Recent advances in layer-by-layer assembly scaffolds for co-delivery of bioactive molecules for bone regeneration: an updated review

Abstract

Orthopedic implants have faced challenges in treating bone defects due to various factors, including inadequate osseointegration, oxidative stress, bacterial infection, immunological rejection, and poor individualized treatment. These challenges profoundly affect both the results of treatment and patients' daily lives. There is great promise for the layer-by-layer (LbL) assembly method in tissue engineering. The method primarily relies on electrostatic attraction and entails the consecutive deposition of electrolyte complexes with opposite charges onto a substrate, leading to the formation of homogeneous single layers that can be quickly deposited to produce nanolayer films. LbL has attracted considerable interest as a coating technology because of its ease of production, cost-effectiveness, and capability to apply diverse biomaterial coatings without compromising the primary bio-functional properties of the substrate materials. This review will look into the fundamentals and evolution of LbL in orthopedics, provide an analysis of the chemical strategy used to prepare bone implants with LbL and introduce the application of LbL bone implants in orthopedics over recent years. Among the many potential uses of LbL, such as the implementation of sustained-release and programmed drug delivery, which in turn promotes the osseointegration and the development of new blood vessels, as well as antibacterial, antioxidant, and other similar applications. In addition, we offer a thorough examination of cell behavior and biomaterial interaction to facilitate the advancement of next-generation LbL films for tissue engineering.

Introduction

Bone tissue has a complex, hierarchical, three-dimensional (3D) porous structure and performs a variety of distinct mechanical, chemical, and biological functions. The use of bone grafting material is considered the most effective treatment for such conditions. It is imperative to recognize that this approach has several limitations, including the difficulty of obtaining the required tissues and and the limited availability of the material. Therefore, it is crucial to utilize synthetic bone grafting materials [1, 2]. In orthopedic surgery, the use of bone substitutes and grafts has significantly increased. The objective of bone regeneration is to repair damaged bone tissue to restore normal function and facilitate the healing process. A biodegradable, porous, and bioactive bone scaffold is required for this engineering approach, together with biochemical growth factors and osteogenic cells. The scaffold must have the ability to provide mechanical support and accelerate the development of bones during bone regeneration [3, 4]. A designed bone scaffold must possess mechanical qualities that closely resemble those of the bone tissue of the host. It must be strong enough to withstand failure and rigid enough to stop the resorption of bone tissue [5]. To support the growth of cells and blood vessels, bone scaffolds must have appropriate mechanical strength to withstand the hydrostatic pressures within the body while retainimg the necessary pore space [6]. A significant characteristic of this hierarchy is the presence of an interconnected, extremely porous microarchitecture with an appropriate porosity level (i.e., 75–95 vol%). This microarchitecture is crucial for the development of cancellous bone scaffolds and for attaining the best possible results in vivo and in vitro [7]. The rate of degradation must be carefully regulated and linked with the new bone development rate. For instance, in the case of a spinal fusion scaffold, it takes approximately 9 months, while for craniomaxillofacial applications, it ranges between 3 and 6 months. This slow process enables the gradual development of new bone tissue and eventually the transfer of load to the newly produced bone [8, 9]. After the damaged area has completely healed, the scaffold ought to have completely deteriorated [10]. The interface between the biological environment and the bone substitute material can initiate a diverse range of actions, starting with an early inflammatory response and leading to the eventual remodeling of bone tissue [11]. Hence, the process of scaffold design encompasses a variety of complex factors, which are additionally specific to the repair sites on individual patients [12, 13].

The Layer-by-Layer (LbL) self-assembly technology has been developed to specifically tackle the issues mentioned above. The LbL technology's superiority allows implants to possess the characteristics of biological factors, cells, biomaterials, and drugs. This enables the implants to effectively heal bone defects by closely mimicking the mechanical properties, biological activity, and physicochemical composition of natural bone. The implant possesses adequate mechanical characteristics to facilitate bone growth and enable adhesion, proliferation, and cell migration. Additionally, it exhibits bacteriostatic characteristics and promotes vascular invasion into the scaffold, hence stimulating formation of bone [14, 15]. Moreover, it possesses favorable biocompatibility, limited susceptibility to infection, high modifiability, and without any apparent complications [16]. It has the potential to fulfill clinical requirements, making the advancement of LbL technology is crucial in various aspects of implant development.

LbL assembly

The LbL assembly is a flexible self-assembly method employed to construct multilayer films [17]. Investigations into LbL assembly have shown its effectiveness in a wide variety of applications, such as fuel cell membranes, energy storage, tissue engineering, and drug delivery [18,19,20]. This technique provides multilayer films through the alternating adsorption of multivalent molecules possessing complementary functionalities. A wide range of functionalities can be used to assemble these films, resulting in electrostatic interactions [17], hydrogen bonding [21], hydrophobic interactions [22], covalent interactions [23], and biological interactions [24]. After each adsorption step, an essential washing process is carried out to remove loosely attached species. The alternating functionality at each deposition stage enables ongoing film growth through successive adsorption cycles. Owing to the aqueous conditions commonly used in traditional LbL assembly, the method has proven highly successful in incorporating species that are sensitive to solvents and temperature, such as proteins [25,26,27], peptides [28, 29], and nucleic acids [24, 30, 31], while preserving their bioactivity.

Multilayer films can be constructed using multiple techniques, such as the conventional dip-based LbL assembly [17], as well as spray [32], and spin assembly [33]. The fundamental concept behind LbL technology involves the electrostatic attraction between anionic and cationic polyelectrolytes. This is achieved by immersing the substrate in solutions with opposite charges, permitting the addition of different bioactive macromolecules (e.g. polysaccharides, proteins, drugs, DNA, and polypeptides). Figure 1 presents a schematic representation of the dip LbL assembly process involving a pair of oppositely charged polyelectrolytes. In dip LbL assembly, the adsorption kinetics are governed by the diffusion of species to the substrate surface. In contrast, the spray LbL process employs convective transport, where deposition solutions are aerosolized and followed by spraying onto the substrate, significantly accelerating the assembly. This method reduces the time required to deposit a single layer from several min, typical of dip LbL assembly, to just a few seconds. The growth of LbL films frequently relies on the ability of their components to be diffused within the developing multilayer structure [34]. The manipulation of deposition conditions and the characteristics of the components governs interdiffusion, film structure, and overall growth. Particularly, important variables include pH, which affects the density of charges; ionic strength, which influences charge shielding; and molecular weight, all of which significantly determine the properties of the film [35, 36]. Recent studies have demonstrated that the choice of LbL deposition technique can directly influence film growth. Specifically, spray LbL assembly tends to produce thinner films growing linearly with reduced interdiffusion compared to dip-assembled films with identical architecture. In contrast, dip-assembled films demonstrate significantly greater interdiffusion and may experience highly nonlinear growth patterns [37].

Fig. 1

The schematic representation of the typical methodology employed for the production of LbL multilayers consisting of polyelectrolytes with opposite charges on a flat substrate

LBL assembly enables the coating of diverse substrates without significantly affecting their physical and mechanical properties. For instance, spray LbL assembly has been effectively employed to apply conformal coatings on porous materials, such as porous bandages [38]. Microscale and nanoscale substrates, including microparticles [25] and nanoparticles [39] and mesenchymal stem cells [40] have also been coated with multilayer films. The capability to incorporate diverse species, combined with the tunable chemical and mechanical properties of the films and the capacity to coat a wide range of substrates, enable LbL assembly stand out from other coating techniques and bulk polymerization methods, which often lack one or more of these advantages.Therefore, the LbL approach, with its ability to adjust the physical and chemical properties of various materials by controlling the number of layers deposited, has presented new opportunities and challenges in a diverse range of applications. These applications include biosensing, sensing, bioelectronics, the food industry, drugs, gene, or therapeutic delivery, protein adsorption, biomineralization, cellular inflammation, cell adhesion/proliferation/differentiation, tissue engineering, and regenerative medicine, catalysis, implantable materials, adhesives, separation, conversion, energy storage and more [41]. The feasible assembly process of LbL films enables their use as model surfaces for studying and controlling cell behavior, as well as for developing innovative tissue engineering structures and regenerative medicine treatments.

