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The wet spinning of fibers from regenerated silk fibroin has long been a research goal. Due to the degradation of the molecular structure of the fibroin protein during the preparation of the regenerated silk fibroin solution, fibroin concentrations with at least 10% protein content are required to achieve sufficient viscosity for wet spinning. In this study, a spinning dope formulation of regenerated silk fibroin is presented that shows a rheological behavior similar to that of native silk fibroin isolated from the glands of B. mori silkworm larvae. In addition, we present a wet-spinning process that enables, for the first time, the continuous wet spinning of regenerated silk fibroin with only 4% fibroin protein content into an endless fiber. Furthermore, the tensile strength of these wet-spun regenerated silk fibroin fibers per percentage of fibroin is higher than that of all continuous spinning approaches applied to regenerated and native silk fibroin published so far.
Assuntos
Bombyx , Fibroínas , Animais , Seda , Larva , ReologiaRESUMO
Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.
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Bombyx/química , Fibroínas/metabolismo , Pupa/química , Seda/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida/métodos , Fibroínas/farmacologia , Fibroínas/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura/métodos , Fosfolipídeos/isolamento & purificação , Fosfolipídeos/metabolismo , Reprodutibilidade dos Testes , Seda/farmacologia , Seda/ultraestrutura , Espectrometria por Raios X/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Temperatura , Fatores de TempoRESUMO
A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-ß1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-ß1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.
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Cartilagem Articular/metabolismo , Fibroínas/farmacologia , Engenharia Tecidual/métodos , Idoso , Materiais Biocompatíveis/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem Articular/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Fibroínas/metabolismo , Humanos , Ácido Hialurônico/farmacologia , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Pessoa de Meia-Idade , Porosidade , Seda/metabolismo , Alicerces Teciduais/químicaRESUMO
In this article, the benefits offered by micro-fibrous scaffold architectures fabricated by textile manufacturing techniques are discussed: How can established and novel fiber-processing techniques be exploited in order to generate templates matching the demands of the target cell niche? The problems related to the development of biomaterial fibers (especially from nature-derived materials) ready for textile manufacturing are addressed. Attention is also paid on how biological cues may be incorporated into micro-fibrous scaffold architectures by hybrid manufacturing approaches (e.g. nanofiber or hydrogel functionalization). After a critical review of exemplary recent research works on cell-free fiber based scaffolds for in situ TE, including clinical studies, we conclude that in order to make use of the whole range of favors which may be provided by engineered fibrous scaffold systems, there are four main issues which need to be addressed: (1) Logical combination of manufacturing techniques and materials. (2) Biomaterial fiber development. (3) Adaption of textile manufacturing techniques to the demands of scaffolds for regenerative medicine. (4) Incorporation of biological cues (e.g. stem cell homing factors).
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Têxteis , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Animais , Quitosana , Teste de Materiais , Engenharia Tecidual/métodosRESUMO
The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.
Assuntos
Bombyx/virologia , Catepsinas/metabolismo , Quitinases/metabolismo , Nucleopoliedrovírus/enzimologia , Animais , Bombyx/metabolismo , Catepsinas/genética , Quitinases/genética , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/virologia , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
PURPOSE: Post-interventional hemorrhage can result in serious complications, especially in patients with hemostatic disorders. Identification of safe and efficient local hemostatic agents is important, particularly in the context of an ageing society and the emergence of new oral anticoagulants. The aim of this in vitro study was to investigate the potential of silk fibroin membranes coated with the inorganic polymer polyphosphate (polyP) as a novel hemostatic device in oral surgery. METHODS: Cocoons of the silkworm Bombyx mori were degummed and dissolved. Varying amounts of long-chain polyP (2-2000 µg/mm2) were adsorbed to the surface of silk fibroin membranes. Analysis of the procoagulant effect of polyP-coated silk membranes was performed using real-time thrombin generation assays in human plasma. Increasing concentrations of polyP (0.15-500 µg/ml) served as a positive control, while uncoated silk fibroin membranes were used as negative control. RESULTS: PolyP-coated silk fibroin membranes triggered coagulation when compared to plasma samples and pure silk fibroin membranes. A polyP-dose-dependent effect of thrombin generation could be found with a maximum (ETP = 1525.7 nMâ min, peak thrombin = 310.1 nM, time to peak = 9.8 min, lag time = 7.6 min.) at 200 µg/mm2 of polymer loading on the silk fibroin membrane surface. CONCLUSIONS: In this study, it was demonstrated that silk fibroin membranes coated with polyP have the potential to act as a promising novel hemostatic device.
Assuntos
Bombyx , Fibroínas , Hemostáticos , Procedimentos Cirúrgicos Bucais , Animais , Humanos , Projetos Piloto , Trombina , Hemostáticos/farmacologia , PolímerosRESUMO
Intervertebral disc (IVD) herniation often causes severe pain and is frequently associated with the degeneration of the IVD. As the IVD degenerates, more fissures with increasing size appear within the outer region of the IVD, the annulus fibrosus (AF), favoring the initiation and progression of IVD herniation. For this reason, we propose an AF repair approach based on methacrylated gellan gum (GG-MA) and silk fibroin. Therefore, coccygeal bovine IVDs were injured using a biopsy puncher (â 2 mm) and then repaired with 2% GG-MA as a filler material and sealed with an embroidered silk yarn fabric. Then, the IVDs were cultured for 14 days either without any load, static loading, or complex dynamic loading. After 14 days of culture, no significant differences were found between the damaged and repaired IVDs, except for a significant decrease in the IVDs' relative height under dynamic loading. Based on our findings combined with the current literature that focuses on ex vivo AF repair approaches, we conclude that it is likely that the repair approach did not fail but rather insufficient harm was done to the IVD.
RESUMO
Low back pain is often due to degeneration of the intervertebral discs (IVD). It is one of the most common age- and work-related problems in today's society. Current treatments are not able to efficiently restore the full function of the IVD. Therefore, the aim of the present work was to reconstruct the two parts of the intervertebral disc-the annulus fibrosus (AF) and the nucleus pulposus (NP)-in such a way that the natural structural features were mimicked by a textile design. Silk was selected as the biomaterial for realization of a textile IVD because of its cytocompatibility, biodegradability, high strength, stiffness, and toughness, both in tension and compression. Therefore, an embroidered structure made of silk yarn was developed that reproduces the alternating fiber structure of +30° and -30° fiber orientation found in the AF and mimics its lamellar structure. The developed embroidered ribbons showed a tensile strength that corresponded to that of the natural AF. Fiber additive manufacturing with 1 mm silk staple fibers was used to replicate the fiber network of the NP and generate an open porous textile 3D structure that may serve as a reinforcement structure for the gel-like NP.
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Intervertebral disc (IVD) degeneration (IDD) is the main contributor to chronic low back pain. To date, the present therapies mainly focus on treating the symptoms caused by IDD rather than addressing the problem itself. For this reason, researchers have searched for a suitable biomaterial to repair and/or regenerate the IVD. A promising candidate to fill this gap is silk, which has already been used as a biomaterial for many years. Therefore, this review aims first to elaborate on the different origins from which silk is harvested, the individual composition, and the characteristics of each silk type. Another goal is to enlighten why silk is so suitable as a biomaterial, discuss its functionalization, and how it could be used for tissue engineering purposes. The second part of this review aims to provide an overview of preclinical studies using silk-based biomaterials to repair the inner region of the IVD, the nucleus pulposus (NP), and the IVD's outer area, the annulus fibrosus (AF). Since the NP and the AF differ fundamentally in their structure, different therapeutic approaches are required. Consequently, silk-containing hydrogels have been used mainly to repair the NP, and silk-based scaffolds have been used for the AF. Although most preclinical studies have shown promising results in IVD-related repair and regeneration, their clinical transition is yet to come.
