A review on 3D printing in tissue engineering applications

5.1 Bone

Three-dimensional (3D) printing is a potential technique for bone tissue engineering. Although, printable ink materials with attractive features such as structural interconnectivity, mechanical strength, regulated degradation rates, and the inclusion of bioactive ingredients are crucial for improved bone regeneration. According to the research study, long bone 3D printed construct created by using inkjet bioprinting has shown positive results in the field of tissue engineering. Gao et al. have implemented 3D inkjet printing technology to fabricate bone tissue using polyethylene glycol dimethacrylate (PEGDMA) as bioink and blended with bone marrow-derived mesenchymal stem cells (BM-MSC). To analyze the osteogenic potential of used bioink they further enhanced their study on HA and bioactive glass nanoparticles and evaluated the difference. In addition, they concluded that HA-based scaffold had shown more osteogenic potential as compared to scaffold fabricated with bioglass [34]. However, in human clinical preliminaries, researchers have also worked on tricalcium phosphate (TCP) powdered-based 3D printed scaffolds produced from inkjet printers. The designed scaffold was embedded in 10 different patients which have faced distinctive maxillofacial defects. Therefore, the resultant of the experimental work has shown a decrease in operating time, improvement in the effectiveness of the scaffolding, and its alliance with the surrounding bone along with its overall no unfavorable effect on those patients [35, 36]. Similarly, a Chinese researcher group has disseminated their clinical trial report on the utilization of 3D-bioprinted titanium scaffold to treat diverse maxillofacial bone deformities and mandibular osteotomy. Once more, they revealed shorter operation time and great appealing outcomes related to tissue engineering application [36]. Inzana et al. utilized 3D inkjet printing technology to fabricate collagen-calcium phosphate scaffolds for bone regeneration applications. For that purpose, they implemented enhanced formulations using inkjet printers, by blended collagen and binder solution to form collagen-calcium phosphate scaffolds. The cytocompatibility of the scaffold materials was analyzed with C3H/10T1/2 cells through XTT assay and bone formation was investigated for 9 weeks by implanting the 3D printed scaffold into a critically sized murine femoral defect model. The results showed that the addition of 0.25 wt% Tween into binder solution resulted in enhanced mechanical strength of prepared scaffolds. The Tween 80 incorporation followed by physiologic heat treatment also resulted in reliable inkjet printing of collagen solution and showed enhanced mechanical strength and cell viability. The addition of 1–2 wt% collagen with binder solution also enhanced the flexural strength and cell viability of prepared composites. They concluded that the inkjet-delivered collagen coupled with osteoinductive factors improved the bone healing efficacy of the 3D printed scaffolds evident by in vivo studies [37]. In the same way, Cooper et al. reported the spatial control of osteoblast differentiation of C2C12 cells using inkjet bioprinting technology. The C2C12 myogenic precursor cells were used for determining cell viability seeded with a density of 200,000 cells and cultured in a growth medium. Spatial control of osteoblastic differentiation of C2C12 cells seeded in BMP-2-printed patterns of the dermal matrix was demonstrated using alkaline phosphate (ALP) staining. It was observed that an average of 78.7% BMP and 69% noggin remained across all concentrations after 24 h printing and ALP staining increased with increasing the amount of seeded BMP-2. Moreover, the 3D constructs printed with BMP-2 showed new bone formation within 2 weeks after implantation, evident in in vitro study [38]. Fielding et al. prepared a 3D printed scaffold using TCP incorporated with silica oxide (SiO₂), zinc oxide (ZnO) dopants and investigated its mechanical and biological properties. In this study osteoblast, cells were cultured, and results were evaluated on different days. The results showed that TCP incorporated with dopants showed increased density of pure TCP and an increase in compressive strength compared to pure TCP scaffolds. The MTT assay revealed that developed 3D printed constructs showed enhanced cell proliferation and a higher number of cell attachments to the surface by day 7. Moreover, the addition of SiO₂ and ZnO dopants into TCP favored mechanical properties and also increased cellular proliferation for tissue regeneration [30]. In continuation to their work, they utilized β-tricalcium phosphate (β-TCP) scaffolds for preparing 3D scaffolds incorporated with zinc oxide (ZnO₂) and silicon oxide (SiO₂) dopants. In this study, the tissue formation was evaluated up to 16 weeks by implanting the prepared scaffolds into the bicortical femur defect murine model. It was observed that the mechanical interlocking between the implants and host tissue was enhanced after 4 weeks of implantation for both pure and doped samples. Moreover, the TCP scaffolds incorporated with SiO₂ and ZnO dopants implied increased new bone formation compared to undoped samples owing to enhanced collagen I and osteocalcin formation. They also concluded that the neovascularisation of doped TCP samples was three times higher compared to pure TCP control, as confirmed by FESEM micrographs [39]. In another work, Wu et al. used a modified 3D printing method to develop highly uniform CS (CaSiO₃, calcium silicate) and β-TCP (tricalcium phosphate) scaffolds for osteogenesis tissue engineering. Tissue formation was investigated by implanting CS scaffolds in a rat femur defect model. The results showed that prepared CS scaffolds using this modified 3D method have uniform scaffold morphology with controlled pore size and porous structure. Furthermore, the compressive strength of 3D printed CS scaffolds was found to be 120 times more than conventional polyurethane template CS scaffolds. The 3D printed CS scaffold showed improved bone formation and stronger deposition of type I collagen and ALP in bone matrix compared to the control, demonstrated by hematoxylin and eosin (H & E) staining and immunohistochemical analysis [40]. Therefore, 3D bioprinted constructs using inkjet printing especially for bone regeneration have one of key potential area in the field research that can create more avenues in the field of 3D bioprinting.

5.2 Cartilage

Bone and cartilage transplant-designed tissues are more appealing as new tissue bones and cartilages may eventually be repaired, maintained, and enhanced. But the strategy of tissue cartilage engineering has remained difficult to imitate the characteristics of an extracellular tissue-specific matrix (ECM) and the cellular zone organization. It is reported that inkjet bioprinting is compatible with high-performance and resolution biological systems [41]. Hence, Xu et al. developed 3D printed constructs using a novel hybrid inkjet printing system for cartilage tissue engineering applications. The novel hybrid system had an electrospinning apparatus and printing solution. In this work, cell viability was evaluated. The results showed that the chondrocytes had more than 80% cell viability within the hybrid construct after one week of printing. Further, the printed hybrid scaffolds showed enhanced mechanical properties, cell proliferation, and maintained biological properties compared to conventional printers noted in both in vitro and in vivo experiments [19]. In another work, they studied the effect of fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1) on human neocartilage formation. It was found that the FGF-2/TGF-β1 treated samples showed enhanced cell proliferation of 40% compared to TGF-β1 treated group while the TGF-β1 treated group exhibited higher chondrogenic gene expression at week 1 compared to FGF-2 and FGF-2/TGF-β1 treated groups. Further they suggested that the 3D printed sample with FGF-2/TGF-β1 observed high ECM formation and possessed better chondrogenic properties suitable for tissue formation [42]. The results from the studies on 3D bioprinted cartilage construct using inkjet bioprinting technique revealed that inkjet printing technique played a significant role in bone cartilage and ECM formation including enhancement of mechanical and cell proliferative properties.