Bone regeneration encounters various obstacles, such as the difficulties of growing cells in vitro, replicating the natural process of bone formation, the high expenses and complex nature of production, and the need to develop biomaterial scaffolds that meet the specific mechanical, biological, and structural requirements for bone tissue engineering purposes [42, 43]. The use of LbL assembly for depositing multifunctional coatings offers a promising solution to these challenges. This technique is cost-effective and simple, enabling exact control over the coatings’ composition and structure. It also enables the fabrication of customized hierarchical structures and has the potential to accommodate high loads of bioactive molecules [44]. In addition, the process for developing coatings through LbL assembly is advantageous for the advancement of biocompatible implants intended for applications involving the replacement and repair of bone tissue [45, 46]. In this review, we provided a concise overview of LbL-assembled multifunctional scaffolds, focusing on coatings that possess customized bulk and mechanical properties, biocompatible coatings that facilitate adhesion and protein and cell growth, biodegradable coatings, as well as coatings that incorporate drug delivery systems and bioactive molecules.

Functions of LbL assembled film in bone scaffolds

One of the primary goals in bone tissue engineering is to develop and produce multi-scale scaffold materials that effectively integrate the necessary mechanical properties and porosity with biological requirements (including biodegradability, biocompatibility, and facilitation of protein and cell attachment) [47]. A high level of interconnected porosity is essential to facilitate bone growth within the scaffold. However, an excessive amount of porosity diminishes the mechanical properties. One prospective innovative strategy for addressing this persistent problem involves the ampplication of LbL assembly to apply a rigid and robust coating with a polymer-nanocomposite material on porous template scaffolds [45]. The use of the LbL method for depositing nanocomposite coatings onto resorbable scaffolds enables the development of scaffold materials with tailored biomechanical characteristics that can be gradually absorbed by the body [48]. A promising strategy to address these complex requirements is the application of multifunctional thin films as coatings via LbL assembly. This approach offers mechanical strengthening, regulated porosity, and additional features such as bioactivity. Furthermore, LbL assembly have effectively used to incorporate proteins, nucleic acids, polysaccharides, growth factors, and functional peptides into biomaterials. This technique also facilitates the deposition of coatings with controlled degradation rates, making it highly applicable for bone regeneration (Fig. 2).

Fig. 2

LBL assembly based approaches for effective application in bone regeneration. The LBL assembly is loaded with functional materials to be used in bone regeneration. The assessment of the LBL with materials is thoroughly carried out in-vitro and in-vivo for successful application in bone regeneration.

This section provides a comprehensive overview of the functions of LbL-assembled films. It focuses on how their performance and architecture can be used to customize bulk and surface mechanical characteristics, ensure biocompatibility, control biodegradability, deliver bioactive molecules and pharmacetical drugs, and enhance protein and cell adhesion for applications in bone tissue engineering.

Bulk mechanical properties

To stimulate osteoblasts, an effective bone scaffold must convey enough force to newly produced tissue while maintaining mechanical stability under varying loading conditions [49]. Achieving this requires that the mechanical properties of a bone scaffold closely align with those of the surrounding host bone tissue. In contrast to conventional methods for fabricating composite coatings. In contrast to conventional methods for fabricating composite coatings (such as in situ polymerization, sintering, melt intercalation, and vacuum evaporation), films developed through LbL assembly offer several advantages. These include enhanced mechanical properties due to the ability to incorporate a large amount of well-distributed nanofillers along with the flexibility to modify the material’s structure and composition at the nano- and micro-levels and easily incorporate multiple functions [50, 51]. Using LbL assembly, polymer nanocomposites comprising natural nanoclays, synthetic oxide NPs, carbon nanotubes, and carbon nanofibers have been manufactured with a rigid, reinforcing phase. The application of negatively charged MTM nano clay to reinforce the mechanical integrity of polymer films produced through LbL assembly has been the subject of numerous studies [52, 53]. Although numerous films constructed using LbL assembly have demonstrated remarkable mechanical properties, their microscale thickness poses a problem for macroscale applications that necessitate substantial materials, including scaffolds for bone tissue. The LbL assembly technique enables the application of conformal coatings on highly porous 3D templates, effectively overcoming dimensional limitations. This approach has been recognized as a successful strategy for transferring the mechanical characteristics of microscale coatings to larger-scale structures [54].

Local surface stiffness

Mechanical stimulation is a crucial regulator of bone function and structure; therefore, mechanical loading has a profound effect on bone healing [55]. Numerous approaches to bone tissue engineering depend on the inoculation of MSCs, which are capable of differentiating into a wide array of cell types [56]. MSCs are sensitive to the structure and composition of the substrates onto which they are implanted. It is well-established that the mechanical characteristics of 2D surfaces and 3D scaffolds stimulate the osteogenesis of MSCs, govern the behavior of attached cells, and affect the lineage specification of MSCs in vitro and in vivo [57, 58]. Pre-calcified collagenous bone tissue demonstrated a microenvironment rigidity exceeding 30 kPa, and matrices with comparable stiffness have been shown to enhance bone formation. Engler et al. showed that MSCs can differentiate into osteoblasts when grown on collagen (Col)-coated polyacrylamide (PAM) hydrogels with a stiffness ranging from 25 to 40 kPa [59]. Controlling the process of bone progenitor cell differentiation through mechanical stimuli and directing MSCs towards an osteogenic lineage using scaffold materials of appropriate stiffness in vitro can decrease the time required for cell pre-differentiation and eliminate the requirement for supplements that induce bone formation [60, 61].

The roughness, morphology, and thickness of LbL-assembled films can be adjusted by making slight modifications to assembly parameters including the solution pH, the polyelectrolytes' molecular weight, salt concentration, rinsing time, dipping duration, temperature and frequency of intermittent drying steps. Strong polyelectrolytes maintain a constant charge density in aqueous solutions, while weak polyelectrolytes exhibit varying charge densities based on pH, enabling adjustments in the interactions between charged polymers. According to Yang et al., the specific values for the thickness of 30 PEI/PAA bilayers were determined to be 4740, 890, 350, or 90 nm, which corresponded to distinct combinations of solution pH values (10/4, 8/6, 4/4, and 7/7 for PEI/PAA, respectively) [62]. Previous studies have described the impact of salt ions on the structural characteristics of polyelectrolyte multilayers, but there is a discrepancy in the findings. The thickness of PAH/PAA films exhibited a positive correlation with the NaCl concentration in the aqueous solution of PAA and PAH [63]. Thicker films in PEI/nano clay bilayers were produced by increasing salt concentrations in the PEI solution at pH 8 (1 mM, 10 mM, 100 mM, 1 M). On the other hand, film thickness remained unchanged when salt was added to the clay solution (pH 10.5) and unchanged PEI (pH 10). Mjahed et al. noted, however, that the PLL/HA film mass and thickness showed a decrease when the salt concentration exceeded 0.48 M [64]. A comparable phenomenon was observed in PAH/PAA films, wherein the thickness reduced as the salt content increased [63].

Additionally, the rigidity of LbL coatings can be enhanced by depositing a reinforcing layer with a high stiffness. As a means of enhancing the surface roughness and rigidity of PAM hydrogels, biocompatible MTM nano clay platelets were effectively integrated into PDDA/MTM bilayer coatings as mechanical reinforcement [65]. The hydrogel demonstrated an increased compressive modulus of 190 kPa after being coated with ten bilayers of PDDA/MTM, resulting in a scaffold surface that enhanced the migration and adhesion of epithelial cells. Numerous polyurethane foams with open cells have been employed as substitutes for cancellous bone. Due to its structural similarity to cancellous bone and various materials employed in bone tissue scaffolding, polyurethane foam was selected as a template to study the potential for enhancing the mechanical properties of highly porous bone tissue scaffolds through the application of the LbL coating technique. The technique of employing stiff coatings to strengthen softer substrates is illustrated by the LbL assembly of conformal coatings on porous 3D templates. The compressive elastic modulus of uncoated open-cell polyurethane foam samples, which was initially 95.33 ± 9.8 kPa, increased by up to 1100% to 882 ± 178.1 kPa after the deposition of 15 PEI/PAA/PEI/nano clay multilayers [52, 66].

