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1.
J Biomater Appl ; : 8853282211046800, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34617818

RESUMO

OBJECTIVE: The generation of bio-/hemocompatible cardiovascular patches with sufficient stability and regenerative potential remains an unmet goal. Thus, the aim of this study was the generation and in vitro biomechanical evaluation of a novel cardiovascular patch composed of pressure-compacted fibrin with embedded spider silk cocoons. METHODS: Fibrin-based patches were cast in a customized circular mold. One cocoon of Nephila odulis spider silk was embedded per patch during the casting process. After polymerization, the fibrin clot was compacted by 2 kg weight for 30 min resulting in thickness reduction from up to 2 cm to <1 mm. Tensile strength and burst pressure was determined after 0 weeks and 14 weeks of storage. A sewing strength test and a long-term load test were performed using a customized device to exert physiological pulsatile stretching of a silicon surface on which the patch had been sutured. RESULTS: Fibrin patches resisted supraphysiological pressures of well over 2000 mmHg. Embedding of spider silk increased tensile force 1.8-fold and tensile strength 1.45-fold (p < .001), resulting in a final strength of 1.07 MPa and increased sewing strength. Storage for 14 weeks decreased tensile strength, but not significantly and suturing properties of the spider silk patches were satisfactory. The long-term load test indicated that the patches were stable for 4 weeks although slight reduction in patch material was observed. CONCLUSION: The combination of compacted fibrin matrices and spider silk cocoons may represent a feasible concept to generate stable and biocompatible cardiovascular patches with regenerative potential.

2.
MRS Commun ; : 1-9, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34513262

RESUMO

In vitro thrombogenicity test systems require co-cultivation of endothelial cells and platelets under blood flow-like conditions. Here, a commercially available perfusion system is explored using plasma-treated cyclic olefin copolymer (COC) as a substrate for the endothelial cell layer. COC was characterized prior to endothelialization and co-cultivation with platelets under static or flow conditions. COC exhibits a low roughness and a moderate hydrophilicity. Flow promoted endothelial cell growth and prevented platelet adherence. These findings show the suitability of COC as substrate and the importance of blood flow-like conditions for the assessment of the thrombogenic risk of drugs or cardiovascular implant materials. Supplementary Information: The online version contains supplementary material available at 10.1557/s43579-021-00072-6.

3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209789

RESUMO

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.


Assuntos
Materiais Biocompatíveis/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Meios de Cultura/farmacologia , Adulto , Materiais Biocompatíveis/química , Plaquetas/citologia , Meios de Cultura/química , Endotélio/citologia , Feminino , Humanos , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polímeros/farmacologia , Tecidos Suporte/química
4.
Ann Biomed Eng ; 49(9): 2066-2079, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33483842

RESUMO

The generation of cellularized bioartificial blood vessels resembling all three layers of the natural vessel wall with physiological morphology and cell alignment is a long pursued goal in vascular tissue engineering. Simultaneous culture of all three layers under physiological mechanical conditions requires highly sophisticated perfusion techniques and still today remains a key challenge. Here, three-layered bioartificial vessels based on fibrin matrices were generated using a stepwise molding technique. Adipose-derived stem cells (ASC) were differentiated to smooth muscle cells (SMC) and integrated in a compacted tubular fibrin matrix to resemble the tunica media. The tunica adventitia-equivalent containing human umbilical vein endothelial cells (HUVEC) and ASC in a low concentration fibrin matrix was molded around it. Luminal seeding with HUVEC resembled the tunica intima. Subsequently, constructs were exposed to physiological mechanical stimulation in a pulsatile bioreactor for 72 h. Compared to statically incubated controls, mechanical stimulation induced physiological cell alignment in each layer: Luminal endothelial cells showed longitudinal alignment, cells in the media-layer were aligned circumferentially and expressed characteristic SMC marker proteins. HUVEC in the adventitia-layer formed longitudinally aligned microvascular tubes resembling vasa vasorum capillaries. Thus, physiologically organized three-layered bioartificial vessels were successfully manufactured by stepwise fibrin molding with subsequent mechanical stimulation.