5.3 Microvascular

Although several alternative techniques for the creation of biomimetic cell environments have been established, however, the absence of vascularization in the 3D model is one of the primary concerns related to tissue engineering technology [43, 44]. In addition, a significant proportion of tissues in the body are supplied by nutrients and oxygen to the cells through blood vessels, as oxygen is around 100–200 μm at the diffusion limit [44, 45]. It is reported that microcirculation is the distal functional unit of the vascular system, based on the microvasculature. A vessel with varied sizes includes arterial (±30 μm), venous (±20 μm) and capillary (<8 μm) arterioles [44, 46]. It is also notified that there is a high potential for nerve tissue regeneration and functional restoration for biofabricated nanostructured and microstructured scaffolds. Furthermore, revascularization and transport are important features in the 3D fascicle structure. Unfortunately, the human peripheral nerves do not have a perfect internal fascicle and microvascular model [47]. Therefore, this section explained applications related to microvascular 3D printed scaffolds by using inkjet printing. Nakamura et al. reported the biocompatible inkjet printing technique for seeding individual living cells. In this investigation bovine vascular endothelial cells were used as “bioink” suspended in a culture medium and injected onto a PET sheet culture disk. The cell printing system used in this investigation was static electricity with actuated inkjet printers. The paper concluded that cells adhered to the culture medium within a few hours after incubation and the electrically actuated inkjet printers were biocompatible which does not produce a heat printing process and is suitable for color printing [48]. In an extension study, they utilized thermal inkjet printers for fabricating human microvasculature by printing human microvascular endothelial cells (HMVEC) onto fibrinogen biopaper substrate. The cell viability was analyzed after 24 h, 7, 14, and 21 days of printing. The results showed after 21 days of culture the printed endothelial cells proliferate to form a confluent lining within the fibrin scaffold. The fabricated microvascular cells showed some integrity after being cultured for 14 days and much integrity was observed from the printed cultured for 21 days [49]. In this part, two different types of inkjet printing techniques have been discussed (actuated and thermal), both types have shown optimistic characteristics for the regeneration of microvascular structure.

5.4 3D patches using smooth muscle cells and different growth factors

The capacity to bioengineered 3D tissues is a possible method to treat a range of illnesses such as cancer, tissue loss, or organ failure. However, traditional approaches for tissue engineering have encountered problems in building 3D tissue constructions similar to the microvasculature and microarchitectures of the natural tissue [50]. Growth factors stimulate strong regulations on cell function in strategy for the production of tissues, but it is difficult to spatially shape their presentation in 3D using a single substance [51]. This part defines the utilization of smooth muscle cells and different growth factors to fabricate 3D patches for tissue regeneration applications. Moon et al. developed 3D patches of smooth muscle cells (SMC) using a bio-printer blended with collagen. In this work the bioink used was SMC cells derived from Sprague Dawley rats while the substrate was collagen to deposit the cell-laden structure. The experimental results revealed that the hydrogel constructs were formed at 16.2 µm thick per layer with controlled spatial resolution, high-throughput droplet generation with 160 droplets/s per layer and high cell seeding uniformity about 26 ± 2 cells/µm² at 1 million cells/mL, 122 ± 20 cells/µm² at 5 million cells/mL and 216 ± 38 cells/µm² at 10 million cells/mL respectively. Moreover, it was also found that multilayered 3D hydrogel constructs exhibited high cell viability of about >90% over 14 days in culture [52]. Lee et al. reported the time-release delivery of growth factor (GFs) in hydrogel tissue constructs for neural stem cell culture. In this investigation, the artificial neural tissue was developed by printing neural stem cells (C17.2) within collagen hydrogel and vascular endothelial growth factor (VEGF) releasing fibrin gel using 3D techniques. They examined the morphological changes of C17.2 cells within collagen and its migration towards fibrin gel. They observed that murine NSC C17.2 cells with VEGF fibrin gel exhibited a high degree of morphological changes compared to control samples for up to 3 days. It was observed that the printed C17.2 cells with VEGF fibrin gel showed high viability of about 92.89 ± 2.32% and that cells migrated towards the fibrin gel at a distance of 33.3 ± 18.2 µm on day one, a distance of 34 ± 45.2 µm at day 2 and a total migration distance of 102.4 ± 76.1 µm over 3 days respectively [53].

In the extension study, they printed the human chondrocytes in layer-by-layer (LBL) assembly to repair defects in osteochondral plugs (3D biopaper). The bioink was prepared by chondrocyte cells isolated from human articular cartilage at a density of 5 × 10⁶ cells/mL The prepared bioink was deposited into the OC plug biopaper with a suspension of 5 × 10⁶ cells/mL using a modified HP Inkjet 500 printer. It was observed that the cell viability of 3D printed constructs increased 26% in simultaneous polymerization compared to polymerization after printing and printed 20% w/v PEGDMA showed reduced compressive modulus by 18% compared to non-printed PEGDMA. Further, it was noted that the printed samples exhibited an increase in glycosaminoglycans (GAG) and collagen II formations, and the formation of CAG/DNA and collagen II/DNA by chondrocytes at week 4 were both significantly higher than those of implant constructs without OC plugs (evident in biochemical analysis). They also concluded that the printed human chondrocytes showed enhanced ECM deposition and phenotype maturation in native tissue [49]. In another study, Phillippi et al. developed spatially defined 3D patterns of immobilized growth factors using inkjet and bioprinting. In this study, the bioink used was recombinant human BMP-2 and the substrate was fibrin coated glass. To investigate cell viability two types of cells were analyzed, namely primary muscle-derived stem cells (MDSCs) and C2C12 cells added with growth factors (noggin and FGF-2) and were printed onto a fibrin-coated glass substrate. Before printing the liquid phase BMP-2 patterns were subjected to alkaline phosphate activity (ALP) for 72 h. The results showed that noggin inhibited BMP-2 pattern response in both MDSC’s and C2C12 cells whereas FGF-2 partially inhibited BMP-2 responses in MDSC and inhibition was completed in C2C12 cells. Both MDSC and C2C12 cells showed differentiation towards the osteogenic lineage by ALP activity and concluded that cells with APL activity (on the pattern) differentiated toward the osteogenic lineage whereas cells without APL activity (off pattern) differentiated toward the myogenic lineage [54, 55]. Ferris et al. developed 3D scaffolds using bioink-based novel microgel suspension into the tissue culture medium. In this study, the bioink used was endotoxin-free low acyl gellan gum with a density of 2 × 10⁵ to 2 × 10⁶ cells/mL for microvalve printing and 1–6 × 10⁶ cells/mL for inkjet printing into rat tail collagen I biopaper. In this research study, the cell viability was investigated using C2C12 (CRL-1772), PC12 (CRL-1721), and L929 (CCL-1) murine cell lines derived from ATCC. The results showed that the bioink exposed to C2C12 and PC12 cells does not have any cytotoxic effect. Moreover, the viability of bioink exposed to PC12 cells was significantly higher compared to other cells. Ink-jet printed PC12 cells and both inkjet and microvalve printed C2C12 cells retained >95% viability and proliferated for more than 48 h at a rate comparable to non-printed controls [56].

Cui et al. evaluated the cell viability, apoptosis, and possible cell membrane damage of thermal inkjet printed Chinese hamster ovary (CHO) cells. In this work the bioink used was CHO cells suspended with a density of 2 × 10⁶ to 20 × 10⁶ cells/mL and were loaded into rat tail collagen type I biopaper using thermal inkjet printers. It was found that the thermal inkjet printed CHO cells showed improved cellular viability of about 89%. Further, it was observed that the number of cells printed decreases with an increase in the concentration of bioink and the highest number of cells printed was seen at 8.2 × 10⁶ cells/mL concentration. Further, the apoptosis ratio was found to be 3.5 ± 1.3% for printed cells and 3.2 ± 1.6% for unprinted cells which showed that the thermal printing process would not affect the cells compared to normal handling [57, 58]. Similarly, Zhao et al. investigated the effect of printing parameters on the printability and cell survival rate in fabricated constructs using 3D technology. The cell viability was investigated using A549 cell lines and the cell-laden 3D printed construct was fabricated by suspending the cells in a bioink solution with a density of 10⁶ cells/mL. The results showed that bioink viscoelasticity was increased with the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. Cell survival rate was decreased upon increasing the viscoelasticity of the gelatin-based bioink. It was also found that high cell survival rate and optimum printability were achieved at bioink storage modulus in the range of 154–382 Pa respectively [59].

In extension work, they used a thermal printer to deposit CHO and embryonic motor neuron cells into a hydrogel-based substrate. In this research, two hydrogel-based substrates were used as a biopaper, named soy agar gel and collagen gel. The cell viability was evaluated. It was found that the 3D printed samples using both cells showed cell viability of >90% and that the cells were not damaged during nozzle firing in the inkjet printing process [60, 61]. The above different experimental examples have shown a broad impact of inkjet bioprinting technique using smooth muscle cells and different growth factors in the area of 3D bioprinting for tissue engineering applications. Therefore, 3D patches based on smooth muscles cells and growth factors using inkjet bioprinting is an important area that needs to do more work to create new ways of research in the tissue engineering region.

5.5 Complex heterogeneous 3D tissue construct

The capacity to produce functioning tissue additives is now providing novel alternatives to existing tissues and organs shortages needed for transplantation [62]. However, the development of artificial tissues and organs completely functional has for many reasons which are not fulfilled expectations [63]. Therefore, Xu et al. developed complex heterogeneous 3D tissue constructs using inkjet printing of multiple cell types. In this study the bioink used was human amniotic fluid-derived stem cells (hAFSCs), canine smooth muscle cells (dSMCs), and bovine aortic endothelial cells (bECs). They were suspended with a density of 2–3 × 10⁶ cells/mL and deposited onto sodium alginate biopaper using thermal inkjet printers. The biological functions of the 3D printed constructs were evaluated in vitro and in vivo. The in vitro results showed that 3D printed cells survived and proliferated with normal cellular function in spatial registered regions. The viability of cells was 90% over 7 days and there was no significant difference in viability between printed and unprinted samples [63]. The author introduced the novel cellular hydrogel biopaper approach with improved cell culturing, handling, and assembling features which can lead to help in the improvement of studies for 3D bioprint scaffolds for future developments in tissue engineering.

5.6 Organ printing

3DP, recognized as the most exciting bio-artificial organ production technique, has offered remarkable adaptability when supplying multifunctional cells together with other biomaterials through accurate spatial placement control. The continual development of 3D printing technology is the integration of biomaterials in biology, chemistry, physics, mechanics, and medicine with other related methods. In the support of cell and biomolecular (or bioactive agent) activities before, during, or following 3D printing, synthetic polymers played an important role. Biodegradable synthetic polymers are particularly preferred options for the production of bioartificial organs with good mechanic characteristics, adjustable chemical structures, non-toxic degradation products, and controlled rates of breakdown [64], [65], [66]. Therefore, this section has described 3D organ printing applications using inkjet bioprinter. Hajdu et al. developed a 3D printed construct based on self-assembling tissue spheroid and fusion kinetics for organ printing. In this work, the tissue spheroid biofabrication was done using bone marrow cells from periostin-null mice and adult murine cardiac valve interstitial cells from C57BL/6 mice. The result showed that both TGFβ1 and serotonin treatment exhibited fibrogenic and maturogenic effects in a tissue spheroid assay compared to controls. It was also found that the length of the fusing aggregates and the interface between them was decreased much faster in tissue spheroids fabricated from periostin-null fibroblasts compared to wild-type mice [67]. Hence, organ printing using inkjet printing has acquired positive results on organ printed constructs, especially due to its favorable microenvironment for new tissue regeneration.

5.7 Topologically designed structures for tissue engineering

Recent advancements have led to the notion of bioprinting becoming an attractive alternative to established methods associated with tissue technology. Biopaper is an important component in the bioprinting process and biomimetic hydrogel [68]. Hence, this part described the applications related to biopaper hydrogel using the inkjet bioprinting technique. Lee et al. developed a cellular hydrogel biopaper for patterning 3D cell culture and tissue reconstruction. In this investigation, the cellular biopaper was prepared using 0.5% sodium alginate precursor and 5% w/v gelatine in PBS and sterilized overnight by germicidal UV irradiation. The results also showed that during days 2–4 of culturing both Hexa and Flat biopaper showed significantly increased cumulative albumin secretion compared to 2D culture. Also found Hexa biopaper had even higher albumin secretion than that of Flat biopaper [69]. Similarly, Jakab et al. reported the biological self-assembly of cells printed into topologically designed structures for tissue regeneration. In this method, the bioink particles were placed into a bio-compatible environment (biopaper) supported 3D delivery system (bioprinter). The 3D structures were formed through the post-printing fusion of bioink particles into the biopaper. In this study fusion of the bioink particles was done by atrioventricular (AV) cushions extracted from chicken embryonic heart and were deposited into rat tail collagen biopaper using inkjet printers. They concluded that the fusion deposition of bioink particles into the gel-biopaper showed smooth deposition of cellular aggregates and cell movement for tissue regeneration applications [70].

In 3D inkjet printing, the 3D printed constructs were fabricated by printing the bioink suspension into the biopaper. The bioink used was a cell suspension supported by growth factor; enhanced cell viability and proliferation for tissue growth were observed compared to scaffolds without growth factors. The 3D printed constructs with growth factors like FGF-2, TGF-β1, or FGF-2/TGF-β1 showed ECM, collagen content, and high GAG for cell growth and new tissue formation. Many researchers used sodium alginate gel and collagen I as biopaper and utilization of other hydrogels was found less common. Some studies have investigated the fusion of bioink particles before post-printing of the cell suspension into biopaper; a tissue spheroid development and smooth deposition of cellular aggregates and cell movement for tissue regeneration applications have been observed [71]. Inkjet bioprinting is commonly used more as compared to other printing techniques. However, from the investigated results inkjet bioprinting technique in different applications of 3D printed scaffold design. It provides better growth factors, mechanical strength, bone healing efficiency, increases cell viability, improved cell morphology, maintains biological properties, improved biocompatibility, and enhanced ECM deposition.

6.1 Heterogeneous tissue regeneration

New types of printers have been developed to achieve functional engineered tissue constructions as the field of tissue engineering advances. However, new 3D biomanufacturing methods for hard tissue and organ engineering are uncommon, and there are numerous constraints in terms of equipment. One of the most promising tissue engineering methods, according to recent research, is the creation of heterogeneous 3D structures. However, the creation of heterogeneous tridimensional scaffolds is heavily reliant on a variety of biomanufacturing methods and technologies [73]. Pati et al. developed 3D cell-laden scaffolds for heterogeneous tissue regeneration using a 3D printing technique. In this work, complex ear tissue constructs were fabricated using synthetic biomaterial polycaprolactone (PCL) as a framework and PEG as a sacrificial layer. The live/dead assay was used to examine the viability of encapsulated cells in the hydrogels. The results showed that the 3D printed constructs were prepared using biomaterials signaled enhanced thermal stability and also improved cell viability of >95% in all cases of hydrogels [74].

In another work, Lee et al. utilized a sacrificial layer process via a 3D technique to regenerate damaged auricular cartilage and fat tissue. In this work, the main framework was fabricated using PCL to deposit the cell-laden hydrogel and further PEG was also deposited as a sacrificial layer to support the main structure. The chondrocytes (CLH) and adipocytes (ALH) are derived from adipose-derived stromal cells. The results showed that the CHL-AHL incorporated hydrogel 3D structure exhibited enhanced collagen formation and also increased the co-regeneration of cartilage and fat tissue compared to individual printed CHL and AHL structures suitable for ear regeneration [75]. In the future, we aim to utilize other hydrogels for fabricating 3D printed constructs that favor tissue regeneration.

6.2 Bone

The regeneration of bone and bone impairment has remained one of the primary challenges in clinical medicine, despite progress in the therapeutic methods since the healing and repair processes are long term and extremely intricate [76]. To solve these issues, research work reported the limited biological functionality of typical mode synthetic materials. In this study, human turbinate mesenchymal stromal cells (hTMSCs) mineralized with ECM were deposited into the 3D printed scaffolds prepared from synthetic materials of PCL, poly (lactic-co-glycolic acid) PLGA, and β-tricalcium phosphate (TCP). The cell viability was evaluated with a cell density of 2 × 10⁵ cells/mL deposited onto a PCL/PLGA/β-TCP scaffold using a multiheaded deposition system (MHDS). The in vivo results showed that ECM-ornamented scaffolds showed higher biological functionality and bone formation compared to bare 3D printed scaffolds. The new hTMSC mineralized ECM showed higher calcium deposition compared to bare 3D printed scaffolds. They concluded that ECM ornamented scaffolds exhibited both osteoinductive and osteoconductive properties for increased tissue formation and bone regeneration [77]. In the continuation work, they fabricated a 3D bioprinted construct with improved mechanical properties for osteochondral tissue regeneration. In this work, thermoplastic PCL was used as a framework and the cells encapsulated with alginate hydrogels were deposited into the pores of the PCL framework using an MHDS. The cell viability was tested by a Live/dead assay kit with two types of cells, chondrocytes and MG63-osteoblast cells derived from a human cell line. The results showed that the 3D constructs deposited with chondrocytes and osteoblast cells remained viable for 7 days without fusion. The research study also revealed that the fabricated 3D printed scaffold using PCL and alginate hydrogel improved the mechanical stability of prepared 3D printed constructs and had the potential for the regeneration of heterogeneous tissues [78]. In an extension study, they also fabricated guided bone regeneration (GBR) using 3D printing technology. The GBR membrane was based on PCL/PLGA/β-TCP composites and investigated the profile release of recombinant human BMP-2 (rhBMP-2). It was observed that the hybrid composites showed sustained release of rhBMP-2 for up to 28 days. The implantation of rhBMP-2 with GBR membrane resulted in the entire healing of calvaria defects and new bone formation within 8 weeks [79]. Holmes et al. developed 3D printed micro and nano featured scaffolds for vascularized bone tissue repair. Polylactic acid (PLA) was used as a framework and the PLA scaffold was printed on a solidoodle fused deposition modeling (FDM) printer layer by layer with 250 μm diameter, hexagonal pore size, and a 375 μm layered height. The results showed that the modification of nHA on both cells enhanced cell adhesion and calcium and collagen I content during osteogenic differentiation. It was found that enhanced human mesenchymal stem cells (hMSC) adhesion and growth were observed on scaffolds with small microchannel and nHA modification while scaffolds with large channels promoted the greatest HUVEC growth [80]. Fedorovich et al. developed a cell-laden hydrogel scaffold by the 3D fiber deposition technique. In this study, the hydrogel scaffold was prepared using high viscosity alginate powder crosslinked. The results showed that increased scaffold porosity resulted in a decrease in elastic modulus of the prepared scaffolds. The 3D printed MSC showed uniform cell distribution and high viability of about 88 ± 6% and 89 ± 7% in the printed group and the unprinted group, respectively, after 24 h in culture. They also suggested that osteochondral grafts exhibited heterogeneous ECM formulation within the scaffold, evident by the presence of a high amount of collagen matrix in cellular grafts that resulted in enhanced osteogeneic tissue formation [81]. Tarafder et al. reported the biological performance of interconnected macroporous TCP scaffolds with the addition of SrO and MgO dopants and their effects in TCP. In this research, the scaffolds were prepared on different three different interconnected pore sizes through 3D technology followed by microwave sintering. An in vivo study was performed by implanting a developed macroporous scaffold in a rat distal femoral bone. It was observed that the sintered pure and Sr-Mg doped TCP scaffolds showed pore sizes of about 311 ± 5.9 µm and 245 ± 7.5 µm. The maximum compressive strength of 12.01 ± 1.56 MPa was obtained for Sr-Mg doped with 500 µm interconnected designed pore sizes. Also, SrO and MgO doped 3D printed scaffolds have shown improved type I collagen and osteocalcin formation that resulted in a significant increase in bone formation, evident by histomorphology and histomorphometric analysis [82]. In the extension work, they fabricated and investigated microwave sintered TCP scaffolds of 27, 35, and 41% designed macroporosity with different pore sizes. The in vitro testing was investigated using human fetal osteoblast cells (hFOB) at 3, 7, and 11 days of incubation and revealed that the smallest designed pore size (500 µm) shows significantly high living cell density. It was observed that the sintered 3D printed scaffolds have shown enhanced compressive strength and maximum compressive strength for scaffolds designed with 500 µm pore size. Further, the sintered 3D printed TCP scaffolds promoted osteoid-like new bone formation evident in histomorphologic analysis and suitable for bone tissue repair and regeneration [83]. In the same way, Santos et al. studied the characteristics of sintered β-TCP scaffolds encapsulated with human osteoblast cells. In this study, the fabricated (β-TCP) scaffolds were sintered at four different temperatures. The results showed that sintered (β-TCP) 3D printed scaffolds have shown increased biocompatibility with enhanced mechanical properties and cell scaffold interactions of human osteoblast cells revealed by SEM images and in vitro or LBL cytotoxicity assay [84].

