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Self-assembling Peptides: From Bio-inspired Materials to Bone RegenerationCenter for Biomedical Engineering, NE47-383, Biological Engineering Division, Massachusetts Institute of Technology, 500 Technology Sq., Cambridge, MA 02139, USA; semino{at}mit.edu
In recent years, the development of new biomaterials with specifications for tissue and organ functional requirements—such as proper biological, structural, and biomechanical properties as well as designed control for biodegradation and therapeutic drug-release capacity—is the main aim of many academic and industrial programs. Hence, the concept of molecular self-assembly is the driving force for the development of new biomaterials that support the growth and functional differentiation of cells and tissues in a controlled manner. The discovery, properties, and development of self-assembling peptides to be used as three-dimensional (3D) scaffolds based on their similarity (in structure and mechanical features) to extracellular matrices are described. Self-assembling peptides can be used for in vitro applications for cell 3D culture as well as in vivo for tissue regeneration such as bone and optical nerve repair, as well as for drug delivery of mediators to improve therapy, as in the case of myocardial infarction. Finally, the use of self-assembling materials in combination with a bioengineering platform is proposed to assist functional bone regeneration in cases of larger bone defects, including exposed fractures due to trauma and spinal disorders dealing with high loadings, as well as replacement of big bone structures due to tumors.
Key Words: periodontal • regeneration • oral • tissue repair • healing • growth • cell culture • osseous
The self-assembly of biomolecules is a well-known phenomenon in biology, from DNA self-complementary double-helix annealing, through protein aggregation, and lipid membrane development, as well as polysaccharide interaction. Self-assembly in the most basic form consists of the spontaneous organization of molecules under thermodynamic equilibrium conditions into structurally stable arrangements by the driving force of non-covalent interactions, including hydrogen bonds, ionic bonds, electrostatic interaction, van der Waals interaction, etc. The direct consequence of the self-assembling process in biological systems is the formation of highly organized and stable macromolecular entities with specific functions, such as informational (i.e., DNA, RNA), structural and mechanical (i.e., cellular cytoskeleton), as well as instructive (i.e., basement membrane). For instance, basement membrane (BM) assembly is an impressive example of different extracellular matrix proteins undergoing self-organization (mainly collagen type IV, laminin I, and nidogen) to develop a lattice of about 150 nm thick that not only structurally separates two different tissue compartments, but also modulates differentiation and proliferation by providing instructive information to cells next to each boundary (Beck et al., 1990; Engel, 1992; Yurchenco and O’Rear, 1994; Kreis and Vale, 1999). For example, the skin basement membrane that connects the endodermis with the ectodermis is the classic example of the largest continuous supramolecular structure residing underneath our skin. The same self-assembling property also occurs in proteins forming the extracellular matrix of connective tissues, such as collagens, laminins, and fibronectins (Charonis et al., 1985; Beck et al., 1990; Engel, 1992; Yurchenco and O’Rear, 1994). Because of their capacity to form gels, some of these molecules have been extensively used as natural biomaterials for study of the behavior of mammalian cells outside the body in a variety of culture systems that mimic the extracellular milieu. The most common are collagen type I as well as components of the basement membrane (MatrigelTM, BD Biosciences, San Jose, CA, USA) that form hydrogel scaffolds with good properties for cell culture.
