D to create GF gradients inside hydrogels: (a) concentration gradient of a single single Trk custom synthesis biomolecule (GF1), (b) sequential delivery of three unique biomolecules (GF1, GF2, GF3), and molecule (GF1), (b) sequential delivery of 3 various biomolecules (GF1, GF2, andand GF3), and (c) encapsulation of biomolecule(s) polymeric micro- and nanocarriers; and (C) solutions for (c) encapsulation of biomolecule(s) in polymeric micro- and nanocarriers; and (C) solutions for graded graded biomaterial fabrication: (a) 3D bioprinting, (b) microfluidics, (c) layer-by-layer scaffolding, biomaterial fabrication: (a) 3D bioprinting, (b) microfluidics, (c) layer-by-layer scaffolding, and (d) and (d) magnetically (electrically) driven distribution of GFs. CreatedBiorender.com. magnetically (electrically) driven distribution of GFs. Produced applying using Biorender.com.Among the techniques for sequential GF delivery assumes the incorporation of many All currently made use of approaches for engineering and fabrication of graded tissue scafnanoparticles regeneration are guided by the same principles: (1) to mimic native bone folds for bone with encapsulated development variables into polymeric scaffolds [49] (Figure 9(Bc)). Several research have reported the fabrication bone remodeling, (2) to create complicated tissues and to adhere to the ordered sequence of of PLGA (poly(lactic acid-co-glycolic acid)) capsules loaded with distinctive growth factors and after that immobilized in hydrogel matrices. Sequential VEGF delivery and BMP-2 were accomplished by the inclusion of alginate microcapsules embedded with GF-containing PLGA NPs into the collagen matrix [163]. Regardless of its complexity, this method allowed for the powerful transport of biomolecules and their functional synergism in bone regeneration. Wang et al. [164] utilized microencapsulation within a hydrogel matrix for the generation of a single concentration gradient along with a dual reverse gradient of bone morphogenetic protein two (rhBMP-2) and insulin-like growth element I (rhIGF-I) to induce osteochondral differentiation of hMSCs. Microsphere GF carriers fabricated from silk and PLGA were additional incorporated in silk fibroin or alginate scaffolds. The hMSCs had been differentiated into osteoblast-like (cuboidal) and chondrocyte-like (spherical) cells along the concentration gradients. Due to the fact silk microspheres turned out to be far more effective GF cars than PLGA microcapsules, the authors proposed a silk-based platform for delivery of a number of biomolecules that allows for regulation in the spatial manage over distribution and temporal manage more than sequestration of GFs. In a study by Yilgor et al., wet-spun chitosan and chitosan-PEO scaffolds were embedded with PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules containing BMP-2 and BMP-7, respectively [165]. The sequential delivery with the growth factors enhanced alkaline phosphatase activity, which was an early indicator of MSC differentiation into chondroblasts and osteoblasts.Int. J. Mol. Sci. 2021, 22,18 ofHettiaratchi et al. developed a BMP-2-delivering method based on the sturdy affinity interactions between heparin microparticles (HMPs) and bone morphogenic proteins embedded inside an alginate/polycaprolactone scaffold. By binding BMP-2 to HMPs, the authors 5-HT Receptor Agonist Accession decreased the rate of biomolecule diffusion of BMP-2 by generating its long-term gradient and by controlling spatial localization [105]. In a different study, heparin-conjugated superparamagnetic iron oxide nanopartic.