Development of polymeric functionally graded scaffolds: a brief review

Solvent-based techniques

Several researchers have fabricated bilayer scaffolds by particulate leaching and by freeze-drying techniques, in particular for osteochondral defects. The bilayer architecture offered good mechanical stability and maintained structural integrity while providing a porous architecture that supported cell ingrowth in chondrogenic and osteogenic layers, together with integration with the surrounding environment.

Among these researchers, Qi et al incorporated mesenchymal stem cells (MSCs) into a bilayer PLGA scaffold obtained by leaching gel microspheres. The bilayer system was prepared by solution casting of PLGA in a mold containing gel particles with 2 different size distributions (79). Similarly, Giannoni et al realized highly porous PCL scaffolds combining solvent-casting and particulate-leaching. The porogen agents were sucrose crystals for the bone-facing layer which contains hydroxyapatite (HAp) and mannitol crystals for the cartilage-facing layer (80). Duan et al investigated in vivo the effect of pore size in bilayered PLGA scaffolds fabricated by solvent-casting of PLGA filled with sodium chloride (NaCl) and subsequent water-leaching (81). The pore size distribution of each layer was controlled by sieving the NaCl particles in 2 different ranges. The solvent-casting and NaCl leaching technique was adopted by Chen et al to prepare a bilayer scaffold composed of biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was a COL sponge, while the lower layer for bone engineering was a composite sponge made of PLGA and naturally derived COL (82).

Reem et al demonstrated the enhanced regenerative properties of a hydrogel designed for subchondral repairs obtained by freeze-drying. The scaffold was filled in the upper and lower regions with 2 different grow factor types to induce the differentiation of MSCs (83). Martínez Ávila et al fabricated a bilayer bacterial nanocellulose (BNC) scaffold for auricular cartilage composed of a dense nanocellulose layer joined with a macroporous layer of BNC/alginate (84). The bilayer scaffold was assembled by solvent interdiffusion among the layers before the freeze-drying step. Niederauer et al prepared 4 types of PLGA-based bilayered implants. To vary stiffness and chemical properties of the constructs, they used different additives at different concentrations (85). In this case, the layers were assembled by using a solvent as a glue.

Bi et al developed a heterogenous bilayer scaffold for osteochondral regeneration by solvent-casting and freeze-drying. They used a CS-COL composite for the chondral phase and a bioactive glass-COL for the osseous region. The interlayer adhesion was ensured by interconnecting the COL via cross-linking agents (86). For the same purpose, Schleicher et al fabricated bilayer scaffolds was made up of either HAp/COL or allogeneic sterilized bone/COL via solvent-casting plus freeze-drying. The integration of the 2 layers was ensured by the COL that acted as a glue for the different phases. The scaffolds’ efficiency was evaluated in a sheep model, and the sample based on allogeneic sterilized bone/COL displayed more stability and higher capacity to be integrated in the short term (87). A 3D heterogeneous bilayer scaffold was prepared by Deng et al to repair large defects in rabbit joints (88). The scaffold consisted of 2 distinct and integrated layers corresponding to the cartilage and bone tissue. The upper layer, made with gelatin, chondroitin sulphate and sodium hyaluronate, and the lower layer, which consisted of gelatin and ceramic bovine bone, were assembled using cross-linking agents. The scaffolds investigated showed an encouraging efficacy in repairing large defects in rabbit joints.

Marrella et al adopted a procedure combining centrifugation and freeze-drying techniques, to prepare porous scaffold made by PCL and COL type I (89). A functional gradient was combined to the morphological one by adding HAp powders to mimic the bone mineral phase. Results showed that 3D bioactive morphologically and chemically graded grafts could be properly designed and realized, in agreement with the theoretical model. Monteiro et al presented a novel approach to developing bilayer scaffolds for skin tissue engineering, able to combine certain specific properties of the epidermis and the dermis (90). They used the spray-assisted layer-by-layer assembly technique to deposit a polyelectrolyte multilayer film made by HA and poly-L-lysine on a porous HA scaffold. The bilayer film promoted cell adhesion, thus contributing to regeneration of the epidermal barrier functions of skin.