Crosslinking is also a valuable method for altering the mechanical characteristics of LbL-coated substrates. Different chemical (such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and Gnp) and physical (such as photo-crosslinking) methods have been used to enhance the rigidity of LbL-assembled films. The application of, a water-based crosslinker (EDC), can result in the synthesis of amide cross-linking bonds after the polyelectrolytes assembly containing carboxyl and/or amine groups [67, 68]. Ren et al. demonstrated that the EDC concentration used in crosslinking after assembly was directly proportional to the rigidity of hyaluronic acid (HA)/PLL coatings applied to glass slide substrates. As rigidity increased, skeletal muscle cell adhesion (C2C12) also increased. The stiffest films (>320 kPa) were also discovered to stimulate cell proliferation, indicating that cell differentiation is dependent on film rigidity [69]. A different investigation on the crosslinking of PLL/HA multilayers using EDC revealed a variety of stiffness gradients ranging from 200 to 600 kPa [70]. Crosslinking PAA/PAH films with EDC has yielded stiffness values between 0.5 and 100 MPa [71]. Both the PAA/PAH material and PLL/HA systems resulted in enhanced cell proliferation and cell spreading, as cells adhered preferentially to the stiffer portions of the film [72].

Biodegradation

Biodegradation of scaffolds designed for bone tissue engineering should be carefully optimized to ensure the gradual transfer of mechanical loads to the newly formed and adjacent bone tissue [73]. When using cell-seeded scaffolds, it is important to regulate the degradation rate to prevent excessive deterioration of the scaffold during the early incubation stage in vitro. The rate of degradation of LbL-assembled films can be regulated based on the choice of polyelectrolytes employed [47]. Multilayered films composed of natural polyelectrolytes, such as polysaccharides, are susceptible to degradation by a range of enzymes, including Lys. Furthermore, the degree of crosslinking can be adjusted to control the rate of degradation. Schneider et al. established that the biodegradation of HA/CH and HA/PLL films in vitro can be modulated by varying the EDC concentration during crosslinking therapy, particularly when these films interact with macrophages and hyaluronidase [74]. When comparing untreated films to crosslinked films, it was observed that the crosslinked films had higher surface roughness, were 10 times more rigid, and showed a slower rate of degradation. Garza et al. showed that the rate of decomposition can be regulated by modifying the biodegradable layers' thickness [75]. Spray-casting was employed to integrate biodegradable PLGA barrier layers in between bilayers of PLL/HA that promoted exponential growth and served as reservoir layers in the films. Films consisting of one PLGA barrier and two PLL/hyaluronan bilayers were quickly degraded (within one hour) by murine bone marrow cells.

Furthermore, when applied to a biodegradable substrate, LbL-assembled coatings can be employed to regulate the rate of degradation of the underlying substrate material [76]. Mg offers the characteristics of being osteoconductive, biodegradable, and biocompatible. Additionally, it possesses a compressive strength and elastic modulus similar to that of bone, making it an remarkably suitable material for fabricating implants and scaffolds for bone repair applications. However, the restricted bioactivity of Mg implants in vivo restricts their common applications and results in the release of large volumes of Mg, hydrogen, and other ions due to their rapid degradation rate [77]. The recent introduction of metal-organic framework-CH coatings for surface modification of Mg alloys has provided a novel method to regulate the rate of degradation in Mg implants used for bone regeneration [78]. To improve the bioactivity of magnesium substrates, multilayer coatings composed of PLL and alginate were applied, incorporating fibronectin that was immobilized via EDC crosslinking. Based on an in vitro biodegradation study, coated substrates exhibited enhanced resistance to corrosion and decreased cytotoxicity in MC3T3-E1 osteoblast cell lines; however, the Mg alloy substrates' degradation kinetics remained unaffected [79].

Protein adhesion and cell interactions

The surface properties of implants significantly influence tissue and cellular processes, including protein adsorption and cell behavior, which are particularly crucial for the regeneration of bone tissue [80]. Significant attempts have been focused on enhancing the surface properties of bone scaffolds to provide additional functionality and establish an optimal setting for the growth of cells and the production of tissues [81]. LbL-assembled multilayer coatings provide significant potential in regulating cell attachment behavior on surfaces due to the significant influence of surface features on cell function [82]. Recently, considerable endeavors have been focused on enhancing the surface characteristics of LbL assembly films, including roughness, surface bioactivity, hydrophilicity, and other related features [53]. Further research has demonstrated that polyelectrolyte multilayer films (PEMs) effectively immobilize biomolecules while maintaining their bioactivity.

Various approaches to modify the physical, chemical, and mechanical characteristics of PEM films have prompted research on how these aspects affect biological interactions. A crucial requirement for the effe/ctive colonization of engineered bone substitutes is the adherence of cells to the scaffold material [83]. To increase pre-osteoblast MC3T3-E1 cell adhesion to nanofibre poly-L-lactic acid (PLLA) scaffolding, Liu et al. applied a poly(diallyldimethylammonium chloride) (PDAC) and gelatin-coating to the scaffold surface [84]. The quantity of gelatin was regulated by altering the final layer, while the wettability of the scaffold was managed by adjusting the number of deposited layers. At 4 and 24 hours after cell seeding, coated scaffolds contained significantly more MC3T3-E1 cells than uncoated scaffolds, and this number increased with the addition of additional PDAC/gelatin bilayers. Additionally, compared to uncoated scaffolds, the cells demonstrated a faster proliferation rate and a more uniform distribution on the coated scaffolds.

The strong adherence and absorption of proteins to the surface of biomaterials are essential for enhancing cell adhesion and promoting tissue regeneration. A bio-interface between native bone and implanted materials cannot be formed without protein adhesion [85]. The phenomenon of proteins adhering to LbL-assembled films has been extensively studied, leading to the development of a kinetic model that explains the mechanisms of protein adsorption [86]. In the context of osteoblast cell culture experiments, PLL/DNA bilayers were employed to improve biomimetic mineralization and protein adsorption on the Ti implant surface [87]. PLL is a biocompatible cationic polyelectrolyte and may be readily conjugated with bioactive compounds. DNA has been employed in the LBL assembly, together with layers of PDDA, PEI, and PAH [88]. On account of its high affinity for calcium ions, the high phosphate content of DNA was discovered to facilitate calcium deposition during bone formation. Additionally, the optimization of Ti’s bioactivity for applications involving bone tissue repair has been proposed via the LbL assembly of DNA and PLL [89]. Coated Ti surfaces exhibited enhanced cell-to-cell and cell-to-substrate interactions with osteoblasts extracted from neonatal rat calvarias, as confirmed by in vitro research, in comparison to control samples consisting of uncoated Ti [90]. The number of interconnected and overlapping osteoblasts was higher on surfaces coated with PLL/DNA/PLL. PLL as the final layer resulted in a greater surface irregularity of PLL/DNA coatings in comparison to other terminating layers. The biocompatibility and cell adhesion of Ti implants were enhanced through the implementation of tailored surface roughness, as determined by cell viability using the Alamar blue assay.

Hierarchical structure of bone scaffolds

The arrangement and architecture of nano- and micro-scale structures are crucial for cell performance. It has been suggested that bone scaffolds should be engineered to replicate the hierarchical structure of the specific bone being addressed [91]. The function of scaffold architecture is crucial in directing cell interactions through direct contact. To facilitate cell migration, support vascularization, assist in nutrient delivery, and eliminate waste, a functional bone tissue scaffold must possess an interconnected porous architecture [92]. It has been demonstrated that coatings can be effectively applied to porous foam substrates using the LbL assembly method. The materials prepared by the LbL assembly of nanocomposite-coated foams exhibited porosities that varied between 96.6 and 98.8% for uncoated foams and foams with the thickest coatings applied, respectively [52]. Similarly, inverted colloidal crystal (ICC) multilayer materials have been developed by first applying coatings onto colloidal crystals using the LbL method and then removing the crystal. The LbL assembly of ICCs has been employed to develop porous cellular environments that specifically target certain tissues, such as connective tissue, cartilage, and bone marrow, apart from bone.

