntos
Materiais Biocompatíveis/química , Monócitos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Poliuretanos/química , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citocinas/metabolismo , Fibronectinas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Interleucina-10/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Microscopia Eletrônica de Varredura , Monócitos/fisiologia , Monócitos/ultraestrutura , Neovascularização Fisiológica/fisiologia , Óxido Nítrico/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
3.
Acta Biomater ; 9(7): 7191-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23454057

RESUMO

While macrophages have been implicated in the failure of bioprosthetic heart valves, the macrophage response to crosslinked native pericardial collagen has not been previously investigated. Using decellularized bovine pericardium (DBP) as a model for native collagen, this study investigated the response of macrophage-like cells (U937s) to DBP, either: (i) untreated, or (ii) exogenously crosslinked with glutaraldehyde or 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC). We have previously validated the use of U937 cells as models for the response of human monocyte-derived macrophages to decellularized pericardial materials and, per our previous work, differentiated the U937 cells directly on the three material surfaces. After 72h in culture, the cells and medium were analyzed for DNA content, acid phosphatase activity, and cytokine and matrix metalloproteinase release. As well, cell/substrate samples were fixed for SEM. Fewer cells attached to or survived on the glutaraldehyde-treated substrate, and some showed an abnormal morphology compared to cells cultured on the other surfaces. Further, cells on glutaraldehyde-treated surfaces released more pro-inflammatory cytokines, more MMP-1 and less MMP-2 and MMP-9. The poor performance of the U937 macrophage-like cells on the glutaraldehyde-treated surfaces appears to be due to surface characteristics rather than to soluble aldehyde or other components leaching from the crosslinked material. These results provide evidence that crosslinking with glutaraldehyde is cytotoxic to macrophage-like cells, and that crosslinking with a zero-length crosslinker like EDC can be an acceptable alternative crosslinking treatment for biomaterials.


Assuntos
Reagentes de Ligações Cruzadas/química , Matriz Extracelular/química , Macrófagos/citologia , Macrófagos/fisiologia , Pericárdio/química , Engenharia Tecidual/métodos , Animais , Bovinos , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/fisiologia , Sistema Livre de Células , Teste de Materiais , Pericárdio/citologia
4.
Biomaterials ; 33(33): 8316-28, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22940217

RESUMO

Protein adsorption is an important phenomenon influencing the cellular response to biomaterials. Previous studies comparing monocyte activation on a degradable polar hydrophobic ionic polyurethane (D-PHI) indicated a reduced pro-inflammatory monocyte response relative to tissue culture polystyrene (TCPS) and poly(lactide-co-glycolide) (PLGA) substrates. The present study investigated the influence of protein binding in order to gain further insight into the observed differential monocyte activation. Several proteins, identified in different relative amounts within the bound protein layers on D-PHI vs. PLGA and TCPS, were evaluated for their effect on monocyte activation. It was found that, in general, both non-coated and protein pre-adsorbed D-PHI supported a reduced pro-inflammatory response relative to PLGA, as indicated by lower levels of tumor necrosis factor-α (TNF-α) release. An initial increase in TNF-α release occurred when α(2)-macroglobulin (A2M) was pre-adsorbed to D-PHI, which was shown to involve the α(2)-macroglobulin receptor and was active on D-PHI but not on the two other biomaterials. This response was not observed during competitive protein binding in the presence of fetal bovine serum (FBS), suggesting that a more complex arrangement of the bound proteins and their interactions with one another, as well as with the surface chemistry of the individual biomaterials, resulted in the low-activating character of D-PHI when interacting with human monocytes.