Kundu et al. fabricated 3D constructs through the layer-by-layer deposition of PCL and chondrocyte cells encapsulated with alginate hydrogels. In this study, the 3D printed scaffolds of the PCL-alginate gel hydrogel hybrid construct were prepared by additive manufacturing (AD) with an MHDS. The results showed that the cell-laden hydrogels had negligible effects on the viability of the chondrocytes. In vitro assessments revealed that the PCL-alginate gels containing growth factor (TGF-β) showed higher ECM and GAG content after 4 weeks of implantation in engineered cartilage. This suggests that TGF-β has a significant effect on tissue response for cartilage regeneration [85].

In another work, Shao et al. reported the mechanical behavior of bioactive glass-reinforced bioceramic scaffolds using a 3D method. In this study, the scaffolds were prepared using bioactive glass (BG) incorporated with CS components. They prepared 1% BG-added CS scaffolds with various pore morphologies. In this study, the 1% BG-added CS scaffolds were printed with rectangular, parallelogram, honeycomb, and Archimedean chords pore structures using extrusion-based three-dimensional printing and characterized using optical microscopy. The results showed that the 1% BG-added CS scaffolds designed with rectangular pore morphology (1080 °C) had very low BG content which readily influenced the apatite formation and significant linear shrinkage (21%) respectively. From experimental results, CS-BG1 scaffolds designed with honeycomb pore structure showed an enhanced compressive strength of 88 MPa compared with the scaffolds with rectangular, parallelogram, and Archimedean chord pore structures. In vitro, SBF experiments revealed that BG reinforced CS scaffolds favored the formation of hydroxyapatite (HA) and contributed to structural and strength reliability in the long term [86]. Calcium silicate (CSi) and TCP were the most widely used materials for the fabrication of 3D scaffolds. To increase the mechanical strength and cell proliferation the addition of dopants like SiO₂ and ZnO was used. It was also concluded that the sintered samples possess high mechanical properties and controlling parameters such as particle distribution, sintering temperature and sintering time, the porosity, bulk density, and compression strength of fabricated scaffolds. These scaffolds significantly enhance new bone formation [87]. Recently Chae et al. worked on 3D cell-printed meniscus constructs, introduced by the new combination of bioink based on polyurethane and polycaprolactone polymers and cell-laden decellularized meniscal extracellular matrix (me-dECM) bioink with improved structural and controlled properties. They reported that (decellularized meniscal extracellular matrix) me-dECM bioink had the deeper capability to deliver a satisfying physiological environment for the cell growth and fibrochondrogenic differentiation of encapsulated human bone-marrow-derived MSCs (hBMMSCs, Catholic MASTER Cells, passage 2). They examined in vivo testing comprehensively on the ectopic mouse and orthotopic rabbit models and verified that the designed 3D cell-printed meniscus construct containing me-dECM bioink with stem cells has shown improved biocompatible features and supported the development of neofibrocartilage with strong mechanical properties which were like the native meniscus. From the outcome of the study, they concluded that the combination of 3D cell-printing technology and me-dECM bioink have developed a better shape and microenvironment characteristics to enable the growth of meniscus [88]. Chae et al. also worked on the tendon-bone interface (TBi). For the regeneration of functional TBi, they have established a new therapeutic system consisting of 3D cell printing and its related bioinks to attain spatially categorized physiology. The spatial composition of cell-laden tendon and bone-related bioinks was used to create an interface like multi-tissue fibrocartilage. The designed 3D TBi printed scaffold presented a cell supportable microenvironment of encapsulated stem cells in terms of cell viability, proliferation, differentiation for in vitro development of TBi. In vivo characteristics were also investigated on rat chronic tear model with 3D TBi constructed patch along with stem cells. The outcome revealed positive sign on TBi healing process. Therefore, the research has proven the new therapeutic approach for functional TBi regeneration using 3D cell-printing and tissue-specific decellularized extracellular matrix (dECM) bioink-based strategy [89]. Different investigations results revealed that utilization of 3D bioprinting techniques have a big impact on the field of tissue engineering using dopants specially to enhance following properties mechanical, biocompatibility, biodegradability, cell viability, cell proliferation, longevity, bone formation, less cytotoxicity.

6.3 Other applications for 3D printing

Despite advances in treatment methods, the healing and repairing of bone injuries and abnormalities has remained one of the most prevalent issues in clinical medicine because the healing and repairing processes are long-term and extremely difficult [90].To this aim, tissue engineering can heal different tissues using artificial biological replacements that restore the form and function of damaged tissue defects or malfunctioning organs [76, 91]. Therefore, this part explained other advanced applications related to extrusion-based 3DP. Shim et al. developed a 3D hybrid scaffold through solid freeform technology containing synthetic biomaterials and natural hydrogel. In this work the hydrogel was deposited into the space of a synthetic biomaterial-based scaffold using a multi-head deposition system (MHDS) and the cellular efficacy of the hybrid scaffold was evaluated using rat primary hepatocytes and mouse pre-osteoblasts (MC3T3 E1 cell line). In this research the synthetic biomaterial used for fabricating the scaffolds were PCL, PLGA, PCL/PLGA (85:15 wt%) and natural hydrogels were hyaluronic acids, gelatine, and atelocollagen. The results showed that the developed 3D hybrid scaffold had a superior effect on cell adhesion, proliferation, and viability of dispensed cells. They also revealed that collagen blended with the PCL/PLGA scaffold showed enhanced adhesion and proliferation of hepatocytes in 3D printed scaffold compared to other hydrogels and also improved cell viability of hepatocytes [92].

Domingo et al. reported the mechanical and biological performances of poly (e-caprolactone) scaffolds using extrusion-based 3D printing technology. In this work, three different lay-down patterns were adopted (0°/90°, 0°/60°/120°, 0°/45°/90°/135°) for fabricating the 3D printed scaffolds thus resulting in quadrangular, triangular and complex polygonal internal geometries. The cell seeding experiments were carried out using hMSC suspended at a density of 17 × 10³ cells/mL and were deposited onto PCL scaffolds. The cell viability and proliferation were investigated for 21 days and the SEM micrographs of PCL scaffolds were characterized by different lay-down patterns. The fabricated 3D rapid prototyped scaffolds had pore sizes and porosity ranges of 245 µm and 49–57%, respectively. Further, the mechanical and compression tests revealed a decrease in scaffold stiffness with increasing the porosity and number of deposition angles (from 0°/90° to 0°/45°/90°/135°). It was also revealed that hMSC viability/proliferation was enhanced after 21 days influenced by the pore size and shape of the scaffold. However, observed increment in cell viability with the decreasing number of deposition angles, larger fiber diameters, and pore sizes [93].

