RESUMO
Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.
Assuntos
Proteínas F-Box/antagonistas & inibidores , Estudo de Associação Genômica Ampla , Heparitina Sulfato/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Algoritmos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Matriz Extracelular/genética , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica/genética , RNA-Seq , Sulfotransferases/antagonistas & inibidoresRESUMO
The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.
Assuntos
Diferenciação Celular , Matriz Extracelular/química , Vidro/química , Células-Tronco Mesenquimais/citologia , Nanopartículas/administração & dosagem , Osteogênese , Proliferação de Células , Células Cultivadas , Colágeno/química , Glicosaminoglicanos/química , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/químicaRESUMO
RATIONALE: The most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J2 (15Δ-PGJ2 ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products. METHODS: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed. RESULTS: HDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ2 . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 µM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations. CONCLUSIONS: In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.
Assuntos
PPAR gama/metabolismo , Ácidos Ftálicos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Simulação de Acoplamento Molecular , PPAR gama/química , PPAR gama/farmacologia , Ácidos Ftálicos/química , Ligação ProteicaRESUMO
Hyaluronan (HA)-based microgels generated in a microfluidic approach, containing an artificial extracellular matrix composed of collagen and high-sulfated hyaluronan (sHA3), were incorporated into a HA/collagen-based hydrogel matrix. This significantly enhanced the retention of noncrosslinked sHA3 within the gels enabling controlled sHA3 presentation. Gels containing sHA3 bound higher amounts of transforming growth factor-ß1 (TGF-ß1) compared to pure HA/collagen hydrogels. Moreover, the presence of sHA3-containing microgels improved the TGF-ß1 retention within the hydrogels. These findings are promising for developing innovative biomaterials with adjustable sHA3 release and growth factor interaction profiles to foster skin repair, e.g., by rebalancing dysregulated TGF-ß1 levels.
Assuntos
Colágeno/química , Ácido Hialurônico/química , Hidrogéis/química , Microgéis/química , Fator de Crescimento Transformador beta1/metabolismo , Animais , Materiais Biocompatíveis/química , Bovinos , Matriz Extracelular/metabolismo , Glicosaminoglicanos/química , Humanos , Microfluídica , Ratos , Pele/metabolismo , Pele/patologia , Streptococcus , Sulfatos/metabolismo , CicatrizaçãoRESUMO
Here, we investigated the synergistic effect of electrospun nanofibrous scaffolds made of gelatin /sulfated hyaluronan (sHA) or native hyaluronan (HA)/chondroitin sulfate (CS) and, keratinocytes (HaCaT)-human mesenchymal stem cells (hMSCs) contact co-culture on epithelial differentiation of hMSCs. The hMSCs were co-cultured in contact with HaCaT cells for 5 days on electrospun scaffold. Results show that electrospun scaffolds containing sulfated glycosaminoglycans (GAGs) stimulate epithelial differentiation in terms of various protein expression markers (keratin 14, ΔNp63α and Pan-cytokeratin) and gene expression of several dermal proteins (keratin 14, ΔNp63α). Electrospun scaffold independent of GAGs alone did not affect the epithelial differentiation of hMSCs but combination of keratinocyte-hMSC contact co-culture and electrospun scaffold promotes the epithelial differentiation of hMSCs.
Assuntos
Comunicação Celular/fisiologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Alicerces Teciduais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Galvanoplastia/métodos , Regulação da Expressão Gênica , Humanos , Teste de Materiais , Nanofibras/química , Alicerces Teciduais/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.
Assuntos
Glicosaminoglicanos/administração & dosagem , Ácido Hialurônico/administração & dosagem , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Sulfatos de Condroitina/administração & dosagem , Sulfatos de Condroitina/química , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/química , Humanos , Ácido Hialurônico/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Inibidor Tecidual de Metaloproteinase-3/química , Cicatrização/efeitos dos fármacosRESUMO
Numerous biological processes (tissue formation, remodelling and healing) are strongly influenced by the cellular microenvironment. Glycosaminoglycans (GAGs) are important components of the native extracellular matrix (ECM) able to interact with biological mediator proteins. They can be chemically functionalized and thereby modified in their interaction profiles. Thus, they are promising candidates for functional biomaterials to control healing processes in particular in health-compromised patients. Biophysical studies show that the interaction profiles between mediator proteins and GAGs are strongly influenced by (i) sulphation degree, (ii) sulphation pattern, and (iii) composition and structure of the carbohydrate backbone. Hyaluronan derivatives demonstrate a higher binding strength in their interaction with biological mediators than chondroitin sulphate for a comparable sulphation degree. Furthermore sulphated GAG derivatives alter the interaction profile of mediator proteins with their cell receptors or solute native interaction partners. These results are in line with biological effects on cells relevant for wound healing processes. This is valid for solute GAGs as well as those incorporated in collagen-based artificial ECM (aECMs). Prominent effects are (i) anti-inflammatory, immunomodulatory properties towards macrophages/dendritic cells, (ii) enhanced osteogenic differentiation of human mesenchymal stromal cells, (iii) altered differentiation of fibroblasts to myofibroblasts, (iv) reduced osteoclast activity and (v) improved osseointegration of dental implants in minipigs. The findings of our consortium Transregio 67 contribute to an improved understanding of structure-function relationships of GAG derivatives in their interaction with mediator proteins and cells. This will enable the design of bioinspired, functional biomaterials to selectively control and promote bone and skin regeneration.
