Fabrication Of Cell-selective Gradient Biomaterials And Their Manipulation Of Cell Migration

Cell migration plays a key role in many biological and physiological processes,such as embryonic development,angiogenesis,tumor metastasis and immune response.The cells in vivo can migrate in response to gradients of stimuli including both biochemical and biophysical signals.Selective cell migration is required for many important physiological processes.Undesired cell migration can cause disease or improper regeneration of tissues.Thus,the surfaces with gradient cues in vitro could guide selective and directional cell migration and provide some guidance in design of advanced biomaterials.An amino group density gradient was fabricated on the polycaprolactone(PCL)membrane surface through gradient aminolysis,which was further transferred-into a gelatin density gradient via covalent coupling by glutaraldehyde.The adhesion,orientation of ECs,migration of ECs,SMCs and FIBs on the PCL membrane with the gelatin gradient were studied.The adhesion of ECs was improved on the gelatin-immobilized surface regardless of the distribution manner(uniform or gradient)of gelatin.ECs also showed significantly higher mobility on the gelatin gradient and better directional migration i.e.90%of cells moved towards higher gelatin density with aspeed of 11 μm/h.SMCs and FIBs also showed directional cell migration with a preferential direction.However,the migration speed was not promoted.ECs,SMCs and FIBs showed highly directional migration on the gelatin gradient with a proper gelatin density.However,on this simple gelatin density gradient,no selectivity to different kinds of cells could be achieved.Next,a more simple and convenient method was used to build a cell-selective surface on biomaterials.PCL film was coated with a thin polydopamine(PDA)layer on which methacrylate-functionalized hyaluronic acid(MA-HA)molecules were covalently immobilized.The thiol(cysteine)-functionalized REDV peptides were further clicked onto the MA-HA layer via a thiol-ene reaction in a gradient manner,which was manipulated by continuous injection of the REDV peptide solution into the container of MA-HA-modified substrate.The REDV gradient on the MA-HA layer could selectively enhance the adhesion,mobility and directional migration of ECs toward the direction of higher REDV density.The cell number of ECs was 2.5-fold to SMCs on the REDV gradient.The ECs moved with a preferential direction(86%)towards higher REDV density with a speed of 14 μm/h.In contrast,the adhesion of SMCs was significantly weakened on the gradient surface compared with that on TCPS,though the mobility was not obviously influenced.ECs sheet could migrate towards higher REDV peptide density and the distance was 3-fold compared to that on uniform surface.Adventitia fibrosis is mainly caused by undesired adhesion,migration and over proliferation of fibroblasts.In such cases,selective cell adhesion and migration of smooth muscle cells(SMCs)over fibroblasts(FIBs)is desirably required for cardiovascular regeneration.Cell specific peptides could recognize the receptor on the cell surface,hence a cell resistant layer combined with cell specific peptides could realize cell selectivity.Val-Ala-Pro-Gly(VAPG),a peptide sequence derived from elastin,can be used as a biospecific cell adhesion ligand for SMCs.A uniform cell-resistant layer of poly(ethylene glycol)was fixed onto the glass slides,and then VAPG peptides were immobilized via click chemistry.Cell number of SMCs was 2 folds higher than FIBs with VAPG peptide grafted.SMCs migrated faster(22 μm/h)with good directionality(84%)towards the high VAPG peptide density region,while FIBs showed a random migration with a much slower rate.Mimicking the structures and functions of extracellular matrix(ECM)is an important guidance for biomaterials design.A matrix simulating the structure and function of ECM provides a better model system of physiological environment for cell migration study.Matrix metalloproteinases(MMPs)is a protease family extensively involved in tissue development and remodeling.A MMP-2 sensitive peptide with two-SH groups was chosen as a cell-responsive crosslinker.The obtained MA-HA was used as the main structural component of the hydrogel and crosslinked by the peptides through Michael-addition.U937 cells(a mononuclear cell)were used as a signal source and MMP producer to induce the SMCs invasion to mimic the adaptive tissue regeneration process in vivo.In vitro results showed that SMCs could migrate deeply to 120μm in MMP-2 sensitive hydrogel with the existence of U937 cells.Further experiments proved that SMCs cell invasion depended on the deformation or degradation of MMP sensitive hydrogel rather than chemokines signals.In vivo results showed different cell invasion behaviors into the hydrogels with different degradability,and found the cell invaded deepest into the MMP sensitive hydrogel at 21 days with a distance of 70 μm.In this study,cell-selective surfaces were built by using cell-resistant layers and cell-specific peptides,which were further created on a biodegradable PCL film with the assistance of a polydopamine adhesive layer.This design and technology could be transferred to other biomaterials surface,highlighting a new perspective on designing advanced and highly functional biomaterials for targeted tissue regeneration.Furthermore,a hydrogel that mimic native three dimensional ECM was fabricated,and cell migration in three dimensional was investigated,paving a new insight into mechanism of biomaterials design.