Reyes et al prepared a bilayer scaffold consisting of PLGA and alginate for the sustained delivery of 2 different growth factors. A bone-orientated, porous PLGA cylinder was prepared via gas foaming, overlaid with PLGA microspheres containing various amounts of grow factors, and dispersed in an alginate matrix via cross-linking and subsequent freeze-drying. Release kinetics and tissue distributions were determined by in vitro and in vivo experiments (91).

The solvent-based approach presents several issues related to toxic solvent utilization, in terms of solvent removal and environmental risks (92). Nevertheless, it allows the achieving of versatile structures by tailoring the concentration and localization of additives, such as growth factors, enzymes and proteins, via chemical reactions in solution. Furthermore, among the techniques used to create voids, freeze-drying permits us to achieve extremely high degrees of porosity (37).

Melt-based approaches

Scaffaro et al prepared a heterogeneous bilayered scaffold in the melt. The authors combined melt-mixing, compression-molding and particulate-leaching to integrate a PLA layer with pores around 100 µm with a PCL layer with pores around 20 µm. NaCl sieved in 2 fractions (0-40 µm and 90-110 µm) was used as template. The tunable pore architecture combined with the different chemophysical properties of the 2 polymers in a heterogeneous bilayered scaffold presenting well-adhered layers, as demonstrated by scanning electron microscopy (SEM) analysis (Fig. 2) and tensile mechanical tests (50). Similarly, Ghosh et al processed a homogeneous bilayered PLA porous scaffold by compression molding followed by leaching. On the cartilage side, starch was added, while in the bone region, which required greater stiffness and strength, HAp was used as a reinforcement (93).

Basically, the main advantages related to the melt-based approach refer to the absence of toxic solvents and to the possibility of tuning the porosity and pore size by simply tailoring the size and concentration of porogen agents. However, this technique presents some drawbacks, such as the difficulty of achieving complex structures or the impossibility of fabricating continuously graded structures, as will be discussed in further paragraphs below.

Computer-aided techniques

Bilayer scaffolds of appropriate geometry can be realized by computer-aided techniques, such as rapid prototyping, image-based design (IBD) and solid free-form fabrication (SFF). Sherwood et al developed a composite PLGA scaffold using the TheriForm™ printing process where the external and internal architectures were created via IBD. The versatility of the technology allowed for varying the material composition, shapes, porosity and mechanical properties throughout the scaffold structure (94). Shao et al assessed the feasibility and potentiality of a hybrid scaffold in large and high-load bearing osteochondral defect repair (95). The bone component was mimicked by PCL, while the soft component (cartilage part) was made of fibrin glue. The results demonstrated that PCL scaffold is a potential matrix for osteochondral bone regeneration, whereas fibrin glue does not inherit the physical properties to promote cartilage regeneration in a large and high-load bearing defect site. Schek et al fabricated an osteochondral implant for temporomandibular joint repair (96). IBD and SFF were used to generate load-bearing scaffolds capable of matching patient and defect site geometry. Biphasic composite scaffolds were manufactured, simultaneously generating bone and cartilage in discrete regions.

As one of the most advanced and promising technologies in tissue engineering, 3D-bioprinting combines SFF and precise placement of cells and other biological factors to the desired 2D and 3D positions. It is described as a precise approach for delivering biomaterials, cells and supporting biological factors to the targeted locations with spatial control to fabricate functionally graded 3D constructs.

Other methods and hybrid methods

Pu et al have fabricated bilayer homogenous PLA fibrous scaffolds by a novel electrospinning technique which uses 2 slowly rotating parallel disks as the collector (Fig. 3). The structure, consisting of a thin aligned-fiber layer (AFL) and a random-fiber layer (RFL), showed gradual variation of porosity and fiber alignment along the thickness. The dense structure of AFL provided the mechanical strength, while the high porosity of RFL enhanced cell infiltration, proliferation and distribution (97, 98).