In recent years, the concept of molecular self-assembly has become the driving force for the development of new biomaterials (Ball, 1994). Therefore, scientists have identified the key parts of molecules—mainly proteins—responsible for the self-assembling process, to dissect and develop simpler systems. Interestingly, early research in chemical evolution revealed the tendency of copolypeptides with alternating hydrophilic and hydrophobic residues to form water-soluble β-sheet structures by self-assembling in the presence of salts (Peggion et al., 1972; Rippon et al., 1973; Seipke et al., 1974; Brack and Orgel, 1975). These studies have shown that alternating amphiphilic-peptide sequences—such as poly-(Lys-Phe), poly-(Glu-Ala), poly-(Tyr-Glu), poly-(Val-Lys), poly-(Lys-Leu), and others—present β-sheet secondary structures and aggregate, depending on salt concentration and pH (Peggion et al., 1972; Rippon et al., 1973; Seipke et al., 1974; Brack and Orgel, 1975). Almost two decades later, new studies in peptide self-assembly were carried out with the aim of developing new materials. These were focused on a short 24-residue peptide motif (K24) derived from a transmembrane domain of the protein IsK (Aggeli et al., 1996) (Table 1
The peptide RAD16-I self-assembling process is used to illustrate the molecular self-assembling process that ends in the development of a nanofiber network (Fig. 1  ). The self-assembling peptides of this class are good candidates to create artificial cell niches, because their nanoscale network and biomechanical properties are similar to those of natural extracellular matrices (Semino, 2003). Hence, the first biomimetic level, the similarity in structural features with extracellular matrices, has been partly accomplished by these new biomaterials. Extensive data have been published on the characterization of the rheological properties of SAP (Zhang et al., 1994; Aggeli et al., 1997; Leon et al., 1998; Caplan et al., 2002). For instance, the stiffness of RAD16-I and RAD16-II can be controlled by peptide concentration. Interestingly, collagen I gels at these concentrations present similar stiffness values, suggesting that both materials share similar mechanical properties. Thus, cells could "sense" similar stiffness values and respond similarly. In addition, structural similarities between collagen I and RAD16-I gels have been observed (Fig. 1d). In this case, electron microscopy (quick-freeze) of 3 mg/mL of collagen I (Fig. 1d, left) and 2.4 mg/mL of RAD16-I (Fig. 1d, right) has been performed. Similar structural features can be appreciated. Although their nanofiber architecture is not exactly the same, it is also true that, from the "point of view" of the cells, both are 3D environments.
In particular, RAD16-I has been frequently used to culture cells in the laboratory because of its nanostructural and biomechanical properties, becoming commercially available under the name of PuraMatrixTM (3DM, Inc., Cambridge, MA, USA) (Fig. 1  ). Its amino acid composition (R, arginine; A, alanine; D, aspartic acid) makes it very stable in water solutions as well as easy to synthesize, and it can be formulated in water up to concentrations of 5% w/v (50 mg/mL), maintaining its liquid properties. It is stable at room temperature for long period of time, which makes it very convenient for storage and distribution purposes. When desired, it can be easily turned into gels by an increase in the ionic strength (i.e., in physiological solutions) or adjustment of the pH to neutrality (Fig. 1). Also, cells can be easily encapsulated into the nanofiber network in a truly three-dimensional (3D) environment. For these reasons, this class of biomaterial scaffold has often been used to promote growth and proliferation of a variety of cell types, including chondrocytes, hepatocytes, endothelial cells, osteoblasts, and neuronal cells, as well as embryonic and somatic stem cells (Kisiday et al., 2002; Semino et al., 2003, 2004; Narmoneva et al., 2004; Bokhari et al., 2005; Genové et al., 2005; Garreta et al., 2006). In particular, it was demonstrated that only when primary mouse embryonic fibroblasts (MEF) were cultured in this three-dimensional (3D) system (PuraMatrixTM) did they show up-regulation of osteopontin (OPN) as well as two metalloproteinases, MMP-2 and MMP-9, characteristic of an embryonic regenerative system (Garreta et al., 2006). Interestingly, only osteo-induced mouse embryonic fibroblast 3D cultures were able to develop mineralized matrix, as evidenced by von Kossa staining but not 2D cultures, as well as by 3D non-induced cultures. In addition, the system produced collagen type I and up-regulated the expression of the transcription factor Runx2, suggesting that the cells acquired an osteoblast-like phenotype (Garreta et al., 2006). Since the RAD16-I peptide scaffold does not contain any specific peptide-signaling motif, this environment can be defined as "non-instructive" from the point of view of cell receptor recognition/activation (either integrin-or growth-factor-like receptor), suggesting that the 3D environment per se presents intrinsic properties that could promote different cellular responses. These new emerging properties are in fact many. The most significant are that cells can change shape, migrate, elongate, contract, and extend processes in 3D. In addition, they can create 3D networks (cell-cell interactions that therefore could contract the matrix), form 3D clusters, and create molecular gradients. Mechanically the cells could experience different matrix stiffnesses and deformations. Additionally, other groups have shown previously that mesenchymal cells from bone marrow origin, mouse embryonic stem cells as well as mouse embryonic fibroblasts, can differentiate into cartilage-, fat-, and bone-like tissue, but strictly under specific inductive media conditions (Stein and Lian, 1993; Lengner et al., 2004). Thus, self-assembling peptides provide a microenvironment with particular properties that favor the culture of mature or primitive cell types with diverse possibilities: cell maintenance, proliferation, or differentiation (Kisiday et al., 2002; Bokhari et al., 2005; Garreta et al., 2006).
