With the promoted understanding of the underlying mechanism of osteogenesis (Chen et al., 2012), BMP, as a prominent member of the TGF-β superfamily, has always been a favored candidate for bone tissue engineering applications.

Since some hydrogels are believed to possess inferior osteoconductive properties, Olthof et al. (2018) modified an oligo[poly(ethylene glycol) (PEG) fumarate] (OPF) hydrogel with bisphosphate. BMP-2 was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres. The additional BMP-2 and drug-laden PLGA microspheres were entrapped in the hydrogel matrix. The researchers believed that the anionic groups would produce a strong interaction between the matrix and inorganic phase of the bone as well as enhance BMP-2-induced bone formation. The hydrogel matrix could be functionalized by peptides, which might be beneficial to reduce the dose of encapsulated BMP. In addition, nanofibrous mesh-hydrogel hybrid composites have been applied to reach a proper spatiotemporal release profile (Kolambkar et al., 2011). Shekaran et al. (2014) modified a matrix metalloproteinase (MMP)-degradable peptide crosslinked PEG with an α2β1 integrin-specific peptide (GFOGER). The interaction between integrin and collagen I has been proposed to be vital in osteogenic differentiation and mineralization. It was suggested that the modified matrix is able to support cell adhesion and proliferation and upregulate osteogenic gene expression. Laden with the low dose of BMP-2, robust bone healing was achieved. Along with BMP-2, BMP-7 is considered to be a promising GF in bone formation. An injectable chitosan/β-glycerophosphate (CS/β-GP) hydrogel laden with BMP-7 and antibiotic exhibited preferable reparative effects towards infection-induced bone loss (Zang et al., 2019). Growth differentiation factor-5 (GDF-5), also known as BMP-14, regulates the development of numerous tissue and cell types, including limbs and teeth. Bae et al. (2014) mixed different concentrations of GDF-5 with a light-cured hydrogel matrix. The results showed that GDF-5 improved the osteogenic ability in a dose-dependent manner, as the strongest augmentation was achieved by the hydrogel loaded with the highest concentration.

Apart from adsorption or physical entrapment, electrostatic, hydrophobic, or other interactions have been introduced into the systems to prolong the release of BMPs. Heparin was reported to be a strong binder to BMPs, yet the side effects were not negligible. Heparin mimics, which are usually negatively charged, are supposed to be capable of controlling BMP release. Chondroitin sulfate (Anjum et al., 2016), 2-N,6-O-sulfated chitosan (26SCS)-based nanoparticles (Cao et al., 2014), alginate sulfate (Park et al., 2018) were synthesized by researchers, and satisfactory results were achieved both in vitro and in vivo. When higher concentrations of heparin mimics were introduced, the release rate of BMP became slower. Seo et al. (2015, 2017) harnessed the ionic and hydrophobic interactions provided by polyphosphazene nanoparticles. They found that release rate of BMPs were controlled by the types and amounts of pendants. Thus, the optimal release profile and osteogenesis outcomes rely on a reasonable proportion of BMP-tethering molecules.

Genetic engineering is another option to obtain long-lasting BMP release. As Lin et al. (2019) described in a manuscript, the BMP gene was transduced into human bone marrow-derived stem cells (BMMSCs), obtaining a continuous (up to 56 days) and economical BMP supply. Using visible light-based projection stereolithography (VL-PSL) technology to encapsulate the transduced cells, the researchers were able to fabricate more structurally and geometrically compatible constructs for individualized bone defects, which would be conducive to achieving tissue fusion and bone tissue engineering long-term success.

Vascularization plays a crucial role in both bone development and bone regeneration (Collin-Osdoby, 1994; Olsen et al., 2000). Blood vessels do not solely work as substance exchange pathways; they are also regarded as highly active paracrine organs targeting various progenitors during bone formation and reconstruction (Collin-Osdoby, 1994). VEGF, a key angiogenic growth factor (Carmeliet and Jain, 2011), has been widely used in bone tissue engineering.

The cooperation between VEGF receptors and integrin adhesion receptors has been elucidated in angiogenic regulation. Garcia et al. (2016) engineered a protease-degradable, GFOGER-modified PEG hydrogel as a VEGF depot. They found that covalently linked VEGF remained highly bioactive during a prolonged release period. Whereas it was shown that a GFOGER hydrogel augmented neovascularization regardless of exogenous VEGF, micro computed tomography (micro-CT) showed delivering exogenous VEGF failed to enhance critical-sized bone repair. Heterogenous material composites are manufactured by which we can juggle both timed drug release and osteoconduction. Composed of a 3D multichannel calcium phosphate cement (CPC) and alginate/gellan gum (AlgGG) hydrogel, the CPC/AlgGG biphasic scaffold tethers VEGF via the interaction with heparin (Ahlfeld et al., 2019). Despite some remarkable properties observed in vitro, significant enhancement by VEGF on new bone formation has not been detected. Amirian et al. (2015) coated VEGF and BMP-2 separately onto gelatin-pectin-biphasic calcium phosphate composites. The results revealed that composites coated with VEGF mainly aided in woven bone formation, and the percent of new bone formation was not greater than those coated with BMP-2.

Since exclusive delivery of VEGF performed barely satisfactorily in GF-induced osteogenesis, dual or multidrug delivery is warranted. When accompanied by BMP-2, VEGF exhibited a significant improvement in bone formation compared with hydrogels encapsulating BMP-2 alone. VEGF combined with BMP-2 has been used routinely as a GF formula in bone tissue engineering. Similar loading strategies were applied by Barati et al. (2016) and Kader et al. (2019) for spatiotemporal release of BMP-2 and VEGF. MSCs and BMP-tethered nanoparticles were embedded in the outer space, while endothelial colony-forming cells (ECFCs) and VEGF-tethered nanoparticles were dispersed inside the microchannel-patterned hydrogel, as illustrated in Figure 1. Degradation and GF release could be tuned by altering stoichiometric ratio chain-extended molecules and proteolysis. According to the data, the release of VEGF and BMP-2 could last over 10 days and 21 days, respectively. It was observed that the patterned hydrogel dual delivery system performed significantly better than that of single delivery systems, which was attributed to paracrine crosstalk. During bone repair, VEGF expression peaks appear in the early period, while BMP peaks later. Thus, consisting of a PLGA microsphere-incorporating poly(propylene fumarate) (PPF) rod surrounded by a rapidly degrading gelatin hydrogel, the composite was designed as a GF delivery vehicle (Kempen et al., 2009). VEGF was encapsulated in the hydrogel, whereas BMP-2 was immobilized by microspheres inside the rod in order to achieve an ideal GF sequential release pattern. VEGF exhibited a large initial burst release within the first 3 days, and BMP-2 showed sustained release over 8 weeks. Likewise, although VEGF did not induce neo-bone formation, it significantly enhanced BMP-induced osteogenesis. Organic-inorganic modular scaffolds are able to optimally orchestrate dual GF release and serve as an “anatomy-structure-function” trinity system in regenerating weight-bearing bones (Bao et al., 2017). Mesoporous bioactive glass (MBG) with hollowed channels and hierarchical porous structures was introduced in a controlled release system as a scaffold (Tang et al., 2020). VEGF was carried by hydrogel inside the channel, and BMP-2 was adsorbed by the MBG scaffolds. 26SCS acted as an analog of ECM, which exhibits super-affinity to GFs. In vitro experiments showed that 26SCS promoted the bioactivity of BMP-2 and VEGF. It could be assumed that the VEGF hydrogel column in the hollowed channels might induce chemotaxis of vascular endothelial cells, thus regulating cell migration and vascular infiltration. Moreover, increased type H vessels and neotissue ingrowth were observed.

FGF signaling is a dominant regulator during bone development and fracture repair (Bourque et al., 1993; Kronenberg, 2003). However, contradictory results have implied that FGF signaling may exert dual-directional effects on osteogenic procedures, probably in a dose-dependent manner (Kato et al., 1998; Quarto and Longaker, 2006). Thus, sustained release should be achieved when FGF is delivered in bone tissue engineering.

Two Japanese groups encapsulated FGF in gelatin hydrogels for controlled release (Kodama et al., 2009; Furuya et al., 2014). A longer FGF release period may improve cell proliferation, the expression levels of osteogenic markers and BMP-2 as well as bone mineral density (BMD) at defect sites. However, these enhancements vanished, and side effects occurred when a high dose of FGF was delivered (Kodama et al., 2009). In order to achieve bone-like biomechanical properties and slower release of FGF, a stiffer hydrogel matrix, poly(2-hydroxyethyl methacrylate) copolymerized with 2-vinyl pyrrolidone, was engineered (Mabilleau et al., 2008). The data suggested that in the first 4 days, the FGF release rate was approximately 1% per day, which was relevant to hydrogel swelling. Unfortunately, no significant difference between the FGF and control groups was noted in bone mass, but the poorly mineralized woven bone area was significantly larger in the FGF group.

It is a preferable strategy for other GFs to accompany FGF in order to obtain a promising outcome. Chen et al. (2016) chose gelatin microspheres as BMP-2 and basic FGF (bFGF) carriers, which were further embedded in a commercialized injectable thermal-sensitive hydrogel. The hydrogel was injected into a porous cell-loading scaffold before use. Micro-CT revealed that the dual-loaded composites achieved the best reparative results. As expected, composites loading bFGF alone regenerated less bone and neobone at the margin of the defect areas, while the dual-loaded composites showed much more central area bone formation. FGF9 has been indicated to be a stabilizing factor for neovessels, thus, Yuan et al. (2016) introduced FGF9 as an assistant for VEGF, exerting synergetic effects on angiogenesis in bone tissue engineering. A specific peptide segment was fused to VEGF and FGF9 to obtain a covalent connection with the fibrin hydrogel. BMP-2 was transfected into BMSCs, endowing a greater osteogenic ability and resistance of the osteogenic differentiation inhibition induced by fusion with FGF9. Less bone was formed in the FGF9 groups compared to the groups treated with only VEGF, whereas VEGF/FGF9-loaded composites performed the best among the groups.

Other peptides that regulate the bone regeneration cascade, including osteoprotegerin (OPG) (Jayash et al., 2017), stromal cell-derived factor-1α (SDF-1α) (Ratanavaraporn et al., 2011; Cipitria et al., 2017; Mi et al., 2017), platelet-derived growth factor (PDGF) (Wang et al., 2020), and parathyroid hormone (PTH) (Erten Taysi et al., 2019), etc., might also be worthy of an attempt. The selected studies of hydrogel-based peptide sustained release systems for bone regeneration and their findings are concluded in Table 1.

Since magnesium ions (Mg²⁺) play an important role in bone metabolism and mineralization, a variety of strategies for the sustained delivery of Mg²⁺ have been applied to hydrogel-based scaffolds. Lin et al. (2018b) coated MgO nanoparticles with PLGA and an alginate hydrogel, constructing a monodisperse core-shell delivery system. The release profile of Mg²⁺ revealed a significant suppression of the initial burst, and its release rate was stable and programmed. Enhancement of progenitor cell viability and proliferation, upregulation of osteogenic gene expression levels, and increased bone regeneration volume in vivo were attributed to the stable and precise Mg²⁺ supply. Bisphosphonates (BPs) possess two adjacent phosphonic groups, which are propitiously bind to divalent metal ions. Zhang and colleagues (Zhang et al., 2017) developed acrylated-BP-Mg nanoparticles to deliver Mg²⁺ as well as strengthen the acellular hydrogel composite, which serves as a matrix for in situ bone formation, via multivalent crosslinked domains. They also utilized Mg²⁺ to fulfill on-demand intelligent drug release in bone tissue engineering (Zhang et al., 2018). Intriguingly, Mg²⁺ played multiple roles in this research. First, BP-Mg nanoparticles enabled hydrogel formation and stabilized the prodrug. Second, Mg²⁺ promoted osteogenic differentiation, resulting in increased alkaline phosphatase (ALP) expression. However, and more importantly, Mg²⁺ is also a critical cofactor of ALP. ALP enzymatic hydrolysis was promoted; thus, more bioactive drug molecules were generated, which introduced positive feedback (Figure 2). According to the results, this strategy significantly enhanced bone regeneration.

Other metal ions, such as strontium ions (Sr²⁺) and cobalt ions (Co²⁺), may act synergistically in bone reconstruction. A Sr²⁺-crosslinked RGD-alginate hydrogel combined with Sr-doped hydroxyapatite microspheres was engineered, showing a sustained release of Sr²⁺ from two sources (Lourenco et al., 2019). The researchers elaborated that this Sr-hybrid system facilitated MSC osteogenic differentiation, inhibited the functions of osteoclasts and modulated the inflammatory response. As a pro-vasculogenic factor, Co²⁺ was incorporated into the alginate hydrogel shell, while BMP-2 was laden into the collagen core (Perez et al., 2015). Co²⁺ released relatively rapidly, as expected. VEGF secretion and qPCR revealed that Co²⁺ not only stimulated angiogenesis but also elevated osteogenic gene expression. These results indicated an appealing prospect for applying metal ions bone tissue engineering in the future.

A range of pharmaceutical molecules were designed or discovered to be effective in bone regeneration. Highlighted as chelating agents, BPs are utilized as antiresorptive drugs frequently in clinics. BPs mainly target osteoclasts, impeding the differentiation and maturation of osteoclast progenitors. Increasing evidence has shown that BPs directly or indirectly take part in other bone-forming mechanisms and are capable of targeting various cells (Corrado et al., 2017). Since bone healing and regeneration is known to consist of three consecutive phases of inflammation, repair, and remodeling, a proper scale of immune response is indispensable (Claes et al., 2012). However, excess or aberrant immune activation may jeopardize bone repair procedures (Claes et al., 2012; Gibon et al., 2017). Therefore, immunomodulatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), have been applied in bone tissue engineering. Evidence has shown that aspirin elevates MSC osteogenic potency by inhibiting the tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) pathways (Liu et al., 2011). Statins are inhibitors of a key enzyme of cholesterol synthesis and are widely used to lower serum lipids. Researchers have reported that osteogenesis was enhanced concomitant with promoted BMP-2 expression in bone cells when treating cells and rodents with statins (Mundy et al., 1999). Localized and sustained delivery of these drugs via hydrogels has pointed to a new direction in bone tissue engineering. Many relevant studies are listed and outlined in Table 2.