Uma Maheshwari et al prepared bilayer composites based on polyvinyl alcohol (PVA), HAp and PCL for bone tissue engineering. Both polymers were loaded with nanoparticles and then electrospun to form a bilayer composite mat, consisting of a hard component (PVA-HAp) and a soft layer (PCL-HAp) (99). Zhang et al prepared bilayer COL/microporous electrospun nanofibrous scaffolds for enhancing the osteochondral regeneration (100). PLA fibers were produced via electrospinning. COL layer was deposited onto PLA nanomat, and bilayer microporous scaffolds (COL-nanofiber) were produced via freeze-drying.

To repair oromaxillary defects, Giannitelli et al studied different foaming conditions to determine the best performance of PU graded foams consisting of a dense shell and a porous core for cell infiltration (38). Salerno et al fabricated the 2 layers of an osteochondral biomimetic PCL/HAp scaffold, by melt-mixing and compression-molding, followed by a foaming process with carbon dioxide (CO₂) and NaCl leaching (101). Deepthi et al developed CS-HA hydrogel–coated PCL multiscale bilayer scaffolds for ligament regeneration (102). They fabricated either aligned or random multiscale fibers, observing that the coated scaffolds showed improved protein adsorption and cytocompatibility, and higher cell retention.

Solvent-processing, freeze-drying and sintering were combined by Oliveira et al to prepare HAp/CS bilayered scaffolds for osteochondral tissue engineering applications (103). In more detail, the HAp layer was created by impregnating a PU sponge with HAp powder. The composite sponge was then burned to remove the polymer, and finally sintered. The CS layer was prepared by dissolving the CS in acetic acid, transferring the CS solution onto HAp scaffolds placed into cylindrical molds and then finally freeze-dried. The materials showed no cytotoxic effect. For the same purpose, Jiang et al prepared bilayered stratified polymer ceramic-hydrogel scaffolds (104). In this case, the technique adopted was a combination of water/oil/water emulsion and sintering methods. Specifically, scaffolds based on agarose hydrogel and composite microspheres of PLGA and bioactive glass (BG) were fabricated and optimized for chondrocyte density and microsphere composition. Although this approach involves complex protocols, it represents a viable strategy to tune the properties of each single layer and achieve high regenerative performances.

Particulate leaching

Scaffaro et al prepared homogenous 3-layered scaffolds in PCL and in PLA presenting pore size distribution that gradually changed from 20 µm to 110 µm (Fig. 4). The authors combined melt-mixing, compression-molding and particulate leaching. In brief, PCL or PLA, PEG and NaCl sieved in 3 fractions were melt-blended, melt-assembled and then put in distilled water as shown schematically in Figure 5. For both PLA and PCL, the resulting structures were characterized by well-adherent layers with different pore sizes, without loss of continuity or interface fracture (46, 105). The same authors prepared a heterogeneous 3-layered scaffold integrating PCL (shell) and PLA (core) by adapting the method described above (51).

Tang et al developed (PLGA) scaffolds with graded pores size for osteochondral repairs. Gelatin microspheres with 3 different diameters (350-450 µm, 200-350 µm and 80-200 µm) were used as a template in which a PLGA solution was cast. The system was then freeze-dried and finally put into hot distilled water for 12 hours to leach out the porogen agents. The scaffolds exhibited a porosity as high as about 95% and a gradient of pore sizes across the cylindrical axis (55).

Finally, particulate-leaching allows the tuning of the porosity and pore size by simply tailoring the size and concentration of porogen agents. On the other hand, the pore shape is strongly affected by the porogen geometry, which is difficult to control.