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BACKGROUND: Post-transplantation bone disease is associated with a high degree of morbidity including pain and fractures. Glucocorticoid-induced osteoporosis on top of pre-existing renal osteodystrophy is considered the major pathogenic factor, while the role of non-glucocorticoid immunosuppressants is less well defined. METHODS: In this study, we investigated the influence of sirolimus (SRL) versus calcineurin inhibitor (CI)-based immunosuppressive regimens on biomarkers of bone resorption in renal transplant patients. In addition, the impact of SRL, tacrolimus and mycophenolate mofetil (MMF) on osteoclast activation and function was assessed in cell culture systems. RESULTS: Using this approach, we demonstrated reduced serum levels of bone resorption markers in patients treated with SRL after kidney transplantation compared to a CI-based regimen. In line with this observation, we detected profoundly reduced osteoclast differentiation and subsequently diminished hydroxyapatite resorption in the presence of SRL compared to MMF and tacrolimus in vitro. Moreover, SRL significantly reduced osteoclast precursor proliferation in vitro compared to tacrolimus and led to augmented apoptosis in osteoclast precursors. CONCLUSIONS: Taken together, SRL was shown to inhibit osteoclast formation in vivo and in vitro. SRL thus may have the potential to balance osteoclast promoting effects of glucocorticoids and CI, thereby counteracting the development of accelerated osteoporosis in renal transplant recipients.
Assuntos
Imunossupressores/farmacologia , Transplante de Rim , Ácido Micofenólico/análogos & derivados , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Sirolimo/farmacologia , Tacrolimo/farmacologia , Adulto , Idoso , Reabsorção Óssea , Diferenciação Celular , Estudos Transversais , Feminino , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/farmacologia , Osteoporose/etiologia , Osteoporose/prevenção & controle , Ligante RANK/biossíntese , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Adulto JovemRESUMO
PURPOSE: The aim of this study was to investigate the influence of oxidized regenerated cellulose/collagen matrix on the concentration and activity of gelatinases, elastase, and plasmin in wound exudate. SUBJECTS AND SETTING: The study included 32 patients with diabetic foot ulcers. Ten patients with a mean age of 66 ± 9 years (mean ± SD) were treated with hydrocolloid dressings; 22 patients with a mean age of 57 ± 12 years were treated with oxidized regenerated/collagen matrix and hydrocolloid dressings. METHODS: Wound exudate was collected on days 0, 5, 14, and every 14 days thereafter for 12 weeks. Total protein was determined according to Bradford's technique. The levels of elastase and plasmin were measured spectrofluorometrically. Besides, gelatinase activity and matrix metalloproteinase-2 concentration were analyzed. The surface area of all ulcers was measured by planimetry. RESULTS: Patients treated with oxidized regenerated cellulose/collagen matrix showed a significant decrease in elastase, plasmin, and gelatinase activities in wound exudates. The matrix metalloproteinase-2 concentration was significantly reduced on days 14, 28, 42, and 56 in comparison to day 0. Furthermore, wound size was significantly reduced at days 14 and 28 in oxidized regenerated cellulose/collagen matrix-treated patients (P < .05). CONCLUSION: Our results showed a significant and immediate reduction in the levels of all tested proteases in the wound exudate of diabetic foot ulcer patients treated with oxidized regenerated cellulose/collagen matrix. These patients also experienced a significantly greater reduction in wound size.
Assuntos
Celulose Oxidada/farmacologia , Pé Diabético/terapia , Absorção Cutânea/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Idoso , Assistência Ambulatorial/métodos , Curativos Hidrocoloides , Celulose Oxidada/uso terapêutico , Estudos de Coortes , Pé Diabético/diagnóstico , Pé Diabético/enfermagem , Endopeptidases/análise , Endopeptidases/metabolismo , Exsudatos e Transudatos/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/metabolismo , Pessoa de Meia-Idade , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Medição de Risco , Índice de Gravidade de Doença , Resultado do Tratamento , Cicatrização/fisiologiaRESUMO
BACKGROUND AIMS: Human mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation. METHODS: hMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks. RESULTS AND CONCLUSIONS: No proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.
Assuntos
Proliferação de Células , Meios de Cultura Livres de Soro , Células-Tronco Mesenquimais/patologia , Soro/imunologia , Transplante de Células-Tronco , Animais , Medula Óssea/patologia , Bovinos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Transmissão de Doença Infecciosa/prevenção & controle , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Células-Tronco Mesenquimais/imunologiaRESUMO
BACKGROUND AIMS: The beneficial effect of human (h) mesenchymal stromal cell (MSC) transplantation in a variety of cell-based intervention strategies is widely believed to be because of paracrine mechanisms. The modification of hMSC cytokine and growth-factor expression patterns were studied following exposure to lipopolysaccharide (LPS) and tissue homogenates (representing tissue debris) from normal and pathologic tissues. METHODS: Human bone marrow-derived MSC were stimulated with LPS or exposed to homogenate from normal or pathologic rat spinal cord or heart. The expression profiles of a number of cytokines and growth factors were investigated using quantitative reverse transcription (RT)-polymerase chain reaction (PCR) with human-specific primers. The effects of tissue homogenates on hMSC proliferation and migratory behavior were also investigated. RESULTS: Stimulation of hMSC with LPS resulted in an up-regulation of interleukin (IL)-1ß, IL-6 and IL-8. However, the pattern of up-regulation varied between donor samples. Furthermore, LPS treatment resulted in a donor-dependent alteration of growth factor expression. Induction of a shift in expression pattern was not observed following exposure to homogenates from either normal or pathologic tissues. Tissue homogenates did stimulate cell proliferation, but not migration. CONCLUSIONS: The hMSC expression pattern is apparently stable, even when cells are confronted by debris from different tissue types. However, treatment of hMSC with LPS is able to change the expression of cytokines and growth factors in a donor-dependent manner that may enhance their potential use in regenerative medicine.
Assuntos
Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Traumatismos da Medula Espinal/metabolismo , Extratos de Tecidos/farmacologia , Animais , Medula Óssea/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Ratos , Ratos Endogâmicos Lew , Medula Espinal/metabolismo , Medula Espinal/cirurgia , Traumatismos da Medula Espinal/patologia , Extratos de Tecidos/metabolismoRESUMO
BACKGROUND: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. RESULTS: The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. CONCLUSION: The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.
Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neurogênese/fisiologia , Células Estromais/fisiologia , Biomarcadores/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Receptores de Dopamina D2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sintaxina 1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Biomaterials are used in tissue engineering with the aim to repair or reconstruct tissues and organs. Frequently, the identification and development of biomaterials is an iterative process with biomaterials being designed and then individually tested for their properties in combination with one specific cell type. However, recent efforts have been devoted to systematic, combinatorial and parallel approaches to identify biomaterials, suitable for specific applications. Embryonic and adult stem cells represent an ideal cell source for tissue engineering. Since stem cells can be readily isolated, expanded and transplanted, their application in cell-based therapies has become a major focus of research. Biomaterials can potentially influence e.g. stem cell proliferation and differentiation in both, positive or negative ways and biomaterial characteristics have been applied to repel or attract stem cells in a niche-like microenvironment. Our consortium has now established a grid-based platform to investigate stem cell/biomaterial interactions. So far, we have assessed 140 combinations of seven different stem cell types and 19 different polymers performing systematic screening assays to analyse parameters such as morphology, vitality, cytotoxicity, apoptosis, and proliferation. We thus can suggest and advise for and against special combinations for stem cell-based tissue engineering.
