CHAPTER 1: Applications of Smart Multifunctional Tissue Engineering Scaffolds

Tissue engineering is an attractive approach to restore and replace diseased or defective tissue offering an alternative to other clinical methods such as organ replacement. Conventional tissue engineering approaches involve the use of a scaffold mainly as a structural element with defined physicochemical, mechanical and biological properties and appropriate architecture and porosity to support cell metabolism. However, recent approaches in tissue regeneration combine three key elements: a scaffold as a micro-environment to promote cell adhesion for tissue development, an appropriate cell type, and biomolecules and drugs to guide cell response and function.^1–3  There has been enormous interest lately in the growth of different types of tissues using multifunctional scaffolds that can actively participate in the process to provide the biological signals that guide and direct cell function (proliferation, growth and differentiation). Such scaffolds are derived from novel functional and smart materials that allow tuning of the properties and behavior of the scaffolds and can perform multiple crucial tasks simultaneously i.e. deliver bioactive and pharmaceutical molecules, direct cell growth and differentiation, and control stem cell behavior.⁴ 

Organic, inorganic and hybrid (organic–inorganic) materials have all been explored in the development of multifunctional scaffolds. Basic material requirements for use in tissue engineering include biocompatibility, histocompatibility, non-toxicity and the ability to engineer an appropriate scaffold with the required functionalities.

Multifunctional scaffolds based on smart materials have been applied in different tissue engineering fields. The most frequently studied areas in the literature include the use of multifunctional scaffolds in bone, cartilage and muscle formation, in cardiovascular and endothelium tissue engineering, in the growth of skin and in neural regeneration. Other applications include their use in dental, corneal and retina tissue engineering as well as in wound healing. This chapter will focus on the most extensively studied tissues of which the understanding and knowledge have matured the most. Although multifunctional materials and stimuli-sensitive nanoparticulate drug delivery systems have also shown great therapeutic potential for various cardiovascular and infectious diseases and cancer,⁵  this application will not be discussed here. In the following, the sections are divided based on the respective tissue of interest, for which the material characteristics and the multifunctionality of materials and scaffolds are discussed (Figure 1.1). The potential clinical applications of the multifunctional scaffolds are also considered.

Bone is a remarkably organized, hierarchical connective and vascularized tissue that provides mechanical support and serves various biological functions. Degenerative diseases, cancer or injury can cause bone defects. Despite the impressive ability of bone to heal spontaneously after trauma or fractures, a significant need still exists to develop strategies that promote the healing of non-spontaneously healed defects as a result of sufficiently large fractures or diseases with poor healing ability (i.e. osteoporosis, cancer). Bone tissue regeneration is a physiological and complex procedure that involves a well-orchestrated participation of various bioactive molecules. Bone extracellular matrix (ECM) comprises different proteins such as collagen fibronectin (FN), osteocalcin (OC), osteopontin (OPN), and bone sialoprotein (BSP). Different bone morphogenetic proteins (BMPs) and growth factors, like transforming growth factor-beta (TGF-β), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), are actively involved in the process of bone regeneration, in a spatiotemporal and concentration-controlled manner.^6–8  Multifunctional scaffolds, based on smart materials, are capable of promoting new bone formation, and have received particular attention in the field of bone tissue engineering lately. These scaffolds must indulge a series of different requirements such as bioactivity, biocompatibility, controllable biodegradability, appropriate mechanical strength, architecture and porosity, sustained delivery of chemical and biological cues (growth factors, genes, peptides, small bioactive molecules and ions) to eliminate infection from pathogens and reduce immune response, while promoting cell attachment and growth and stimulating osteo-differentiation and angiogenesis.^9–12 

Natural polymers have been extensively employed as multifunctional materials in bone and cartilage tissue engineering. This is driven by their superior biological response and their behavior that closely mimics tissue replacement, as well as the inherent non-toxicity and biodegradability of these materials, which renders them particularly attractive for use in biomedical applications. Silk possesses good mechanical properties that can be combined with the adhesive properties of the tripeptide Arg-Gly-Asp (RGD) for the development of robust multifunctional scaffolds exhibiting good cell adhesive properties, high wettability and enhanced biodegradability supporting the attachment, proliferation, and spreading of MC3T3-E1 cells.¹³ 

Enormous effort has been focused on the potential of cells and stem cells to differentiate because it allows the growth of tissues in vitro before their implantation in vivo using a variety of available cells. The design of the scaffold microenvironment, along with the presentation of appropriate cues to induce the differentiation of stem cells, is a highly promising strategy in tissue engineering. The surface functionalization of biomaterial scaffolds with biomimetic proteins is commonly employed in this direction. Collagen is the most frequently used matrix as it is found in the extracellular matrix and provides mechanical strength and supports bone formation. Collagen-based scaffolds have been shown to increase the adhesion, growth, and differentiation of osteoblastic cells and promote tissue formation in vivo. On the other hand, adhesive molecules such as FN can regulate cellular recognition of the scaffold through integrin signaling. OC, a non-collagenous protein with a high affinity for mineral crystals, promotes the biomineralization process and has been reported as the key factor during the late phase of osteoblasts and stem cell differentiation. In a novel strategy, OC-FN possessing a collagen binding domain has been integrated in a collagen fibrillar network to provide multifunctional and highly stable scaffolds.¹⁴  The FN active sites enhance the attachment of mesenchymal stem cells (MSC) onto the hybrid matrix, whereas a rapid cell confluence and differentiation to a mature and osteogenic phenotype is driven by OC, leading to significantly improved in vivo bone formation in calvarial defects.

BMPs are an important protein family used extensively for the differentiation of pre-osteoblasts and MSCs into osteogenic and chondrogenic cells in bone and cartilage tissue engineering. Among them, BMP-2 exhibits high osteoinductive capacity.¹⁵  However, the delivery mode of BMP-2 from the carrier affects the efficacy of bone regeneration. A sustained in vivo delivery of BMP-2 has been shown to favour bone formation compared to the burst release of the protein.^16,17  Site specific binding and regulated delivery of BMP-2 can prolong its delivery and maintain a higher local concentration at the bone injury site. Vehicles based on different biomaterials have been used to tackle this challenge, among which, demineralized bone matrix collagen, derived from cancellous bone tissues, is particularly attractive because it resembles the human bone structure and composition. The specific conjugation of a monoclonal antibody containing six histidine tags on the collagen scaffold followed by BMP-2 binding using orthogonal chemistries increases the loading capacity of the scaffold for BMP-2 and its ability to control the release in vitro.¹⁸  The multifunctional scaffolds show increased osteogenic differentiation due to the presence of BMP-2 and more ectopic bone formation. Another strategy employs multifunctional porous or nanoparticulate materials that can deliver single or multiple growth factors in a controllable manner. Alginate is a particularly attractive matrix due to its inertness and lack of interference in the signaling molecules–cells interactions. Macro-porous alginate scaffolds functionalized with both the TGF-β1 chondrogenic-inducing factor and the RGD peptide strongly affect the MSC morphology, viability and proliferation as well as cell differentiation and the appearance of committed chondrocytes, leading to more effective chondrogenesis compared to the scaffolds functionalized solely with TGF-β1.¹⁹  This is attributed to the effective cell–matrix interactions promoted by the immobilized RGD peptide, which result in a better cell accessibility to the TG-Fβ1 inducer. The regulatory role of TGF-β1 in the osteogenic activity of BMP-2 has been further confirmed in collagen sponge scaffolds. Regulation of the osteoblast and osteoclast generation in the early stages of bone formation induce a five-fold greater bone volume upon the co-delivery of the two growth factors, compared to that induced by BMP-2 alone.²⁰  Moreover, gelatin sponges comprising a biodegradable three-dimensional hydrogel porous structure, and incorporating both BMP-2 and Wnt1 inducible signaling pathway protein 1 (WISP1), exhibit a higher bone formation capacity for mice with reduced ability to regenerate bone compared to the scaffolds incorporating BMP-2 or WISP1 alone.²¹  This indicates that WISP1 enhances the BMP-2-induced osteogenesis and leads to an increased expression of the osteopontin gene in vivo, facilitating human bone marrow stromal cell migration to the defective zone. Similar gelatin scaffolds allow the controlled and sustained delivery of a stromal cell-derived factor-1 (SDF-1) and BMP-2 and promote angiogenesis and bone regeneration in vivo compared to the release of either of the two proteins alone.²²  The synergistic effect of SDF-1, which induces stem cell migration and inflammatory cell and stem cell recruitment,^23,24  and BMP-2 is attributed to the enhanced expression level of the CXC chemokine cell-surface receptor-4 (Cxcr4), Runt-related transcription factor 2 (RUNX2), and OC genes activating the process of cell recruitment, angiogenesis, and osteogenesis. Apart from porous scaffolds, nanoparticulate carriers have several advantages in terms of prolonging the release of actives. Nanoparticles based on chitosan and chondroitin sulfate are used to deliver proteins, growth factors and platelet lysates to cells.²⁵  The nanoparticles exhibit high encapsulation efficiencies due to the interactions of the proteins with the polysaccharides and control the release of their cargo for over one month. The platelet-loaded nanoparticles enhance the osteogenic differentiation of human adipose-derived stem cells in vitro and exhibit an increased level of mineralization. An interesting approach employs genetically modified plant virus particles as multivalent, low cost and low toxicity nanosized carriers for the presentation of the RGD sequence to enhance bone differentiation of stem cells in osteogenic media containing xenogeneic proteins and growth factors.²⁶  The virus particles with the RGD peptide extended from the carboxy end of the tobacco mosaic virus coat protein are immobilized onto glass slides pre-treated with two polyelectrolytes, polyallylamine and poly(styrene sulfonate), to form a stable layer-by-layer (LbL) assembly on the substrate. These surfaces induce the rapid onset of several bone differentiation markers, OC, BMP-2 and calcium, in bone-marrow-derived MSC culture, leading to rapid bone replacement.

Finally, self-assembling peptides have lately appeared as a particularly attractive system to replicate the regulatory role of the extracellular matrix and facilitate osteogenic cell differentiation and bone deposition. Bioactive peptide nanofibers presenting histidine moieties on the fiber periphery exhibit multifunctional matrix-regulatory and catalytic properties supporting osteogenesis.²⁷  This is enabled by the alkaline phosphatase-like behaviour of the imidazole-functionalized peptide fibers that is involved in controlling phosphate homeostasis and in promoting the formation of hydroxyapatite (HA) by the nonspecific cleavage of phosphate esters on the fiber surface. Similar, multifunctional, amphiphilic peptides containing a carboxyl-rich peptide domain and a peptide sequence with binding affinity for BMP-2 are co-assembled with negatively charged spacer molecules into a scaffold exhibiting improved osteogenic efficacy in a rat model (Figure 1.2). A 10-fold decrease of the BMP-2 dose and a 100% and 42% spinal fusion rate in the presence of exogenous and endogenous BMP-2 is recorded.²⁸ 

A very important feature of a functional scaffold is its ability to induce vascularization of the tissue following implantation. This is supported by the porosity of the scaffold as well as its spatiotemporally controlled bioactive release properties. Polysaccharide hydrogel bead scaffolds based on κ-carrageenan can incorporate the PDGF, which induces the production of VEGF and FGF by smooth muscle cells, and support angiogenesis.²⁹  The great encapsulation efficiency and the sustained release kinetics of PDGF lead to the formation of a highly functional vascular network, whereas the temperature-induced gelling of κ-carrageenan renders these materials attractive for use in injectable systems, requiring minimally invasive procedures.

Synthetic polymers can be appropriately designed to incorporate multiple osteoinductive agents and are very effective in inducing bone formation. Functionalized synthetic micro/nanoparticles and fibers carrying reactive groups and possessing large surface areas for grafting multiple BMPs have been extensively employed to prolong the release of the proteins, reduce their diffusion away from the injury site and maintain sufficient protein concentration for cell differentiation in bone tissue engineering. BMP-7 coupled with BMP-2 in 3-D scaffolds replicates the in vivo bone regeneration conditions. The controlled and sequential release of BMP-2 and BMP-7 from nanoparticles incorporated in poly(ε-caprolactone) (PCL) 3-D scaffolds increase the osteoinductive properties of the multifunctional construct compared to the release of BMP-2 alone or the simultaneous delivery of the two growth factors.³⁰  Electrospun PCL nanofibers have also been employed for the covalent immobilization of liposomes loaded with RUNX2 acting simultaneously as a gene delivery platform and tissue engineering scaffold and supporting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).³¹  The high contact area of the cells with the liposomes facilitates the internalization of the latter by the cells, thus maximizing the delivery of the gene and leading to a long-term gene expression and an early onset of other osteoblastic marker expressions relevant for bone homeostasis.

Multilayer films are also attractive for the efficient loading and controlled release of multiple biological cues. Recombinant human (rh) BMP-2, and an angiogenic growth factor, rhVEGF165, have been incorporated in a polyelectrolyte multilayer film. The degradable LbL assembly induces a dosage correlation effect on the differentiation of MC3T3-E1S4 pre-osteoblasts and increases human umbilical vein endothelial cells’ (HUVECs) proliferation leading to a 33% higher bone density compared to the BMP-2 alone.³²  Preorganization of the interactions between multivalent scaffolds bearing immobilized cues is nowadays an attractive means to amplify the signaling pathways and control the cell fate. The spatially controlled activation of TGF-β by a self-assembled monolayer displaying peptide ligands to TGF-β receptors, allows the sensitization of the adhered cells to very low amounts of endogenous TGF-β and can serve as a platform for controlling cell decisions.³³  Fibrous materials bearing appropriate functionalities (propargyl, alkene, alkoxyamine, and ketone groups) of controlled surface density enable the use of orthogonal bioconjugation chemistries to prepare multifunctional fibers. Multifunctional coextruded fibers have been modified using a photochemical process to incorporate the functional groups, and then decorated with cell-responsive peptides of spatially controlled surface density.³⁴  The combined cellular response to two synergistic peptides, the RGD sequence and the osteogenic growth peptide (OGP) sequence induces increased adhesion and differentiation of pre-osteoblast cells on the scaffolds.

Another important family of smart polymers used in tissue engineering is that of electroactive polymers, which allow the stimulation of cell adhesion, proliferation, and differentiation by an electrical stimulus (ES). The incorporation of aniline moieties in polymeric materials can provide scaffolds with electroactive properties. LbL films based on a poly(l-glutamic acid)-graft-tetraaniline/poly(l-lysine)-graft-tetreaniline assembly can serve as scaffolds of high stiffness, roughness and electroactivity.³⁵  These characteristics favour the growth and differentiation of pre-osteoblast MC3T3-E1 cells into maturing osteoblasts when applying the ES, presenting a novel approach to improve osteogenesis.

Shape memory materials have also been employed as a novel class of stimuli-responsive materials with great potential for the realization of smart tissue engineering constructs using minimal invasive implantation. Their intrinsic shape recovery properties enable the delivery of a bulky device in a small sized shape through a narrow passage in the body, and the recovery of its original shape when actuated by an external stimulus such as temperature, ultrasound, etc. The use of such materials to enhance the efficacy in repairing bone defects has been demonstrated. In one example, rat calvarial osteoblasts were cultured on thermo-responsive and biodegradable poly(d,l-lactide-co-trimethylene carbonate) fibrous scaffolds fabricated by electrospinning.³⁶  The fibers exhibit biomimicking properties and excellent shape memory properties thus enhancing bone tissue repair with minimal invasive implantation. However, despite their great advantages and potential, injectable, dual-purpose grafts are still limited in the literature.

Nowadays, much research effort has been focused on dual purpose bone grafts delivering locally an antibiotic to prevent bacterial biofilm formation and a growth factor to induce vascularization and bone healing at biologically relevant timescales.³⁷  Hybrid scaffolds comprising poly(lactic-co-glycolic acid) (PLGA) microspheres, containing BMP-2, in poly(β-amino ester) (PBAE) hydrogel particles loaded with ketoprofen, a pharmaceutical molecule, have been developed.³⁸  The fast-degradation of the PBAE particles induces a rapid drug release over the first 12 h, while at the same time these particles act as a porogen to the PLGA scaffold producing a porous microarchitecture which sustains the BMP-2 release. Another dual growth factor delivery system combing a viscoelastic gellan xanthan gel with bFGF- and BMP-7-loaded antibacterial chitosan nanoparticles has been employed to induce the differentiation of human fetal osteoblasts.³⁹  The injectable biomaterial with in situ ionic- and temperature-induced gelation exhibits enhanced cell growth and differentiation attributed to the prolonged delivery of the two growth factors as well as powerful antimicrobial properties against the most common pathogens in implant infections, and is proposed for minimally invasive bone regeneration. An interesting approach employs a programmable dual therapy multilayer implant coating for single-stage revision in orthopedic applications based on a LbL film (Figure 1.3).⁴⁰  The self-assembled, hydrolytically degradable multilayers enable the time-dependent co-delivery of the antibiotic gentamicin and BMP-2 in an independently controlled fashion leading to more effective bacteria-free and rapid bone healing. Other studies have also shown the dual-purpose sustained release of BMP-2 and vancomycin,⁴¹  gentamicin,⁴²  or nanosilver⁴³  to control infection and promote bone formation. In all these cases, understanding the molecular interactions of bacteria and cells with the scaffolds in the presence of drugs is essential in the design of the optimal strategy for open fracture healing. The selection of appropriate antibiotics, the tuning of the release kinetics of both pharmaceuticals, as well as more challenging preclinical studies in larger animals, will advance the application of this concept at a clinical level.

Inorganic scaffolds for bone tissue engineering are mainly based on bioactive calcium phosphates and bioactive glasses. Biocompatible and osteoconductive tricalcium phosphate (TCP) and HA are typical forms of bioactive calcium phosphates with a chemical composition close to that of the inorganic component of the native bone tissue.⁹  Bioactive glasses (BG) include calcium containing silica glasses which upon contact with the body fluids develop a hydroxycarbonated apatite surface deposit. This surface layer induces bone formation and binds strongly to the surrounding tissues and vessels in vivo. BG can also stimulate the secretion of angiogenic growth factors in vitro.^6,10 

Multifunctional scaffolds based on these inorganic materials have been developed by the incorporation of growth factors, small drugs, ions (such as copper and cobalt), stem cells or a combination of these bioactive cues. Porous scaffolds, functionalized with such signaling molecules and cells, display the inherent osteoconductivity of the inorganic material in addition to enhanced angiogenic capacity, osteostimulation and antibacterial properties. Several studies have reported the combination of growth factors with metal ions or cells or two different protein growth factors for the development of multifunctional scaffolds in tissue engineering. Specific ions (Li⁺, Sr²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Co²⁺) enhance bone regeneration by stimulating osteogenic differentiation; others (Cu²⁺and Co²⁺) promote vascularization and some (Cu²⁺, Ce³⁺ and Ga³⁺) exhibit antibacterial properties.^44,45  These cations are often loaded in HA or bioactive glasses during synthesis to enhance and ameliorate their ability for bone regeneration. A hierarchically macro/mesoporous biodegradable silica doped with calcium/magnesium and loaded with BMP-2 enhances osteogenesis and bone growth both in vitro and in vivo.⁴⁶  BMP-2 has also been combined with bone marrow stromal cells (BMSCs) in a mesoporous CaO–P₂O₅ silica scaffold.⁴⁷  The combination of BMP-2 and BMSCs increases new bone formation significantly compared to the scaffold alone or the scaffold containing only BMP-2. FGF, a mitotic growth factor that stimulates cellular growth and promotes angiogenesis and wound healing, has been combined with mesenchymal stem cells (MSCs) in mesoporous calcium containing silica-based microparticles.⁴⁸  The particles exhibit enhanced in vitro formation of HA due to the calcium doping and improved adhesion and proliferation of the MSCs induced by the FGF.

An alternative strategy to enhance bone regeneration is by the sequential release of two growth factors with different binding affinities for the scaffold. Porous β-TCP coated with a thin nanoporous mineral layer has been loaded with a mineral-binding version of BMP-2 and VEGF.⁴⁹  The higher mineral binding affinity of BMP-2 retards its release compared to VEGF and increases blood vessel density tissue infiltration to the scaffold, however, the low concentration of BMP-2 does not induce ectopic bone formation. Small pharmaceutical drugs have been used extensively to enhance the angiogenic and osteogenic properties of the inorganic scaffolds. By incorporating dimethyloxallyl glycine (DMOG), a small pharmaceutical molecule that inhibits the hypoxia-inducible factor 1a, in a mesoporous bioactive glass (MBG) scaffold, the angiogenic capacity and osteogenic differentiation of human bone marrow stromal cells (hBMSC) have been enhanced.⁵⁰  Magnetite nanoparticle decorated HA containing clodronate (CL), a drug for the treatment of osteoporosis that inhibits osteoclast activity, has been shown to possess improved osteoconductivity function and bone formation arising from the HA and CL, while the magnetic nanoparticles are believed to contribute to the rate of bone cell proliferation and differentiation.^51,52 

An attractive platform lately combines scaffolds with antimicrobial properties and high angiogenic and osteogenic differentiation capacity. The former is induced by the incorporation of antibiotics in scaffolds charged with metal ions that promote angiogenesis and bone formation. Levofloxacin has been loaded in Sr doped HA/β-TCP porous granules to enhance pro-osteoblastic proliferation and osteoblastic differentiation of MG63 cells.⁵³  Multifunctional scaffolds with high angiogenic and antibacterial capacity and antimicrobial properties are also obtained by incorporating cobalt (Co²⁺) or copper (Cu²⁺) ions and antibiotics, ampicillin or ibuprofen, in hierarchical mesoporous bioactive glasses (MBG).^54,55  The increased osteogenesis and angiogenesis is attributed to the hypoxia function of the scaffolds induced by the metal ions, whereas the sustained release of the antibiotics and the presence of Cu²⁺ reduces bacterial survival significantly. Moreover, the incorporation of Si and Zn elements, in the form of silica dioxide and zinc oxide, in 3-D printed β-TCP scaffolds has been shown to enhance bone and blood vessel formation and a more vascular branching morphogenesis compared to the undoped scaffolds.⁵⁶ 

Bone tissue itself is a nanocomposite material consisting of an organic and an inorganic component. The organic phase is mainly based on collagen I, while the inorganic phase is nanocrystalline HA. Therefore, organic–inorganic hybrid materials, comprising a soft polymer and a hard inorganic component, can better mimic the structure and chemical composition of bone and are particularly advantageous in bone tissue engineering.

Multifunctional hybrid scaffolds comprising a bioactive inorganic material combined with two sequentially delivered growth factors resemble more closely the physiological ECM cues in natural bone tissue regeneration. The sequential delivery of the therapeutic growth factors FGF2 and FGF18 from core PEO-shell PCL hollow fibrous scaffolds increases considerably osteogenesis and new bone formation with 35% new bone volume.⁵⁷  Control over the release profile of the growth factors can be achieved by the incorporation of FGF2 in the core of the fibers (fast release) and encapsulation of FGF18 within the MBG also loaded in the core (slow release). Similar behavior is observed for a chitosan sponge loaded with both PDGF, that is released quickly and acts during the first phase of bone regeneration, and VEGF-containing alginate microspheres, which exhibit a slow release of their cargo and are involved in the latter stages of bone formation.⁵⁸ 

Therapeutic small molecules with controlled release profiles have been extensively incorporated in multifunctional hybrid scaffolds to enhance bone tissue formation. Dexamethasone (DEX), an osteogenic agent, has been loaded in PLGA microspheres immobilized on the surface of a porous HA scaffold to ameliorate both bone volume and quality and a similar effect has been reported for alendronate-containing HA microspheres cross-linked in a chitosan matrix.^59,60  Porous PLGA/TCP scaffolds containing icaritin, a phytoestrogenic molecule from the herb Epimedium, can also stimulate both osteogenesis and angiogenesis, leading to an enhanced ALP activity and increased osteogenic marker expression in vitro and increase bone formation and vascularization in vivo.⁶¹  The combination of osteoconductive and antibacterial properties in a hybrid scaffold requires, as mentioned above for the other types of materials, a scaffold that promotes bone formation and the use of drugs with anti-inflammatory properties. Multifunctional hybrid poly(3-hydroxybutyrate)/bioglass scaffolds with gentamicin, an aminoglycoside antibiotic,⁶²  and Sr-loaded calcium polyphosphate scaffolds containing erythromycin as the antibiotic and coated with poly(vinyl alcohol) (PVA) have been shown to inhibit bacterial growth both in vivo and in vitro.⁶³  On the other hand, the combination of drugs and protein growth factors provides scaffolds with novel properties. A porous collagen-HA scaffold with BMP-2 and zoledronic acid (ZA) exhibits both anabolic (bone forming) and anti-catabolic (minimal bone-resorbing) behavior and a significantly increased bone volume in rats, compared to the scaffold with the BMP-2 alone.⁶⁴ 

An indirect way to deliver growth factors and favor bone formation involves the incorporation of genes, capable of expressing the growth factors, in the scaffolds. Such a gene therapy approach for the sustained release of growth factors can employ mesoporous bioglass/silk fibrin scaffolds, containing adenovirus for both PDGF-b and BMP-7, which leads to effective treatment of osteoporotic fractures.⁶⁵  Often the combined delivery of growth factors and cells is advantageous in bone reformation and vascularization. Multifunctional nanocomposite scaffolds of PLGA/PCL/HA loaded with vascular stents of PLCL/Col/HA, as a carrier for bone marrow mesenchymal stem cells (BMSCs), and a sodium alginate hydrogel to deliver two growth factors, BMP-2 and the bFGF, increase the ALP activity and the vascularized bone formation significantly.⁶⁶  Core–shell fibrous scaffolds consisting of MSCs entrapped in a collagen core and an alginate hydrogel encapsulating BG enriched with Ca and Si ions in the shell enhances the osteogenic differentiation of the MSCs significantly and promotes cellular invasion around the scaffold due to the release of Ca and Si, which act as cues for the surrounding and encapsulated cells.⁶⁷ 

Triple functional scaffolds displaying three different functions are particularly attractive since they can combine cell adhesion, osteoconductive and osteoinductive properties.⁶⁸  Cell adhesion RGD moieties and osteoinductive BMP-2, immobilized on a porous hybrid scaffold based on PLGA and HA, exhibit high density new bone formation when combined with MSCs encapsulated within collagen hydrogels.⁶⁹  Modern approaches in bone tissue engineering employ hybrid shape memory scaffolds based on chemically cross-linked PCL and HA particles, with a shape transition from a deformed shape to a recovered shape at body temperature, combined with the BMP-2 growth factor to promote new bone formation under minimally invasive surgery.⁷⁰ 

However, despite the enormous work involving growth factors for bone formation, their use is still controversial, and thus great attention has been paid lately to growth-factor-free approaches, which employ dynamic and bioactive hybrid materials in bone tissue engineering. The hybrid scaffolds combine enhanced stiffness, enzymatic stability, porosity, injectability and good cell adhesion in the presence of RGD domains, with high osteogenic differentiation of pre-osteoblast cells, in the absence of any osteoinductive factor, driven by the inorganic component.⁷¹ 

Another family of hybrid scaffolds is that based on synthetic/natural polymers, which aim to better mimic the natural bone environment. Synthetic/natural polymer scaffolds with multifunctional properties include those used for the delivery of two growth factors or a combination of a drug and a growth factor. Hybrid scaffolds comprising pullulan nanogels as the natural polymer and four-arm thiol-functionalized polyethylene glycol as the synthetic component have been applied for the combined release of FGF18 and BMP-2 and exhibit almost perfect healing, much higher than the scaffolds with only one of the two growth factors (Figure 1.4).⁷²  DEX and BMP-2 have been combined in core–shell microcapsules comprising a synthetic PLGA core and a natural alginate shell.⁷³  The controlled release of the two active molecules can be tuned by varying their position in the core or the shell of the capsule, however, the osteogenic activity of rat BMSCs increases in all cases. In a related work, multifunctional hybrid scaffolds based on poly(l-lactide-co-ε-caprolactone)/collagen nanofibers have been loaded with bovine serum albumin (BSA)-stabilized BMP-2 and DEX.⁷⁴  The scaffold with both bioactive agents in the core and the shell of the fibers shows the highest ALP activity compared to that incorporating BMP-2 in the core, suggesting that the osteogenic activity is regulated by the concentration of BMP-2 in the cell culture. In order to further control the release of BMP-2 in the medium, electrospun nanofibers based on poly(ε-caprolactone)-co-poly(ethylene glycol) (PCL-co-PEG) and loaded with DEX and chitosan-stabilized BSA nanoparticles containing BMP-2 (BNPs) have been developed.⁷⁵  The sustained delivery of BMP-2 from the BNPs improves further the ALP activity and supports in vivo osteogenesis in calvarial bone defects.

Finally, growth-factor-free approaches have employed semipermeable capsules based on a LbL coating of polylysine, alginate, chitosan and alginate, to encapsulate poly(l-lactide) (PLLA) microparticles functionalized with collagen I and a co-culture of adipose stem cells (ASCs) and endothelial cells (ECs). These microcapsules increase the ALP activity and the surface mineralization and enhance the BMP-2, RUNX2 and BSP markers.⁷⁶  The observed self-regulation and differentiation of the cells without the need of growth factors is attributed to the liquefied environment of the capsules, which allows the transfer of nutrients, and the spatial coexistence of the cells, which can communicate and self-organize within the capsule.

Skeletal muscle tissue engineering employs different cell sources and bioactive molecules to activate skeletal muscle regeneration and can be applied as a promising therapeutic tool to treat patients with large-scale muscle atrophy. Biomaterial scaffolds have been shown in animal models to play a key role in increasing the therapeutic potential of the cells and growth factors. The function of the biomaterial is to serve as a matrix providing a suitable micro-environment for the cells, to guide tissue reorganization and to deliver and release bioactive factors. Biomaterials in different forms, ranging from porous three-dimensional scaffolds to patterned surfaces, have been employed in muscle tissue engineering to induce, among others, vascularization, innervation, and contractility. Multifunctional and hybrid biomaterials that enhance muscle regeneration have attracted particular attention. Such materials can release multiple bioactive molecules and provide surface signals to activate, recruit and rearrange the cell populations (Figure 1.5).⁴  The sustained co-delivery of VEGF, to promote angiogenesis, and IGF-1, to directly stimulate muscle regeneration, from an injectable and biodegradable alginate gel has been investigated.⁷⁷  The combined release of VEGF and IGF-1 leads concurrently to angiogenesis, reinnervation, and myogenesis in ischemic hindlimbs of mice models. Moreover, appropriate functionalization of the above macroporous alginate scaffold with the cell adhesion oligopeptide G4RGDSP prevents apoptosis of the cells upon transplantation and promotes their proliferation and differentiation to myoblasts, resulting in increased muscle mass and contraction function.⁷⁸ 

Electroactive shape memory polymers are particularly attractive in muscle regeneration. Shape memory polymer networks comprising a six-arm polylactide (PLA) star and amino-capped aniline trimers as the electroactive functionalities have been developed and used for the enhanced proliferation and differentiation of C2C12 myoblast cells.⁷⁹  The scaffolds displayed good mechanical properties at body temperature, biocompatibility, degradability and excellent shape memory properties with a recovery time of a few seconds, recovery ratio >94%, and fixity ratio ∼100%, characteristics that render them excellent candidates in muscle tissue engineering.

A unique tissue interface and a highly specialized region in the muscle–tendon unit, is the muscle–tendon junction (MTJ). Tailor-made materials to support composite MTJ tissue engineering are employed in the fabrication of scaffolds that exhibit different mechanical profiles at different parts of the scaffold mimicking the strain profiles of the native MTJ. Such a mandrel-shaped scaffold, comprising co-electrospun PCL/collagen and PLLA/collagen fibers at the two opposite ends, formed a continuous scaffold with high stiffness/low compliance at one end and low stiffness/high compliance in the other.⁸⁰  This scaffold supports both tendon tissue, at the PLLA end, due to its high stiffness and low ductility, and skeletal muscle at the end of electrospun PCL, where the scaffold is less stiff, but has a greater ductility. Another interesting approach in muscle tissue engineering has been introduced based on a soft cell-culture-platform comprising a biocompatible low-modulus polydimethylsiloxane substrate onto which ultrathin stretchable gold nanomembrane sensors and patterned graphene nanoribbons were deposited.⁸¹  The modulus of the system has been designed to match that of muscle tissue and the device has been employed for the unidirectional orientation of C2C12 myoblasts. The platform presents various functions such as enhanced cell proliferation and differentiation and alignment of the cells on the patterned graphene nanoribbons, mimicking the structure of the native muscle tissue.

Finally, an essential aspect for proper in vivo skeletal muscle development is the electrical stimulation of the tissue-engineered constructs to mimic the electrical impulses of the muscles from the central nervous system. Although this field is still in its infancy, the pioneering work of Ito et al. shows that optimization of the electrical pulse stimulation during myogenic differentiation can lead to functional skeletal muscle tissues.⁸² 

Skin is the largest human tissue, which consists of three layers—epidermis, dermis and hypodermis—with a complex network of vessels and nerves that under physiological conditions are inherently self-renewable. Skin possesses multiple functions as a physical barrier that protects the host from bacteria and infections and from mechanical or chemical damage. Skin has the ability to regenerate when the injury affects the epidermal layer and the top layer of the dermis. However, skin regeneration and healing of massive and deep dermal loss due to acute injuries and chronic diseases is still a challenge today. There are several treatments to repair skin trauma including autografts, allografts and xenografts. However, these methods have several downsides such as immune rejections, donor limitations and scarring.^83–86  An alternative approach for skin replacement is skin tissue engineering, which focuses on promoting the regeneration of lost skin by stimulating the ability of the skin to self-regenerate.

Wound healing is an intricate process that consists of three overlapping phases: initial inflammation, proliferation and tissue remodeling. There are numerous protein growth factors (epidermal growth factor, (EGF), PDGF, TGF-β and FGF), cells (fibroblasts, keratinocytes and leukocytes) and cytokines that are involved in the cascade processes of skin regeneration and healing. Current efforts in skin tissue engineering are aimed at the successful development of smart multifunctional scaffolds with the ability to simulate the ECM micro-environment of native skin and to promote the right cues that can direct and promote cell proliferation, differentiation, migration and organization leading to skin regeneration, recovery of its full function and at the same time prevent scars. Bioactive elements that can be embedded within the scaffolds to induce skin regeneration are protein growth factors, cells, or therapeutic molecules with antibacterial and anti-inflammatory properties. These signaling molecules can be incorporated directly within the scaffold or they can be first introduced in polymeric vesicles to control their delivery.^85,86 

The materials used to fabricate scaffolds for skin tissue engineering should be biocompatible, non-cytotoxic, biodegradable, possess appropriate mechanical and physical properties, and promote favorable cell interactions. Natural and synthetic polymers and hybrid materials have all been used for skin regeneration. Natural materials include collagen, gelatin, fibrin, hyaluronic acid, alginate and chitosan (CS), whereas synthetic analogues are PCL, PLGA, PEO and PLA.^85–88  There are several examples of scaffolds incorporating two or more growth factors to enhance their skin regeneration capacity. Electrospun CS/PEO nanofibers containing fast-released VEGF and PDGF-containing PLGA nanoparticles to slow down the release of PDGF, can stimulate angiogenesis, increase re-epithelialization and control granulation tissue formation (Figure 1.6).⁸⁹  Similar biological functions are attained by EGF- and VEGF-loaded chitosan microparticles embedded within a dextran hydrogel,⁹⁰  while PEO/PLGA nanofibers containing rhEGF- and recombinant human basic fibroblast growth factor (rhbFGF)-loaded PLGA microspheres enhance the proliferation rate of human skin fibroblasts due to the synergistic effect of the two growth factors.⁹¹  Enormous progress has also been made in the simultaneous incorporation of multiple growth factors—VEGF, PDGF, bFGF and EGF—in collagen/hyaluronic acid nanofibers.⁹²  Control over the sequential release of bFGF and EGF at the first stages of healing to increase epithelialization and vasculature spouting, followed by the slow release of VEGF and PDGF, pre-loaded within gelatin nanoparticles, to induce blood vessel maturation, increases the proliferation rate of endothelial cells and results in a better network formation, enhanced wound healing rates, increased collagen deposition and improved vessel maturation upon implantation in diabetic rats.

The combination of the artificial dermal matrix Integra® with hMSCs and platelet-derived rich plasma (PRP) in multifunctional scaffolds offers additional advantages in skin regeneration due to the high plasticity of the hMSCs, which can differentiate into other cell linkages, and the wound healing properties of PRP, by the release of PDGF, TGF-β, EGF, IGF-1, IGF-2 and VEGF.⁹³  Bioactive molecules, such as vitamins C and D, the steroid hormone hydrocortisone, the peptide hormone insulin, the thyroid hormone triiodothyronine, retinoic acid and EGF, incorporated within collagen/PLGA or core–shell nanofibers consisting of gelatin/poly(l-lactic acid)-co-poly(ε-caprolactone) enhance human dermal fibroblast and keratinocyte proliferation as well as ADSCs proliferation and differentiation to epidermal lineages.^94,95  Antibiotics are also important to prevent wound infection. An interesting approach involves the use of collagen-coated poly(3-hydroxybutyric acid)/gelatin nanofibers containing a bioactive extract from the Coccinia grandis plant to increase the antibacterial activity of the scaffold against both Gram positive and Gram negative bacteria, as well as the adhesion of both NIH 3T3 fibroblasts and human keratinocytes.⁹⁶  Gentamicin- and rhVEGF-containing PLGA microparticles have also been incorporated within collagen/chitosan porous scaffolds to sustain their release and thus promote the antibacterial and angiogenic properties of the scaffold.⁹⁷ 

Multifunctional, composite, electrospun nanofiber scaffolds comprising cationic gelatin/hyaluronan/chondroitin sulfate with incorporated sericin, an antioxidant, antibacterial, and anticancer factor, and glycosaminoglycans, have been fabricated.⁹⁸  The presence of sericin increases the proliferation of human foreskin fibroblasts, human keratinocytes and hMSC and stimulates the hMSCs’ epithelial differentiation when co-cultivated with keratinocytes. Gene therapy approaches have been extensively used to induce the local release of growth factors during skin regeneration and to enhance their regenerative potential in vivo. Polyethylenimine-coated plasmids coding for VEGF, introduced in a FDA-approved collagen scaffold, increase the release of VEGF and the non-adherent cells significantly, especially erythrocytes, presenting a novel method for scaffold bioactivation.⁹⁹  In a similar approach, polyethylenimine polyplexes of a basic fibroblast growth factor-encoding plasmid (pbFGF) incorporated in core–shell PLA–PEG increases skin healing, vascularization and collagen deposition significantly.¹⁰⁰  A novel strategy towards the development of a multifunctional scaffold combines a trimethyl chitosan/siRNA complex in a porous membrane bilayer dermal analogue comprising collagen–chitosan and silicone. The complex architecture promotes skin regeneration in the porous collagen–chitosan scaffold with silicon acting as a temporal epidermis to prevent infections, while the trimethyl chitosan/siRNA complex interrupts the TGF-β1 signaling pathway leading to scar-free new skin formation.¹⁰¹ 

The number of patients with cardiovascular diseases (CVDs) has risen worldwide and these diseases are the main cause of mortality.¹⁰²  Typical CVDs include coronary artery disease, and the malfunction of myocardium or heart valves. Healthy autologous vascular grafts, which could be a therapeutic approach in clinical use, have the limitation of a shortfall in demand.¹⁰³  Potential solutions to overcome existing challenges caused by autologous grafts are based on the development of synthetic materials for vascular and myocardium tissue replacement. Although synthetic polymeric and metallic vascular grafts have a broad clinical use, there are certain limitations introduced by the material’s thrombogenic ability, caused due to protein adsorption and platelet adhesion and activation on the surface of the grafts.^104,105 

While the implantation of stents is the major therapeutic approach to cure coronary artery malfunctions,¹⁰⁶  it can give rise to cellular and biochemical actions inducing pathological diseases.^107,108  The series of actions involves inflammation and smooth muscle hyperplasia that can lead to thrombosis upon further inflammation or rupture of the plaque. If the thrombotic event does not occlude the artery, the cycle occurs again.^109–111  The thickening of the artery wall may be due to poor re-endothelialization on the stents.¹⁰⁷  Thrombosis and restenosis are two serious challenges to control as they are major obstacles for commercially available vascular stents.¹¹²  The physiological vascular endothelium comprises a monolayer of endothelial cells (ECs) acting as the natural anticoagulant of the artery wall.¹¹²  This structure is essential to preserve vascular homeostasis and adjust the growth of smooth muscle cells (SMCs). Seeding of endothelial cells (ECs) onto the stent surface has been investigated as a means to avoid direct contact of the blood with the synthetic material producing thrombosis,¹¹³  and to regulate the phenotype and proliferation of the SMCs.¹⁰⁷ 

Numerous studies have reported the delayed or absent endothelialization in late stent thrombosis (LST).¹¹⁴  The bare stent surface is a risk for the formation of LST.¹¹²  Drug-eluting stents (DES) are important tools to treat CVDs as they decrease the restenosis rate significantly.^108,115,116  Current drug-eluting stents aim to prevent the proliferation of vascular SMCs, though they simultaneously inhibit the growth of ECs, resulting in a delayed endothelialization.¹¹⁷  Thus, using effective techniques for the endothelialization of vascular grafts is of paramount importance in the inhibition of restenosis and the long-term efficacy of the grafts.^106,113 

The inflammatory response is another important issue in the endovascular implantation that is often disregarded.¹¹⁸  Particularly, the response of SMCs and macrophages to vascular injury and endothelial cell death is critical for the ECs’ malfunctions, thrombosis and inflammation.¹¹⁹  In recent years, there has been a need to develop physical or chemical methods that enable the introduction of multifunctional cues on the materials’ surfaces that can simultaneously function as inductive material substrates for increased endothelialization, inhibition of SMCs’ hyperplasia, and possess anti-coagulant, anti-restenotic, anti-inflammatory and antibacterial action.

Various multifunctional approaches to enhance endothelialization, a major strategy for the improvement of vascular biocompatibility, will be described in this subsection. As mentioned previously, a vascular stent is important to selectively increase the proliferation of ECs, reduce the growth of SMCs and simultaneously suppress blood coagulation. Research effort has¹²⁰  focused recently on the importance of the competitive growth of ECs versus SMCs for the development of a functional endothelium on a stent.^120–122  These strategies employ the binding of bioactive peptides and gene engineering,¹²³  ECM proteins or catechols with EC selectivity, and TiO₂ nanotube coatings as promising platforms to enhance the selectivity of the stents for ECs,^124,125  however, these surfaces still lack effective anticoagulant properties.