Freeze-drying

The versatility of freeze-drying lies in the possibility of obtaining multilayer constructs via an iterative technique that allows the material composition and scaffold microarchitecture to be specifically tailored in each region of the scaffold. Levingstone et al adopted this method to prepare 3-layered scaffold mimicking the native composition of subchondral bone ECM. The articular cartilage-mimicking layer, was composed of a rich glycosaminoglycan content and type II COL. The cartilage-mimicking layer of the scaffold was made of type I COL, type II COL and HA. Finally, the bone-mimicking layer was prepared with type I COL and HA (61). Zhu et al used a similar approach to fabricate a CS-based 4-layered scaffold presenting a porous structure able to mimic the cartilage ECM. The COL/CH–PCL/CS scaffolds presented hierarchically distributed average pore sizes and porosity, as well as an interconnected porous structure (57).

Kon et al, Yusong et al and Algul et al prepared 3-layered scaffolds made of CH, gel and COL, respectively, by overlapping layers with increasing amounts of mineral phases. The assembled layers were then freeze-dried obtaining a biomimetic interconnected porous structure (59, 60, 106). Similarly, Kim et al prepared a human COL-based 4-layered scaffold with a tailored material composition that was designed for the repair of the tendon-to-bone region. Different materials were chosen to build each layer as follows: (i) COL for the tendon layer; (ii) COL and chondroitin sulfate for the uncalcified fibrocartilage layer; (iii) COL and a small amount of apatite for the calcified fibrocartilage layer; (iv) COL and apatite made up the bone layer. The 4 layers were then assembled by an iterative freeze-drying method (62).

Wang et al used COL type I to prepare a 3-layer scaffold designed for the regeneration of human dermis. Solutions containing COL type I at different concentrations were separately frozen at -80°C and then freeze-dried together. The mean pore size of the scaffolds increased upon increasing the COL concentration. The authors demonstrated that the “sandwich” COL scaffolds significantly promoted wound healing, especially if compared with homogeneous scaffolds displaying a uniform pore size (54). Tampieri et al developed a composite osteochondral scaffold organized in different integrated COL-based layers. The authors piled up the layers onto each other; thereafter, a knitting procedure was used at each interface to ensure good layer integration, thus avoiding delamination at the interfaces. Finally, freeze-drying under vacuum conditions was performed to obtain the porous structure (63).

The main advantage of freeze-drying lies in the possibility of obtaining highly porous, multilayer constructs through an iterative technique that can be adopted for a wide range of polymers. On the other hand, the method is not easy to scale up to an industrial level, and it is usually adopted for small prototypes.

Electrospinning

Multilayer electrospun scaffolds are usually developed by consecutively electrospinning different materials. Wang et al used this technique to prepare a 3-layer scaffold for cranial defects. The inner PLA layer was designed to reduce tissue adhesion on the brain. The middle layer, made of a PLA/PCL blend, was designed to avoid water infiltration. Finally, the COL-based outer layer was designed to promote cell adhesion. Mechanical and in vivo biological tests demonstrated that the multilayer scaffold had sufficient mechanical strength and adequate biochemical properties to be potentially used for dural cranial repairs (67). Bye et al used the same approach to prepare poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/PCL or PLA multilayer scaffolds demonstrating the reliability of these systems as biobarriers for preventing cell penetration (64).

Electrospinning is a versatile technique for fabricating nanoscale and microscale fibers, and has great potential for mimicking the microenvironment of natural ECM by controlling a large number of parameters such as flow rate, potential, distance between syringe and collector and so on. Moreover, this approach allows the preparation of scaffolds consisting of different layers more easily compared with the other technique. On the other hand, the intrinsic morphology of the porous structures is often difficult for cells to permeate, without specific precautions.

Other methods and hybrid methods

Mi et al prepared a small-diameter vascular scaffold composed of 3 different layers: a porous outer layer made of thermoplastic PU, a woven silk filament middle layer and a nanofibrous inner layer again in PU. The FGS was fabricated by a multiple-step protocol involving thermally induced phase separation, braiding and electrospinning techniques. The different layers provided the scaffold with better mechanical properties when compared with those observed in each single layer taken separately (107).