Seyednejad et al. fabricated 3D printed scaffolds based on hydroxyl functionalized polyester (poly (hydroxyl methyl glycoside-co-ε caprolactone) [PHMGCL]) through fiber deposition. In this study, 3D printed scaffolds of PHMGCL and HMG: CL (8:92 wt%) were prepared by fiber deposition (melt-plotting) and compared with poly(ε-caprolactone) PCL scaffold. The biodegradation and biocompatibility of PHMGCL and PCL scaffolds were investigated after 4 and 12-weeks post-implantation in balb/c mice. The in vitro enzymatic assay revealed that the degradation weight loss of PHMGCL 3D printed scaffolds were more than 60% within 3 months while PCL scaffolds showed no weight loss in the time frame. Further, the PHMGCL scaffold was degraded within 50 h while the PCL scaffold degradation was observed after 72 h, as noted in NMR analysis. Further, the PCL scaffolds showed an increased number of cells after 3 months of implantation and indicated milder tissue response to PCL scaffolds compared to PHMGCL scaffolds. The research concluded that the fast degradable PHMGCL scaffold showed good biocompatibility and could be a promising biomaterial for tissue engineering applications [94]. Billiet et al. reported the cell viability of a macroporous 3D printed scaffold using gelatin methacrylamide. Three-dimensional porous scaffolds were produced by sequential fiber deposition using the plotter pneumatic dispensing system. It was also observed that the cell viability of conical-type needles was found to be higher than cylindrical-shaped needles and a further decrease in viability with an increase in pressure level. The 3D printed constructs were fabricated using VA-086 initiator displayed high cell viability >97%, which can be suitable for tissue regeneration [95]. Rajaram et al. reported the effect of hyaluronic acid (HLA) and polycation polyethyleneimine (PEI) on alginate hydrogels in terms of physical properties. The cell culture was analyzed using ATDC5 chondrogenic cells derived from mouse genic and rat Schwann cells. It was suspended in alginate-hydrogel solution at a density of 1.6 × 10⁶ cells/mL to generate a cell-laden hydrogel construct using Bioplotter. The observed results showed that encapsulated Schwann cells and ATDC5 maintained cell viability for long periods and tended to show reduced cell survival at higher PEI concentrations. The addition of HLA to alginate increased the viscosity of hydrogel and helped to attain good strands during 3D plotting. They reported that there was no significant increase in mechanical properties even after increasing the PEI concentration by five-fold. Further, they concluded that increasing the concentration of PEI in crosslinking solution resulted in a decreased percentage loss or degradation of scaffolds [96]. Skardal et al. fabricated photo-crosslinked alginate-HLA 3D printed constructs were based on HLA-MA: GE-MA hydrogels for tissue engineering. In this work, the HA was synthesized using methacrylic hyaluronic acid to form HLA-MA, whereas the gelatine was cross-lined with methacrylate ethanolamide to form GE-MA. As a result, gel-like fluid was extruded by partial photochemical co-crosslinking of GE-MA with HLA-MA. The cells used for investigation were HepG2 C3A, Int-407, and NIH3T3 and they were seeded into HLA-MA: GE-MA hygrogels with a density of 25,000 cells per 100 µL hydrogel to develop cell laden constructs using Fab@ Model 1 Bioprinter. In this system the partially crosslinked hydrogels were extruded from a syringe into a designed base layer and irradiated again to create a firmer cellular tubular structure with cell-free core and cell-free structural halo. The printed cells with HLA-MA: GE-MA hydrogels showed enhanced presence of collagen and procollagen compared to cell free hydrogels. Moreover the fabricated HLA-MA: GE-MA hydrogels by two step photo crosslinking showed both cytocompatible and biocompatible properties in vitro and in vivo [97]. Zhang et al. fabricated a strontium-mesoporous bioactive glass (Sr-MBG) scaffold using 3D printing technology. In this study, a 3D bioplotter was used to print the Sr-MBG scaffold and the cell response of the MC3T3-E1 cell line on the Sr-MBG scaffold was investigated. The cell culture study was performed by seeding 1 × 10⁵ cells/mL on each Sr-MBG scaffold. The cell viability of MC3T3-E1 cells was determined by the Cell Counting Kit-8 assay. The study result reported that printed 3D Sr-MGB scaffolds had uniform interconnected macropores and higher porosity. The number of cells attached to the Sr-MGB scaffolds was observed to be higher than that of MGB scaffolds. Further, the Sr-MBG scaffolds showed a sustained drug delivery property with dexamethasone (DEX) drugs owing to its mesoporous structure. It was also observed that 75–85% of the loaded DEX was released from Sr-MBG scaffolds on day one which was followed by a very slow release thereafter [98].

Huang et al. utilized the 3D printing technique for designing complex drug release profiles and compared them with conventional mold techniques. The prepared drug implants were characterized using SEM and UV absorbance spectrophotometry. It was found that the implants prepared by 3D printing achieved both distinct bi-modal and pulsed release profiles in a single implant compared to conventional techniques. Moreover, the pulse release appeared from day 5 to 25 with a steady-state phase of 25 days. The next pulse release phases began at day 50 and ended at day 80; the steady-state profile release was observed at 5 µg/ml of LVFX [99].