Assuntos
Materiais Biocompatíveis , Glicosaminoglicanos/química , Animais , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Modelos Animais , Ressonância de Plasmônio de SuperfícieRESUMO
Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.
Assuntos
Alginatos , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacologia , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacologia , Diferenciação Celular , Metilcelulose/metabolismo , Metilcelulose/farmacologia , Taninos/metabolismo , Taninos/farmacologiaRESUMO
The WNT signaling pathway is a central regulator of bone development and regeneration. Functional alterations of WNT ligands and inhibitors are associated with a variety of bone diseases that affect bone fragility and result in a high medical and socioeconomic burden. Hence, this cellular pathway has emerged as a novel target for bone-protective therapies, e.g. in osteoporosis. Here, we investigated glycosaminoglycan (GAG) recognition by Dickkopf-1 (DKK1), a potent endogenous WNT inhibitor, and the underlying functional implications in order to develop WNT signaling regulators. In a multidisciplinary approach we applied in silico structure-based de novo design strategies and molecular dynamics simulations combined with synthetic chemistry and surface plasmon resonance spectroscopy to Rationally Engineer oligomeric Glycosaminoglycan derivatives (REGAG) with improved neutralizing properties for DKK1. In vitro and in vivo assays show that the GAG modification to obtain REGAG translated into increased WNT pathway activity and improved bone regeneration in a mouse calvaria defect model with critical size bone lesions. Importantly, the developed REGAG outperformed polymeric high-sulfated hyaluronan (sHA3) in enhancing bone healing up to 50% due to their improved DKK1 binding properties. Thus, rationally engineered GAG variants may represent an innovative strategy to develop novel therapeutic approaches for regenerative medicine.
Assuntos
Doenças Ósseas , Regeneração Óssea , Glicosaminoglicanos , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Camundongos , Osso e Ossos/metabolismo , Glicosaminoglicanos/metabolismo , Via de Sinalização WntRESUMO
Hyaluronan, the extracellular matrix glycosaminoglycan, is an important structural component of many tissues playing a critical role in a variety of biological contexts. This makes hyaluronan, which can be biotechnologically produced in large scale, an attractive starting polymer for chemical modifications. This review provides a broad overview of different synthesis strategies used for modulating the biological as well as material properties of this polysaccharide. We discuss current advances and challenges of derivatization reactions targeting the primary and secondary hydroxyl groups or carboxylic acid groups and the N-acetyl groups after deamidation. In addition, we give examples for approaches using hyaluronan as biomedical polymer matrix and consequences of chemical modifications on the interaction of hyaluronan with cells via receptor-mediated signaling. Collectively, hyaluronan derivatives play a significant role in biomedical research and applications indicating the great promise for future innovative therapies.
RESUMO
Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.
RESUMO
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.
RESUMO
Sustained inflammation associated with dysregulated macrophage activation prevents tissue formation and healing of chronic wounds. Control of inflammation and immune cell functions thus represents a promising approach in the development of advanced therapeutic strategies. Here we describe immunomodulatory hyaluronan/collagen (HA-AC/coll)-based hydrogels containing high-sulfated hyaluronan (sHA) as immunoregulatory component for the modulation of inflammatory macrophage activities in disturbed wound healing. Solute sHA downregulates inflammatory activities of bone marrow-derived and tissue-resident macrophages in vitro. This further affects macrophage-mediated pro-inflammatory activation of skin cells as shown in skin ex-vivo cultures. In a mouse model of acute skin inflammation, intradermal injection of sHA downregulates the inflammatory processes in the skin. This is associated with the promotion of an anti-inflammatory gene signature in skin macrophages indicating a shift of their activation profile. For in vivo translation, we designed HA-AC/coll hydrogels allowing delivery of sHA into wounds over a period of at least one week. Their immunoregulatory capacity was analyzed in a translational experimental approach in skin wounds of diabetic db/db mice, an established model for disturbed wound healing. The sHA-releasing hydrogels improved defective tissue repair with reduced inflammation, augmented pro-regenerative macrophage activation, increased vascularization, and accelerated new tissue formation and wound closure.
