Bone, as the main part of the body’s skeletal system, is responsible for a variety of crucial functions such as providing locomotion, protecting soft tissues, and acting as a storage site for minerals and bone marrow [1–5]. It’s ability to provide these functions is a testament to its structurally dynamic and complex architecture that is highly adaptive based on mechanical demands [1, 2, 6]. One of the most common traumatic injuries within the musculoskeletal system are bone fractures, which are caused by exertions of significant force that overpower what the bone can withstand, leading to a crack or break [7–10]. The intricate activities and organization of the native tissue, however, allows for a built-in complex healing mechanism for these scenarios, in which fractures can repair themselves following damage without scarring [7, 9, 11–15].

In contrast to this, critical size defects, another source of bone related injuries, are defined by their inability to spontaneously heal despite the introduction of surgical stabilization [16–20]. This category often stems from large bone loss as a result of traumas, infections, diseases, or non-unions, and represents a major clinical challenge in the domain of orthopedics [16, 17, 20–23]. Consensus on the exact definition of this term is not currently well established in the field, as many considerations play a factor including the condition of the soft tissue, anatomical location, presence of adequate blood supply, and overall patient medical history [16, 17, 21, 23]. However, it is generally accepted in the literature that if the defect length is larger than 2 cm, with a concurrent loss of more than 50% of bone circumference, the terminology can be applied, and a surgical intervention is needed for effective bone healing [16, 17, 24].

A variety of treatment methods for these defects have been explored clinically, both through the incorporation of different grafting materials such as autografts and allografts, as well as surgical techniques including distraction osteogenesis or the induced membrane technique. While the effective management of these defects has been shown to be complex, the current clinical gold standard approach for treatment is the autologous grafting technique [13, 16–18, 22, 25, 26]. This method boasts many advantages including its low cost, high incorporation rate, lack of disease transmission and immunocompatibility, as it is taken directly from a patient’s own body at a distal site from the defect [17, 22, 24, 27, 28]. Despite these beneficial traits, autologous grafts are often associated with numerous disadvantages as well accompanying its usage, with the most important one being its limited graft volume [13, 16, 17, 22, 23]. Other limitations to these grafts include large rates of complications, donor site morbidity, infection, and long hospitalization times [16, 17, 19, 22–24, 29, 30].

The evident limitations associated with clinical treatments have led researchers to use the established principles of engineering and biology to design and fabricate artificial bone specific microenvironments capable of encouraging repair in large defects [31–38]. These substitutes are expected to act similarly to the native tissue in terms of both architectural structure as well as intended function [34, 35, 39]. Thus far, no specific design of these biomaterials has been able to successfully satisfy the complex criteria needed to be considered ideal for bone tissue regeneration [37]. The most important of these requirements is the notion of biocompatibility, meaning that the implanted material should elicit an appropriate host response that is specific for the intended application [33, 35, 37, 40–44]. This new definition of the term considers the material’s functionality as well as its intended target for regeneration, which was not previously included. Triggering of the foreign body response post-implantation can lead to specific fibrin adsorption onto the scaffold’s surface, ultimately leading to faster material resorption and possible harmful effects on the patient [37, 42].

To successfully accomplish the design of these biomaterials, researchers have set forth a polytherapy framework called the “Diamond Concept”, implementing five main components: osteoconductive scaffolds, osteogenic cells and osteoinductive mediators within a suitable mechanical environment incorporating an appropriate vascularization strategy [18, 32–34, 41, 43, 45, 46]. The biomaterial itself in this context is typically composed of either metals, ceramics, polymers or composites, which act as a 3D microenvironment that provides structural support and promotes the functions of encapsulated cells [25, 43, 47, 48]. Among these substitutes, injectable hydrogels, composed of a cross-linked hydrophilic polymer network, have recently gained interest in these clinical applications due to their advantageous properties, including the potential for less invasive surgeries and decreased hospitalization times [19, 49–51]. In our laboratory, we have previously developed one of these injectable hydrogel scaffolds, which forms through electrostatic attractions between anionic phosphate groups of a guanosine diphosphate (GDP) purine crosslinker and cationic amine groups of the chitosan polymer [19, 49, 52–57]. Results found that crosslinking occurs rapidly (<1.6 seconds) in this scaffold without the need for external stimuli, such as pH or temperature changes, thus limiting unwanted solution diffusion into surrounding tissues, ensuring localization at the defect site, and allowing for an easy and efficient encapsulation of cells and factors [58–60].