5.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478148

RESUMO

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.


Assuntos
Pesquisa Biomédica , Doenças Cardiovasculares/terapia , Células Endoteliais da Veia Umbilical Humana/citologia , Artérias Umbilicais/citologia , Implantes Absorvíveis , Citoesqueleto de Actina/metabolismo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Doenças Cardiovasculares/etiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Medicina Regenerativa/métodos , Medicina Regenerativa/tendências , Engenharia Tecidual/métodos , Engenharia Tecidual/tendências , Artérias Umbilicais/metabolismo , Fator de von Willebrand/metabolismo
6.
Int J Artif Organs ; 44(2): 130-138, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32611278

RESUMO

Vascularization of tissue engineered implants is crucial for their survival and integration in the recipient's body. Pre-vascularized, fibrin-based implants offer a solution since low concentration fibrin hydrogels (1 mg/mL) have been shown to promote tube formation of endothelial cells in co-culture with adipogenic stem cells. However, higher fibrinogen concentrations (> 20 mg/mL) enabling the fabrication of stable implants are necessary.We here characterized fibrin gels of 1-30 mg/mL for their rheological properties and whether they support tube formation of endothelial cell-adipogenic stem cell co-cultures for up to 7 days. Moreover, 20 mg/mL gels containing preformed channels and endothelial cell-adipogenic stem cell co-culture were perfused continuously in a customized flow chamber with 3.9 dyn/cm2 for 12 days and analyzed for capillary formation.Rheology of fibrin gels showed increasing stability proportional to fibrinogen concentration with 20 mg/mL gels having a storage module of 465 Pa. Complex tube networks stable for 7 days were observed at 1-5 mg/mL gels whereas higher concentrations showed initial sprouting only. However, perfusion of 20 mg/mL fibrin gels resulted in endothelialized pore formation in several layers of the gel with endothelial cell-adipogenic stem cell co-culture.Thus, perfusion supports the formation of capillary-like structures in fibrin gels that are too dense for spontaneous tube formation under static conditions. Future studies are necessary to further increase pore density and to investigate proper nutrition of tissue-specific target cells in the scaffold.


Assuntos
Fibrina/farmacologia , Regeneração Tecidual Guiada/métodos , Hidrogéis/farmacologia , Reepitelização/fisiologia , Engenharia Tecidual , Tecidos Suporte , Implantes Absorvíveis , Capilares/crescimento & desenvolvimento , Humanos , Perfusão/métodos , Próteses e Implantes/normas , Reologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
7.
Ann Biomed Eng ; 48(3): 913-926, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30815762

RESUMO

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-ß, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10 days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.


Assuntos
Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/citologia , Túnica Média , Adipogenia , Adulto , Idoso , Reatores Biológicos , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Colágeno Tipo I , Feminino , Fibrina , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Estimulação Física , Pressão , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Estresse Mecânico , Engenharia Tecidual , Tecidos Suporte , Fator de Crescimento Transformador beta/farmacologia
8.
Tissue Eng Part A ; 25(13-14): 936-948, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30648499

RESUMO

IMPACT STATEMENT: We here showed that even under optimized conditions for biochemical differentiation of adipose-derived stem cells (with respect to a pronounced marker protein expression for a reasonable period of time) it was not possible to obtain functional smooth muscle cells from all donors. Moreover, an underestimated role may play the effect of the scaffold material on smooth muscle cell functionality. Both aspects are crucial for the successful tissue engineering of the vascular medial layer combining autologous cells with a suitable scaffold material and thus should be thoroughly addressed in each individualized therapeutic approach.


Assuntos
Adipogenia , Células-Tronco Mesenquimais/citologia , Desenvolvimento Muscular , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Colágeno/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fenótipo , Ratos , Transdução de Sinais , Doadores de Tecidos
9.
J Tissue Eng Regen Med ; 13(4): 537-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30552734

RESUMO

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30 µg/ml as tested with no effect on proliferation. TGFß3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14 days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.