We have learned the importance of characterizing and evaluating the self-assembling peptides in vitro to estimate their potential use in vivo. Recently, some distinct studies carried out in vivo have demonstrated that self-assembling peptides present some advantages over other biomedical platforms (Table 2 ). For instance, RAD16-I applied in small bone defects (3 mm) in mice calvaria promoted bone regeneration by inducing the expression of bone-related genes such as alkaline phosphatase, Runx2, and Osterix in adjacent tissue cells (Misawa et al., 2006). In comparative experiments, where either RAD16-I or MatrigelTM was injected independently in similar bone defects, x-ray radiographs indicated the formation of bony bridges with favorable development of mature bone tissue. Moreover, the newly formed tissue presented cortical bone with medullary cavities in the case of RAD16-I, but not with MatrigelTM (Misawa et al., 2006). Consequently, the strength of the regenerated bone was higher in RAD16-I than in MatrigelTM, at least for this small defect model. In another example, a self-assembling peptide similar to DN1, P11-4 (Table 1), was used in skeletal tissue engineering for the treatment of dental caries-like lesions. In this study, low-viscosity peptide solutions were injected into caries-like lesions in enamel in ex vivo simulated intra-oral conditions, to promote hydroxyapatite nucleation (Kirkham et al., 2007). Interestingly, the peptide treatment increased mineralization of the lesions by inducing hydroxyapatite nucleation de novo as well as by inhibiting demineralization (Table 2). Although enamel remineralization was demonstrated when the caries-like lesions were treated with P11-4 peptide solutions, the exact mechanisms triggering this process are not yet known. These investigators speculated that the peptide solution would be expected to form a fibril network within the pores of the lesion, where the anionic groups of the side-chains could attract calcium, inducing de novo precipitation of the respective phosphate salts (Kirkham et al., 2007). These examples indicate that self-assembling peptides are promising osteoconductive materials for future applications in bone-tissue regeneration.
In a third relevant publication, the use of RAD16-I has been described for brain repair and axon regeneration in a functional experimental model of the optic track in the hamster, where the animal’s vision returned after acute injury (Ellis-Behnke et al., 2006a). There are several important barriers to be overcome to achieve axonal regeneration after injury in the central nervous system (CNS), including scar tissue formation, gaps in nervous tissue, failure of mature neurons to initiate axon regrowth, and the presence of factors that inhibit axon growth in mature mammalian CNS. The use of self-assembling peptides allowed for the creation of a permissive environment for axonal extension by means of a synthetic biological nanofiber network material that connected the two faces of the lesion, allowing movement of cells and processes into the scaffold and, at the same time, preventing the scar formation that normally occurs in early stages of CNS injury. As a consequence, by reducing or largely overcoming the first two obstacles in axonal regeneration (scar formation and gap in the nervous tissue), the investigators demonstrated the possibility of reconnecting disconnected parts of the CNS—in particular, the optic nerve—after trauma (Ellis-Behnke et al., 2006a) (Table 2 ).