Assuntos
Materiais Biocompatíveis/química , Monócitos/metabolismo , Poliuretanos/química , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Ligação Proteica
5.
Curr Vasc Pharmacol ; 10(3): 347-60, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22239637

RESUMO

Cardiovascular disease (CVD) is a leading cause of death and hospitalization worldwide. The need for small caliber vessels ( < 6mm) to treat CVD patients has grown; however the availability of autologous vessels in cardiac and peripheral bypass candidates is limited. The search for an alternative vessel source is widespread with both natural and synthetic tissue engineered materials being investigated as scaffolds. Despite decades of exhaustive studies with decellularized extracellular matrices (ECM) and synthetic graft materials, the field remains in search of a commercially viable biomaterial construct substitute. While the previous materials have been assessed by evaluating their compatibility with fibroblasts, smooth muscle cells and endothelial cells, current materials are being conceived based on their interactions with stem cells, progenitor cells and monocytes, as the latter may hold the key to repair and regeneration. The graft's ability to recruit and maintain these cells has become a major research focus. The successful tissue engineering of a small caliber vessel graft requires the use of optimal material chemistry and biological function to promote cell recruitment into the graft while maintaining each functional phenotype during vessel tissue maturation. The discussion of these significant research challenges constitutes the focus of this review.


Assuntos
Prótese Vascular , Doenças Cardiovasculares/cirurgia , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Implante de Prótese Vascular/métodos , Doenças Cardiovasculares/fisiopatologia , Matriz Extracelular/metabolismo , Humanos , Alicerces Teciduais
6.
Acta Biomater ; 8(1): 89-98, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21963405

RESUMO

Tissue culture polystyrene (TCPS) is a ubiquitous substrate used by many researchers in the biomedical and biological sciences. Different parameters involved in the production of TCPS, including the treatment time and the use of reactive gases and chemical agents, can have a significant influence on the ultimate surface properties achieved. The assumption that they will all yield a consistent and controlled product has not proven to be true. To provide a better insight into the bioactivity differences in TCPS supplied by different manufacturers, TCPS from three different companies (Sarstedt, Wisent Corp., and Becton Dickinson (BD)) were analyzed for their surface properties, protein adsorption characteristics, and interactions with human monocytes. Marked differences were observed in terms of surface wettability and surface chemistry. Furthermore, Wisent TCPS adsorbed more than twice the amount of serum proteins compared with BD and Sarstedt TCPS. Sarstedt showed significantly more cell retention (more DNA) compared with both BD and Wisent TCPS brands over a 7 day culture period. Cytokine release from monocytes adherent on the three different TCPS also differed significantly, suggesting that the differences in the surface properties were sufficient to differentially mediate monocyte activation. These results have important implications for TCPS research use, in terms of appreciating the interpretation of the data when TCPS is used as a control substrate as well as when it is used where a pre-conditioned state would influence the outcome of the study.


Assuntos
Técnicas de Cultura de Células/instrumentação , Citocinas/metabolismo , Monócitos/metabolismo , Poliestirenos/química , Adsorção , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Cromatografia Líquida/métodos , Humanos , Espectrometria de Massas/métodos , Monócitos/citologia , Espectroscopia Fotoeletrônica , Ligação Proteica , Propriedades de Superfície , Molhabilidade
7.
Acta Biomater ; 8(2): 488-501, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21971418

RESUMO

Activated monocytes can promote inflammation or wound repair, depending on the nature of the implant environment. Recent work showed that a degradable, polar-hydrophobic-ionic polyurethane (D-PHI) induced an anti-inflammatory monocyte phenotype. In the current study it is hypothesized that wound-healing phenotype monocytes (activated by D-PHI material chemistry) will promote human vascular smooth muscle cells (hVSMC) to attach and migrate into porous D-PHI scaffolds. hVSMC migration is necessary for hVSMC population of the scaffold and tissue formation to occur, and then, once tissue formation is complete, the monocyte should promote contractile phenotype markers in the hVSMC. hVSMC were cultured for up to 28 days with or without monocytes and analyzed for cell viability, attachment (DNA) and migration. Lysates were analyzed for the hVSMC contractile phenotype markers calponin and α-smooth muscle actin (α-SMA) as well as urokinase plasminogen activator (uPA; pro-migration marker) using immunoblotting analysis. Histological staining showed that hVSMC alone remained around the perimeter of the scaffold, whereas co-culture samples had co-localization of monocytes with hVSMC in the pores, a more even cell distribution throughout the scaffold and increased total cell attachment (P<0.05). Co-culture samples had higher cell numbers and more DNA than the addition of both single cell cultures. The water-soluble tetrazolium-1 data suggested that cells were not dying over the 28 day culture period. Calponin, also linked to cell motility, was maintained up to 28 days in the co-culture and hVSMC alone, whereas α-SMA disappeared after 7 days. Co-cultures on D-PHI showed that monocytes were activated to a wound-healing phenotype (low TNF-α, elevated IL-10), while promoting uPA expression. In summary, this study showed that, by co-culturing monocytes with hVSMC, the latter showed increased total cell attachment and infiltration into the D-PHI scaffold compared with hVSMC alone, suggesting that monocytes may promote hVSMC migration, a condition necessary for ultimately achieving uniform tissue formation in porous scaffolds.