Microscale structures have been extensively demonstrated to be crucial for the first attachment in the bio-interface of scaffolds and to enhance cell interaction. On the other hand, nanoscale organization has a more pronounced impact on cell differentiation and adhesion [93]. Apatite nanocrystals situated inside a Col matrix constitute the mineral component of bone. The remarkable mechanical qualities displayed by bone are largely due to the nanometer-sized HA crystals [94]. It is well-documented that nanostructured surfaces facilitate osteoblast adhesion, and the inclusion of an about 10-nm-scale dispersed nano-topographic surface has been shown to stimulate cell proliferation [95]. Cell behavior can be affected by interactions with nano- and microscale components of the extracellular matrix (ECM), as stem cells are responsive to the physical characteristics of the ECM [96]. Comprised of fibronectin, Col, and laminin, the ECM traverses multiple length scales, stores cytokines, and growth factors, transmits physical and complex biochemical signals, and stimulates cell–matrix, and cell-cell interactions [97]. Therefore, bioinspired 3D printed bone scaffolds with nano- and microscale characteristics that replicate ECM structures are among the latest approaches in bone tissue engineering. Nanostructured scaffolds of bone tissue, consisting of hydrogels, nanofibers, and NPs, are engineered to replicate the ECM and provide structural support similar to that of bone tissue. LbL assembly can result in functional biomolecules at precisely controlled dosages and multilayer structures with a wide range of potential compositions [98]. According to studies, LbL assembly may effectively use ECM components including growth factors, proteins, polysaccharides, and cells. The moderate deposition conditions of these components also preserve their bioactivity [98, 99].

Growth factors and bioactive molecules delivery

Biomolecule loading, such as growth factors and/or drugs, can improve the performance of bone scaffold materials, making them more effective in treating bone diseases while promoting bone regeneration through the stimulation of proliferation, cell adhesion, and differentiation [100, 101]. The intended site of action, appropriate concentration, and predetermined duration are all important considerations for incorporated drugs. Surfaces were further modified utilizing the LbL method to enable the production of drug delivery carriers with even better-targeting effects. In addition to facilitating localized delivery of the drug to the tissue and protecting against physiological media exposure, LbL-assembled films can assist in fulfilling these criteria [102, 103].

Regulating the number of growth factors administered to healing areas helps optimize their effectiveness and prevent adverse consequences such as bone loss, immune response, and tumor formation [all of which are associated with excessive doses of bone morphogenetic proteins (BMPs)] [104]. Growth factors can be incorporated into a multilayer film through electrostatic LbL assembly, or they can be added to crosslinked films after LbL assembly to provide reservoirs for the proteins [105]. Poly-β-aminoester 2 and PAA coatings were employed as delivery methods with adjustable regulation over the release of growth factors such as vascular endothelial growth factor (VEGF) and BMP-2. BMP-2, a well-known osteoinductive agent, has been widely employed to enhance the biological activity of bone implants or scaffolds by stimulating the proliferation, migration, and differentiation of MSCs [106]. Shah et al. investigated the deposition of LbL-assembled coatings comprising poly-β-aminoester 2-PAA/VEGF/PAA and poly-β-aminoester 2-PAA/BMP-2/PAA onto microporous PCL/β-tricalcium phosphate (3D) osteoconductive scaffolds [107]. The efficacy of the growth factors released from the LbL films, a number of in vitro tests were performed to quantify and visualize the effect of growth factor on a cell line with a physiological response to the growth factors. The two growth factors were introduced into the system in response to a pH change from 5.0 to 7.4, which led to charge imbalance and destabilization in the coating, as well as poly-β-aminoester 2's hydrolytic degradation. Several studies have investigated different methods for delivering BMP-2 using LbL-assembled materials. These methods include depositing a chemically crosslinked PLL/HA film onto TCP/HA porous ceramics to deliver rhBMP (recombinant BMP-2), graphene oxide onto Ti implants, and poly-β-aminoester 2/BMP-2 onto 3D printed β-TCP/PCL. These techniques were examined in a mouse model with calvarial defects in vivo [108, 109]. The use of LbL assembled films containing BMPs on a substrate permits precise and regulated administration, thereby potentially mitigating the primary limitation of the present clinical delivery approach for BMP-2 employing Col sponge, namely its rapid and uncontrolled release [110].

The potential benefits of antibiotic coating deposition onto orthopedic implants via the LbL assembly technique include the acceleration of osteogenesis and the reduction of implant-associated infections [111]. Compared to other coating techniques, the primary benefits of employing this technological method are its relatively simple operation and lack of apparatus requirements. Poly-β-amino ester/PEI/gentamicin sulfate films, developed using the LbL technique, were used to treat a pre-existing infection in a porcine model (pigs) by applying them to Ti implants [112]. The coating exhibited a 70% release of gentamicin in vitro and effectively reduced the number of viable bacteria in vivo. To achieve regulated release, bone growth factor BMP-2 with PDDA/CH, gentamicin-loaded PAA, and a multidrug coating on Si substrates were combined with clay barrier layers [113]. The purpose of this coating is to prevent implant failure caused by post-operative infection and to accelerate bone regeneration. The clay barrier obstructs interlayer diffusion, thereby facilitating tailored release behavior and accelerating the growth factor release rate. Wang et al. described a method for delivering both growth factors and cell adhesion peptides using a dual system [98]. In addition to loading BMP-2 with BSA-BMP-2 NPs, they employed oxidized alginate (OAlg) combined with CH. Grafting the resultant films onto Ti scaffolds produced an optimal microenvironment that stimulated the ingrowth of bone tissue. Because of the bovine-serum albumin, the coatings maintained BMP-2 release for 28 days. Both biomolecules facilitated MSC adhesion, growth, and differentiation while also improving the development of bone tissue in rabbits, hence the authors concluded the osteogenic differentiation of BMSCs in vitro and bone tissue regeneration in vivo is greatly enhanced.

To promote bone tissue regeneration, osteogenic growth factors and exosomes are also incorporated into LbL-assembled films. As part of a dual growth factor-release system, A core-shell silk fibroin (SF), polyvinyl alcohol (PVA), and PCL nanofibrous film was established by Cheng et al. via LbL assembly and co-axial electrospinning [114]. The nanofibers were modified to include BMP2, and a film assembled with LbL facilitated the attachment of connective tissue growth factor (CTGF) to the surface. In vitro and in vivo (nude mice) investigations demonstrated better osteogenesis and angiogenesis, and the combination of LbL and electrospinning methods offered a successful platform for CTGF and BMP-2 delivery. In addition to simulating the interactions among exosomes, cells, and ECM, the LbL-assembled film composed of glycosaminoglycan HA and polycationic poly L-lysine (PLL) has been demonstrated to exhibit a significant increase in affinity towards cells representing various lineages [115]. Recent studies have revealed that integrating osteogenic growth factors and functional exosomes into LbL-assembled films is a promising technique to more accurately replicate the host structure, improve growth factor delivery, and increase biological capabilities as they observed the increased bone regeneration in rats (radial defect model) [116].

Orthopedic applications of LbL assembly scaffolds

Scaffolds with numerous advantageous properties, such as osteogenic, anti-inflammatory, antibacterial, angiogenesis, and stability, have been improved via LbL self-assembly technology. Implant scaffolds designed to repair severe bone defects should possess these characteristics. The gradual release of biological agents, the pharmacological qualities of the drugs transported by LbL assembly scaffolds, or their inherent traits could all be responsible for these properties. Orthopaedics faces an immense obstacle in treating large bone defects; therefore, scaffolds must possess antibacterial, osseointegration, anti-inflammatory, and antioxidant properties to aid blood vessel function. However, ongoing efforts are being made to attain the desired result of diverse properties.

Angiogenesis

Angiogenesis is a critical process in the formation, evolution, and remodeling of bone, which is an exceptionally vascularized organ. Angiogenesis is an essential requirement for the healing of bone defects [117]. For bone remodeling and regeneration to take place at the site of bone defects, the vascular network must transport oxygen, soluble growth factors, nutrients, and various cell types to the area. Thus, it is crucial to incorporate a biological function into LbL implants to enhance their vascularization potential throughout the preparation process. Recently, significant research efforts have been focused on enhancing the bone regeneration and angiogenesis properties of bone scaffolds [118]. Several ways have been developed to incorporate metal ions or growth factors to control the processes of osteogenesis and angiogenesis. However, the decrease in biological activity and the possibility of causing cell damage restrict its practical use. Hence, it is imperative to investigate alternate approaches for bone formation that possess strong osteogenic potential.

