Type 1 diabetes (T1DM) affects one in every 400 children and adolescents in the US. Due to the limitations of exogenous insulin therapy and whole pancreas transplantation, pancreatic islet transplantation has emerged as a promising therapy for T1DM. However, this therapy is severely limited by donor islet availability and poor islet engraftment and function. We engineered an injectable bio-synthetic, polyethylene glycol-maleimide hydrogel to enhance vascularization and engraftment of transplanted pancreatic islets in a mouse model of T1DM. Controlled presentation of VEGF-A and cell adhesive peptides within this engineered material significantly improved the vascularization and function of islets delivered to the small bowel mesentery, a metabolically relevant site for insulin release. Diabetic mice receiving islets transplanted in proteolytically degradable hydrogels incorporating VEGF-A exhibited complete reversal of diabetic hyperglycemia with a 40% reduction in the number of islets required. Furthermore, hydrogel-delivered islets significantly improved weight gain, regulation of a glucose challenge, and intra-islet vascularization and engraftment compared to the clinical standard of islet infusion through the hepatic portal vein. This study establishes a simple biomaterial strategy for islet transplantation to promote enhanced islet engraftment and function.
The ability to efficiently isolate undifferentiated human induced pluripotent stem cells (UD-hiPSCs) as colonies from contaminating non-pluripotent cells is a crucial step in the stem cell field to maintain hiPSC survival, purity, and karyotype stability. Here we demonstrate significant differences in ‘adhesive signature’ among UD-hiPSCs, parental cells, partially reprogrammed cells, and differentiated progeny. The distinct adhesive signature of hiPSCs was exploited to rapidly (~10 min) and efficiently isolate fully reprogrammed bona fide hiPSCs as intact colonies from heterogeneous reprogramming cultures and differentiated progeny using microfluidics. hiPSCs were isolated in a label-free fashion and enriched to > 95–99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells. This rapid and label-free strategy is applicable to isolate UD-hPSCs (hiPSCs, hESCs) from heterogeneous cultures during reprogramming and routine cultures and can be expanded to purify stem cells of specific lineages, such as neurons and cardiomyocytes.
reprogramming; cell adhesion; fibronectin; integrin; focal adhesion; microfluidics
Implant-associated inflammation is a major cause for the reduced performance/lifetime and failure of numerous medical devices. Therefore, the ability to non-invasively and quantitatively monitor implant-associated inflammation is critically important. Here we show that implant-associated inflammation can be imaged via fluorescence imaging using near-infrared hydrocyanine dyes delivered either locally or intravenously in living mice. This imaging strategy allowed quantitative longitudinal monitoring of inflammation by detecting reactive oxygen species (ROS) released by inflammatory cells in response to implanted poly(ethylene terephthalate) (PET) disks or injected poly (lactic-co-glycolic acid) (PLGA) microparticles, and exhibited a strong correlation to conventional analysis of inflammation. Furthermore, modulation of inflammatory responses via controlled release of the anti-inflammatory agent dexamethasone was detected using this sensitive imaging approach. Thus, hydrocyanine-based fluorescence imaging of ROS could serve as a surrogate measure for monitoring implant-associated inflammation as well as evaluating the efficacy of therapeutic approaches to modulate host responses to implanted medical devices.
Biocompatibility; Foreign body response; Inflammation; Free radical; Superoxide
Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.
Biomaterial-associated infections represent a significant clinical problem, and treatment of these microbial infections is becoming troublesome due to the increasing number of antibiotic-resistant strains. Here, we report a naturally functionalized bacterial polyhydroxyalkanoate (PHACOS) with antibacterial properties. We demonstrate that PHACOS selectively and efficiently inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in vivo. This ability has been ascribed to the functionalized side chains containing thioester groups. Significantly less (3.2-fold) biofilm formation of S. aureus was detected on PHACOS compared to biofilms formed on control poly(3-hydroxyoctanoate-co-hydroxyhexanoate) and poly(ethylene terephthalate), but no differences were observed in bacterial adhesion among these polymers. PHACOS elicited minimal cytotoxic and inflammatory effects on murine macrophages and supported normal fibroblast adhesion. In vivo fluorescence imaging demonstrated minimal inflammation and excellent antibacterial activity for PHACOS compared to controls in an in vivo model of implant-associated infection. Additionally, reductions in neutrophils and macrophages in the vicinity of sterile PHACOS compared to sterile PHO implant were observed by immunohistochemistry. Moreover, a similar percentage of inflammatory cells was found in the tissue surrounding sterile PHACOS and S. aureus pre-colonized PHACOS implants, and these levels were significantly lower than S. aureus pre-colonized control polymers. These findings support a contact active surface mode of antibacterial action for PHACOS and establish this functionalized polyhydroxyalkanoate as an infection-resistant biomaterial.
Functionalized polyhydroxyalkanoate; Antimicrobial polymers; Methicillin-resistant Staphylococcus aureus; Biofilm; Biomaterial infection
The repair of large nonunions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells (BMSCs) to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause BMSCs to lose their differentiation ability. To overcome these limitations, we have genetically engineered BMSCs to constitutively overexpress the osteoblast-specific transcription factor Runx2. In the present study, we examined Runx2-modified BMSCs, delivered via polycaprolactone scaffolds loaded with type I collagen meshes, in critical-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds, and empty defects. Runx2 expression in BMSCs accelerated healing of critical-sized defects compared to unmodified BMSCs and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects, which may reduce recovery time and the need for external fixation of critical-sized defects.
Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of bio- and non-biological applications because of its chemical inertness, non-toxicity, ease of handling, and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion, and it has proven difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion, and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.
deformable substrate; fibronectin; cell mechanics; focal adhesion
Inflammatory responses to implanted biomedical devices elicit a foreign body fibrotic reaction that limits device integration and performance in various biomedical applications. We examined chronic inflammatory responses to microgel conformal coatings consisting of thin films of poly(N-isopropylacrylamide) hydrogel microparticles cross-linked with poly(ethylene glycol) diacrylate deposited on poly(ethylene terephthalate) (PET). Unmodified and microgel-coated PET disks were implanted subcutaneously in rats for 4 weeks and explants were analyzed by histology and immunohistochemistry. Microgel coatings reduced chronic inflammation and resulted in a more mature/organized fibrous capsule. Microgel-coated samples exhibited 22% thinner fibrous capsules that contained 40% fewer cells compared to unmodified PET disks. Furthermore, microgel-coated samples contained significantly higher levels of macrophages (40%) than unmodified PET controls. These results demonstrate that microgel coatings reduce chronic inflammation to implanted biomaterials.
foreign body response; macrophage; hydrogel; polyethylene terephthalate; fibrous capsule
Actin-myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum-free cultures. Inhibition of myosin light chain kinase or Rho-kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum-induced enhancements in strengthening. Blebbistatin-induced inhibition of myosin II reduced serum-induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin-null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin-dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum-induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK-dependent, vinculin-containing focal adhesion assembly.
actin-myosin; focal adhesion kinase (FAK); vinculin; contractility
Cell-based therapies represent promising strategies for tissue repair, particularly in cases in which host cells, due to disease, age, or excessive trauma, are unable to repair the defect or deficiency alone, even with additional delivered therapeutics. Current cell therapies fail to address long term engraftment or delivery timing and location and result in modest improvements with long term engraftment rates of less than 1%. In many cell therapy applications, an appropriate carrier must be used to deliver transplanted cells and promote cell engraftment and function for a successful outcome by providing the appropriate microenvironment for the interactions between transplanted and host cells. This review highlights important considerations for engineering the microenvironment for cell delivery and engraftment in tissue repair.
Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression, and function during hMSC commitment along osteogenic, chondrogenic, and adipogenic lineages. Using self-assembled monolayers of ω-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long term functional differentiation of adult stem cells and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.
Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography technique to create 2-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current μ-CP approaches rely on non-specific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a non-fouling background. This technique is limited by insufficient control over ligand density. We present a modified μ-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell-material interactions.
micropatterning; lithography; microcontact printing; self-assembled monolayers
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration.
fibronectin; osseointegration; coating; integrins; biomimetic; implant
Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocytes/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-α, IL-1β, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.
cell adhesion; cytokine; foreign body response; hydrogel; macrophage; polyethylene terephthalate
Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling.
collagen; fibronectin; cell adhesion; focal adhesion; integrin
Synthetic polymer coatings are used extensively in modern medical devices and implants because of their material versatility and processability. These coatings are designed for specific applications by controlling composition and physical and chemical properties, and they can be formed into a variety of complex structures and shapes. However, implantation of these materials into the body elicits a strong inflammatory host response that significantly limits the integration and biological performance of devices. Biomaterial-mediated inflammation is a complex reaction involving protein adsorption, leukocyte recruitment and activation, secretion of inflammatory mediators, and fibrous encapsulation of the implant. Significant research efforts have focused on modifying material properties using various anti-inflammatory polymeric surface coatings to generate more biocompatible implants. This minireview provides a brief background on the events of biomaterial-mediated inflammation and highlights various approaches used for modifying material surfaces to modulate inflammatory responses. These include both passive and active strategies, such as nonfouling surface treatments and delivery of anti-inflammatory agents, respectively. Novel approaches will be needed to extend the in vivo lifetime and performance of devices and reduce the need for multiple implantation surgeries.
biomaterial; polymer; coating; host response; implant; anti-inflammatory
Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.
Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the α5β1-integrin-specific FN fragment FNIII7–10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII7–10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.
Cell–extracellular matrix (ECM) and cell–cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell–cell contact while maintaining constant cell–ECM areas. Bowtie-shaped micropatterned areas (75–1600 µm2) were engineered to either permit or prevent cell–cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell–cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell–cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell–cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell–cell contact regulates early keratinocyte differentiation independently from changes in cell shape.
Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell-cell and cell-matrix interactions. For example, Madin-Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured 3-dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3-D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3-dimensional culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviors as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.
cell adhesion; extracellular matrix; differentiation; laminin; RGD
Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer.
fibroblast; genetic engineering; gene therapy; gene transfer; self-assembly; alkanethiol
Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin conditional knock-out mice, we demonstrate that plasma fibronectin modulates the foreign body response to polyethylene terephthalate discs implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of plasma fibronectin compared to controls. In contrast, deletion of plasma fibronectin did not alter acute leukocyte recruitment to the biomaterial, indicating that plasma fibronectin modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of plasma fibronectin while macrophage numbers were not different, suggesting that plasma fibronectin regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular fibronectin was present in the capsules of both normal and plasma fibronectin-depleted mice, suggesting that cellular fibronectin could not compensate for the loss of plasma fibronectin. These results implicate plasma fibronectin in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.
Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the GFOGER collagen-mimetic peptide, selectively promoting α2β1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone-implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone-implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant coating strategy that enhances bone repair and orthopaedic implant integration.
biomimetic material; cell adhesion; collagen; osseointegration; integrin
Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.
Cells regulate adhesion in response to internally-generated and externally-applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin–integrin α5β1 bond switch the bond from a short-lived state with 1-s lifetime to a long-lived state with 100-s lifetime. We term this phenomenon “cyclic mechanical reinforcement” as the bond strength remembers the history of force application, accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin–integrin α5β1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor–ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.