Assuntos
Técnicas de Cocultura/métodos , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Monócitos/citologia , Miócitos de Músculo Liso/citologia , Poliuretanos/farmacologia , Alicerces Teciduais/química , Contagem de Células , DNA/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/ultraestrutura , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
8.
J Cell Biochem ; 112(12): 3762-72, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21826703

RESUMO

Potential benefits of co-culturing monocytes (MC) with vascular smooth muscle cells have been reported on for tissue engineering applications with a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI). Since the interaction of MC and endothelial cells (EC) within the blood vessel endothelium is also a process of wound repair it was of interest to investigate their function when cultured on the synthetic D-PHI materials, prior to considering the materials' use in vascular engineering. The co-culture (MC/EC) in vitro studies were carried out on films in 96 well plates and porous scaffold disks were prepared for implant studies in an in vivo subcutaneous mouse model. After 7 days in culture, the MC/EC condition was equal to EC growth but had lower esterase activity (a measure of degradative potential), no pro-inflammatory TNF-α and a relatively high anti-inflammatory IL-10 release while the ECs maintained their functional marker CD31. After explantation of the porous scaffolds, a live/dead stain showed that the cells infiltrating the scaffolds were viable and histological stains (May-Grunwald, Trichrome) demonstrated tissue in growth and extracellular matrix synthesis. Lysates from the implant scaffolds analyzed with a cytokine antibody array showed decreased pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF), increased anti-inflammatory cytokines (IL-10, IL-13, TNF-RI), and increased chemotactic cytokines (MCP-1, MCP-5, RANTES). The low foreign body response elicited by D-PHI when implanted in vivo supported the in vitro studies (EC and MC co-culture), demonstrating that D-PHI promoted EC growth along with an anti-inflammatory MC, further demonstrating its potential as a tissue engineering scaffold for vascular applications.


Assuntos
Materiais Biocompatíveis , Prótese Vascular , Endotélio Vascular/citologia , Modelos Animais , Monócitos/citologia , Poliuretanos/metabolismo , Animais , Western Blotting , Técnicas de Cocultura , Citocinas/metabolismo , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Monócitos/metabolismo
9.
Biomaterials ; 32(26): 6034-44, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21641638

RESUMO

A degradable, polar/hydrophobic/ionic polyurethane (D-PHI) scaffold was optimized in in vitro studies to yield mechanical properties appropriate to replicate vascular graft tissue while eliciting a more wound-healing phenotype macrophage when compared to established materials. The objectives of this study were to characterize the biodegradation (in vitro and in vivo) and assess the in vivo biocompatibility of D-PHI, comparing it to a well-established, commercially-available scaffold biomaterial, polylactic glycolic acid (PLGA), recognized as being degradable, non-cytotoxic, and showing good biocompatibility. PLGA and D-PHI were formed into 6 mm diameter disk-shaped scaffolds (2 mm thick) of similar porosity (∼82%) and implanted subcutaneously in rats. Both PLGA and D-PHI scaffolds were well-tolerated at the 7 d time point in vivo. In vitro D-PHI scaffolds degraded slowly (only 12 wt% in PBS in vitro after 120 d at 37 °C). In vivo, D-PHI scaffolds degraded at a more controlled rate (7 wt% loss over the acute 7 d implant phase and subsequently a linear profile of degradation leading to a 21 wt% mass loss by 100 d (chronic period)) than PLGA scaffolds which showed an initial more rapid degradation (14 wt% over 7 d), followed by minimal change between 7 and 30 d, and then a very rapid breakdown of the scaffold over the next 60 d. Histological examination of D-PHI scaffolds showed tissue ingrowth into the pores increased with time whereas PLGA scaffolds excluded cells/tissue from its porous structure as it degraded. The results of this study suggest that D-PHI has promising qualities for use as an elastomeric scaffold material for soft TE applications yielding well integrated tissue within the scaffold and a controlled rate of degradation stabilizing the form and shape of the implant.