Recent evidence indicates that deferoxamine (DFO), an iron chelator, can stimulate the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, which plays a role in promoting stem cell angiogenic response and osteogenic differentiation, thus facilitating tissue regeneration and wound repair [119]. The traditional administration of DFO is hindered by its short vascular retention half-life and limited agent availability, which reduces its effective action. Consequently, Ran et al. used the LbL assembly method to coat TiO₂ nanotube arrays with DFO before depositing CH and Gel multi-layer films [120]. A substrate consisting of deferoxamine-TiO₂-LbL was acquired. The findings demonstrated that the controlled release of DFO from TiO₂ arrays had a substantial effect on the growth patterns of human umbilical vein endothelial cells (HUVECs). This release also facilitated vascular differentiation and enhanced the proliferation, adhesion, and osteogenesis of MSCs. In a comparable investigation, Yu et al. employed LbL technology to load DFO onto TiO₂ substrates to treat bone defects; the results similarly demonstrated noteworthy angiogenic differentiation in vitro [121]. Growth factors play a critical role in the processes of angiogenesis and osteogenesis, and it has been demonstrated that bone BMP-2 is an essential osteoinductive growth factor. Multiple growth factors, including vascular fibroblast growth factor (bFGF), VEGF, and Tissue Growth Factor (CTGF), are frequently required for the repair of bone defects [122]. To impart their pro-angiogenic biological function, these growth factors have been utilized in the fabrication of LbL implants in several investigations. LbL assembly was used to incorporate VEGF and BMP-2 into hydroxyapatite composites to produce bone implants and used them to repair critical-sized calvarial defects in rabbit with the size of 15 mm in diameter. The findings showed that innovative composite scaffolds made of BMP-2 and VEGF exhibited exceptional angiogenic and osteogenic potential, thereby facilitating the formation of new bone as shown in Fig. 3 [123]. Shah et al. conducted a comparable investigation wherein they employed LbL technology to fabricate polyelectrolytes with angiogenic rhVEGF165 and rhBMP2 coatings [107]. surprisingly the findings showed a more thorough remodeling of the bone and an expansion of the local vascular network.

Fig. 3

Angiogenesis coupled with osteogenesis during intramembranous ossification: A Physiological VEGF levels maintain bone homeostasis. Low levels of VEGF disrupt osteoblast differentiation, while high levels increase osteoclast recruitment, leading to bone resorption; B Migration and proliferation of endothelial cells during bone repair, and secretion of BMP-2 and BMP-4.

Osseointegration effect

Orthopedic clinics emphasize the significance of the osseointegration effect as the principal scaffold property. It refers to the direct and organized functional and structural relationship between the surrounding new bone tissue and the scaffold. Following implantation, several biological processes surrounding the implant start to alter. These processes primarily involve osteocyte proliferation, adhesion, differentiation, and bone regeneration to accomplish osseointegration of the defect site and the scaffolds’ relative stability [124]. The osteogenic effect of scaffolds assembled with LbL is accomplished primarily via two distinct pathways. First, loaded agents (biological substances, including drugs) are released and diffused at the defect site. By their distinct biological or pharmacological properties, these agents facilitate bone integration; this is also the most prevalent method in LbL-assembled scaffolds. Second, the physical characteristics of the implant surface, such as its hydrophilicity, roughness, and wettability, have a direct impact on the behavior of cells, namely their proliferation, adhesion, and differentiation. Consequently, these parameters ultimately influence the effect of osseointegration.

To facilitate osseointegration, anabolic substances stimulate the activity of osteoblasts, thereby promoting osteogenesis. The incorporation of SV into the LbL bone scaffold led to a substantial increase in cell proliferation, mineralized osteoblast differentiation, and ALP. Furthermore, the group treated with SV-loaded multilayer implants exhibited a significant reduction in the enzyme tartrate-resistant acid phosphatase (TRAP), which serves as a marker for osteoclast development [125]. Vitamin D produces VD3, an active compound that facilitates the intestinal absorption of phosphorus and calcium. Nevertheless, the use and healing impact of VD3 is greatly restricted because of its limited ability to dissolve in water. Chen et al. [162] used β-cyclodextrin (β-CD) as a vehicle for VD3 to accomplish β-CD/VD3 coupling through VD3's interaction with its interior. This approach resolved the issue of VD3's limited solubility in water through the LbL self-assembly and enabled loading onto a Ti6Al7Nb substrate [126]. Under osteoporotic conditions, osseointegration can be substantially improved by the scaffold. The main types of anti-catabolic drugs included in LbL scaffolds are calcitonin, β-estradiol, and bisphosphonates. These drugs primarily work by inhibiting osteoclast activity to promote osseointegration, leading to a decrease in the bone resorption process. BMPs function as stimulators of bone development. It greatly contributes to the process of bone and cartilage formation. Studies have indicated that BMP-2-induced Smads pathway activation might raise the expression of the following osteogenic genes, such as OCN, ALP, or OPN transcription, which in turn promotes the differentiation of osteoblasts [127]. However, in patients with osteoporosis, BMP-2 can enhance the osteogenic differentiation of MSCs by upregulating the osteocalcin mRNA and DLX5 expression. However, it is crucial to maintain the dosage and activity of BMP-2. To regulate the release of the drug, Hu et al. developed multilayer Gel/CH coatings on TiO₂ nanotubes that they had fabricated as reservoirs for BMP-2 [128]. In addition to promoting the osteoblastic differentiation of MSCs in vitro, the layer maintains the drug's biological activity and release conditions.

The LbL assembly method, along with the characteristics of biomolecules or polyelectrolytes, significantly influences the structure and surface attributes of LbL bone scaffolds, such as hydrophilicity and surface roughness. The biological characteristics of bone cells and the tissues in their environment are directly influenced by the physical properties of LbL scaffolds; therefore, these properties cannot be disregarded. Rough surfaces possess an increased surface area, which not only facilitates drug release but also provides the opportunity for a larger quantity of drugs to interact with cells, thereby increasing the probability of contact. The LbL-based multifunctional (TA@HA/Lys)_n coating was developed by Wang et al. as shown in Fig. 4 [129]. The (TA@HA/Lys)_n coating's rough and porous surface facilitated the rapid adhesion and growth of MC3T3-E1 cells during the first phase, hence surface roughness has a dual effect of increasing bone production in rabbits (in vivo), and speeding up osteogenesis in vitro. The enhancement of the hydrophilic properties of the implant surface might lead to an increase in the binding of proteins, hence facilitating cell attachment, growth, and differentiation. Furthermore, this phenomenon might also be influenced by the effect of hydrophilicity on drug release. The LbL technique was used by Ao et al. to develop a novel, stable Col/HA multilayer modified Ti coating (TCs) [130]. The findings indicated that Col/HA multilayers with favorable hydrophilicity had a greater ability to enhance peri-implant osteogenesis in a rabbit model with femur condyle defects. This effect can be partially attributable to the hydrophilicity of the Col/HA multilayers. To simulate the extracellular matrix of bone, Greguec et al. coated self-assembling biopolymer films made of alginic acid and Col with Ti dioxide [131]. Sodium alginate-capped membranes exhibited greater cell viability and improved osteoblast adhesion, according to the findings.

Fig. 4

A schematic representation depicting the molecular structures of (A) Lys, (B) tannic acid (TA), (C) hydroxyapatite (HA), (D) the TA_pHs@HA forming process, and (E) the fabrication of (TA@HA/Lys)_n. (1) The substrate was initially coated with PEI, then (2) TA@HA, and (3) Lys were deposited; (4) this process was repeated a desired number of times for (2) and (3).

Antioxidant

Aside from infection, inadequate osseointegration, and neovascularization, which are crucial for the success of implants, the role of oxidative stress, particularly those induced by excessive generation of ROS, is increasingly recognized as an essential component. Normal ROS metabolism produces second messengers, which control various physiological and biological processes [132]. The equilibrium of osteolysis and osteogenesis, which has long been associated with bone defects, osteoporosis, and arthropathy, can be disturbed by an excess of ROS.