In a fourth case, self-assembling peptides were used for the delivery of insulin-like growth factor 1 (IGF-1), a known factor that increases cardiac stem cell number and growth, to improve cell therapy in myocardial infarction (Davis et al., 2006). The elegant strategy in this case was to develop a prolonged delivery system consisting of a self-assembling peptide nanofiber (peptide RAD16-II, AcN-RARADADARARA DADA-CNH2) and a "biotin sandwich" approach. Basically, the peptide nanofiber contains biotin groups extending outside of the nanofiber that specifically bind biotinylated IGF-1 via tetravalent streptavidin working as a bridge molecule. This process does not interfere with self-assembling and nanofiber network development. The system allowed for the specific and highly controlled delivery of IGF-1 to the local myocardial environment, providing sustained delivery of the growth factor for 28 days and promoting improvement in the therapy (Davis et al., 2006). The treatment decreased caspase-3 cleavage by 28% and increased the myocyte cross-section area by 25% (cell proliferation). Moreover, cell therapy with the peptide nanofiber IGF-1 delivery system was tested, indicating that systolic function was improved after an experimental myocardial infarction, clearly demonstrating that the developed bioengineering platform can substantially improve cellular therapies (Table 2
Structural, biophysical, and biomechanical similarities to extracellular matrices—such as true three-dimensionality (nanofiber network), diffusion coefficient, and matrix deformation, respectively—are good features to have in a biomaterial. The next level in biomimetic accomplishment is to develop a new generation of biomaterials that carry instructive capacity for cells—in other words, with specific binding as well as signaling ability. In particular, the extracellular matrix can regulate specific cell signaling in an extensive spectrum of biological processes. This section will focus on how it could be possible, with rational functionalized biomaterials, to regulate two main processes related to bone tissue development: angiogenesis (the generation of a new vasculature from an existing blood vessel) and biomineralization. A good place to learn about signaling capacity from a protein is the basement membrane (BM), a three-dimensional network composed mainly of laminins and collagens (Beck et al., 1990; Engel, 1992; Yurchenco and O’Rear, 1994; Kreis and Vale, 1999). For instance, it has been well-established that the maintenance and function of endothelial cells (the cells covering the internal walls of arteries and veins) are critically influenced by their interaction with the basement membrane (Kubota et al., 1988; Grant et al., 1989; Sakamoto et al., 1991; Bell et al., 2001). Laminin 1 is mainly exposed to the lumen of the walls in intimate contact with the endothelial cells, while collagen IV, as well as collagen I and fibronectin, are more internally located in the basement membrane structure in contact with smooth-muscle cells. Interestingly, sequences present in laminin 1 are anti-angiogenic and promote endothelial monolayer development (but not sprouting), maintaining the blood vessel integrity. Instead, the protein sequences present in collagen IV and collagen I are pro-angiogenic, suggesting that when angiogenic factors diffuse to the vessel from the surrounding tissue (i.e., a growth factor like VEGF, produced by a tumor) and reach the endothelial monolayer, they promote the secretion of proteases (membrane-anchored metalloproteinases, or MMPs). These MMPs degrade the laminin 1 layer, exposing new binding sites from collagens to endothelial cell receptors involved in adhesion and migration, creating a positive cascade of events that promote angiogenesis (Stupack and Cheresh, 2002).
Two sequences present in the laminin-1 β1 chain, YIGSR and RYVVLPR, have been shown to promote cell adhesion, cell migration, and endothelial cell monolayer development and the inhibition of angiogenesis (Iwamoto et al., 1987; Kleinman et al., 1989; Skubitz et al., 1990; Sakamoto et al., 1991). In addition, type IV collagen, the major component of the basement membrane, forms a network structure that involves its interaction with other components of the basement membrane, including laminin, nidogen, and heparan sulphate proteoglycan (Charonis et al., 1985; Paulsson et al., 1987; Poschl et al., 1996). The peptide TAGSCLRKFSTM, present in the
The peptide scaffold RAD16-I was also tailor-made with laminin motifs, to develop a basement membrane analog that enhances endothelial cell maintenance and function in vitro. Investigators obtained new peptide scaffolds by extending RAD16-I at the amino terminal by direct solid-phase synthesis with three short peptide sequence motifs present in three main components of the extracellular matrix, including YIGSR and RYVVLPR from laminin 1, TAGSCLRKFSTM from collagen IV, and RGD from fibronectin (Genové et al., 2005). Therefore, the generic modified peptide obtained (AcN-X -GG-[RADA]4-CONH2), where X corresponds to each sequence motif, represented a new type of functionalized and biologically active nanofiber scaffold. The molecular model of the functionalized nanofiber obtained after self-assembling peptide RAD16-I (90%) was blended with YIGSR-GG-RAD16-I indicates that the minimum recognition domain for the 67-kDa laminin receptor can properly bind at the nanolevel scale (Fig. 2