Assuntos
Materiais Biocompatíveis/efeitos adversos , Materiais Biocompatíveis/metabolismo , Poliuretanos/metabolismo , Engenharia Tecidual/métodos , Animais , Masculino , Microscopia Eletrônica de Varredura , Poliuretanos/efeitos adversos , Porosidade , Ratos , Ratos Wistar
10.
Biomaterials ; 32(21): 4816-29, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21463894

RESUMO

There are few synthetic elastomeric biomaterials that simultaneously provide the required biological conditioning and the ability to translate biomechanical stimuli to vascular smooth muscle cells (VSMCs). Biomechanical stresses are important physiological elements that regulate VSMC function, and polyurethane elastomers are a class of materials capable of facilitating the translation of stress induced biomechanics. In this study, human coronary artery smooth muscle cells (hCASMCs), which were seeded into a porous degradable polar/hydrophobic/ionic (D-PHI) polyurethane scaffold, were subjected to uniaxial cyclic mechanical strain (CMS) over a span of four weeks using a customized bioreactor. The distribution, proliferation and contractile protein expression of hCASMCs in the scaffold were then analyzed and compared to those grown under static conditions. Four weeks of CMS, applied to the elastomeric scaffold, resulted in statistically greater DNA mass, more cell area coverage and a better distribution of cells deeper within the scaffold construct. Furthermore, CMS samples demonstrated improved tensile mechanical properties following four weeks of culture, suggesting the generation of more extracellular matrix within the polyurethane constructs. The expression of smooth muscle α-actin, calponin and smooth muscle myosin heavy chain and the absence of Ki-67+ cells in both static and CMS cultures, throughout the 4 weeks, suggest that hCASMCs retained their contractile character on these biomaterials. The study highlights the importance of implementing physiologically-relevant biomechanical stimuli in the development of synthetic elastomeric tissue engineering scaffolds.


Assuntos
Vasos Coronários/anatomia & histologia , Miócitos de Músculo Liso/fisiologia , Poliuretanos/química , Poliuretanos/metabolismo , Estresse Mecânico , Alicerces Teciduais/química , Actinas/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Antígeno Ki-67/metabolismo , Teste de Materiais , Proteínas dos Microfilamentos/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Resistência à Tração , Engenharia Tecidual/métodos , Calponinas
11.
Biomaterials ; 32(14): 3584-95, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21345489

RESUMO

Strategies to optimize biomaterial chemistry for applications in vascular tissue engineering attempt to promote endothelial and smooth muscle cell recruitment into porous material constructs. The primary objective is to facilitate relevant tissue formation in a wound healing versus pro-inflammatory manner. The present work investigated the interactive co-cellular response of human monocytes and human vascular smooth muscle cells (VSMCs) with a novel degradable, polar/hydrophobic/ionic (D-PHI) polyurethane and compared it to a commercially available biomaterial, poly-lactic-glycolic acid (PLGA) as well as tissue culture polystyrene (TCPS). D-PHI triggered a smaller pro-inflammatory response (acid phosphatase, esterase, tumor necrosis factor-α) at later time points (>14 d) than PLGA suggesting that monocytes may be transitioning to a more wound-healing phenotype on the D-PHI surface. When D-PHI was coated with collagen, monocyte cell attachment did not differ with the native D-PHI; however, PLGA showed significant differences between collagen coated versus uncoated surfaces. There were more VSMCs and monocytes attached in co-culture to D-PHI when compared to PLGA. Co-cultures on D-PHI released more IL-10 (anti-inflammatory) than monocytes cultured alone, while the VSMCs retained the expression of its marker protein calponin. Together the above data suggest that co-culturing monocytes with VSMCs may aid in stimulating the attachment of VSMCs to D-PHI while eliciting the desired functional phenotypes for both monocytes (i.e. low inflammation based on IL-10 values) and VSMCs (expressing calponin, a marker of contractility). Moreover, the results of this study demonstrated that D-PHI performed equally or better to PLGA in terms of the assayed biological parameters.


