The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro – Correlation between contraction force and electrophysiology

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential.We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.     

Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.     

In vitro generation of differentiated cardiac myofibers on micropatterned laminin surfaces

Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.     

p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes

Adult mammalian cardiomyocytes are considered terminally differentiated and incapable of proliferation. Consequently, acutely injured mammalian hearts do not regenerate, they scar. Here, we show that adult mammalian cardiomyocytes can divide. One important mechanism used by mammalian cardiomyocytes to control cell cycle is p38 MAP kinase activity. p38 regulates expression of genes required for mitosis in cardiomyocytes, including cyclin A and cyclin B. p38 activity is inversely correlated with cardiac growth during development, and its overexpression blocks fetal cardiomyocyte proliferation. Activation of p38 in vivo by MKK3bE reduces BrdU incorporation in fetal cardiomyocytes by 17.6%. In contrast, cardiac-specific p38alpha knockout mice show a 92.3% increase in neonatal cardiomyocyte mitoses. Furthermore, inhibition of p38 in adult cardiomyocytes promotes cytokinesis. Finally, mitosis in adult cardiomyocytes is associated with transient dedifferentiation of the contractile apparatus. Our findings establish p38 as a key negative regulator of cardiomyocyte proliferation and indicate that adult cardiomyocytes can divide.     

Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.     

The influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth muscle cell phenotype

This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary artery SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle α-actin, calponin, and SM-22α. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype.     

Alginate type and RGD density control myoblast phenotype

Alginates are being increasingly used for cell encapsulation and tissue engineering applications; however, these materials cannot specifically interact with mammalian cells. We have covalently modified alginates of varying monomeric ratio with RGD-containing cell adhesion ligands using carbodiimide chemistry to initiate cell adhesion to these polymers. We hypothesized that we could control the function of cells adherent to RGD-modified alginate hydrogels by varying alginate polymer type and cell adhesion ligand density, and we have addressed this possibility by studying the proliferation and differentiation of C2C12 skeletal myoblasts adherent to these materials. RGD density on alginates of varying monomeric ratio could be controlled over several orders of magnitude, creating a range of surface densities from 1-100 fmol/cm(2). Myoblast adhesion to these materials was specific to the RGD ligand, because adhesion could be competed away with soluble RGD in a dose-dependent manner. Myoblast proliferation and differentiation could be regulated by varying the alginate monomeric ratio and the density of RGD ligands at the substrate surface, and specific combinations of alginate type and RGD density were required to obtain efficient myoblast differentiation on these materials.     

Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering

Poly(ethylene glycol) (PEG) hydrogels were investigated as encapsulation matrices for osteoblasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of rat calvarial osteoblasts. When attached to hydrogel surfaces, the density and area of osteoblasts attached were dramatically different between modified and unmodified hydrogels. A concentration dependence of RGD groups was observed, with increased osteoblast attachment and spreading with higher RGD concentrations, and cytoskeleton organization was seen with only the highest peptide density. A majority of the osteoblasts survived the photoencapsulation process when gels were formed with 10% macromer, but a decrease in osteoblast viability of approximately 25% and 38% was seen after 1 day of in vitro culture when the macromer concentration was increased to 20 and 30wt%, respectively. There was no statistical difference in cell viability when peptides were added to the network. Finally, mineral deposits were seen in all hydrogels after 4 weeks of in vitro culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network.     

The functional properties of nephronectin: an adhesion molecule for cardiac tissue engineering

Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development, and studies suggest that it plays an important role in regulating myocyte proliferation. Here, the effects of fetal, neonatal and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro are studied. At 24 h, overall cell attachment was lowest on fetal ECM; however, ∼80% of the cells were cardiomyocytes, while many non-myocytes attached to older ECM and poly-l-lysine controls. After 5 days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared with other substrates at 5 days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.     

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.     

Formation of three-dimensional fetal myocardial tissue cultures from rat for long-term cultivation.