Since collagen I provides a good angiogenic environment, promoting capillary morphogenesis in the presence of VEGF (Fig. 3B ), it is therefore feasible to prepare a new series of self-assembling peptides carrying functional peptide motifs that activate angiogenic responses in endothelial cells. Hence, this could be used as a strategy to design two new sets of self-assembling peptide scaffolds, one that prevents angiogenesis (i.e., to promote non-vascularized tissue development, such as cartilage, skin, or cornea) and another that supports it (i.e., to promote vascularized tissue development, such as liver, muscle, or bone). Another kind of matrix function, which does not require direct cell interaction and instruction, but instead will modulate the establishment of a proper cell microenvironment, is the process of matrix mineralization. The process is naturally induced in the bone by extracellular matrix proteins that provide adequate conditions for ordered crystals of calcium hydroxyapatite [HAP, generically Ca10(PO4)6(OH)2] nucleation and development. The components in the extracellular matrix responsible for the biomineralization process are collagenous and non-collagenous proteins containing bioactive motifs that have been used for the design of self-assembling peptides with enhanced activity of promoting HAP crystal growth. The bone-like tissue of the teeth, dentin, is basically a mineralized matrix composed of proteins such as collagens, osteopontin (Hunter et al., 1994), bone sialoprotein (Chen et al., 1992), dentin sialoprotein (Butler, 1998), and dentin phosphoprotein (George et al., 1993). For instance, dentin phosphoprotein is highly associated with collagens and may be responsible for most of the dental mineralization process. This matrix contains more than 200 potential phosphorylation residues, mainly located in serine-rich domains (in the form of phosphoserine) with β-sheet configurations containing an extensive negatively charged surface that acts as a Ca2+ attracter, promoting HAP crystal development. Interestingly, the β-sheet configuration provides a planar surface where the distance between two phosphoserine side-changes (6.6–6.9 Å) is around the same as that between two Ca2+ atoms of HAP, therefore promoting mineralization by providing zones with a degree of surface match (epitaxy) favoring both crystal nucleation as well as their orientation (Addadi et al., 2001). In particular, dentin matrix protein 1 (DMP1) is an acidic phosphoprotein present in bone and dentin and is highly involved in biomineralization. The protein undergoes intermolecular bridging (or self-assembling) in the presence of Ca2+, forming aggregates where apatite crystallization is initiated (He et al., 2003). Moreover, it was demonstrated that two acidic domains of DMP1 (ESQES and QESQSEQDS) have the capacity of self-assembling developing β-sheet nanofibers when they are combined as soluble peptides (1:1 molar ratio) in the presence of Ca2+. The peptide nanofibers induced plate-shaped apatite crystals similar to those obtained in vitro with the parental protein DMP1, suggesting that the intermolecular β-sheet interaction present in the protein template could be the functional structural cause of biocomposite self-assembling (He et al., 2003). Peptide-mediated initiation of nanocrystals might offer a new platform for obtaining self-assembling of peptides with tailor-made sequences that promote biomineralization.
Other protein members of the dentin, also present in bone, such as bone sialoprotein, exhibit similar characteristics to enhance HAP nucleation. In this case, the protein contains serine- and tyrosine-phosphorylation sites, also in a β-sheet configuration, to promote HA crystal formation (Tye et al., 2003). This information was used to design biomaterials containing these bioactive peptide sequences that induce HAP crystal growth in vitro—for instance, de novo-designed peptide-amphiphiles (PA) that self-assemble into nanoscale fibers in water solutions and are also capable (by design) of promoting HAP nucleation as well as cell attachment (Hartgerink et al., 2001). The peptide structure and its self-assembling principle are different from those of the β-sheets previously described (such as RAD16-I). The peptide presents 5 basic structural sequential features: (1) a hydrophobic alkyl chain, (2) 4 cysteine residues, (3) 3 glycines, (4) a phosphoserine group, and (5) an integrin-binding motif RGD (Fig. 4A