Assuntos
Materiais Biocompatíveis/farmacologia , Monócitos/metabolismo , Miócitos de Músculo Liso/metabolismo , Polímeros/farmacologia , Células Cultivadas , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Microscopia Eletrônica de Varredura , Monócitos/efeitos dos fármacos , Monócitos/ultraestrutura , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/ultraestrutura , Espectroscopia Fotoeletrônica
12.
Biomaterials ; 32(2): 439-49, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20933269

RESUMO

The monocyte-derived macrophage (MDM), present at biomaterial implantations, can increase, decrease or redirect the inflammatory and subsequent wound healing process associated with the presence of a biomaterial. Understanding MDM responses to biomaterials is important for improved prediction and design of biomaterials for tissue engineering. This study analyzed the direct differentiation of monocytes on intact, native collagen. Human monocytes were differentiated on decellularized bovine pericardium (DBP), polydimethylsiloxane (PDMS) or polystyrene (TCPS) for 14 d. MDMs on all surfaces released high amounts of MMP-9 compared to MMP-2 and relatively little MMP-1. MDMs differentiated on DBP released more MMP-2, but less acid phosphatase activity. MDMs on all three surfaces released low amounts of cytokines, although substrate differences were found: MDMs on DBP released higher amounts of IL-6, IL-8, and MCP-1 but lower amounts of IL-10 and IL-1ra. This research provides evidence that MDMs on decellularized matrices may not be stimulated towards an activated, inflammatory phenotype, supporting the potential of decellularized matrices for tissue engineering. This study also demonstrated that the differentiation surface affects MDM phenotype and therefore study design of macrophage interactions with biomaterials should scrutinize the specific macrophage culture method utilized and its effects on macrophage phenotype.


Assuntos
Materiais Biocompatíveis , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Macrófagos/citologia , Macrófagos/metabolismo , Pericárdio , Fosfatase Ácida/metabolismo , Animais , Bovinos , Diferenciação Celular/genética , Polaridade Celular/genética , Células Cultivadas , Citocinas/metabolismo , Gelatinases/metabolismo , Humanos , Immunoblotting , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase
13.
Acta Biomater ; 7(1): 115-22, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20728587

RESUMO

A degradable, polar, hydrophobic, ionic polyurethane (D-PHI), with physical properties comparable to those of peripheral arterial vascular tissue, was evaluated for monocyte interactions with two different physical forms: two-dimensional films and three-dimensional porous scaffolds. Monocytes, isolated from human whole blood, were seeded onto D-PHI films and scaffolds, and differentiated to monocyte-derived macrophages (MDM) for up to 28 days. The effect of surface structure on the MDM phenotype was assessed by assaying: cell attachment (DNA), activation (intracellular protein expression, esterase and acid phosphatase (AP) activity) as well as pro- and anti-inflammatory cytokines (TNF-α and IL-10, respectively). The cells on scaffolds exhibited an initial peak in total protein synthesized per DNA at 3 days; however, both substrates generated similar protein levels per DNA at all other time points. While scaffolds generated more esterase and AP per cell than for films, the cells on films expressed significantly more of these two proteins relative to their total protein produced. At day 7 (acute phase of monocyte activation), cells on films were significantly more activated than monocytes on the scaffolds as assessed by cell morphology and tumor necrosis factor-α and interleukin-10 levels. Histological analysis of scaffolds showed that cells were able to migrate throughout the three-dimensional matrix. By inducing a low inflammatory, high wound-healing phenotype monocyte, the negative effects of the foreign body reaction in vivo may be controlled in a manner possible to direct the vascular tissue cells into the appropriate functional phenotypes necessary for successful tissue engineering.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Monócitos/citologia , Poliuretanos/farmacologia , Alicerces Teciduais/química , Adulto , Fosfatase Alcalina/metabolismo , Adesão Celular/efeitos dos fármacos , Extratos Celulares , DNA/metabolismo , Esterases/metabolismo , Feminino , Humanos , Interleucina-10/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/ultraestrutura , Proteínas/metabolismo , Propriedades de Superfície/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Adulto Jovem
14.
Acta Biomater ; 6(11): 4218-28, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20601230