Assuntos
Cartilagem Articular/citologia , Diferenciação Celular , Movimento Celular , Condrócitos/citologia , Fibronectinas/sangue , Adulto , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Feminino , Fibronectinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Proteoglicanas/metabolismo
10.
Tissue Eng Part A ; 24(5-6): 432-447, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978275

RESUMO

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.


Assuntos
Prótese Vascular , Células Endoteliais/imunologia , Sangue Fetal/imunologia , Engenharia Tecidual , Adulto , Células Endoteliais/citologia , Sangue Fetal/citologia , Inativação Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Masculino
11.
Innov Surg Sci ; 3(3): 215-224, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31579785

RESUMO

Introduction: We have recently reported about a novel technique for the generation of bioartificial vascular grafts based on the use of a compacted fibrin matrix. In this study, we evaluated the effects of a dehydration process on the biomechanical properties of compacted fibrin tubes and whether it allows for their long-term storage. Materials and methods: Fibrin was precipitated from fresh frozen plasma by means of cryoprecipitation and simultaneously with a thrombin solution applied in a high-speed rotating casting mold. Subsequent dehydration of the fibrin tubes (29/38) was performed in dry air with a dilator inside the tube to prevent the collapse of the lumen. Dehydrated fibrin tubes were stored for six (n=9) and 12 months (n=10) at room temperature. Comparative analysis was done on initially generated and dehydrated fibrin tubes before and after storage to evaluate the effects of the dehydration process and storage on the biomechanical properties and structure of the tubes. Results: Thirty-eight fibrin tubes were generated by high-speed rotation-molding from 142±3 mg fibrinogen with an inner diameter of 5.8±0.1 mm and a length of 100 mm. A centrifugal force of nearly 900×g compacted applied fibrin, while fluid was pressed out of the matrix and drained from the mold via holes resulting in a 16-fold compaction of the fibrin matrix. Dehydration was characterized by shrinkage of the tubes to a diameter of 3.2±0.2 mm, while the length remained at 100 mm equivalent to a further two-fold compaction. The biomechanical strength of the dehydrated fibrin tubes significantly increased to values comparable to that of native ovine carotid arteries and maintained during the first 6 months of storage. After 12 months of storage, only five of 10 tubes were intact, and only one showed maintained biomechanical strength. Discussion: Compaction of a fibrin matrix in high-speed rotation-moulding and subsequent dehydration enables for the construction of small-caliber fibrin grafts. Over and above, the dehydration process allows their storage and stockpiling as a prerequisite for clinical use.

12.
Ann Biomed Eng ; 43(11): 2630-41, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25921001

RESUMO

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts.


Assuntos
Prótese Vascular , Artérias Carótidas/fisiologia , Tecidos Suporte , Animais , Fenômenos Biomecânicos , Sobrevivência Celular , Colágeno/análise , DNA/análise , Detergentes , Elastina/análise , Células Endoteliais , Glicosaminoglicanos/análise , Cavalos
13.
Regen Med ; 9(6): 759-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25431912

RESUMO

AIMS: To evaluate the impact of human plasma-derived fibronectin (FN) on human subchondral mesenchymal progenitor cells regarding cell migration, proliferation, and chondrogenic differentiation. MATERIALS & METHODS: Human subchondral mesenchymal progenitor cells were analyzed for their migration capacity upon treatment with human plasma-derived FN. Proliferation activity was evaluated by DNA content. For chondrogenesis, cells were cultured in high-density pellet cultures in the presence of FN, TGFß3, and a combination thereof. RESULTS: Treatment of progenitors with FN significantly increased the number of migrating cells and elevated proliferative activity. Histological staining indicated formation of an extracellular matrix with type II collagen. Gene expression analysis gave no evidence for chondrogenic differentiation mediated by FN, but revealed a significant induction of type II collagen expression. CONCLUSION: FN has a potential to recruit human subchondral mesenchymal progenitor cells, possibly supporting proliferation and matrix assembly in cartilage repair procedures using bioactive implants after microfracture treatment.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Fibronectinas/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Idoso , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Células Cultivadas , Quimiotaxia , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
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