Studies have indicated that during oxidative stress, ROS enhances the activity of osteoclasts by accelerating nuclear factor kappa B’s (NF-κB) migration to trigger the RANKL pathway. This leads to disruption in osteolysis or bone resorption. Injuries and foreign body reactions during bone implantation can lead to scaffold failure or delayed scaffold osseointegration with the adjacent tissue. Foreign body reactions stimulate the recruitment of inflammatory cells and generate a substantial amount of ROS [133]. Using exogenous antioxidants is a successful strategy for addressing oxidative stress. Enhancing the antioxidant capacity of the scaffold surface by LbL technology can enhance the functionality of osteoblasts and promote increased bone formation [134]. Tannic acid, a widely distributed, biodegradable polyphenolic compound containing numerous phenolic groups, scavenges ROS by converting phenols into quinone groups. Using the LbL approach, Yang et al. developed tannic acid/Gel multilayer films by combining Gel’s biocompatibility and tannic acid's antioxidant properties [135]. Tannic acid/Gel multilayer films demonstrated favora antioxidant properties, effectively minimizing damage in ROS-induced stress environments. Furthermore, these films promoted osteogenesis in vitro and supported bone formation in vivo, as demonstrated in a rabbit bone implant model. Therefore, the study suggests that the antioxidant coating play a crucial role in optimizing the performance of bone-related implant. Zhou et al. designed a bone implant system with antioxidant properties by applying PPy-PDA-HA on the surface using an LbL-PED technique [136]. The system used a large number of catechol groups to provide a protective barrier against antioxidants. It also utilizes the stored electrons in PPy to neutralize free radicals. Additionally, the redox capacity of PPy-PDA NPs gives them the ability to scavenge radicals, which enhances the process of bone formation and enables the functionalization of LbL-constructed scaffolds.

Since they are powerful antioxidants, the naturally occurring polyphenolic bioflavonoids known as proanthocyanidins (PACs) are involved in a broad variety of biological processes. Experimental evidence suggests that PAC can regulate osteoclast bone resorption and promote bone growth [137]. LbL technology could be utilized by Tang et al. to inhibit ROS and improve osseointegration. A rat model of femoral intramedullary implantation was used to evaluate the osseointegration and osteoinductive potential of HA/CS-PAC coated titanium implants in vivo. Meanwhile, the murine pre-osteoblast cell line MC3T3-E1 was employed to assess the antioxidant and osteoinductive effects of multilayered HA/CS-PAC coated titanium substrates under conditions of H₂O₂-induced oxidative stress in vitro. As shown in vitro and in vivo, the LbL technology, which employs PAC HA and CH multilayers, inhibits oxidative stress and promotes osteogenesis and implant osseointegration, even under conditions of oxidative stress [138].

Antibacterial

Bacterial infection of LbL-constructed scaffolds can result in the loosening, separation, and failure of osseointegration of the scaffold. This can ultimately lead to the need for reoperation, making it a significant factor in scaffold failure [139]. Nevertheless, systemic administration presents several limitations, including the risk of adverse drug reactions and the difficulty in achieving effective diffusion of drugs and agents to highly calcified bone tissue via blood circulation [140]. Thus, it is imperative to develop a variety of antibacterial systems that may effectively prevent bacterial adhesion, reduce biofilm formation, and ultimately achieve sterilization. Orthopedic implant therapy is the possible and significant process of modifying scaffolds to facilitate osseointegration and prevent infection. The typical agents used in orthopedic bone scaffolds mostly consist of Lys, antibiotics, antimicrobial peptides, metal ions, and various other substances.

Gentamicin, a prevalent antibiotic, effectively impedes bacterial protein synthesis by mediating the binding with 30 s ribosomal subunit., resulting in the death of the bacteria. Additionally, it can hinder the synthesis of proteins by obstructing the production of mRNA complexes and interfering with bacterial membranes [141]. By depositing sodium alginate and CH onto arrays of TiO₂ nanotubes, Liu et al. produced gentamicin-loaded bone scaffolds using LbL technology [142]. The application of this treatment significantly decreased bacterial attachment and enhanced the integration of Ti and alloy scaffolds with surrounding bone tissue. Vancomycin, a frequently used therapy for gram-positive infections, can obstruct bacterial peptidoglycan’s ability to function, hence impacting the integrity of cell membrane components such as phospholipids and peptides. Resistance mechanisms have evolved, involving numerous enzymes, despite its apparent tolerance to resistance because of its particular bactericidal action. Because of this, vancomycin, the last line of defense against severe infections, may no longer be effective in clinical settings [143]. LbL nanocoatings were developed by Dwivedia et al. using vancomycin/PLA-loaded liposomes [144]. The nanocarrier system serves as a protective barrier for the agents, thereby promoting their decomposition, inactivation, and immune response. Bone plates coated with vancomycin liposomes demonstrated prolonged antibacterial efficacy without causing any harm to normal cells. These results offer a viable strategy for preventing Gram-positive bacteria and C. difficile from colonizing serious infections and forming biofilms. Other antibiotics, including metronidazole, tobramycin, clindamycin, and others, have been successfully incorporated into bone scaffolds using LbL for antibacterial applications, yielding more favorable results [145, 146].

Lys is an effective hydrolase with potent antibacterial properties. It can specifically break the β-(1, 4)-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan, which is the primary constituent of the bacterial cell wall. To ensure the continuous action and sustained activity of Lys, Li et al. [12] utilized LbL technology to fabricate ultrathin (GO/Lys) 8 films measuring tens of nanometers in thickness. To achieve this, they alternately deposited GO and Lys, given the sensitivity of Lys to changes in physical and chemical characteristics [147]. This method exhibits a highly potent bactericidal impact on Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). The LbL approach, which involves loading Lysine onto bone scaffolds, has demonstrated great potential in improving the antibacterial characteristics of bone implants [12]. Wang et al. [148] fabricated multifunctional coatings (TA@HA/Lys)_n using a LbL technique. The resulting hybrid coating, which includes Lys, has remarkable antibacterial capabilities and enhances bone formation in rabits (in vivo) as a result of its antibacterial impact.

Silver ion (Ag⁺) is commonly employed as an alternative antibiotic therapy because of its capacity to inhibit bacterial development and replication. Ag NPs possess a broad range of antibacterial effects and exceptional characteristics [149]. Ag ions can, however, aggregate and lose their antibacterial activity when exposed to oxidation. Furthermore, excessive amounts of Ag ions can induce toxicity in human cells and tissues [150]. To ensure the sterilization of Ag NPs while preventing adverse effects, controlled-release vehicles are therefore necessary. Studies have indicated that CH-heparin PEMs loaded with AgNPs can enhance antibacterial properties, facilitate cell adhesion and proliferation, and accomplish multi-path sterilization through modifications in surface hydrophilicity and roughness [151]. Deng et al. designed a dual-therapeutic implant coating by utilizing the LbL self-assembly technique with Ag⁺ and Zn⁺ [152]. The coating, formulated using microporous/nanoporous sulfonated polyetheretherketone, exhibited exceptional potential for osteogenic differentiation and bacteriostatic effects. A crucial trace element in bone tissue, zinc performs biological functions with antibacterial properties. Nevertheless, neither Zn nor ZnO exhibits potent bactericidal properties. Ag incorporation into the zinc surface may protect the osteogenic activity of the implants while substantially enhancing their bactericidal properties. By reducing the harmful effects of Ag, it is possible to enhance its antibacterial properties and support Zn in the process of osteogenesis, resulting in the combined effect of antibacterial osteogenesis [152].

Bioprinting in scaffold fabrication and application in bone regeneration

Three-dimensional (3D)-printed biomaterials and scaffolds produced through additive manufacturing for tissue engineering applications facilitate a more organized and systematic approach to scaffold/biomaterial design [153]. The emergence of 3D bioprinting technology has recently offered an innovative solution for fabricating three-dimensional tissue constructs with geometrically precise structures for the effective application in tissue regeneration [154]. 3D bioprinting combines tissue engineering, 3D printing, developmental biology, and regenerative medicine to develop biomimetic tissues, offering precise control over complex architectures, diverse compositions, and spatial distributions compared to traditional scaffold-based methods [155]. Both 3D printing as well as 3D bioprinting employ a LBL approach to fabricate anatomically shaped constructs from computer-aided design (CAD) models. However, 3D bioprinting specifically incorporates bioactive molecules and cell-laden bioinks for the purpose of forming the biomimetic tissue constructs, whereas traditional 3D printing does not involve cells or bioactive substances [155, 156]. Hence, the LbL concept can be effectively integrated with such modern additive approache, e.g. 3D bioprinting. This combination of LbL assembly with 3D bioprinting platforms offers significant benefits for fabricating the functional LbL nanocomposites, specifically designed for applications in regenerative medicine and pharmaceuticals (Fig. 5) [157, 158]. Guduric et al. [159] employed PLA/chloroform solutions as printing inks to fabricate PLA membranes through extrusion-based 3D printing. This method involved the LbL assembly of cellularized sheets, incorporating human bone marrow MSCs along with endothelial progenitor cells. Evaluation of MSC viability, phenotype, and proliferation showed evidence of osteoblastic differentiation after 7 days in culture media, indicating that the LbL assembly technique successfully supports even cell distribution within the scaffold. Furthermoe, Chen et al. [160] developed hydroxyapatite–gelatin scaffolds through extrusion-based 3D bioprinting for applications in bone tissue engineering. Subsequently, they applied LbL-assembled coatings of sodium hyaluronate and chitosan onto the scaffolds. Applying LbL coatings to hydroxyapatite–gelatin scaffolds resulted in a lower swelling ratio, improved compressive strength, and a slower degradation rate. Moreover, no significant cytotoxic effects were observed on MC-3T3E1 cells, and the scaffolds provided suitable conditions for cell proliferation as well as adhesion. These results suggest that the integration of 3D bioprinting with LbL assembly approaches holds substantial promise for bone tissue engineering.