RESUMO
In order to restore the regeneration capacity of large-size vascularized tissue defects, innovative biomaterial concepts are required. Vascular endothelial growth factor (VEGF165) is a key factor of angiogenesis interacting with sulfated glycosaminoglycans (sGAG) within the extracellular matrix. As this interplay mainly controls and directs the biological activity of VEGF165, we used chemically modified sGAG derivatives to evaluate the structural requirements of sGAG for controlling and tuning VEGF165 function and to translate these findings into the design of biomaterials. The in-depth analysis of this interaction by surface plasmon resonance and ELISA studies in combination with molecular modeling stressed the relevance of the substitution position, degree of sulfation, and carbohydrate backbone of GAG. Acrylated hyaluronan (HA-AC)/collagen (coll)-based hydrogels containing cross-linked acrylated, sulfated hyaluronan (sHA-AC) derivatives with different substitution patterns or an acrylated chondroitin sulfate (CS-AC) derivative function as multivalent carbohydrate-based scaffolds for VEGF165 delivery with multiple tuning capacities. Depending on the substitution pattern of sGAG, the release of biologically active VEGF165 was retarded in a defined manner compared to pure HA/coll gels, which further controlled the VEGF165-induced stimulation of endothelial cell proliferation and extended morphology of cells. This indicates that sGAG can act as modulators of protein interaction profiles of HA/coll hydrogels. In addition, sHA-AC-containing gels with and even without VEGF165 strongly stimulate endothelial cell proliferation compared to gels containing only CS-AC or HA-AC. Thus, HA/coll-based hydrogels containing cross-linked sHA-AC are biomimetic materials able to directly influence endothelial cells in vitro, which might translate into an improved healing of injured vascularized tissues.
Assuntos
Colágeno/química , Glicosaminoglicanos/química , Ácido Hialurônico/química , Hidrogéis/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Glicosaminoglicanos/metabolismo , Hidrogéis/farmacologia , Microscopia de Fluorescência , Ligação Proteica , Sulfatos/química , Suínos , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
High serum levels of Wnt antagonists are known to be involved in delayed bone defect healing. Pharmaceutically active implant materials that can modulate the micromilieu of bone defects with regard to Wnt antagonists are therefore considered promising to support defect regeneration. In this study, we show the versatility of a macromer based biomaterial platform to systematically optimize covalent surface decoration with high-sulfated glycosaminoglycans (sHA3) for efficient scavenging of Wnt antagonist sclerostin. Film surfaces representing scaffold implants were cross-copolymerized from three-armed biodegradable macromers and glycidylmethacrylate and covalently decorated with various polyetheramine linkers. The impact of linker properties (size, branching) and density on sHA3 functionalization efficiency and scavenging capacities for sclerostin was tested. The copolymerized 2D system allowed for finding an optimal, cytocompatible formulation for sHA3 functionalization. On these optimized sHA3 decorated films, we showed efficient scavenging of Wnt antagonists DKK1 and sclerostin, whereas Wnt agonist Wnt3a remained in the medium of differentiating SaOS-2 and hMSC. Consequently, qualitative and quantitative analysis of hydroxyapatite staining as a measure for osteogenic differentiation revealed superior mineralization on sHA3 materials. In conclusion, we showed how our versatile material platform enables us to efficiently scavenge and inactivate Wnt antagonists from the osteogenic micromilieu. We consider this a promising approach to reduce the negative effects of Wnt antagonists in regeneration of bone defects via sHA3 decorated macromer based macroporous implants.
RESUMO
The aim of this work was to establish improved cultivation conditions for human keratinocytes (HUKORS) and melanocytes (HUMORS) from the outer root sheath (ORS) of human hair follicles for purposes of generating an epidermal graft. To this end, the cells were cultivated on artificial extracellular matrix coatings composed of collagen (COLL) and hyaluronan (HA) with varying sulfation degrees. HUKORS and HUMORS were characterized based on their morphology and proliferation, marker gene expression, protein expression and melanin content in melanocytes. Depending on the sulfation degree, the matrices provided a favorable proliferation environment for HUKORS and improved the balance between proliferation and the exertion of melanotic phenotype (gene expression and melanin content) in HUMORS. Based on the increased gene expression of microphthalmia-associated transcription factor, as well as the downstream-affected melanotic genes premelanosome protein and tyrosinase in HUMORS cultivated on collagen matrices with high-sulfated HA, we assume that sulfated HA enhanced melanotic phenotype either by directly binding the CD44 receptor or by concentrating signaling mediators on site. Being a promising cultivation environment for both HUKORS and HUMORS, collagen matrices with sulfated HA have a potential of significantly improving the development of ORS-based epidermal grafts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1640-1653, 2019.
Assuntos
Matriz Extracelular/química , Folículo Piloso/citologia , Ácido Hialurônico/farmacologia , Queratinócitos/citologia , Melanócitos/citologia , Sulfatos/farmacologia , Adulto , Animais , Biomarcadores/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Hialurônico/química , Queratinócitos/efeitos dos fármacos , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Fenótipo , Poliestirenos/farmacologia , Ratos , Adulto JovemRESUMO
Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72â¯h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings.
Assuntos
Colágeno/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/farmacologia , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Sulfatos/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Colágeno/química , Preparações de Ação Retardada/farmacologia , Epiderme/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Sulfatos/química , Suínos , TermodinâmicaRESUMO
Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.
Assuntos
Glicosaminoglicanos/metabolismo , Ácido Hialurônico/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sítios de Ligação , Sulfatos de Condroitina/farmacologia , Simulação por Computador , Glicosaminoglicanos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Imobilizadas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neovascularização Fisiológica , Fosforilação , Domínios Proteicos , Esferoides Celulares , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. METHODS: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. RESULTS: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. CONCLUSION: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies.