Scaffold Fabrication Techniques of Biomaterials for Bone Tissue Engineering: A Critical Review

Abstract

Bone tissue engineering (BTE) is a promising alternative to repair bone defects using biomaterial scaffolds, cells, and growth factors to attain satisfactory outcomes. This review targets the fabrication of bone scaffolds, such as the conventional and electrohydrodynamic techniques, for the treatment of bone defects as an alternative to autograft, allograft, and xenograft sources. Additionally, the modern approaches to fabricating bone constructs by additive manufacturing, injection molding, microsphere-based sintering, and 4D printing techniques, providing a favorable environment for bone regeneration, function, and viability, are thoroughly discussed. The polymers used, fabrication methods, advantages, and limitations in bone tissue engineering application are also emphasized. This review also provides a future outlook regarding the potential of BTE as well as its possibilities in clinical trials.

Keywords: 4D printing; additive manufacturing techniques; biocompatibility; bone tissue engineering; clinical trials; electrohydrodynamic behavior; fabrication.

Figure 1

Schematic diagram of bone hierarchical structure. Reprinted with permission from Ref. [8] Copyright 2016, John Wiley & Sons, Ltd.

Figure 2

Schematic representation of traditional fabrication methods and microscopic images. (A2) Solvent casting and its microscopic image showing (a1) salt leached, (a2) salt-PEG 200 leached, (a3) salt-PEG 600 leached, and (a4) salt-PEG 1000 leached PCL scaffolds. Reprinted with permission from Ref. [43]. Copyright 2014, John Wiley & Sons, Ltd (B2) Freeze-drying method. Reprinted with permission from Ref. [44]. Copyright 2006, Elsevier Ltd.; and its SEM results of (b1) pure CS, (b2) pure ZN (zein), (b3) pure nHAp (nanohydroxyapatite), (b4) composite COM-10 i.e. ZN:CS:nHAp in 45:45:10, (b5) composite COM-15 i.e. ZN:CS:nHAp in 45:45:15, (b6) composite COM-20 i.e. ZN:CS:nHAp in 45:45:20. Reprinted with permission from Ref. [45]. Copyright 2018, Elsevier Ltd. (C2) Hydrogel formation and its (c1) surface and (c2) cross-sectional view. Reprinted with permission from Ref. [46]. Copyright 2019, Elsevier Ltd. (D2) Cryogel. Reprinted with permission from Ref. [47]. Copyright 2013, Korean Academy of Periodontology; FE-SEM images of developed biocomposites (chitosan-gelatin-hydroxyapatite and chitosan-gelatin-zinc doped hydroxyapatite). Reprinted with permission from Ref. [48]. Copyright 2020, Elsevier Ltd. (E2) Phase separation (a) NIPS based scaffolds and surface view of scaffolds (PCL, PD64 (PCL/DMSO in 60:40), PD55 (PCL/DMSO in 50:50) and PD46 (PCL/DMSO in 40:60) with its average pore size of the developed scaffolds. Reprinted with permission from Ref. [49]. Copyright 2020, RSC, and (b) TIPS and its SEM images of (e1) PLLA/HA scaffold in 50:50, (e2) PLGA/HA scaffold (50:50) with 2.5% (w/v) concentration of polymer and −18 °C quenching temperature (e3) PLLA scaffold and (e4) PLGA scaffold with 10% (w/v) polymer concentration; quenching condition: liquid nitrogen; volume ratio of dioxane and water at 87:13. Reprinted with permission from Ref. [50]. Copyright 2014, John Wiley & Sons, Ltd. (F2) Gas Foaming and its SEM micrograph representation of macroporous alginate foams showing well separated pores, among which MAF5 (Sr2+/Ca2+ molar ratio at 0:100) depicts interconnected porous structure. Reprinted with permission from Ref. [51]. Copyright 2018, Elsevier Ltd.; Reprinted with permission from Ref. [52] Copyright 2010, World Scientific (all adapted from [53,54]).

Figure 3

Illustration of electrohydrodynamic techniques and their scanning electron microscope images. (A3) Electrospray method. Reprinted with permission from Ref. [80]. Copyright 2018, Taylor & Francis; and its macroporous ZrO2 foams structure formed by the combination of (a1) electrospraying and (a2) slurry dipping, respectively. Reprinted with permission from Ref. [81]. Copyright 2006, John Wiley & Sons, Ltd. (B3) Horizontal electrospinning. Reprinted with permission from Ref. [82]. Copyright 2016, Penerbit UTM Press, Universiti Teknologi Malaysia; and SEM micrographs of (b1) cellulose acetate (CA) scaffolds (9%) and (b2) regenerated cellulose scaffold (9%). Reprinted with permission from Ref. [83]. Copyright 2019, Elsevier Ltd. (C3) Core-shell electrospinning. Reprinted with permission from Ref. [84]. Copyright 2019, John Wiley & Sons, Ltd; SEM morphology of core-shell PCL-PLA/HA electrospun fibres at (c1) 2:3 and (c2) 3:3 core:shell flow rate ratio (marker bars at 20 μm) including (c1.1) and (c2.1) depicting the histograms of the fibre diameters at 2:3 and 3:3 flow rates. Reprinted with permission from Ref. [85]. Copyright 2019, IOP Sceince (D3) Emulsion electrospinning. Reprinted with permission from Ref. [86]. Copyright 2018, Elsevier Ltd; FE-SEM images of: (A) PLCL/HA (poly (L-lactic acid-co-ϵ-caprolactone)/hydroxyapatite), (B) PLCL/lam ((poly(L-lactic acid-co-ϵ-caprolactone)/hydroxyapatite/laminin), and (C) PLCL/HA/Lam nanofibers; and (D) TEM images of HA loaded PLCL/HA/Lam nanofibers. Reprinted with permission from Ref. [87]. Copyright 2013, Taylor & Francis (E3) Melt electrospinning. Reprinted with permission from Ref. [88], Copyright 2012, doiSerbia; SEM images of (e1) solvent-based electrospun fibers and (e2) melt-based electrospun fibers. Reprinted with permission from Ref. [89]. Copyright 2013, RSC (F3) Electrospinning using rotating collector Reprinted with permission from Ref. [90]. Copyright 2019, MDPI; FE-SEM of (f1–f3) randomly-oriented and aligned (f4–f6) electrospun nanofibers at weight ratios of PLGA/gelatin of (f1,f4) 10: 0, (f2,f5) 9:1, (f3,f6) 7:3. Reprinted with permission from Ref. [91]. Copyright 2010, Elsevier Ltd. (G3) Rotary/centrifugal jet spinning. Reprinted with permission from Ref. [70]. Copyright 2017, John Wiley & Sons; Morphology and distribution of fiber diameter obtained before and after annealing and by spinning at two different speeds 7000 rpm and 9000 rpm, respectively as depicted in (g1–g4) and (g6–g8). (g1) Polyvinylpyrrolidone-barium titanate (PVP–BaTiO3) fiber spun at 7000 rpm, (g2) BaTiO3 nanofiber calcined at 850 °C spun at 7000 rpm, (g3) fiber diameter distribution of PVP–BaTiO3 spun at 7000 rpm, (g4) BaTiO3 fiber diameter distribution annealed at 850 °C spun at 7000 rpm, (g5) PVP–BaTiO3) fiber spun at 9000 rpm, (g6) BaTiO3 nanofiber calcined at 850 °C spun at 9000 rpm, (g7) fiber diameter distribution of PVP–BaTiO3 spun at 9000 rpm, (g8) fiber diameter distribution of BaTiO3 annealed at 850 °C spun at 9000 rpm. Reprinted with permission from Ref. [79]. Copyright 2014, Elsevier Ltd.

Figure 4

Schematic representation of different types of 3D printing methods and their microscopic images: (A4) Extrusion method. (a1) The 3D printing procedure, (a2,a3) Camera images showing 3D printed hydrogel construct. (a4,a5) High magnification images of the surface of the construct showing pores and struts morphology (scale bars at 1000 and 500 µm, respectively). (a6) The elemental composition of the surface of the mesoporous silica-calcia nanoparticles containing hydrogel supported by a SEM image of the construct. Reprinted with permission from Ref. [107]. Copyright 2021, Elsevier Ltd. (B4) inkjet 3D bioprinting. SEM microstructure displaying spherical powder sintered scaffolds (b1–b4), in addition to micropores on air jet milling powders sintered scaffolds surface (b5–b8), nano-sized grains sintered scaffolds showing many cracks (b9–b12). Reprinted with permission from Ref. [108], Copyright 2018, Elsevier Ltd. (C4) laser-assisted bioprinting (c1) Design of experiment, (c2) Optical microscopy at day 0 (bar = 150 μm). (c3) Fluorescence microscopy showing cell migration and proliferation on day 3 (bar = 200 μm). (c4) Fluorescence microscopy showing a complete covering of the initial nHA pattern at day 6 (bar = 200 μm). (c5,c6) Scanning electron microscopy of the nHA surface. HOP cells spread onto the material on day 3. (c7,c8) Scanning electron microscopy of the nHA surface. HOP cells spread onto the material on day 6. (c9) ALP activity assay showing that HOPs maintain their osteoblastic phenotype at day 6. Reprinted with permission from Ref. [109] Copyright 2011, IOPscience (all adapted from [110]).

Figure 5

Schematic illustration of (A5) FDM. Reprinted with permission from Ref. [53]. Copyright 2020, Elsevier Ltd.; (F) The developed PCL/HA 3D artificial bones. (G) Surface view of PCL/HA 3D artificial bone, in which upper-right image is magnified. (H) Cross-sectional view of PCL/HA 3D artificial bones, in which the upper-right image is magnified from the corresponding area. Reprinted with permission from Ref. [114]. Copyright 2014, ACS Publications and (B5) SLS (adapted from [53]); (a) The developed sintered scaffold of cuboid-shaped morphology and highly ordered porous structure. (b) SEM image showed the detailed morphology of pores in a representative 10% HA/PCL scaffold. (c–e) SEM micrographs verified the microspheres were well connected via laser sintering in PCL scaffolds (c), 10% HA/PCL (d) and 20% HA/PCL (e). (f) The porosity analysis (g) mechanical properties of the scaffolds. All data represented the mean ± SD; n = 5, * p < 0.05 (data compared with other two groups). Reprinted with permission from Ref. [115]. Copyright 2015, Elsevier Ltd.

Figure 6

(A6) Schematic illustration and scanning electron microscope images of BJ. Reprinted with permission from Ref. [125], Copyright 2021, CSI; SS316- Tricalcium phosphate (left) and its morphology (right). Reprinted with permission from Ref. [126], Copyright 2017, Conference Reviewed Paper. (B6) Schematic representation of injection molding and (a) microscopic observations of the surface of zirconia toughened alumina at the various steps of the selective etching process, demonstrating the formation of fluoride precipitates during hydrofluoric acid etching and their subsequent removal in HCl; (b) FE-SEM observations of the surface of injection molded samples with different surface topographies before and after selective etching. Low, Medium and High surfaces attained from increasingly rough molds. Polished entitles the surface of samples that were polished after sintering. Scale bars: 5 μm. Reprinted with permission from Ref. [127], Copyright 2016, Elsevier Ltd.