Assuntos
Materiais Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/toxicidade , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Feminino , Temperatura Alta , Humanos , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) modifies wound microenvironments by binding and inactivating excess levels of proteases such as elastase, plasmin and gelatinases in wound exudates. To compare levels of the gelatinases matrix metalloproteinase 2 (MMP-2), elastase and plasmin in wound exudates collected from chronic venous insufficiency patients with venous leg ulcers treated with either an ORC/collagen matrix or a standard control therapy. During a 12-week treatment period, wound exudate samples were obtained from a control group of 10 patients treated with a hydrocolloid dressing and a treatment group of 17 patients treated with a combination of ORC/collagen matrix and hydrocolloid dressing. On admission and days 5, 14 and every subsequent 14th day, ulcers were photographed to determine healing rate and changes in ulcer appearance, and MMP-2 concentration and the gelatinase, elastase and plasmin activities were analysed from wound exudates. The patients treated with ORC/collagen matrix showed a significant decrease in elastase, plasmin and gelastinase activity as compared with the control group, with no significant difference in the MMP-2 concentrations between the two groups. The results show a significant and immediate reduction in protease activity in wound exudates from venous leg ulcers treated with ORC/collagen.
Assuntos
Celulose Oxidada , Colágeno , Exsudatos e Transudatos/enzimologia , Hemostáticos , Peptídeo Hidrolases/metabolismo , Úlcera Varicosa/enzimologia , Idoso , Bandagens , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Úlcera Varicosa/terapia , Cicatrização/fisiologiaRESUMO
Growth factors play a crucial role in wound healing in general and are promising tools for the treatment of chronic wounds as they can restore the physiological wound healing process. In growth factor-loaded wound dressings, human epidermal growth factor (EGF) is released in a burst and washed out quickly. The developed matrix consists of recombinant EGF produced in transgenic silkworms as a fusion protein with the fibroin light chain. The covalent linkage prevents EGF from draining into the surrounding tissue while presenting the growth factor on the surface. EGF-functionalized silk membranes and nonwovens lead to a 2.5-fold increase in the cell number of fibroblasts, while retaining full bioactivity even after e-beam sterilization. EGF is long-term presented without burst release and significantly reduces the wound area by 15% in an in vitro wound model. Hence, the cost-effective production of a biomaterial using transgenic silkworm larvae in combination with a growth factor paves the way for a promising new multifactorial wound cover for chronic wound healing. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2643-2652, 2018.
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Bombyx/química , Fator de Crescimento Epidérmico/farmacologia , Seda/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Partículas beta , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Fibroínas/farmacologia , Humanos , Larva/efeitos dos fármacos , Masculino , Camundongos , Modelos Biológicos , Proteínas Recombinantes/farmacologia , Propriedades de SuperfícieRESUMO
Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor ß3 (TGF-ß3, TGF-ß3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1324-1333, 2018.
Assuntos
Anel Fibroso/citologia , Engenharia Genética/métodos , Fator 6 de Diferenciação de Crescimento/farmacologia , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais , Fator de Crescimento Transformador beta3/farmacologia , Diferenciação Celular/efeitos dos fármacos , DNA/análise , Humanos , Mitocôndrias/fisiologiaRESUMO
(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.
RESUMO
Additive manufacturing technologies are a promising technology towards patient-specific implants for applications in regenerative medicine. The Net-Shape-Nonwoven technology is used to manufacture structures from short fibers with interconnected pores and large functional surfaces that are predestined for cell adhesion and growth. The present study reports on a modeling approach with a particular focus on the specific structural properties. The overall porosities and mean pore-sizes of the digital models are simulated according to liquid-displacement porosity in a tool implemented in the modeling software. This allows adjusting the process parameters fiber length and fiber diameter to generate biomimetic structures with pore-sizes adapted to the requirements of the tissue that is to be replaced. Modeling the structural and porosity properties of scaffolds and implants leads to an efficient use of the processed biomaterials as the trial-and-error method is avoided.