The fact that self-assembling peptides could promote matrix biomineralization in vitro as well as in vivo indicates a potential application for their use in bone-related therapies as biomaterial themselves, or in more complex tissue-engineering platforms. In summary, this class of materials presents the following features: (1) They are made by rational design and can be easily synthesized, producing chemically defined biomaterials; (2) the peptides are injectable, gelling after interaction with body fluids and adopting the geometry of the tissue defect; (3) chemical functionality can be controlled, as can biomechanics; (4) as far as it is known, they produce no apparent immune responses (based on described in vivo data from Table 2 ); (5) they are biodegradable, breaking down into amino acids (based on tryptic digestions in vitro; Semino, unpublished observations); and (6) they share many properties with natural extracellular matrices, such as truly three-dimensional nanofiber matrices to support cell maintenance, proliferation, and differentiation. Although it is a clear step forward in biomaterial development, it is also true that self-assembling peptides are a simplified form of β-amyloids. Proteins undergoing amyloid structures (like β-sheet self-assembling peptides) develop into long fibers, several microns in length. Analysis of recent data indicates that amyloid fiber growth occurs by way of intermediate structures, with distinct structures such as ellipsoid or toroid (Lashuel et al., 2002; Yong et al., 2002). Interestingly, these intermediate structures are suspected to be neurotoxic species in Alzheimer’s and Parkinson’s diseases, rather than the final fibril (Volles et al., 2001). Therefore, more research should be done to ensure that β-sheet self-assembling peptides are safe for human use. To develop new strategies for bone tissue regeneration, one must consider the development of not only new biomaterials, but also an entire bioengineering platform that could assist in the regeneration of bone defects of considerable size. In addition, the platform should promote not only biomineralization, but also functional osteoblast niche establishment and angiogenic capacity, as well as the maintenance of biophysical and biomechanical properties of tissues undergoing regeneration. It must be taken into account that bone is a tissue with peculiar properties, a mineralized extracellular matrix wherein reside multiple cell types (mesenchymal stem cells and osteoprogenitors, osteoblasts, osteocytes, osteoclasts, etc.), highly vascularized and, importantly, mechanically hard, but with a degree of flexibility to cause sufficient matrix deformation. In addition, bone undergoes constant matrix remodeling, where the equilibrium between synthesis and degradation plays an important role in calcium homeostasis (bone-calcium mobilization). These features are not easy to obtain with a single biomaterial, and therefore a multidisciplinary approach is required to develop a platform capable of promoting tissue bone development in situ by inducing the intrinsic regenerative capacity of adult tissues. It could be postulated that the creation of the correct microenvironment would be sufficient to induce injured tissues to regenerate. But how can we induce tissue regeneration in an injured tissue using a bioengineering approach? Materials sciences and engineering would be able to develop such technology only with a thorough knowledge of the physiological processes required to promote regeneration. Hence, for small defects (non-mechanically compromised), the application of an injectable scaffold capable of promoting mineralization and cell niche establishment (and eventually vascularization) may be enough. This was clearly the case when RAD16-I self-assembling peptide scaffold was compared with MatrigelTM, where RAD16-I self-assembling peptide scaffold performed better than MatrigelTM in facilitating bone regeneration in small (3 mm) bone defects of mouse calvaria (Misawa et al., 2006). The reason a synthetic scaffold performed better in this particular experiment could be the consequence of some intrinsic properties of this material. First, the peptide scaffold does not present an instructive capacity for cells, such as specific peptide sequence motifs (i.e., integrin-binding domains, laminin receptor domain, etc.), suggesting that it is a nanofiber network without chemical signaling per se, providing an environment similar only in structure, but not in signaling, to the natural ECM, as previously described (Caplan et al., 2002; Genové et al., 2005; Garreta et al., 2006; Sieminski et al., 2008). Second, the synthetic scaffold would allow for binding of secreted extracellular matrix proteins and growth factors from the environment, as previously demonstrated in vitro, to interact with proteins deposited by the cells or from the medium (Sieminski et al., 2008). Finally, the self-assembling peptide RAD16-I presents a biomechanically permissive matrix that allows for cell migration and proliferation in a proper microenvironment, with viscoelastic properties similar to those of soft natural scaffolds such as collagens, as previously described (Leon et al., 1998; Caplan et al., 2002; Genové et al., 2005; Sieminski et al., 2008). Therefore, once the material is applied to the bone defect, local cells would migrate and proliferate into this permissive scaffold (structurally and mechanically similar to the natural ECM), secreting specific types and amounts of growth factors, cytokines, and ECM proteins, switching the balance from repair to innate regeneration. Instead, the natural matrix