The notion of this polytherapy approach follows the more recent progression from the implementation of bioinert to bioactive materials within bone applications, with research showing that osteoconductivity and osteoinductivity are equally important design parameters, and that the scaffold should have the ability to interact with cells in the local microenvironment [31, 33, 38, 39, 42, 61–64]. The combination of these five aspects is expected to more carefully mimic the natural healing environment of bone, thereby encouraging cell and tissue growth as well as bone regeneration. Incorporation of cells within biomaterial designs has been evidenced to significantly improve healing outcomes through the cells’ ability to secrete extracellular matrix proteins as well as different growth factors and cytokines [33, 34, 65]. Different studies in the literature have indeed confirmed this benefit, demonstrating a significant lack in cellular ingrowth from the surrounding host tissue in non-cell-loaded scaffolds [66].

Osteoinductive mediators, such as bone morphogenetic proteins (BMP), platelet derived growth factors (PDGF), and biologically active ions, have also long been explored as encapsulants in biomaterial scaffold design due to their well-established ability to promote cellular growth, differentiation and bone formation [33, 45, 67–74]. While BMPs, particularly BMP2, remain gold standards in osteoinduction, recent research has shifted towards finding alternative approaches that can overcome some of BMPs’ limitations, namely their high cost and possibilities for ectopic bone formation. Usage of glycogen synthase kinase 3 (GSK3) inhibitors, for example, are one of these newer approaches, that have been shown to have a positive effect on both osteogenic differentiation and subsequent bone formation. Some studies in the literature have indeed reported significant increases in the early osteoblastic differentiation marker, alkaline phosphatase (ALP) and osteogenic gene expression levels with this addition in cultures of different cell types compared to respective controls [75–83].

The overall success of these GSK3 mediators can be heavily attributed to their well-established role as agonists to the Wnt/β-catenin signaling pathway, which plays a critical role in osteogenic processes, including both differentiation and bone formation [84–92]. Canonical Wnt signaling is initiated when Wnt proteins bind to lipoprotein receptor-related protein 5 and 6 (LRP5/6) and frizzled co-receptors, triggering an intracellular cascade that activates bone formation [87, 93]. As the loss of function of the LRP5 co-receptor has been shown to be linked to a decrease in bone mass, similar to cases of osteoporosis, this further highlights the importance of this pathway in skeletal development [87, 94, 95]. The binding of Wnt proteins to these receptors results in a disbanding of the destruction complex, which in turn prevents the phosphorylation and subsequent degradation of cytoplasmic β-catenin [84, 87, 93]. Due to this action, the stabilized β-catenin that has accumulated in the cytoplasm can then translocate into the nucleus, where it binds to transcription factors such as lymphoid-enhancer binding factor (LEF) and T-cell specific transcription factor (TCF) to successfully upregulate osteogenic target genes [84, 87, 93]. Interestingly, activation of Wnt signaling has also been demonstrated to regulate osteoclastogenesis, by increasing osteoprotegerin expression, which can aid further in healing [94–97]. The dual function of Wnt signaling renders it an attractive target for enhancing the osteoinductive properties of biomaterials, offering a promising potential as a mediator for improving bone repair and regenerative outcomes.

Therefore, in this review, the landscape of materials for scaffold design is discussed with respect to bone regenerative applications in defect healing specifically, emphasizing the recent shift from single material scaffolds to those made of composites, or modified using coatings and nanoparticles (Figure 1A). Additionally, we provide a summary of previous work on the usage of purine crosslinkers, for the first time, within our laboratory’s chitosan based crosslinked scaffold with an aim of improving bone repair and regeneration. The current advancements on the incorporation of Wnt signaling mediators in biomaterial scaffold design are also presented, reflecting the growing interest in these strategies for promoting osteoinduction in these biomaterials. Although only 42 articles have been published on this topic over the past decade, the increasing number of publications each year, along with promising findings, suggest a clear trajectory for future research (Figure 1B, Figure 1C).