RESUMO

High porosity and pore interconnectivity are important features of a successful tissue engineering scaffold. The objective of this work was to optimize the pore interconnectivity and to increase the porosity of an elastomeric degradable/polar/hydrophobic/ionic (D-PHI) polyurethane porous scaffold while maintaining its mechanical integrity in order to allow for the transfer of mechanical stimulus to vascular smooth muscle cells (VSMCs) seeded onto the scaffold. The effect of varying porogen (sodium bicarbonate (salt) and polyethylene glycol (PEG)) composition and concentration on the mechanical properties, degree of swelling and porosity of the scaffolds was investigated. It was found that the use of 10 wt.% PEG and 65 wt.% salt in scaffold fabrication (D-PHI-75T) resulted in micropore (1-5 µm) formation, a high porosity (79 ± 3%) and mechanical properties (elastic modulus=0.16 ± 0.03 MPa, elongation-at-yield = 31 ± 5% and tensile strength=0.04 ± 0.01 MPa) required to withstand the physiologically relevant mechanical strain experienced by VMSCs in vivo. This study also investigated the influence of cyclic mechanical strain (CMS) on select molecular markers of A10 VSMCs when seeded into the optimized D-PHI scaffold. To study the interaction of A10 cells with the optimized D-PHI-75T scaffold in the presence of uniaxial strain (10%, 1 Hz), a CMS bioreactor was designed and constructed. Molecular marker studies showed a statistical increase in DNA mass and calponin expression after 3 and 7 days of CMS when compared to static samples, indicating that the translation of mechanical loading from the novel polyurethane elastomeric scaffold onto VSMCs will be important to consider with regard to modulating cell phenotype.


Assuntos
Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Poliuretanos/química , Poliuretanos/farmacologia , Estresse Mecânico , Alicerces Teciduais/química , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Linhagem Celular , DNA/metabolismo , Análise de Elementos Finitos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Immunoblotting , Microscopia Eletrônica de Varredura , Peso Molecular , Proteínas Musculares/metabolismo , Polietilenoglicóis/farmacologia , Porosidade , Ratos
15.
J Biomech Eng ; 132(3): 031002, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20459190

RESUMO

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.


Assuntos
Envelhecimento/fisiologia , Colágeno/química , Colágeno/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Tendões/química , Tendões/fisiologia , Animais , Bovinos , Colágeno/ultraestrutura , Simulação por Computador , Humanos , Estresse Mecânico , Tendões/ultraestrutura , Suporte de Carga/fisiologia
16.
J Biomed Mater Res A ; 93(1): 280-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19562747

RESUMO

Decellularized tissue-derived heart valves are an example of biomaterials derived from natural scaffolds. These types of implants are increasing in popularity although their in vivo performance is still only poorly understood and has, at times, been catastrophic. It is apparent that better understanding is required before these biomaterials can be used safely. In this study, the human monocyte-derived macrophage (MDM) response to decellularized bovine pericardium (DBP) was used as a model to predict the biological performance of these materials on implantation. Human monocytes differentiated on tissue culture polystyrene (TCPS) for 14 days were trypsinized and reseeded onto DBP, TCPS, and polydimethylsiloxane (PDMS) for 48 h. The MDMs on DBP contained less intracellular and extracellular esterase activity compared with MDMs on TCPS and PDMS, as well as less acid phosphatase activity than on TCPS. As well, morphologically, MDMs on DBP were less spread, less multinucleated and did not display many lamellipodia. Taken together, these data represent the first evidence of the MDM response to intact, native extracellular matrix, demonstrating that these cells reacted with an altered, possibly reduced foreign body response on this natural scaffold compared with the two control surfaces. This in vitro MDM cell model may provide a novel method for predicting and elucidating the biological performance of tissue-derived biomaterials, thereby directing a more rational design of biomaterials for tissue regeneration purposes.