Three-dimensional cardiomyocyte cultures offer new possibilities for the analysis of cardiac cell differentiation, spatial cellular arrangement, and time-specific gene expression in a tissue-like environment. We present a new method for generating homogenous and robust cardiomyocyte tissue cultures with good long-term viability. Ventricular heart cells prepared from fetal rats at embryonic day 13 were cultured in a scaffold-free two-step process. To optimize the cell culture model, several digestion protocols and culture conditions were tested. After digestion of fetal cardiac ventricles, the resultant cell suspension of isolated cardiocytes was shaken to initialize cell aggregate formation. In the second step, these three-dimensional cell aggregates were transferred onto a microporous membrane to allow further microstructure formation. Autonomously beating cultures possessed more than 25 cell layers and a homogenous distribution of cardiomyocytes without central necrosis after 8 weeks in vitro. The cardiomyocytes showed contractile elements, desmosomes, and gap junctions analyzed by immunohistochemistry and electron microscopy. The beat frequency could be modulated by adrenergic agonist and antagonist. Adenoviral green fluorescent protein transfer into cardiomyocytes was possible and highly effective. This three-dimensional tissue model proved to be useful for studying cell-cell interactions and cell differentiation processes in a three-dimensional cell arrangement.     

The effects of heparin releasing hydrogels on vascular smooth muscle cell phenotype

Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds were engineered to promote contractile smooth muscle cell (SMC) phenotype via controlled release of heparin. The scaffold design was evaluated by quantifying the effects of free heparin on SMC phenotype, engineering hydrogels to provide controlled release of heparin, and synthesizing cell-adhesive, heparin releasing hydrogels to promote contractile SMC phenotype. Heparin inhibited SMC proliferation and up-regulated expression of contractile SMC phenotype markers, including smooth muscle α-actin, calponin, and SM-22α, in a dose-dependent fashion (6 μg/ml to 3.2 mg/ml). Heparin release from PEGDA hydrogels was controlled by altering PEGDA molecular weight (MW 1000–6000) and concentration at polymerization (10–30% w/w), yielding release profiles ranging from hours to weeks in duration. Heparin released from PEGDA gels, formulated for optimized heparin loading and release kinetics (30% w/w PEGDA, MW 3000), stimulated SMCs to up-regulate contractile marker mRNA. A cell-instructive scaffold construct was prepared by polymerizing a thin hydrogel film, with pendant RGD peptides for cell attachment, over the optimized hydrogel depots. SMCs seeded on these constructs had elevated levels of contractile marker mRNA after 3 d of culture compared with SMCs on control constructs. These results indicate that RGD-modified, heparin releasing PEGDA gels can act as cell-instructive scaffolds that promote contractile SMC phenotype.     

Polyurethane films seeded with embryonic stem cell-derived cardiomyocytes for use in cardiac tissue engineering applications

Cardiomyocytes are terminally differentiated cells and therefore unable to regenerate heart tissue after infarction. The successful engraftment of various cell types resulting in improved cardiac function has been reported, however methods for improving the delivery of donor cells to the infarct site still need to be developed. The use of bioengineered cardiac grafts has been suggested to replace infarcted myocardium and enhance cardiac function. In this study, we cultured embryonic stem (ES) cell-derived cardiomyocytes on thin polyurethane (PU) films. The films were coated with gelatin, laminin or collagen IV in order to encourage cell adhesion. Constructs were examined for 30 days after seeding. Cells cultured on laminin and collagen IV, exhibited preferential attachment, as assessed by cellular counts, and viability assays. These surfaces also resulted in a greater number of contracting films compared to controls. A degradable elastomer seeded with embryonic stem cell-derived cardiomyocytes may hold potential for the repair of damaged heart tissue.     

Mechanisms underlying the attachment and spreading of human osteoblasts: From transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.     

Control of cell adhesion on poly(methyl methacrylate)

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG-PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.