Figure 7

Mechanisms of various types of photolithography with microscopic images: (A7) SLA. poly (trimethylene carbonate) microporous (PTMC) scaffolds with 20 and 40% of hydroxyapatite (HA) (a) Model design (b) macroscopic and (c) microscopic SEM images of all scaffolds (d) 3D architecture (e) strut thickness (f) pore diameter (g) porosity of all SLA fabricated scaffolds. Reprinted with permission from Ref. [141], Copyright 2022, Frontiers (B7) DLP. Surface morphology, surface and cross-sectional view of all scaffolds with PDA (polydopamine) modification dipped in Tris-HCL at concentration of 2 mg/mL, 4 mg/mL and 8 mg/mL. (b1) Biphasic calcium phosphate (BCP) (b2) 2PDA-BCP. (b3) 4PDA-BCP. (b4) 8PDA-BCP. Deposition of Ca-P/PDA is showed by white stars, white arrows designate the PDA layer and amorphous Ca-P nanoparticles. Green arrows illustrated by green arrows. the size of newly formed amorphous Ca-P nanoparticles is showed by yellow circle. Reprinted with permission from Ref. [142], Copyright 2020, Frontiers ((A7,B7) adapted from [143]), (C7) CLIP. Reprinted with permission from Ref. [144], FE-SEM images of (c1) Pristine nHA and (c2–c6) Fractured surface of of poly(ethyleneglycol)diacrylate containing 0 wt%, 0.5 wt%, 1.0 wt%, 1.5 wt%, and 2.0 wt% n-HA. Copyright 2021, RSC; Reprinted with permission from Ref. [145], Copyright 2018, IOPsceince (D7) the 2PP process. (d1) Large area over-view (d2) side-view tilted at 30 °C (d3) Top view and (d4) closer top view of IP-L780 photopolymer. Reprinted with permission from Ref. [146], Copyright 2008, Taylor & Francis; Reprinted with permission from Ref. [147], Copyright 2019, ACS and (E7) the MPL process. Reprinted with permission from Ref. [148], Copyright 2008, Elsevier Ltd.

Figure 8

SEM images of microsphere based sintered scaffolds, using PCL/0.5%TNT (TiO2 Nanotube) at 100 µm and 500 µm. Reprinted with permission from Ref. [159], Copyright 2021, AIP publishing.

Figure 9

4D-printed scaffolds with its microscopic image. (a) Representation of development of mineralized, microchanneled collagen scaffold and in vivo assessment of osteogenesis and angiogenesis. SEM morphology of (b) PCL/PVA, (c) PVA-leached fibrous PCL, (d) collagen-embedded fibrous PCL, (e) collagen microchannels after leaching fibrous PCL, and (f) SBF-treated microchanneled collagen. Reprinted with permission from Ref. [160], Copyright 2020, Elsevier Ltd.

Figure 10

Fabrication of freeze-dried scaffolds via lyophilization technique using C3CA scaffolds. (a–c) Camera images and (d–f) cross-sectional view of C3CA scaffolds processed at −20, −40, and −80 °C depicting porous structures, and the pores were bigger in C3CA20 compared to those in C3CA40 and C3CA80. Scale bar: 100 μm. Masson’s trichrome staining of C3CA scaffolds sections after 28 and 35 days of implantation. Red arrows depicting blood vessels; yellow arrows depicting collagen deposition. Scale bar: 100 μm. Positive staining of CD31 verified the vascularization in the implants. The occurrence of mild staining of osteocalcin-positive cells designates the unconfined cells to osteoblastic lineage. Fibrous tissue in-growths into the pore spaces of implants as directed with no apparent alteration between peripheral tissues (Pt) and implant material (S). Scale bar: 500 μm. Reprinted with permission from Ref. [166], Copyright 2018, MDPI.

Figure 11

Composites comprising PCL developed by an electrospinning-based thermally induced self-agglomeration (TISA) technique. SEM morphology displaying representative PCL-3D scaffolds in low (A–C) and high (D–F) magnification. Rows top to bottom visualize neat PCL, step 1 coating, and step 2 SBF coating. FE-SEM image and contact angles of NF PCL mats before immersing in either SBF step (G), after step 1 (H), and after both steps (I). ATR spectra of NF PCL mats prior to and after SBF treatment (J). Confocal microscopy of PCL/HA scaffolds of individual layers (K–M) and 30 μm 3D-cross sections (N–P). Fluorescent dyes were introduced into step 1 SBF with rhodamine B in red colour and FITC-BSA in green. C2C12 morphologies on TISA (top row) and TISA/HA composite (bottom row) scaffolds, after 24 h (Q,T) and 72 h (R,U) of culture. (S) and (V) show a 100 μm z-stack of cell morphologies after 72 h of culture in individual scaffold. Reprinted with permission from Ref. [174], Copyright 2021, Elsevier Ltd.