Assuntos
Materiais Biocompatíveis/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Pericárdio/citologia , Fosfatase Ácida/metabolismo , Animais , Células Cultivadas , DNA/metabolismo , Esterases/metabolismo , Humanos , Immunoblotting , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Macrófagos/enzimologia , Macrófagos/ultraestrutura , Microscopia Confocal
17.
Biomacromolecules ; 10(10): 2729-39, 2009 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-19754121

RESUMO

In tissue engineering, the ability to manipulate scaffold design characteristics is important to achieve functional tissue regeneration. In this study, degradable polar hydrophobic ionic polyurethane (D-PHI) porous scaffolds were synthesized using a lysine-based divinyl oligomer (DVO). Optimization studies on the DVO and D-PHI scaffold synthesis were conducted to maximize isocyanate and methacrylate monomer conversion, respectively. D-PHI scaffold properties were manipulated through the introduction of a lysine-based cross-linker. Specifically, increasing D-PHI cross-linker concentration resulted in an increase of the elastic modulus (0.5-21 MPa), a decrease of the elongation-at-yield (45-5%) and a reduction of scaffold swelling (170-100%). Based on a preliminary study with A10 vascular smooth muscle cells, D-PHI scaffolds demonstrated the ability to support cell adhesion and growth during 2 weeks of culture, suggesting their potential suitability for longer term vascular tissue engineering. The versatility of the D-PHI properties may allow for the tailoring of cell-material interaction and ultimately functional tissue regeneration.


Assuntos
Vasos Sanguíneos/citologia , Poliuretanos/síntese química , Engenharia Tecidual , Animais , Adesão Celular , Proliferação de Células , Células Cultivadas , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Ratos
18.
Biomaterials ; 30(29): 5497-504, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19635633

RESUMO

Tissue regeneration alternatives for peripheral vascular disease are actively being investigated; however, few studies in this area have probed the role of the wound healing monocyte-derived macrophage (MDM). Inflammatory MDMs transition to wound healing MDMs as the relative levels of tumor necrosis factor-alpha (TNF-alpha) decrease and IL-10 increase. TNF-alpha has been linked to the regulation of HMGB1 (high mobility group box 1 protein), a nuclear protein that upon macrophage stimulation can be secreted and act as a pro-inflammatory cytokine. This study investigated the influence of a degradable polar hydrophobic ionic polyurethane (D-PHI) on MDM cell expression of pro- versus anti-inflammatory markers, when the material was uncoated or pre-coated with collagen prior to cell studies. Effects were compared to similar groups on tissue culture polystyrene (TCPS). Collagen coated TCPS and D-PHI had significantly more DNA than the uncoated TCPS after 7d (p=0.001 and p=0.006 respectively); however, there was significantly less esterase activity from cells on D-PHI (+/-collagen) than for cells on TCPS after 7d (p=0.002, p=0.0003 respectively). No significant differences in esterase activity were observed between collagen coated and non-coated D-PHI surfaces. Analyses of pro-inflammatory cytokines (TNF-alpha, IL-1beta and HMGB1) secreted from differentiating monocytes adherent to D-PHI demonstrated a decrease whereas anti-inflammatory IL-10 increased over time when compared to TCPS, suggesting that D-PHI was less inflammatory than TCPS. Since D-PHI maintains cell attachment while aiding in the transition of MDM to a wound healing phenotype, this material has qualities suitable to be used in tissue engineering applications where MDM play a key role in tissue regeneration.