In another research work, Chung et al. fabricated 3D scaffolds using additive biofabrication (3D bio-printing). In this study alginate (Alg) was preferred as a bioink for printing the cells. The structure of the printed Alg-gel scaffold. It was found that pre crosslinked alginate formulation, consisting of alginate and CaCl₂, was not stable during extrusion and resulted in liquid-like scaffolds with inconsistent pore diameters. The added gelatin improved the printability with well-defined structures and pore diameters. The in vitro degradation tests showed a 50% drop in modulus after 4 days which further decreased to more than 80% after 14 days, indicating the enhanced stability of printed cells. Live/dead cell assay showed that myoblast viability of about 90% was maintained within Alg-Gel scaffolds for 48 h and was unaffected by all extrusion pressure levels [100]. Suwanprateeb et al. used hydroxyapatite and Bis-GMA (bisphenol A diglycidyl ether dimethacrylate) for making composites via 3D printing. In vitro cell toxicity was performed using human osteoblast cells by seeding 10,000 cells/mL into HA/Bis-GMA scaffolds. The results showed that the sintered 3D samples possessed higher hydroxyapatite content, high density, and greater modulus but the strength and strain at break points were found to be lower compared to the green 3DP (nontoxic) sample. In vitro cell toxicity revealed that both HA-Bis- GMA-g and HA-Bis-GMA-s were non-toxic to osteoblast cells and osteoblast cells were observed attached and attained morphology on the surface of composites as evidenced by SEM imaging [101]. In the extension work, they investigated the mechanical properties of high-density polyethylene (HDPE) structure fabricated using a 3D printing technique. In this investigation, two heat processes were followed in making 3D printed scaffolds, namely primary heating. It was observed that the heat treatment of HDPE scaffolds showed the highest tensile modulus and strength of about 0.7 GPa and 14.8 MPa compared to unsintered samples. The sintered scaffolds at 160 °C showed decreased porosity and increased tensile parameters compared to samples sintered at 180 °C for 15 min. While the thermogravimetric analysis results revealed that low weight residue produced by HDPE whereas large amount of residue produced by the 3D printed polyethylene sintered at 180 °C for 60 min. This indicates improved mechanical properties of sintered 3D printed constructs [101]. Leukers et al. fabricated hydroxyapatite (HA-S) granulate scaffolds using 3D printing followed by heat treatment. Approximately 82% cell viability was measured through tetrazolium assay (WST-1) which shows good seeding efficiency of HA’s scaffold w. Cells seeded on HA-S. However, cultured dynamically showed higher cell viability and proliferation over 7 days than statically cultured cells either on plastics or HA structures. Moreover, the HA-S scaffolds made by 3D printing showed no cytotoxicity after heat treatment [102]. In another study, Ge Gao et al. fabricated a new in vitro atherosclerosis model, which was comprised of a triple-layered artery equivalent (AE) with adjustable geometries and improved structural characteristics using a novel promising platform 3D in-bath coaxial cell printing method. It was observed that the novel AE constructed model has produced operational tissues that retorted to many stimulations presented inside the 3D printed scaffold due to the induction of endothelial dysfunction. Moreover, an occurrence of repeated hallmark events before atherosclerosis was notified in the presence of various vascular tissues under complicated and stenotic flows. However, the 3D printed construct model was further examined individually and combinedly to see the effect of cell co-culture and local disturbed flows as the initiation of regulating atherosclerosis and consequence of atorvastatin as dose dependent. The outcomes from the novel proposed research suggested that constructed AE model spotlighted the importance of 3D printing techniques and expanded the version of enhanced biomedical applications. It can create a big breakthrough in pathophysiology related to cardiovascular diseases (CVDs) and their associated drugs for effective treatments [72]. Although, 3D printing is a potential technique, which produces geometrically different porous structures in a 3D orientation and might effectively regulate the tissue microenvironment. Notably, by printing essential tissues, this technique increases physiological relevance and overcomes key constraints of traditional fabrication procedures, such as decreased repeatability, compatibility difficulties with porogens, and organic solvents. Indeed, using different polymers and efficiently controlling the microstructure of materials, this technology can quickly and accurately fabricate individualized or patient-specific tissue or organ models, avoiding the deformities of immune response and allograft rejection caused by traditional organ transplantation.

6.4 Heart valve

The use of hydrogel materials in tissue engineering heart valve techniques is being vastly investigated [103], [104], [105], [106]. In addition, hydrogels have recently been used to create starter scaffolds that are quickly remodeled by cells before being decellularized to form implantable valves, to imitate the native ECM environment and good grip cells inside electrospun engineered valves; and to integrate regional matrix or mechanical differences directly into engineered valves [107, 108]. Moreover, hydrogels are utilized to regulate and investigate mechanical and biochemical signals in the heart valve cell response [109], [110], [111]. As new material combinations are produced or materials are employed unprecedentedly, a key issue is to adjust hydrogel technology to optimal cell survival methods [107]. Automated 3D bioprinting of cells into the structure of the hydrogel valve allows controlled cell deposition inside the structure [103]. Although, a great degree of geometric control and shape closeness of a hydrogel implant scale may be obtained by employing photocrosslinks and 3D printed constructs [107]. Hence this part defines the direct and photocrosslinks 3D printed construct applications specifically used for a heart valve. Duan et al. fabricated living aortic valve conduits based on an alginate/gelatin hydrogel system via 3D printing. In this study, the cells investigated were aortic valve root cells (SMC) and leaflet cells (VIC) bioprinted within alginate/gelatin hydrogel scaffold using a dual syringe system. The MTT assay was performed on VIC and SMC encapsulated in alginate/gelatine hydrogel discs at both low (2 × 10⁶ cells/mL) and high cell density (1 × 10⁷ cells/mL). The tensile stress and modulus of SMC encapsulated constructs were found to be about 1.74 ± 0.29 MPa and 1.98 ± 0.15 MPa while hydrogels encapsulated with VIC showed reduced tensile strength and moduli compared to a cell free sample. The cell viability results showed that seeded SMC cells and VIC cells were viable over 7 days in culture and the viability of cells was 81.4 ± 3.4% for SMC and 83.2 ± 4% for VIC [112]. In their continuation, they developed 3D printed trileaflet valve constructs using hydrogels and human valve interstitial cells. In this work the hydrogels HA and gelatin were synthesized using metha acrylic anhydride to form Me-HA and Me-GA structures and 0.05% w/v 2-hydroxy-1(4-(hydroxyethoxy) phenyl)-2-methyl-1propanone was added to form gel precursors. Next, the cell-laden 3D printed constructs were fabricated by seeding human aortic valvular interstitial cells (HAVIC) in hydrogels at a density of 5 × 106 cells/mL and the viability of cells was determined using Live/Dead assay. The results showed that an increase in Me-Gel and Me-HA concentration resulted in lower stiffness and higher viscosity, facilitated cell spreading, and better maintained HAVIC fibroblast phenotype. It was found that the incorporated VIC in Me-HA/Me-Gel hydrogels were almost alive after 3-day and 7-day culture and showed the viability of >90% in all hydrogel conditions. Further, they also remodeled the matrix with enhanced collagen and high glycosaminoglycans for new tissue growth [113]. Hockaday et al. developed native anatomic and axis-symmetric aortic valve geometrical using 3D printing technology. In this study, they implemented a 3D printing strategy combining UV photo crosslinking with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels to fabricate a complex and heterogeneous valve scaffold at different inner diameters of 12–22 mm. The printing system was performed using an extrusion-based 3D printing device. The cell viability was studied by seeding porcine aortic valve interstitial cells (PAVIC) into the PEG-DA scaffolds and culturing them for up to 21 days. The incorporated PEG-DA scaffolds showed a high elastic modulus of about 5.3 ± 0.9 to 74.9 ± 1.5 kPa compared to the control. The results showed that the 22 mm ID showed a larger printed shape fidelity as compared to the 12 mm ID where the shape fidelity was in the range respectively. Moreover, the PAVIC-incorporated scaffolds maintained viability of 100% over 21 days [114]. It can be concluded that photo crosslinking approach showed better results in terms of mechanical and cell viability in comparison with direct 3D printing of cells in hydrogels. However, UV light intensities and its exposure time is a big challenge while constructing 3D construct based on hydrogels. There is a need to explore more studies based on optimization of UV photo crosslinking-based 3D printing constructs.