Aydin et al produced a structure for the regeneration of defects of articular cartilage composed of 3 distinct layers. The bone-mimicking layer was fabricated from a PLA/PCL polymeric blend dissolved in chloroform and mixed with NaCl. In a second stage, scCO₂ was used to enhance the interconnectivity of the monoliths. The PLGA-based upper layer was a nonwoven felt. Finally, vertical channels were formed, starting from the bone layer to the intermediate layer, using stainless steel pins. The channels were designed to facilitate the delivery of osteogenic and chondrogenic clues from bone marrow to then eventually be transformed into Haversian channels (58).

Nie et al prepared a homogeneous 3-layered scaffold made of PLA, combining TIPS and sugar particle leaching. The macropore size gradient was obtained by controlling the sugar particle sizes from 300 µm to 600 µm as well as porosity and interconnectivity. The scaffolds were prepared by packing the sugar spheres in a gradient shape and then casting PLA/dioxane solution under vacuum. Subsequently, the system was freeze-dried, and finally the sugar was leached out in distilled water. The average pore size, as well as the average interpore opening size, was controlled by the average diameter of the sugar spheres (4).

Combining different technologies to obtain a multilayer scaffold offers the possibility of tuning the characteristics of each single region with an high degree of control in terms of mechanical, structural and chemical properties. Conversely, this approach requires complex protocols, thus hindering any industrial scalability of these approaches.

Solvent-based techniques

Liu et al reported an in situ diffusion method for the fabrication of compositionally graded COL/HAp composite scaffolds (108). Chemical and microstructural analyses revealed a gradient of the Ca/P ratio across the width of the COL scaffold template, resulting in the formation of a calcium-enriched side and a calcium-depleted side of scaffold. Dormer et al fabricated a FGS by encapsulating proteins in polymer droplets, achieved by ultrasonication (109). The resulting polymer microspheres, containing a graded amount of proteins, were then sintered and freeze-dried to achieve the final structure. The results highlighted that continuous gradients in bioactive signals allowed the temporal and spatial release of appropriate growth factors for the osteochondral regeneration.

A similar approach was previously adopted by Wang et al (110). In this case, 2 growth factors, bone morphogenetic protein 2 and insulin-like growth factor I, were incorporated as a single concentration gradient or reverse gradient combining 2 factors in the scaffolds. Bai et al fabricated biomimetic scaffolds by freeze-casting a slurry of HAp particles (111). The slurry, containing nanoparticles and various reactants such as surfactants and polymers (i.e., PEG and Aquazol), was ball-milled and freeze-casted. During the freezing process, lamellar ice crystals grew preferentially from the copper rod outward to the plastic mold, generating a gradient in their thickness. At the same time, HAp particles were expelled and assembled to replicate the structure of ice crystals. The authors were able to achieve interconnected gradient channels mimicking the porous network of natural bone.

Harley et al adopted a “liquid-phase co-synthesis” technique that was found to enable the production of porous, layered scaffolds mimicking the composition and structure of articular cartilage on one side, subchondral bone on the other side, and a continuous, gradual or soft interface between the 2 tissues (i.e., articular joints) (5). Liquid-phase co-synthesis interdiffusion of unmineralized, type II COL-glycosaminoglycan (CG) and mineralized (reinforced with CaP nanoparticles), type I COL-glycosaminoglycan (CG-CaP) suspensions were subsequently freeze-dried.

Mannella et al developed a porous scaffold with a pore size gradient by TIPS, achieved by imposing a different thermal history on the 2 sides of a polymeric solution, consisting of PLA, dioxane and water (112). The authors demonstrated the possibility of tuning the pore size range by controlling and manipulating some key parameters, such as demixing temperature and residence time in the miscibility gap region.

In some cases, the solvent-based technique is combined with UV-assisted reactions, such as photopolymerization or cross-linking to fabricate continuous graded scaffolds (113). Two different macromers (each one in its own appropriate solvent) and photocatalyst solutions were used to prepare a functionalized star polydimethylsiloxane, processable in dichloromethane, and a modified PEG. The continuous gradient of the scaffold was achieved by a gradient maker, whereas the integration of the 2 polymers was ensured by UV-assisted cross-linking. The results demonstrated spatial variation in morphology, bioactivity, swelling and elastic modulus.