Figure 12

3D-printed hybrid scaffolds. Top view depicting positively stained scaffolds for Alizarin Red S in all cases except pure polycaprolactone case. Middle image showing magnified images of stained scaffold struts describing the punctate stain of the mineralized particles within the PCL. Bottom view showing SEM of strut surfaces displaying rougher surface topographies in the more concentrated hybrid scaffolds. Histological studies of excised constructs. Cellularity under H & E stain (left) as well as bone (black/dark brown) and osteoid (red) formation under the von Kossa and van Gieson stains (right) is obvious. Asterisks represent scaffold struts. In the von Kossa and van Gieson stains, note the presence of both osteoid (red, arrowheads) and mineralized tissue (red/brown, arrows), signifying active mineralization appearing within the constructs. Anatomical shape printing of pure and hybrid scaffolds. Middle image showing human temporomandibular joint condyle was obtained and printed into anatomically shaped, porous scaffolds. Scaffolds were subject to ARS staining to confirm and visualize the presence of mineralized particles in the hybrid scaffold. Bottom view represents the MicroCT scans to check the presence of mineralized particles in the 30% DCB:PCL scaffolds. There were no mineral particles present in pure PCL scaffold. Reprinted with permission from Ref. [180], Copyright 2019, Elsevier Ltd.

Figure 13

Three-dimensional printed scaffolds of PLLA, PD-PLLA, and Qu/PD-PLLA with their stereomicroscope images. (A) Camera images, (B) stereomicroscope images, and (C) SEM images of the 3D-printed scaffolds of PLLA, PD-PLLA, 100Qu/PD-PLLA, 200Qu/PD-PLLA, and 400Qu/PD-PLLA. (D) Printing reproducibility and accuracy were studied by quantification of thread diameter, pore size, and porosity determined by image analysis from stereomicroscope pictures. (E) Live/dead fluorescent staining images of MC3T3-E1 cells on the PLLA, PD-PLLA, and Qu/PD-PLLA scaffolds after 1, 4, and 7 days of culture. The confocal laser scanning microscopy images of (F) morphology and (G) quantification of spreading area of the MC3T3-E1 cells after culturing on the PLLA, PD-PLLA, and Qu/PD-PLLA scaffolds for 48 h. The OD value of the MC3T3-E1 cells culturing on the PLLA, PD-PLLA, and Qu/PD-PLLA scaffolds for (H) 1, 4, and 7 days. (I) ALP staining, and (J) ALP activity of MC3T3-E1 cells after culturing on the PLLA, PD-PLLA, and Qu/PD-PLLA scaffolds for 7 and 14 days. (K) Alizarin red staining and (L) quantitative result of MC3T3-E1 cells after culturing on the PLLA, PD-PLLA, and Qu/PD-PLLA scaffolds for 21days. Reprinted with permission from Ref. [185], Copyright 2019, Taylor & Francis.

Figure 14

Bone substitute formed by structuring injection molding. FE-SEM images showing crystalline morphology of (a1,a2) structured HA/PE, (b1,b2) structured BG/HA/PE, (c1,c2) normal HA/PE, and (d1,d2) normal BG/HA/PE. MC3T3-E1cells adhesion and spreading on (e1) structured PE, (e2) structured HA/PE, and (e3) structured BG/HA/PE after culturing for 7 days. (e4) CCK-8 results, (e5) ALP results and (e6) Western blot analysis. The intensities of each test protein bands in (f) were normalized. SEM images of the surface of (f1,f3) structured HA/PE and (f2, f4) structured BG/HA/PE after immersion in SBF for 21 days: magnification of (f1,f2) 1000× and (f3,f4) 10,000×. The insets of c and d are the EDX spectrum of the sample surface. Reprinted with permission from Ref. [186], Copyright 2020, SAGE.