Growth factors in the transforming growth factor-beta (TGF-β) superfamily are unified by polypeptide structure, functional with a broad range of target cells that facilitate cellular proliferation, differentiation, extracellular matrix production, and embryonic development. The TGF-β family, particularly isoforms 1–3 (TGFβ1–3), are implicated in cartilage and bone regeneration ^[1][6].

TGFβ1 is explicitly an inducer of osteoblast differentiation during bone reformation. The downstream signaling of TGFβ1 involves the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) pathways, all active mediators in mesenchymal stem cell differentiation ^[2][7]. When released from platelets after blood clot formation at the site of bone injury, TGFβ1–3 binds to type 2 heteromeric receptors, which phosphorylate type 1 heteromeric receptors. Subsequent phosphorylation of specific Smad proteins acting as transcription regulators can induce collagen type 2 and proteoglycan synthesis or secretions associated with ossification of the soft callus ^[3][4][8,9].

N-cadherin expression is also induced by the activation of TGFβ1–3, promoting mesenchymal progenitor cell differentiation into chondrocytes during extracellular matrix development. Conflicting studies have examined whether all TGFβ isoforms are equally stimulatory in chondrogenesis, so further elucidation may be necessary. TGFβ1–3 are important regulators of the TGFβ subfamily BMPs ^[5][10].

BMPs are highly established osteogenic factors in skeletal research, with BMP-2, -4, and -7 showing clinical promise in bone and cartilage regeneration. Both homodimer and heterodimer configurations of the 15 BMP isoforms are active in mesenchymal stem cell differentiation into osteoblasts or chondrocytes and are inducers independent of a stimulus ^[6][11]. BMPs are also implicated in embryogenesis and cell homeostasis in other tissues ^[7][12].

Platelets secrete selected isoforms in various bone healing phases, such as BMP-2, -6, and -9 that are involved in osteoblast formation from MSCs and others in osteocyte maturation. BMP-2 release is localized to the injury site but accepted as an angiogenic factor in contribution to bone regeneration ^[8][13]. Although BMP-3 inhibits the functions of other BMPs, it is the amplest isoform in adult bone. As with all TGF-β superfamily growth factors in chondrogenesis, the BMP/heteromeric receptor complexes cause Smad messenger phosphorylation for altered transcription and increased collagen type 2 or proteoglycan, therefore inducing cartilage and associated extracellular matrix development ^[9][14].

Amongst the most studied BMPs, the BMP-7 isoform, when overexpressed in MSCs, has been shown to increase the release of vascular factors as well as induce osteogenesis from MSCs secretions. There does, however, appear to be an optimal dose of delivery to rats at 50 ug for this function, whereas a more potent dosage does not increase efficacy ^[10][15]. A BMP-2/7 heterodimer combination may be a more effective engineered facilitator of bone repair as opposed to either BMP-2 or -7 isoforms. The impact of this engineered growth factor on inflammation should be elucidated further before human clinical use ^[11][16]. The regulation of the pleiotropic function of BMPs between osteogenesis, chondrogenesis, and angiogenesis needs to be established experimentally to optimize the best dose and combination of these factors.

Mediators of angiogenesis, such as the dimeric vascular endothelial growth factor (VEGF), aid the success of bone and cartilage regeneration. VEGF is particularly implicated in neovascularization and, remarkably, the recruitment of osteogenic cells that differentiate into osteoblasts in bone formation. Its recruitment function is supported by VEGF release during callus formation and resorption ^[12][17].

