Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair.Collagen biomaterials that can host endothelial c...Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair.Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues.Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions,followed by chemical cross-linking which maintains their stability.However,cross-linking scaffolds renders their biological activity suboptimal for many cell types,including human umbilical vein endothelial cells(HUVECs),by inhibiting cell–collagen interactions.Here,we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides(THPs).These are ligands for collagen-binding cell-surface receptors(integrins or discoidin domain receptors)or secreted proteins(SPARC and von Willebrand factor).THPs enhanced HUVEC adhesion,spreading and proliferation on 2D collagen films.THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days.This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.展开更多
Collagen-based biomaterials are used widely as tissue engineering scaffolds because of their excellent bioactivity and their similarity to the natural ECM.The regeneration of healthy bone tissue requires simultaneous ...Collagen-based biomaterials are used widely as tissue engineering scaffolds because of their excellent bioactivity and their similarity to the natural ECM.The regeneration of healthy bone tissue requires simultaneous support for both osteoblasts and,where angiogenesis is intended,endothelial cells.Hence it is important to tailor carefully the biochemical and structural characteristics of the scaffold to suit the needs of each cell type.This work describes for the first time a systematic study to gain insight into the cell type-specific response of primary human osteoblast(hOBs)and human dermal microvascular endothelial cells(HDMECs)to insoluble collagen-based biomaterials.The behaviour was evaluated on both 2D films and 3D scaffolds,produced using freeze-drying.The collagen was cross-linked at various EDC/NHS concentrations and mono-cultured with hOBs and HDMECs to assess the effect of architectural features and scaffold stabilization on cell behaviour.It was observed that 3D scaffolds cross-linked at 30%of the standard conditions in literature offered an optimal combination of mechanical stiffness and cellular response for both cell types,although endothelial cells were more sensitive to the degree of cross-linking than hOBs.Architectural features have a time-dependent impact on the cell migration profile,with alignment being the most influential parameter overall.展开更多
X-ray micro-computed tomography(μ-CT)can be used to provide both qualitative and quantitative information on the structure of three-dimensional(3D)bioactive scaffolds.When performed in a dry state,μ-CT accurately re...X-ray micro-computed tomography(μ-CT)can be used to provide both qualitative and quantitative information on the structure of three-dimensional(3D)bioactive scaffolds.When performed in a dry state,μ-CT accurately reflects the structure of collagen-based scaffolds,but imaging in a wet state offers challenges with radiolucency.Here we have used phosphotungstic acid(PTA)as a contrast agent to visualise fully hydrated collagen scaffolds in a physiologically relevant environment.A systematic investigation was performed to understand the effects of PTA on the results of μ-CT imaging by varying sample processing variables such as crosslinking density,hydration medium and staining duration.Immersing samples in 0.3% PTA solution overnight completely stained the samples and the treatment provided a successful route forμ-CT analysis of crosslinked samples.However,significant structural artefacts were observed for samples which were either non-crosslinked or had low levels of crosslinking,which had a heterogeneous interior architecture with collapsed pores at the scaffold periphery.This work highlights the importance of optimising the choice of processing and staining conditions to ensure accurate visualisation for hydrated 3D collagen scaffolds in an aqueous medium.展开更多
基金supported in the Department of Biochemistry by grants from British Heart Foundation(SP/15/7/31561,FS/15/20/31335 , RG/15/4/31268)funded by EPSRC fellowship(EP/N019938/1)+2 种基金supported by BHF grants FS/18/46/33663 , RM/17/2/33380funding from the Wellcome Trust and MRC to the Wellcome-Medical Research Council Cambridge Stem Cell InstituteConfocal imaging of scaffolds was performed at the Microscopy Core facility at the Sainsbury Laboratory,Cambridge,UK and was supported by the Gatsby Charitable Foundation.
文摘Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair.Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues.Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions,followed by chemical cross-linking which maintains their stability.However,cross-linking scaffolds renders their biological activity suboptimal for many cell types,including human umbilical vein endothelial cells(HUVECs),by inhibiting cell–collagen interactions.Here,we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides(THPs).These are ligands for collagen-binding cell-surface receptors(integrins or discoidin domain receptors)or secreted proteins(SPARC and von Willebrand factor).THPs enhanced HUVEC adhesion,spreading and proliferation on 2D collagen films.THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days.This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.
基金supported by the Engineering and Physical Sciences Research Council(EPSRC)and Geistlich Pharma AG.R.E.C.and S.M.B.acknowledge funding from an EPSRC Professorial Fellowship(EP/N019938/1)which also supported the research undertaken by D.V.B.D.V.B.would like to thank the Cambridge Royce facilities grant EP/P024947/1 and Sir Henry Royce Institute-recurrent grant EP/R00661X/1.
文摘Collagen-based biomaterials are used widely as tissue engineering scaffolds because of their excellent bioactivity and their similarity to the natural ECM.The regeneration of healthy bone tissue requires simultaneous support for both osteoblasts and,where angiogenesis is intended,endothelial cells.Hence it is important to tailor carefully the biochemical and structural characteristics of the scaffold to suit the needs of each cell type.This work describes for the first time a systematic study to gain insight into the cell type-specific response of primary human osteoblast(hOBs)and human dermal microvascular endothelial cells(HDMECs)to insoluble collagen-based biomaterials.The behaviour was evaluated on both 2D films and 3D scaffolds,produced using freeze-drying.The collagen was cross-linked at various EDC/NHS concentrations and mono-cultured with hOBs and HDMECs to assess the effect of architectural features and scaffold stabilization on cell behaviour.It was observed that 3D scaffolds cross-linked at 30%of the standard conditions in literature offered an optimal combination of mechanical stiffness and cellular response for both cell types,although endothelial cells were more sensitive to the degree of cross-linking than hOBs.Architectural features have a time-dependent impact on the cell migration profile,with alignment being the most influential parameter overall.
基金supported by Engineering and Physical Sciences Research Council(EP/N019938/1).
文摘X-ray micro-computed tomography(μ-CT)can be used to provide both qualitative and quantitative information on the structure of three-dimensional(3D)bioactive scaffolds.When performed in a dry state,μ-CT accurately reflects the structure of collagen-based scaffolds,but imaging in a wet state offers challenges with radiolucency.Here we have used phosphotungstic acid(PTA)as a contrast agent to visualise fully hydrated collagen scaffolds in a physiologically relevant environment.A systematic investigation was performed to understand the effects of PTA on the results of μ-CT imaging by varying sample processing variables such as crosslinking density,hydration medium and staining duration.Immersing samples in 0.3% PTA solution overnight completely stained the samples and the treatment provided a successful route forμ-CT analysis of crosslinked samples.However,significant structural artefacts were observed for samples which were either non-crosslinked or had low levels of crosslinking,which had a heterogeneous interior architecture with collapsed pores at the scaffold periphery.This work highlights the importance of optimising the choice of processing and staining conditions to ensure accurate visualisation for hydrated 3D collagen scaffolds in an aqueous medium.