Nemir et al used PEG dimethacrylate (PEGDM) hydrogels, with varying polymer chain length, and photolithographic patterning techniques to provide substrates with spatially tunable mechanical properties in both gradients and distinct patterns (114). The hydrogels were patterned to produce anisotropic structures exhibiting patterned strain under mechanical loading. Similarly, Chatterjee et al prepared PEGDM hydrogel, which gradient was imparted by mixing two PEGDM solutions (at different concentration) containing suspended cells in the stock chambers of a gradient maker (115). The prepolymer solution was then polymerized through the glass slide of the mold by exposure to UV. Hill et al fabricated photocrosslinkable dextran hydrogels containing a gradient of siRNA, using a dual-programmable syringe pump system, and showed a differential gene silencing in incorporated cells sustained over time (116). More recently, selective photopolymerization of PEGDM and epoxy monomers was used to provide tunable elasticity in a multimaterial hydrogel (117).

Electrospinning

He et al fabricated nanofibrous scaffolds with continuous structure and composition gradients in 2 electrospinning steps (118). In the first step, a PLGA solution was moved back and forth along the axis of the collector, rotating at high speed to produce aligned nanofibers. Two hours later, the PLGA loaded with HAp nanoparticles was electrospun onto the exposed aligned nanomat previously realized. In this case, the collector was kept at low rotation speed and 30 degree swinging to produce a randomly oriented PLGA-HAp mat. The different distance between needle and cylindrical surface in the 2 steps enabled the formation of a thickness gradient at the boundary of the aligned and random nanomats along the circumferential direction of the collector. Owing to these features, the FGS possessed a structural and chemical gradient. Lipner et al created PLGA nanofibrous scaffolds by electrospinning (119). Thereafter, the nanomats were cut into small pieces, plasma-treated to enhance their hydrophilicity and coupled with CS to promote the attachment of calcium ions, thus enabling mineral nucleation.

Tzezana et al prepared electrospun PCL fibers loaded with all-trans-retinoic acid (ATRA), designed to release ATRA at a predetermined rate, due to morphogenic gradients throughout the thickness of hydrospun scaffolds (120). The structures were then exposed to flow conditions in a bioreactor, and gradient formation was verified by a convection-diffusion mathematical model, which proved a continuous solute gradient across the scaffold.

Zou et al prepared electrospun fibers with graded contents of amino groups, with the latter being generated through an aminolysis process using a microinfusion pump (121). Gelatin grafts allowed the creation of fibrous scaffolds with gradients in HAp contents, crystal size and mechanical properties through in situ mineralization. Plasmid DNA (pDNA) was included during the mineralization process, and gradations in pDNA loading contents were created on fibrous scaffolds, strongly depending on HAp gradients. Gradients in the amount of pDNA released and the expression of target proteins provided a temporally and spatially controlled delivery of growth factors in scaffolds. Hence, after cell seeding, the fibrous mats displayed gradients in cell density, osteoblastic differentiation and COL deposition along the longitudinal axis.

Other methods and hybrid methods

Erisken et al fabricated functionally graded nonwoven meshes of PCL incorporated with tricalcium phosphate (TCP) nanoparticles using a hybrid twin-screw extrusion/electrospinning process. For this purpose, the multichannel spinneret die of the twin-screw extruder was connected to a high-voltage supply, to permit the time-dependent feeding of various solid and liquid ingredients and their melting, dispersion, deaeration and pressurization together with electrospinning in continuum (122).

Oh et al used a centrifugation-based method to realize a scaffold presenting a gradient pore size distribution increasing along the longitudinal direction. In particular, the scaffolds were prepared in 2 steps: (i) centrifugation and (ii) heating fibril-like PCL in a cylindrical mold. The FGS was achieved by exploiting the gradual increment of the centrifugal force along the axis. Morphological analysis showed a gradual increase of the pore size distribution from 88 to 405 µm that could be easily controlled by tuning the rotational speed (53).