The hypoxia-inducible factor-1 is activated under oxygen environments of low partial pressure post-tissue damage. Osteoblasts and pre-osteogenic cells release VEGF, binding to VEGF tyrosine kinase receptors 1 and 2 (VEGFR1–2) on endothelial cells. VEGFR1–2 activation induces notch, RAS-Raf-ERK1/2, and Phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (AKT) signaling and endothelial cell growth and migration ^[13][18]. The resultant blood vessels formed to stimulate the recruitment of osteoblasts to the injury site and provide localized oxygen, nutrients, and growth factors relevant to bone maintenance. Usually, cartilage lacks blood vessels. However, chondrocytes have the capability to release VEGF during bone repair ^[12][17]. Not only do factors in the TGFβ superfamily mediate VEGF release, but the PDGF also exhibits the same role ^[14][19].

PDGF is observed to induce chemotaxis and mitosis amongst MSCs, chondrocytes, and inflammatory cells. Specifically, the synthesis of hyaline cartilage and its extracellular matrix is induced via PDGF ^[15][20]. A role in osteogenesis is also suggested by studies on the PDGF-BB of the five isoforms. Usually, hours to 3 days post-bone injury, platelets are trapped between a hematoma formed post-injury and release PDGF, binding to its respective PDGF receptor (PDGFR) ^[14][19]. G-protein coupled receptor kinase interacting protein-1 expression is also increased with PDGF ^[16][21]. PDGFR-AA, -AB, and -BB binding of the complementary PDGF isoform initiates ERK1/2 and PI3K/AKT signaling downstream of the growth factor/receptor complex. Proliferative growth is subsequently stimulated in MSCs and inflammatory cells during the inflammatory and early soft callus phase ^[17][22].

Both insulin-like growth factors 1 and 2 (IGF1–2) polypeptide isoforms are imperative in the later stages of bone repair post-inflammatory phase. IGFs can induce osteoblast and chondrocyte development from MSCs or osteoclasts from myeloid precursors. IGFs, therefore, mediate anabolic and catabolic processes in bone repair. Both isoforms bind to IGF binding proteins (IGFBP1–6) and IGF1 or IGF2 receptors. Intracellular signaling in relevant target cells induces bone matrix synthesis via type 1 collagen release, although IGF-1 is also implicated in osteoblast chemotaxis and function via target cell interaction ^[18][23]. Osteoblast differentiation relates to IGF-1 via activation of the rapamycin complex 2 (Mtorc2)/AKT pathway for the hedgehog and Gli-2-regulated transcription ^[19][24]. The release of IGF-1 is also apparent during impaired bone matrix resorption via osteoclasts, the catabolic link to IGFs in bone repair ^[20][25]. This conflicting effect of IGFs makes it tricky to use for bone regeneration.

IGF1 and 2 mediate chondrocyte and cartilage extracellular maintenance via signaling for matrix synthesis and inhibition of enzymes in extracellular matrix degradation. Signaling for PI3K/AKT and ERK 1/2 pathway activation is implicated in these events. With IGF-1 the more abundant isoform in skeletal tissue, MSC chondrogenic differentiation is observed with IGF-1 signaling ^[21][26]. However, the IGF-1 activity in bone and cartilage tissue is augmented when combined with other growth factors, such as TGF-β.

Twenty-two homologous polypeptides encompass the fibroblast growth factor (FGFs) family, which is associated with the renewal of many tissues. In terms of skeletal rejuvenation, FGFs are released from MSCs, osteoblasts, inflammatory chondrocytes, endothelial, and macrophage cells ^[22][27]. Subsequent endothelial and osteoblast proliferation via FGFs cell surface binding also indirectly promotes angiogenesis; hence FGFs are recognized as angiogenic factors. FGF transmembrane receptors (FGFRs) contain an intracellular tyrosine kinase domain that is phosphorylated with tyrosine residues under ligand binding and induce a cascade of target protein phosphorylation to induce phospholipase C3-kinase/AKT, Ras/MAPK, phospholipase C, and protein kinase C signaling. The transcription of the gene involved in cell proliferation is also mediated by FGFs via the signal transducers and activators of the transcription 1 (STAT1)/p21 pathway ^[17][22].