6.5 Soft tissues

Appropriate material for soft tissue replacement can meet a wide range of therapeutic demands from cosmetic, reconstruction, and correctional reasons. It is reported that there is a rising demand for new procedures and materials that can improve soft tissue regeneration each year, with five million reconstruction surgeries [14, 115]. In this connection, Ting et al. reported the mechanical characterization of bioprinted 3D soft tissue constructs. In this study, the scaffold was prepared using gelatine/polysaccharide alginate hybrid materials as the matrix and C₂C1₂ mouse myoblast cells were seeded into developed scaffolds at a density of 6 × 10⁶ cells/mL. The cell viability was evaluated using a Live/Dead assay kit. The live/dead staining results showed that the average viability of cells was 54.72% within 2 h of printing. However, the value increased greatly after 24 h and very few dead cells were observed after two days. So, it was concluded that increasing porosity resulted was due to a decrease in strength of the construct. Moreover, the fabricated 3D printed constructs have shown a decreasing trend for both compressive modulus and strength during the first week, especially after 3 days of culture [116]. In extrusion-based 3D printing, the composite framework in the form of biomaterials was used for depositing the cell-laden hydrogels. In most articles, the biomaterials used were PCL, PLGA, and a combination of PLC/PLGA [117]. The use of biomaterials has been preferred for the fabrication of complex structures. Therefore, it would be a good choice to consider other biomaterials in developing a new complex structure. The cell-laden hydrogels 3D constructs supported by biomaterials also possess significant thermal stability and the enhanced cell viability of dispensed cells for new bone formation. Some articles have revealed that cell-laden hydrogels subjected to UV radiation resulted in enhanced cell viability and proliferation.

7.1 Maxillofacial

Bioprinting is an innovative technique in medical applications with strong potential. Amongst the existing bioprinting techniques LAB is a significant approach because of its excellent resolution, high cell viability, and capacity to deposit high-viscous bioink. The LAB technique can precisely regulate the cells to rebuild organ life [118]. It is a fact that serious facial asymmetry has a negative physical and spiritual impact on patients [119]. Therefore, this section described the maxillofacial application based on Laser-assisted 3D bioprinting. Michael et al. developed 3D skin constructs using 3D LAB. In this research NIH3T3 fibroblast and HaCaT keratinocytes were used for making skin constructs. The in vivo study was performed using dorsal skinfold chambers in the nude mice. The in vitro results showed that multilayered tissue was formed with collagen-producing fibroblasts but did not show any differentiation in keratinocytes (HaCaT) cells. The results showed that printed keratinocytes formed a multilayered epidermis with the start of differentiation and stratum corneum. Moreover, the proliferation of keratinocytes was found in suprabasal layers and the start of differentiation of keratinocytes in vivo was due to the presence of growth factors present in mice that were absent in vitro [120]. Bergmann et al. have designed customized implants for remodeling of maxillofacial and craniofacial defects in bone replacement. The customized implants were printed through a 3D printing technique consisting of β-TCP (40 wt%) and BGH glass (60 wt%). The experimental results revealed that the sintered 3D samples have shown four-point bending strength of about 14.9 MPa compared to unsintered samples. Furthermore, XRD analysis concluded that the sintered 3D samples exhibited a phase composition change to wollastonite (CaSiO₃) and rhenanite (CaNaPO₄) and both were biocompatible and biodegradable [121]. In another work, Klammert et al. prepared 3D implants using powdered calcium phosphate for the reconstruction of cranial and maxillofacial defects. In this work dilute phosphoric acid (H₃PO₄) was printed into tricalcium phosphate powder, resulting in the formation of dicalcium phosphate hydrate (brushite) while hydrothermal treatment of brushite implants led to the formation of dicalcium phosphate anhydrous (Monetite, CaHPO₄). The developed 3D printed calcium phosphate implants showed an adequate accuracy of suitable biocompatibility and adjustable resorption behavior. They concluded that the mechanical properties of brushite and monetite matrices were less than that of titanium implants and would be suitable as placeholders for ingrowing bone tissue [122]. Using a 3DP technology and a precise selection of material during maxillofacial surgeries for complicated facial asymmetry might help to create newly developed 3D scaffolds with improved operation designs and more exact manipulation for tissue engineering applications.

7.2 Other 3D printing applications

LAB is an emergent tissue engineering tool that can recreate the anisotropy of live tissues by printing cells and fluid materials with a cell-level precision [123, 124]. Therefore, this part highlighted some other prominent applications of 3D printed constructs created by LAB. Catros et al. developed 2D and 3D tissue engineering constructs using LAB. In this work, they investigated the laser printing parameters for patterning and assembling nano-hydroxyapatite (nHA) and human osteoprogenitors with LAB. The cell-laden 3D printed construct was developed by seeding HOPs isolated from human bone marrow stromal cells (HBMSC) nHA bioink with a density of 30,000,000 cells/mL. The cell viability was characterized using Live/Dead Assay Kit. SEM, fluorescence microscopy and immunochemistry analysis showed that LAB did not alter the physicochemical properties of nHA nor the viability, proliferation, and phenotype of HOPs over time (up to 15 days) [125]. In their continuation, they developed 3D scaffolds using LAB. In this research, the PCL scaffolds were stacked using LBL techniques and seeding was on a single locus of the scaffolds. The prepared scaffolds were characterized using SEM, live/dead assay, and histology. The in vivo and in vitro studies were performed in NOD-SCID mice and the number of cells was quantified by a photon imager for 21 days in vitro and 2 months in vivo. The results showed that the LBL constructs had enhanced cell viability over 21 days. The integrity of printed papers was maintained by the use of PCL biopaper and the circular shape of MG63 cells was maintained after 4 days. Further, the LBL organization showed enhanced cell numbers favoring cell proliferation and tissue growth evident in histological sections [126]. Yu et al. designed a novel doughnut multi-layered drug delivery device (DDD) using the 3D technique. The new novel drug contained acetaminophen as a model drug, hydroxyl propyl methylcellulose as a matrix, and ethyl cellulose (EC) as a release-retardant material. It was revealed that fabricated multilayered DDDs using a 3D printing system showed strong adhesion between the barrier layer and the drug-contained region and offered linear profile for all different diameters, heights, concentrations of EC, and central hole diameters. The research paper also concluded that 3D printing had the potential to develop DDDs with complex design features which allows dosage adjustment of the DDD independently by changing the heights of the DDD [127].

Although Laser-assisted 3D printing has numerous advantages like good biocompatibility, high resolution, and great efficiency but the fabrication of 3D printed scaffolds by laser-assisted printing was not commonly useful due to some drawbacks. First of all, the use of many polymeric biomaterials in developing scaffolds was limited in laser-assisted printing where it requires additional chemical modifications to make the materials photopolymerizable. Since it has no nozzles (used in the inkjet and extrusion printers), it was difficult to deliver the material to the desired region for fabrication and the photopolymers filled the entire reservoir and had no intended space for polymerization, resulting in wastage of materials and increased costs. As discussed above, the use of laser-assisted 3D printing was found to be less useful and there is a need to utilize a combination of methods to overcome the drawbacks in the future.