Fig. 5

Integration of bioprinting with LBL assembly for bone tissue engineering. The computer programming is used for structure of the 3D printing. The feed materials are incorporated in the printer containing functional materials which creates LBL assembly for specific applications in bone regeneration

Furthermore, advanced bone scaffolds must posssed enough ability to regulate cellular behavior and mimic the native structure of bone tissue. Cui et al. addressed this by employing CAD to fabricate a 3D bioprinted bone construct with fluid-perfused microvascular networks and incorporated a nanoscale release system for dual growth factors (BMP-2 and VEGF) for enhanced tissue regeneration [161]. Following culture in co-cultured dynamic fluid systems, the 3D bioprinted scaffolds incorporating dual growth factors along with sequential release profile demonstrated remarkable bioactivity and significant potential for promoting vascularized bone formation (Fig. 6). Furthermore, Chen et al. developed polydopamine-modified calcium silicate (PDACS)/poly-caprolactone (PCL) constructs incorporating Wharton’s jelly MSCs combined with HUVEC-laden bioink [162]. The combination of synthetic materials with cellular components not only promoted osteogenesis but also facilitated the development of vascular networks during angiogenesis, highlighting the potential of 3D bioprinting to significantly enhance bone tissue regeneration across multiple dimensions. Increased levels of proteins associated with bone formation were also detected in the composites. Moreover, Chiesa et al. established an in vitro vascularized bone model by developing a capillary-like network using a gelatin-nanohydroxyapatite 3D bioprinted scaffold integrated with HUVECs and MSCs [163]. The investigation revealed that a sequential-induction strategy effectively achieved vascular-osteogenic finidings within a unified culture system. Initially, the approach involved promoting osteogenesis in hMSCs that were incorporated into a 3D bioprinted gel-nanohydroxyapatite (nHA) scaffold. Following this, the generation of a stable vascular network was stimulated within the hydrogel, which was enriched with HUVECs to fill the scaffold's macropores. This methodology concluded in a biologically relevant in vitro model of bone vascularization, mirroring the de novo formation of capillary vessels characteristic of tissue development. Furthermore, Dental pulp stem cells (DPSCs) ffer considerable advantages over traditional BMSCs and present a promising approach as seed cells for bone engineering bioprinting. This premise led to a study aimed at exploring the osteogenic potential of DPSCs within bioprinted scaffolds made from methacrylate gelatin (GelMA) hydrogels, both in vitro as well as in vivo [164]. Initially, GelMA was successfully bioprinted at varying concentrations, with and without the DPSCs incorporation. Afterwards, the osteogenic DPSCs differentiation in the bioprinted constructs containing varying GelMA concentrations were compared. For the assessment of the influence of DPSCs-loaded GelMA bioprinted structures on bone regeneration in vivo, a cranial defect model was established in immunodeficient mice, where the printed constructs were transplanted. After 28 days of printing, DPSCs in all three groups involved in bioprinted structures demonstrated high cell viability, exceeding 90%. Particularly, DPSCs in the 10% GelMA group revealed an increased expression of osteogenic markers, along with significantly activated ephrinB2/EphB4 signaling, playing a crucial role in bone homeostasis. In vivo experiments indicated that DPSC showed higher survival rates in 10% GelMA, leading to increased formation of new bone in the DPSC-laden 10% GelMA group as compare to the constructs with other concentrations. Therefore, bioprinted DPSC-laden 10% GelMA appears to be a more suitable option for bone regeneration applications.

Fig. 6

A Schematic representation showing the fabrication process for nanocoating modified 3D bioprinted scaffolds. The biomimetic scaffold was meticulously engineered to seamlessly integrate robust bone support with intricate vascular channels, utilizing advanced FDM printing techniques. Through strategic surface modification, the scaffold was transformed into a bioactive construct capable of promoting vascularization within bone tissue, thereby enhancing its functionality and effectiveness in biomedical applications. B Sequential adsorption of growth factors: rhBMP-2 was incorporated in the first 15 dual-layers, followed by rhVEGF in the top 5 dual-layers with genipin crosslinking. In a dynamic fluid, MMP2 secretion from HUVECs triggered growth factor release, leading to vascularized bone structure formation after 4 weeks of co-culture.

Current limitations and future perspective

LbL assembly offers a new method for advancing the combination of multilayer material systems that each fulfill specific requirements and can collectively fulfill all of the different demands in a coating material system applied to bone tissue engineering. The advancement towards this objective holds great promise for significant technological advances and substantial influence in the treatment of bone defects and various other biomedical applications. Although substantial advancements have been made in the last two decades, it remains important for researchers to carefully consider both the benefits and challenges of LbL assembly technology to facilitate its continued improvement and growth. It is particularly important to note that the main drawbacks of LbL assembly are associated with the inherent limitations of the molecular interactions that govern the assembly process. For instance, electrostatic interactions are only effective for constructing charged, water-soluble multilayer systems that are responsive to external stimuli. Hydrogen bonding tends to provide multilayers with lower stability, while charge-transfer interactions are confined to nonionic compounds. Host-guest interactions require precise molecular recognition, coordination chemistry interactions are constrained by the choice of materials, and covalent bonding is feasible for only certain molecules. These limitations significantly influence the formation of stable and durable multilayer structures. Although the current LbL assembly technique faces certain limitations, it presents various challenges and opportunities that invite further exploration of its significant potential in the near future, as outlined below. Firstly, various studies have concentrated on fabricating LbL multilayer assemblies using conventional electrostatic interactions, while the development of multilayer systems through other intermolecular or combined interactions remains inadequate. In this context, it is anticipated that advanced LbL assembly techniques employing various intermolecular interactions could open up new pathways for the precise regulation of multilayer film growth, thereby facilitating the development of well-defined nanoarchitectures with innovative functionalities. Secondly, a thoughtful integration of the LbL assembly technique with various bottom-up or top-down approaches (including photolithography and printing techniques) may enhance the methodologies available for LbL assembly construction. This combination would certainly contribute to the development of a wide range of novel nanoarchitectures with high performance at a reduced cost, facilitating their broader practical applications.