FGF-2 is the best studied of this growth factor family in bone and cartilage regeneration and is identified as an inflammatory cytokine followed by FGF-1 in only bone regeneration. In terms of bone tissue, both growth factors induce soft-callus formation after the inflammatory phase, osteoblast function in bone reformation, and MSC proliferation ^[23][28].

Cartilage joint development and matrix homeostasis via chondrocyte production is facilitated by FGFs, particularly FGF-2. The priming mechanism for chondrogenic differentiation is induced prematurely by FGF-2. Specific induction of F-actin element structural changes during monolayer cartilage expansion aids chondrogenesis, confirming FGF-2 function ^[23][28].

As with the previous growth factors discussed, FGF delivery in combination with other growth factors in skeletal regeneration enhances results; however, the coupling should be carefully selected. BMP-6 has been shown to suppress selected FGF-2 pathways in chondrogenic differentiation ^[24][29]. In turn, FGF-2 could inhibit the TGF-β effect in MSCs, prohibiting its crucial function in cartilage regeneration ^[25][30].

2. Layer by Layer Assembly Technology for Growth Factor Delivery

Layer by layer is a simple and easy technique for the formation of polyelectrolyte multilayer. Porous scaffolds are generally modified through this process for the controlled delivery of growth factors because it performs well in preventing growth factor function loss and sequestering high growth factor concentration in a moderate aqueous environment. In the formation of this assembly, hydrogen bonding, electrostatic and covalent interactions are typically utilized ^[26][71]. The polyelectrolyte multilayer properties can be adjusted to regulate the growth factor release. Therefore, this assembly technique enhances loading capacity and desired bio-factor release can be used for the delivery to the target site. Vectors for gene delivery and DNA can be introduced into the layers without any alteration to their native conformation. 3-D bioprinting is a novel LBL technique that combines materials with growth factors and forms a 3-D scaffold, thus, manifesting capability in the framework of regulated drug delivery ^[27][72]. Ansboro et al. worked on the LBL technique and reported that TGF-B-3 binds the HA microsphere, presents an appropriate drug delivery system, and enhances the chondrogenic gene expression ^[28][73]. Another study exhibits that on the PLGA membrane, an LBL nanolayer coating of BMP-2 and PDGF resulted in better bone regeneration than BMP-2 alone delivery in mice models ^[29][74].

3. Hydrogel Technology for Growth Factor Delivery

Hydrogels are compounds with a high-water content and are among the few biomaterials that can be utilized to make ECM-like scaffolds. Hydrogels can be utilized for controlled drug delivery to target sites in bone defects ^[30][75]. Drug encapsulation in the hydrogel is one of the common and simple strategies to produce a 3-D drug delivery system. Hosting of drugs, proteins, and DNA within hydrogel can be done by mixing the polymer matrix before any kind of crosslinking. For hydrogel carriers, both natural and synthetic materials can be used ^[31][76]. Hydrogel releases drugs on the desired sites and possesses tissue-compatible substrates for better cell growth and attachment. Hydrogels could preserve the growth factor bioactivity for a long duration preventing them from environmental degradation. It has been reported that BMP-2 incorporated inside HA hydrogel remained bioactive even after 28 days. Another study demonstrated that BMP-2 had been encapsulated in the gelatin implants, and they remained active for six weeks in the cell culture ^[32][77]. In another study, a hydrogel coating on the surface of the PLC scaffold was utilized for BMP-2 delivery for checking its effect on bone regeneration. The results showed enhanced bone mineralization and regeneration in contrast with scaffolds without containing hydrogel ^[33][78]. Hydrogel also has some limitations. A molecule’s entrapment directly depends upon which encapsulation method is used. In the physical encapsulation method, usually, diffusion is used for the controlled release, while in chemical encapsulation, the release depends on polymer degradation or gel matrix dissociation. Another disadvantage is that the hydrogel network mainly consists of water, so its tensile strength may be lower, hindering its capacity to bear the load ^[34][79].