counterpart MatrigelTM is of complex composition, containing not only extracellular matrix components (such as proteoglycans, laminin 1, and collagen IV), but also epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and others that could elicit signals that impede regeneration. Interestingly, and in agreement with this idea, another kind of biopolymer, such as fibrin gel, is an attractive alternative for tissue equivalent fabrication, because it contains several advantages, including cell entrapment in the forming gel and cell and fibrin alignment, as well as biomechanical properties similar to those of the ECM (Grassl et al., 2002; Neidert et al., 2002). Most importantly, fibrin gels do not inhibit ECM synthesis by resident cells, as occurs in collagen gels (and probably MatrigelTM), suggesting that the tissue undergoing remodeling or regeneration could proceed more naturally in terms of degrading the transient fibrin scaffold and producing a new ECM (Neidert et al., 2002; Grassl et al., 2003; Long and Tranquillo, 2003; Ross and Tranquillo, 2003). Moreover, it is also true that researchers have just begun to understand the concept of cellular microenvironment and how this knowledge can be used to assist tissue regeneration. Therefore, the scenario would be different for larger bone defects involving high mechanical loadings, including exposed fractures due to trauma, as well as replacement of large sections due to tumors (Prolo and Rodrigo, 1985). In this case, an injectable material would not be enough, and a mechanically resistant holder, cage, or scaffold would be required. The classic materials used for artificial bone repair are calcium phosphate ceramics such as hydroxyapatite, tri-calcium phosphate, and biphasic calcium, which would provide significant strength and loading capacity, but would not induce bone formation on their own (Burg et al., 2000). Bioglasses (composed of SiO2, Na2O, CaO, and P2O5) are biocompatible ceramics with high bonding capacity to the adjacent bone. However, their applications in humans have been restricted to low-stress zones, due mainly to their mechanical weakness, low fracture toughness (amorphous two-dimensional glass network). Although their Young’s modulus is approximately 30 GPa (similar to that of cortical bone), their bending strength is around 40–60 MPa, which is not enough for load-bearing applications. Thus, improvement in the mechanical properties of biomaterials for applications in bone repair/regeneration is imminent, and new strategies should be developed to achieve this goal.
Previous experiments have been conducted in vitro to study the enhancement of osteoblast growth and differentiation on a peptide hydrogel RAD16-I combined with poly-HIPE polymer (hybrid material), with promising results (Bokhari et al., 2005). The peptide hydrogel provided a nanostructure network environment, while the poly-HIPE polymer provided a micro-scale scaffold. The results suggested that the presence of RAD16-I creates a permissive environment for osteoblast growth and enhances osteoblast differentiation (Bokhari et al., 2005). This type of strategy needs to be developed to attain the high standards required for large bone defects. In another example, bone growth was induced in rats by means of a 3D-hybrid scaffold with sustained release of basic fibroblast growth factor (bFGF) (Hosseinkhani et al., 2007). The strategy in this case was to fabricate a hybrid scaffold by the combination of self-assembling peptide-amphiphile (PA) nanofibers containing bFGF with collagen sponge reinforced with the incorporation of poly(glycolic acid) (PGA) fibers. In vitro release of bFGF was evaluated, as well as ectopic bone formation induced by the release of the growth factor after subcutaneous implantation of the constructs into the backs of the animals (Hosseinkhani et al., 2007). Homogeneous bone formation was histologically observed through the hybrid scaffold, confirming that this procedure could be used to improve bone regeneration (Hosseinkhani et al., 2007). Based on this concept, new biomaterial platforms could be developed by combining instructive injectable scaffolds (functionalized self-assembling peptide nanofibers) with a polymer matching the biomechanical properties of the bone, such as a microporous scaffold made of poly(lactic acid) (PLA), only for cases where low stress is required (Fig. 5
I gratefully acknowledge Lisa Spirio, Nerea Gallastegui de la Rosa, Núria Marí-Buyé, and Roger Kamm for their scientific discussions and Eileen Shakespear for her editing assistance during the preparation of this manuscript. The research conducted by the author was supported by the Translational Centre for Regenerative Medicine, University of Leipzig, Germany, Award 1098SF, and by NIH research grant 1-RO1-EB003805-01A1 to CES. Received for publication October 4, 2007. Revision received February 29, 2008. Accepted for publication March 19, 2008.
Journal of Dental Research, Vol. 87, No. 7,
606-616 (2008)
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ABSTRACT
SELF-ASSEMBLING PEPTIDES ARE...
1(IV) chain of collagen IV, was found to bind specifically to heparin in a dose-dependent manner (