To overcome these limitations, the development of TiO₂ nanotube arrays modified by a mussel-inspired polydopamine ad-layer for the controlled loading of the thrombin inhibitor, bivalirudin (BVLD), which was chosen as an eluted anticoagulant and provided multiple functions, such as enhanced hemocompatibility and selectivity for ECs in a competitive growth with SMCs, has been proposed.¹²⁶  Similarly, the development of a multifunctional surface comprising hyaluronic acid and dopamine via conjugation onto 316L stainless steel led to better hemocompatibility compared to the bare material controls (Figure 1.7),¹²⁷  while an electropolymerized polydopamine coating on the surface of complex-shaped cardiovascular stents facilitates the immobilization of vascular endothelial growth factor, enhancing the desired cellular response of ECs and preventing neointima formation after stent implantation.¹²⁸  Moreover, co-immobilization of serum albumin and peptide aptamer, with specific activity for endothelial progenitor cells (EPCs) and anti-platelet adhesion, on a polydopamine-coated titanium surface enhances in situ self-endothelialization.¹²⁹ 

Recent advances in vascular tissue engineering apply multifunctional nanotechnology strategies to promote better endothelialization and microvessel regeneration (Figure 1.8).¹³⁰  A biodegradable urethane-loaded polyester multifunctional nanoparticulate scaffold has been developed with two ligands, one for glycoprotein 1b to target the damaged arterial endothelium and the second for anti-CD34 antibodies to trap endothelial progenitor cells for endothelium regeneration.¹³¹  Among the synthetic polymeric vascular scaffolds, PCL has been used for the conjugation of the multifunctional REDV short peptide to zwitterionic polycarboxybetaines exhibiting increased antibacterial activity, anti-thrombogenic capacity and endothelial cell growth,¹⁰³  while co-immobilization of adhesive peptides and VEGF on a vinylsulfone-modified dextran matrix proved to be selective to ECs over SMCs.¹³²  Since the mechanical properties of the polymer should match the elasticity of blood vessels, soft poly(n-butyl acrylate) networks with tunable mechanical properties can be utilized to angiogenically stimulate intermediate CD163+ monocytes/macrophages as a cellular cytokine delivery system to accomplish functional endothelialization,¹³³  whereas metallic cardiovascular stents with elastic degradable co-polyetheresterurethane can increase endothelial cell adhesion.¹³⁴  Another efficient strategy involves the modification of the titanium surface by forming an endothelial coating on a hyaluronic acid micro-pattern.¹⁰⁶  A multifunctional ligand comprising a cyclic RGD peptide, a tetraethylene glycol spacer, and a gallate group is used to regulate the adhesion of human ECs and serum proteins to bioceramics.¹³⁵ 

Moreover, natural biomaterial matrices have been applied as the basis for multifunctionality to enhance angiogenesis and selective endothelialization, as exemplified in the use of porous biodegradable adhesives made with hexanoyl group-modified gelatin,¹³⁶  silk heparin biomaterials,¹³⁷  and resilin-based hybrid hydrogels.¹³⁸  Using biomimicking nanofilaments and microstructured scaffolds, multi-scaled and multifunctional hybrid MSC constructs are produced, which enable vascularized adipose tissue engineering.¹³⁹  Another concept, based on a nitric oxide catalytic bioactive coating, which mimics the endothelium function, has been employed in multifunctional vascular stents to promote re-endothelialization and reduce restenosis of stents,¹⁴⁰  while an endothelium-mimicking matrix comprising peptide amphiphiles increases endothelialization, prevents inflammatory responses and promotes vasodilation.¹⁴¹ 

Tissue engineered approaches for efficient cardiovascular devices such as heart valves and cardiac patches using multifunctional materials have been reported recently. Despite the recent advances in the development of functional living heart valves, a clinically viable product does not exist yet. The next step in engineering living heart valves requires a better insight into how natural multi-scale structural and biological heterogeneity ensures their function.¹⁴²  A blend of collagen (Type I), silk fibroin and the synthetic polymer poly(glycerol-sebacate) has been used to create multifunctional electrospun nanofibrous materials tailored for heart valve replacement.¹²⁸ 

Embryonic stem cells have been extensively investigated in the production of human cardiomyocytes (CMs). Important recent advances for the clinical translation of this approach have pursued the cardiac differentiation of the stem cells at high yield without any genetic modifications. Functional, mature cardiomyocytes that are able to support fast action, conduction and high contractile stresses have been developed. As an example, a tissue engineered cardiac patch that can induce the functional maturation of human ESC-derived cardiomyocytes has been described.¹⁴³  Moreover, functional cardiac patches have been engineered from carbon-nanotube-embedded photo-cross-linkable gelatin methacrylate sheets,¹⁴⁴  or collagen covalently linked with VEGF,¹⁴⁵  while Thai silk fibroin and gelatin hydrogels functionalized with simvastatin have been used for a vascular patch.¹⁴⁶  Integration of soft and rigid components such as PEG diacrylate and PCL by a novel bioprinting method has shown great potential for the formation of functional hybrid tissue engineering constructs.¹⁴⁷ 

Neural tissue engineering is an emerging field for the treatment of various disorders of the central nervous system (CNS), which includes the brain and the spinal cord, degenerative diseases, and traumatic injuries of the peripheral nerves. Due to the limited regenerative potential of the CNS, there is an unmet need for efficient strategies to replace the damaged neural tissues. To this end, reliable methodologies and systems with multifunctional capabilities that control the guidance of neural tissue formation, the efficient delivery of soluble molecules and the differentiation of neural stem cells are essential (Figure 1.9).^148,149 

Stem-cell-based approaches have shown great promise towards the repair of destroyed neural tissue. Nanoparticulate carriers allow the effective release of biochemical cues to regulate neural differentiation, and offer multifunctional capabilities for delivery, imaging and therapy,¹⁵⁰  while a multifunctional nanocomplex has been reported that simultaneously induces the self-activation of the mRNA sequential expression and allows spatiotemporal imaging of the differentiation of the neural stem cells.¹⁵¹ 

Patterning of multifunctional biomaterials to direct neural precursor cells in a tunable system can provide a unique tool for the investigation of cells in niche-like environments. In this direction, hydrogel-based scaffolds such as heparin and PEG-peptide conjugates,¹⁵²  alginate constructs for growth factor delivery,¹⁵³  injectable PNIPAAm-PEG for the release of neurotrophins,¹⁵⁴  a poly(ethyl acrylate-co-2-hydroxyethyl acrylate) copolymer combined with an injectable self-assembling polypeptide (RAD16-I),¹⁵⁵  multifunctional conducting polymer nanoparticles,¹⁵⁶  elastin-like protein hydrogels,¹⁵⁷  colloidal particles formed by a ‘click’ reaction of bisepoxide and polyetheramine comonomers,¹⁵⁸  offer useful platforms that can act as guidance conduits and promote neurostimulation. Additionally, thiol-functionalized reduced graphene oxide with poly(methacrylic acid) microcapsules integrate surface topography and electrical stimulation for 3-D neuron-like cell growth,¹⁵⁹  whereas nanoporous gold can affect the surface topography and achieve close physical coupling of the neurons.¹⁶⁰  For the sustained delivery of biochemical cues, electrospun biodegradable PCL-co-poly(ethyl ethylene phosphate) copolymer fibers¹⁶¹  to mediate the release of retinoic acid and brain-derived neurotrophic factor¹⁶²  have been reported. Moreover, carbon nanotube composites¹⁶³  have been employed as multifunctional substrates for the differentiation of human neural stem cells, and soft carbon nanotube fiber microelectrodes for the safe stimulation and recording of neural activity.¹⁶⁴ 

Multifunctional fibers enabling the simultaneous optical, electrical and chemical stimulation of neural circuits have been developed using a thermal drawing process,¹⁶⁵  and were assembled into synthetic nerve guidance scaffolds that can replace nerve autografts to repair damaged tissue following peripheral nerve injury.¹⁶⁶  Fiber reinforced conductive polymer composites,¹⁶⁷  as well as lysinated molecular organic semiconductors, are innovative materials offering optoelectronic functionalities with improved biocompatibility and bidirectional communication of neural cells.¹⁶⁸  Finally, scaffolds with metal-ion binding agents can provide multiple targets as therapeutic agents in neurodegenerative diseases.¹⁶⁹ 