Although LbL allows for precise control over layer thickness at the nanoscale, achieving uniformity in coating thickness over complex 3D structures remains a challenge. Irregular surfaces and porous scaffolds, common in bone regeneration materials, can result in uneven coatings, leading to inconsistent drug release profiles and reduced overall efficacy. This has been a point of concern in biomaterial research, where non-uniform films can affect cellular responses and bone tissue integration. Furthermore, The sequential deposition process involved in LbL assembly can be time-intensive, particularly when constructing multilayered films with precise control over thickness and composition. This might limit large-scale or rapid production for clinical applications. The remaining challenges in the field involve optimizing process parameters to achieve rapid and more stable coatings, which are essential for the long-term viability of multilayer systems. There is a pressing need to develop an efficient coating strategy that facilitates the production of innovative bone medical devices at reduced costs while ensuring high reproducibility. In this context, integrating the LbL method with nano-drug delivery technology can provide a comprehensive strategy for addressing various bone disorders, such as osteoporosis and osteomalacia. Furthermore, the integration of LbL assembly techniques with other fabrication methods, such as additive manufacturing, holds the significant potential for transformative innovations. This synergy may enhance the formation of distinctive multilayer properties and accelerate deposition processes, thereby addressing the persistent challenges related to scaling up for industrial production. Over the past decade, all films synthesized through the LbL method have involved the alternating immersion of the substrate in solutions containing the interacting polyelectrolytes, along with rinsing steps using the solvent to prevent cross-contamination between the two charged solutions. Therefore, various successful attempts have been made to effectively eliminate the intermediate rinsing steps to enhance the deposition rate of polyelectrolyte multilayers. In this context, spin coating and spray coating result in reduced production time, which also decrease the manufacturing costs. In such circumstamces, spray coating and spin coating result in reduced production times, consequently lowering manufacturing costs. The shortened production and layering durations not only accelerate the layer formation rate but also limit the biomolecule’s diffusion from the coating surface during deposition (5–10 min per layer). It has been shown that spray coating reduces production time by a factor of 250 compared to traditional dip coating [165]; however, the layer formation can vary significantly, particularly with the deposition of weakly charged substances. For instance, vancomycin was previously applied using both dip or spray coating methods. Vancomycin, being a weakly charged molecule, experienced considerable interdiffusion during the dip coating process [37]. However, a single spray cycle occurs on a shorter time scale than the interdiffusion process, allowing the drug to remain at the layered structure surface. Therefore, dip coating leads to the formation of thick films with low drug concentrations, whereas spray coating yields thinner films with higher concentrations of drugs; this variation significantly influences their release profiles [37]. This example demonstrates the benefits of spray coating while emphasizing the importance of thoroughly understanding the kinetics of deposition in developing controlled-release devices. Moreover, employing microfluidics for microcapsule fabrication offers an alternative approach to enhance efficiency in terms of both time and cost. A microfluidic chip facilitated the deposition of six hydrogen-bonded layers of polyelectrolyte multilayers (PEM) on an oil core in under three min [166]. These chips are advantageous due to their scalability, reduced material consumption, potential for automated production, and ability to integrate in-process quality control screening (e.g. UV absorbance for content verification and DLS for size analysis).

While LbL assembly allows for the co-delivery of multiple bioactive molecules, precise control over their release profiles remains challenging. This can result in suboptimal therapeutic outcomes, particularly in the time-sensitive healing processes of bone regeneration. For instance, the cumulative release from multilayered constructs can lead to premature or delayed therapeutic activity, affecting the regeneration process. For example, growth factors like BMP-2 need to be released at specific stages to induce osteogenesis, but without precise control, these factors may be released too early or too late, reducing their efficacy in promoting bone formation. Future research should focus on enhancing the precision of release kinetics for bioactive molecules, possibly by fine-tuning layer thickness or incorporating stimuli-responsive elements that allow for dynamic control of delivery.

LBL technology has been effeiciently employed to assemble polyelectrolytes, growth factors, natural polymers, pharmaceutical compounds, and proteins for diverse healthcare applications. Nonetheless, the structural complexity of proteins restricts their application in LBL assembly. During LbL assembly, proteins may undergo structural changes, denaturation, and loss of biological activity as they interact with the partner subtances. Therefore, optimizing assembly conditions while selecting suitable LbL partners is essential for ensuring proper orientation, controlled adsorption, and effective protein’s organization. Extensive efforts have been accomplished to modify the intricate surface charge characteristics of particular type of proteins, including the use of protein-polymer complexes and protein crystals or nanoparticles. Engineered proteins featuring improved stability, specificity, and tunable properties have the potential to address certain limitations of native proteins, thereby enhancing their integration into LbL films. However, the processes of protein production as well as purification can be costly, tedious, and time-consuming. Beyond the cost as well as availability of proteins in large quantities, several challenges must be addressed for commercial-scale applications. These challenges include the difficulty in controlling film uniformity regarding roughness, thickness, and architecture; prolonged build-up times associated with dip coating; batch-to-batch variability; the industrial adaptability and automation of equipment for film production; and the need for effective quality control. Incorporating proteins into LbL films for commercial use can introduce regulatory challenges, particularly concerning safety, contamination, and biocompatibility. To address these issues, it is vital to ensure a sterile environment, such as a clean room, or to implement suitable sterilization techniques as methods like heat or irradiation may adversely affect protein stability.

The biocompatibility of LbL coatings has been well-documented, but their long-term interaction with bone cells and immune responses in vivo requires further exploration. Some studies suggest that LbL films can provoke inflammatory responses or fibrous encapsulation, which can hinder the healing process and reduce the material’s effectiveness in promoting bone growth. Moreover, the complexity of the bone tissue environment may affect the predictability of LbL film behavior once implanted. More comprehensive long-term studies are needed to fully understand the behavior of LbL scaffolds in vivo, including their biodegradation, immune response, and integration with bone tissue over extended periods.

While successful bioprinting of tissue-engineered bone constructs has been reported, several pressing challenges in bioprinting still demand targeted attention. One primary concern is the absence of standardized guidelines for the entire 3D bioprinting process. Furthermore, the structural complexity of bone tissue significantly complicates the fabrication process, especially when compared to avascular cartilage tissue, containing a limited number of chondrocytes. Moreover, distinct regions of bone tissue in the human body possess varying biomechanical properties and microenvironment architectures. Therefore, obtaining accurate imaging data, coupled with computational simulations and mathematical modeling, is essential for effectively designing the structure of the target tissue. Moreover, the fabrication of vascularized tissue-engineered bone presents unique challenges, as it necessitates the use of at least two distinct cell types, unlike some tissues containing only one. Therefore, it is vital to ensure cell viability throughout the bioprinting process while also promoting the exchange of nutrients and bioactive factors between the host and the engineered constructs. Hence, there is a crucial need for more sophisticated and adaptive bioinks with the potential to support cell bioactivity and accurately emulate the properties of natural tissues. Moreover, establishing standardized protocols and securing regulatory approval will be pivotal in transforming LbL assembly-based therapies from the research setting to clinical practice. Achieving this will require effective collaboration among researchers, regulatory agencies, and industry partners.

Although numerous new technologies and procedures have been developed for bone tissue repair and regeneration, there is still significant potential for further advancements. It is anticipated that there will be a substantial rise in the need for industrial and academic applications while using this LbL method to fabricate multifunctional coatings. Due to its adaptability and tunability in producing multilayer coatings with tunable mechanical properties and structures, the LbL assembly method will be regarded as a promising method in the field of bone-regenerating biomaterials. The LbL assembly process offers both precise nanoscale control for surface modification and new opportunities to produce novel deposited scaffolds for biomaterials-related tissue engineering applications. In conclusion, there are numerous opportunities and challenges associated with the use of bone scaffold materials in bone regeneration and repair using the LbL technique. Future research in bone tissue engineering will focus on investigating the effects of perfusion, loading conditions, and coating methods on scaffold materials. Specifically, the goal is to understand how these factors influence the behavior of bone cells and bone repair under hydrated settings. The LbL technology is primarily advancing in the tissue engineering and biomedical field. It is anticipated that there will be a significant rise in the need for industrial and academic applications in designing and manufacturing multifunctional coatings using the LbL approach.

Conclusion

A persistent obstacle in the field of biomaterials is the need to meet the wide-ranging demands for engineered tissue using a single-material system. The LbL assembly process provides the benefit of incorporating a diverse range of components into a single multilayered material system. Furthermore, it is a simple, cost-effective coating method that is applied to a variety of substrate materials. Recently, there has been an extensive exploration of various techniques and substances to produce LbL-constructed materials. These studies have demonstrated the ability to achieve precise thickness control, locally adjust mechanical stiffness, enhance mechanical strength, promote biodegradability, improve cell and protein adhesion, facilitate biologically beneficial electroactivity, develop hierarchical microstructural characteristics, and facilitate drug delivery. Recent studies have documented the use of LbL-assembled coatings consisting of composites of distinct multilayers to generate multifunctional coatings. These coatings achieve their multifunctional properties by integrating layers that fulfill various requirements, such as drug delivery and mechanical reinforcement. The capacity of LbL films to incorporate a diverse array of therapeutics, including small molecules and sensitive biologics, while preserving their bioactivity, presents a promising opportunity for bone regeneration. This approach could pave the way for innovative therapeutic strategies in modified bone implants, fracture healing, and localized, controlled release systems, either passive or actively triggered, to enhance tissue repair and regeneration in targeted bone regions. The continuous advancements in LbL-assembled materials underscore their potential in addressing the complex requirements of tissue engineering and therapeutic applications.