The consideration of the specific clinical application is crucial within the design process as singular situations will necessitate different priorities with respect to scaffold properties [37, 39, 42]. Since bones often serve load-bearing purposes, such as the continued compressive and torsional loading of long bones, the mechanical properties should be one of the key considerations for choosing a material [33, 35]. The intended substitute needs to be able to withstand the expected loading conditions so as not to allow for tissue collapse, and modulus values need to match those of the native tissue surrounding the defect site [33, 37, 38]. In cases where the mechanical properties exceed that of the natural bone tissue, a phenomenon called stress shielding occurs, which correlates with excessive resorption [37].

The natural polymer chitosan is one of the bulk materials that has been widely examined with respect to the growing field of injectable hydrogels and scaffolds, encouraging the need for less invasive surgical procedures and thus a decreased hospitalization recovery period [49–51]. This benefit comes from their inherent ability to solidify into 3D structures following in vivo application through the presence of external stimulants such as changes in pH, temperature, and UV irradiation [19, 56]. The gelation mechanism of injectable scaffolds also provides for an easy encapsulation technique for both cells and osteoinductive mediators, providing them with a hydrated internal environment [19, 50, 51, 205–208].

Usage of crosslinkers, such as genipin [209–211], glutaraldehyde [212–214], and tripolyphosphate [215, 216], has been well established in the injectable scaffold domain, meant to stabilize the chitosan scaffold’s internal structure and improve physical properties [54, 56, 217–219]. These evident benefits on material properties, in the scope of chitosan-based biomaterials for bone tissue engineering, have been thoroughly demonstrated in the literature and reviewed in a recent publication by the authors [204].

However, these gold standard crosslinkers tend to present cytotoxicity and compatibility issues in their usage, rendering a need for alternatives [19, 49, 52–57]. One of the more recently developed chitosan-based crosslinked scaffolds in this field, developed by our laboratory, relies on the addition of a purine as an anionic crosslinker for the first time, to overcome these previously demonstrated limitations [19, 49, 52–57]. Purines, such as adenine and guanine, and their derivatives are considered to be naturally biocompatible as they are involved in different biological processes within the body including the formation of nucleic acids, energy transfers and the actions of various signaling pathways [54, 58, 59, 220, 221]. Composed of a pyrimidine and an imidazole, purines tend to have a negative charge in solution, which helps crosslink the cationic chitosan chains together through mixing without the need of external triggers or stimuli [58–60].

Both GDP and adenosine diphosphate (ADP) have been investigated as crosslinkers for chitosan within the scope of this scaffold. Benameur et al. [56] demonstrated that while a sponge will form with either crosslinker through the ionic interaction between phosphate groups on the purines and NH₂ groups on chitosan, slight differences are observed when GDP is used compared to ADP. GDP crosslinking allows for additional Hoogsten hydrogen bonding between the guanine groups resulting in the formation of G-tetrad-like structures (Figure 5A) [56]. In comparison, ADP crosslinking is relatively linear with each ADP molecule interacting with two chitosan chains, one through its phosphate group and an additional one through its NH₂ group [56].