Over the last few years, increased research effort has been undertaken to develop multifunctional smart 3-D biomaterial scaffolds, containing signaling elements and active molecules for tissue engineering applications. The ability of these scaffolds to promote tissue growth and regeneration has been investigated mainly in vitro and in vivo using different animal models. The translation of the developed technology and acquired knowledge to clinical trials in human patients is not straightforward and requires the appropriate regulation of numerous issues that will guarantee the safe and efficient application of the technology in humans.^170,171 

The use of multifunctional engineered constructs in clinical trials in humans is still in its infancy and studies demonstrating the potential of these scaffolds in this area are scarce. Current clinical studies use biomaterial scaffolds mainly in the fields of skin, blood vessel, cardiac, bone, cartilage and dental regeneration and in tracheal reconstruction.¹⁷⁰  Clinical applications for some bio-based tissue engineering scaffolds, bioactive proteins, and a reservoir of various growth factors (i.e. rhBMP-2, rhBMP-7, PRP) as well as drugs (i.e. gentamicin) and various cell types have been approved.^87,172–176 

Most of the clinical trials in bone and cartilage tissue engineering utilize a bio-based scaffold combined with an appropriate cell type. For example, a HA scaffold seeded with MSCs has been used to fill human bone defects,¹⁷⁷  whereas hyaluronan, collagen and alginate scaffolds combined with autologous chondrocytes have been applied for the treatment of chondral injury.^172,178  There are also very few studies reporting the use of growth factors on an engineered construct to produce a functional scaffold. These mainly involve the incorporation of rhBMP-2 in β-TCP granular scaffolds seeded with ASCs for the reconstruction of maxillary and mandibular bone^179–182  and the combination of β-TCP granular scaffolds with BMSCs and a PRP gel for the treatment of an anterior mandibular defect and alveolar bone atrophy.¹⁸³  Furthermore, BMP-2 and BMP-7 have been introduced in collagen sponges for the treatment of multiple bone defects,¹⁸⁴  β-TCP particles containing PDGF-BB have been applied for the therapy of bone defects caused by periodontal diseases¹⁸⁵  and PDFG/IGF combined with a methylcellulose gel vehicle has been employed for periodontal regeneration.¹⁸⁶  In other related studies, an injectable gel of thrombin-calcium chloride with BMSCs and PRP has promoted bone regeneration in dental implants,¹⁸⁷  and a microscaffold consisting of calcium phosphate cement (CPC) and rhBMP-2 has induced bone regeneration.¹⁸⁸ 

Clinical trials in skin tissue engineering use different cell types with engineered constructs. For example, a collagen sponge scaffold combined with ASCs covered by an artificial dermis can be used to treat knee injuries¹⁷⁹  and an engineered construct consisting of a layer of keratinocytes and a layer of collagen-containing fibroblast is employed in the treatment of foot and venous leg ulcers.¹⁷⁰  Functionalized scaffolds are also used in the treatment of skin injuries. Multi-layers of electrospun fibers serve as patches that are able to control the release of nitric oxide in the treatment of the foot ulcers of diabetes patients and for the therapy of cutaneous leishmaniasis.^189–191  Furthermore, clinical studies have shown an enhancement of the wound healing rate of burn wounds and diabetes ulcers using bFGF, keratinocyte growth factor (KGF) and PDGF.^{86,87,192–194} 

In cardiovascular interventions, the need to treat restenosis following stent implantation has led to the use of drug-eluting stents (DES) made of metallic or biodegradable polymeric materials¹⁹⁵  coated with anti-proliferative and anti-inflammatory agents that are eluted slowly and reduce neointimal formation.¹⁰⁸  Although preliminary studies in humans applying two anti-proliferative agents, paclitaxel¹⁹⁶  and sirolimus (rapamycin) that inhibit SMCs growth,¹⁹⁷  have been reported with promising results, recent studies have shown that DES can lead to late stent thrombosis and thus result in long-term failure. This has occurred when stenting complex lesions, due to localized allergic reactions at the vessel wall that led to chronic inflammation.¹⁹⁸  An additional deterioration can occur by the release of the anti-proliferative drugs sirolimus and paclitaxel, which not only effect SMCs, but also ECs, and in this way impair the wound healing process.¹⁹⁹  The use of covered stents as a treatment option in various vascular complications, as well as the design and materials employed in the stents’ manufacturing process, including nanotechnology approaches, have recently been reviewed.¹⁹⁹  Recent developments in vascular conduits based on the advances in tissue engineering, which are expected to function like real blood vessels with an intact endothelial layer and respond to endogenous vasoactive signals when implanted in clinical applications, are gaining increasing interest for vascular regeneration.²⁰⁰ 

Peripheral nerve reconstruction of gaps in clinical applications utilizes nerve autografts as the gold standard, although they do possess many shortcomings. To improve the outcome, various strategies using collagen, poly(glycolic acid), poly (dl-lactide-ε-caprolactone) and decellularized nerve allografts have been reported in several clinical studies and were reviewed recently by Gerth et al.²⁰¹  Advances in multifunctional approaches, employing conduits seeded with stem cells and providing local delivery of growth factors and neurotropic factors into the lumen of the engineered conduits, have been described to improve nerve regeneration in several recent reports.^202–205  Moreover, a recent multicenter clinical study using a human acellular nerve graft as an alternative to an autogenous nerve has reported on its safety and efficacy for the repair of nerve defects between 1 and 5 cm in size.²⁰⁶ 

Advances in biology related to the understanding of the fundamental mechanisms underlying the regeneration of different tissues has instructed the choice of cell types and their combination in a tissue engineered approach. On the other hand, great progress has been made in the field of 3-D scaffold fabrication and the methodologies for their surface chemical modification enabling the incorporation of appropriate signaling and pharmaceutical molecules and their controlled release as well as the expression of specific markers to elicit favorable cell responses leading to tissue repair. The combination of the above knowledge and expertise is expected to dramatically change clinical therapies in human patients in the upcoming years.

Progress in the applications of smart materials and multifunctional scaffolds in tissue engineering has been great over the last decade. The sustained, sequential and controlled release of multiple chemical and biochemical cues have been tackled and surface engineering in terms of physicochemical properties and topography has been addressed. However, there are still critical aspects and challenges to be overcome before the above results can be transferred to the clinic.

In bone tissue engineering, the challenge is to develop smart multifunctional scaffolds presenting multiple cues and mimicking the functions of the natural ECM environment of bone to promote therapeutic drug delivery and vascularized bone tissue regeneration. Muscle tissue engineering is still in its infancy, due to the peculiar organization of the skeletal muscle, which requires smart scaffolds with multiple functions that will provide the appropriate mechanical and bioactive cues for the cells to adhere, proliferate, form networks and replace the dysfunctional tissue. Electrical stimulation of the scaffolds, in a manner to replicate the natural impulses of the muscles from the neural system, needs particular consideration.

In skin tissue engineering, multicomponent structured materials that can regenerate the complex and hierarchical three-layered structure of skin are important. A key factor in achieving a fully functional skin is to establish constructs that will restore its normal connections with the surrounding nerve and muscle tissues once transplanted into living organisms. In cardiac tissue engineering, the development of multifunctional scaffolds with micro-environmental cues can promote the functional maturity of cardiomyocytes, while appropriate multifunctional materials and approaches that eliminate the inflammation and thrombogenicity of intravascular implants are essential. In neural tissue engineering, multifunctional materials are needed for the sustained delivery of biochemical cues, the synergistic topographical signaling and the design of nerve guidance channels with the capability to stimulate topographic, chemotactic and haptotactic signals that will induce functional nerve regeneration, and increase the axon growth rate.