Assuntos
Colágeno/química , Macrófagos/citologia , Macrófagos/fisiologia , Monócitos/citologia , Monócitos/fisiologia , Poliuretanos/química , Cicatrização/fisiologia , Adolescente , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Feminino , Humanos , Masculino , Teste de Materiais , Adulto Jovem
19.
J Heart Valve Dis ; 18(2): 187-97, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19455894

RESUMO

BACKGROUND AND AIM OF THE STUDY: Decellularized materials, which represent a popular option for a variety of applications in regenerative medicine, including bioprosthetic heart valves, offer the opportunity to study cellular responses to extracellular matrix biochemistry and architecture. The study aim was to investigate the response of U937 macrophage-like cells (a model of the monocyte-derived macrophage, the pivotal cell to the initial and chronic cellular responses to implanted biomaterials) to decellularized bovine pericardium, to explore its expected biological performance in vivo, and to predict any adverse reactions in clinical trials. METHODS: Differentiated U937 cells were cultured on three surfaces: decellularized bovine pericardium (DBP); polydimethylsiloxane (PDMS); and tissue-culture polystyrene (TCPS). Cell lysates were analyzed for DNA (to determine cell attachment and viability), esterase (as a marker of degradative potential) and acid phosphatase activity (as a marker of the innate immune response). Cell morphology was also compared using confocal and scanning electron microscopy. RESULTS: U937 cells cultured on DBP were less spread and had less multinucleation than cells on either control polymer. No significant differences in DNA amount were observed between the substrates at each time point. In addition, cells cultured on DBP contained less acid phosphatase and esterase activity than cells on TCPS (p < 0.05). CONCLUSION: The study results suggested that U937 cells seeded onto DBP reacted with an altered, more mild, foreign body response than cells cultured on either PDMS or TCPS. This U937 cell model provides evidence that the response of macrophages to decellularized materials is not initially aggressive. The present study served as a first step in elucidating the biological mechanisms by which tissue-derived valve replacements fail in the host--an understanding that may direct a more rational design of valvular and decellularized materials.


Assuntos
Bioprótese , Próteses Valvulares Cardíacas , Macrófagos/citologia , Pericárdio/citologia , Fosfatase Ácida/metabolismo , Animais , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Dimetilpolisiloxanos , Esterases/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Poliestirenos , Engenharia Tecidual
20.
J Biomed Mater Res A ; 91(4): 1150-9, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19148928

RESUMO

Phorbol myristate acetate, a protein kinase C activator, inhibited monocyte-derived macrophage (MDM)-mediated degradation of aliphatic (HDI) polycarbonate-based polyurethanes but not degradation of the aromatic polycarbonate-based polyurethane (MDI). The objectives of this study were to determine if reactive oxygen species are involved in the phorbol myristate acetate effect on esterase activity and MDM-mediated polycarbonate-based polyurethane degradation and to find a good marker of material-initiated activation of MDM. The phorbol myristate acetate-dependent effects of the material chemistry on cell activation and degradation were evaluated by adding reactive oxygen species scavengers, catalase plus superoxide dismutase to MDM and assaying possible markers of MDM activation: esterase activity, acid phosphatase activity, and high molecular weight group box 1 protein (HMGB1). All treatments reduced the esterase activity in MDM on HDI but not in MDM on MDI. Acid phosphatase was inhibited in MDM to varying degrees on all surfaces by phorbol myristate acetate or catalase plus superoxide dismutase either alone or together. Secretion of HMGB1 from MDM on HDI431 was higher than MDI; however only secretion from MDM on HDI was inhibited by phorbol myristate acetate. In MDM on HDI, catalase plus superoxide dismutase reduced intracellular HMGB1 levels +/- phorbol myristate acetate; whereas, catalase, superoxide dismutase plus phorbol myristate acetate increased intracellular HMGB1 in MDM on MDI, suggesting that esterase and HMGB1 are more specific markers of activation than acid phosphatase. Manipulation of signaling pathways may provide insight surrounding the mechanism of activation for oxidative and/or hydrolytic degradative pathways in the MDM response to material surface chemistry.


Assuntos
Reação a Corpo Estranho/imunologia , Sequestradores de Radicais Livres/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Poliuretanos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fosfatase Ácida/metabolismo , Ativação Enzimática/efeitos dos fármacos , Reação a Corpo Estranho/induzido quimicamente , Proteína HMGB1/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Macrófagos/enzimologia , Modelos Biológicos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Cimento de Policarboxilato/farmacologia , Proteína Quinase C/metabolismo , Propriedades de Superfície/efeitos dos fármacos
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