Further optimization studies on these designed scaffolds have examined the effect of different chitosan concentrations and solution pH values on the gelation and structure of the scaffolds (Table 2) [49, 52, 56]. Since pH greatly affects chitosan’s solubility and the availability of protonated sites for interaction with anionic purines, maintenance of the pH value below that of the pKa was necessary [56]. In this scenario, pH values of 5 or 6 were selected for scaffold fabrication as they allowed for the most rapid interactions between the chitosan chains and the purine crosslinkers [49]. Similarly to pH optimization, the concentration of chitosan in solution before crosslinking (3 mg/mL and 6 mg/mL) was also important to optimize preliminarily as it was shown to affect the integrity of the formed scaffold [49]. Mekhail et al. [49] observed that when too low of a concentration was used, viable sponges were not able to be produced, and in too high of a concentration, a very viscous chitosan solution was present thereby undermining the injectability of the sponge. Combinations of these two concentrations and pH values were used in early scaffold studies to examine their influence on the scaffold’s material properties [49, 52, 56]. However, results suggested that these factors did not significantly affect the scaffold formation as Fourier transform infrared spectroscopy (FTIR) showed no changes in peak intensities for any of the participating groups and cellular metabolic activity was similar amongst scaffolds with the same crosslinker [56].

Previous literature findings have clearly demonstrated the importance of Wnt signaling in the early stages of fracture healing and bone formation, thus suggesting a new potential avenue for drug and therapeutic targeting [234–237]. As mentioned earlier, binding of Wnt proteins to appropriate receptors activates the canonical Wnt pathway, whereby the destruction complex is disbanded, leading to increased levels of stabilized β-catenin that can translocate and upregulate target genes [84, 87, 93]. In the absence of this stabilization, the differentiation of stem cells into the osteoblastic lineage is inhibited, and thus bone healing is negatively affected [234].

Many publications in the literature have used this knowledge to suggest the targeting of cytoplasmic protein, GSK3, inhibition through small molecules, as an alternative technique to mimic the function of Wnt related proteins and improve the osteoinductive potential of bulk or composite biomaterials (Table 3) [234, 235, 238, 239]. Indeed, work by Comeau-Gauthier et al. [240] successfully demonstrated that the embedding of GSK3 inhibitor, Tideglusib within a surgifoam collagen scaffold in a mouse femoral cortical window defect model yielded a significant increase in the bone volume fraction compared to controls (Figure 7A). This finding was accompanied by an observable increase in ALP activity, which together confirm the benefit of GSK3 inhibitors on the initial stages of osteoblast differentiation [240].

The examination of other GSK3 inhibitors such as lithium chloride [75–81], AZD2858 [82, 83], and 6-bromoindirubin-3'-oxime (BIO) [88, 89, 243, 244, 248–251] in bone applications have all also demonstrated similar positive effects on early markers of osteogenic differentiation such as an increase in ALP levels or RUNX2 gene expression. Interestingly, a study conducted by Li et al. [89] on stem cell like myoblastic C2C12 cells was able to illustrate increased BMP2 induced ALP activity in BIO treated groups compared to those of other GSK3β inhibitors (Figure 7B) [89]. These findings and the adjacent null results presented in cultures of pre-osteoblastic MC3T3 cells suggest a potential additional benefit in a GSK3β independent mechanism at early stages of differentiation [89]. Within the same study, the authors were further able to confirm the significant reductive effect of BIO on the phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) in the MAP kinase pathway but not on p38 phosphorylation, thus concluding a combinatory influence of BIO on both the Wnt and BMP signaling pathway [89].

Indirubins and their derivatives, such as the semi-synthetically derived cell-permeable indirubin, BIO have been more recently established as gold standard agonists of Wnt signaling, along with lithium chloride and are known to derive from edible gastropod mollusks as well as shrubs [237, 254, 255]. Multiple studies exploring the usage of BIO on cell cultures have clearly indicated the improvement of osteogenic differentiation parameters in its presence, yet the importance of dosage on the observation of these positive effects is consistently seen throughout the literature [88, 248, 251]. Indeed, Zhao et al. [88] observed that the addition of BIO to co-cultured bone marrow derived MSCs (BMSC) in doses past 2.5 μM resulted in significant morphological changes to the cells along with a general inhibition of proliferative activity. Further work within the same study incorporating lower doses of 0.5 and 1 μM were successful in increasing the number of ALP positive cells, as well as an enhancement of gene expression levels for early osteogenic markers, COLI and RUNX2 [88]. Another study using the GDP crosslinked chitosan scaffold [252] has also exhibited the dose dependency of BIO incorporation, with findings revealing a marked elevation in both ALP secretion levels as well as RUNX2 gene expression (Figure 7C) for 1 μM doses compared to 10 μM. BIO’s dose dependency is highly expected as it is characteristically known to have cytotoxic effects at higher concentrations, thereby explaining previous literature findings [243].

The general translation of the promising osteoblastic differentiation results to in vivo models has, however, been limited with results showing mixed healing outcomes [234]. Application of these activators in a systemic manner, often used in animal studies, generally faces obstacles due to the non-specificity of the Wnt signaling cascade [234, 237]. This could explain why systemic treatments of these therapeutics tend to result in a relatively poor biodistribution and an increase in the possibility of off-target side effects [234, 236]. These limitations have therefore shifted the focus of bone tissue engineering research in this domain towards the design of localized targeting methods such as drug loading into delivery carriers such as liposomes and nanoparticles, or encapsulation in biomaterial scaffolds and hydrogels (Figure 8) [236].

A recent study by Low et al. [242, 252] effectively demonstrated the benefit of localized treatment by comparing two BIO incorporated aspartic acid octapeptide micellar delivery systems (branched and linear) to a direct injection of free BIO through the tail vein. Results showed that by adding the targeting moiety to these two groups, BIO was able to accumulate within the fractured bone region, prior to subsequent clearance by the kidneys (Figure 7D) [253]. In comparison, the free BIO injection yielded a systemic biodistribution with almost no accumulation visible in the fracture area (Figure 7D arrow A), thereby confirming the benefit of the targeting moiety [253]. Further work by the same research group additionally demonstrated that the implementation of the bone specific targeting mechanism allowed for a significant increase in bone density, volume fraction and trabecular thickness compared to respective free BIO control within their mouse femoral fracture model [242]. Together, these results clearly indicate the importance of a localized treatment method, which allows for the benefits of Wnt signaling agonists, while effectively reducing the necessary drug dosages and limiting unwanted off-target accumulation [234, 236, 237].

The necessity for a localized delivery system to target the Wnt signaling pathway highlights the significant potential of GSK3β inhibitors as osteoinductive mediators in composite scaffold designs. Unlike single bulk biomaterials, composite scaffolds offer the advantage of precise tuning of material properties such as porosity, mechanical strength, and biodegradability. This customization allows the designed scaffolds to more closely mimic the natural bone microenvironment, which ultimately improves their biocompatibility and ability to integrate effectively in the surrounding tissue. These tailored scaffolds can be further designed to incorporate bioactive agents that activate the Wnt signaling pathway, which has been well-established as a key regulator of osteogenesis. By enabling a sustained and localized release of these osteoinductive signals, composite scaffolds provide continuous exposure of the cells in the surrounding tissues to osteogenic cues, that promote both osteoblastic differentiation and mineralization. The prolonged osteoinductive effects, achieved through the encapsulation of these factors in composite scaffolds, can effectively enhance bone formation and have the potential to significantly improve healing outcomes.

The design of an optimal biomaterial for this complex application necessitates a carefully thought-out approach that incorporates the principles of the diamond concept, which is essential for successful healing outcomes in critical size bone defect treatment. As research within this field continues to evolve, future work should continue to focus on developing composite and co-polymeric biomaterials that more accurately resemble the native internal architecture and functionality of the bone tissue they intend to regenerate. Recent studies have increasingly suggested the potential of targeting Wnt signaling through incorporation of specific GSK3β agonists as osteoinductive mediators during the biomaterial design stages. Introduction of a localized approach through scaffold encapsulation facilitates a precise targeting action to the specific tissue of interest and mitigates associated risks of systemic delivery systems. Initial findings in the literature indicate that this targeting strategy has the potential to significantly enhance both osteogenic differentiation and bone formation, further underscoring its promise to achieve more favorable healing outcomes. Thus, the integration of these advancements into the biomaterial design process could improve the material’s osteoinductive potential and lead to the development of a more effective clinical treatment for critical size bone defects.