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Abstract
Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue–to–bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.

Abstract
The treatment of bone defects caused by diseases, trauma, or tumor has always been a great clinical challenge. Implantation of bone biomaterials into bone defect areas is an effective method for bone injury repair. In this study, we used three-dimensional (3D) printing technology to prepare nano-hydroxyapatite (nHA)/sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds loaded with different ratios (0, 0.13, 0.26, 0.39, 0.53, and 0.79‰) of emodin (EM) (EM/nHA/SA/Gel). Scanning electron microscopy showed that the scaffolds had a smooth surface without fracture and nHA was evenly distributed on the surface. The cell proliferation and migration results showed that the 0.39‰ EM group, in particular, could significantly promote the proliferation and migration of mouse embryonic osteoblast precursor (MC3T3-E1) cells and significantly increase the mRNA expression of osteogenic differentiation-related genes (bone morphogenetic protein/BMP-2, BMP-9, osteocalcin). In addition, the 0.39‰ EM group exhibited the best effect on osteogenic differentiation-related proteins (alkaline phosphatase, Runx 2, OSX). The expression of M2 polarization-related genes (arginase-1, CD206) also significantly increased after the treatment with the 0.39‰ EM group. Micro-CT showed that in the rat skull defect model, the EM/nHA/SA/Gel scaffold group significantly promoted bone regeneration after being implanted into the skull for 30 days. Our results indicate that the EM/nHA/SA/Gel hydrogel scaffolds can not only directly promote the proliferation and differentiation of osteoblasts but also indirectly promote osteogenic differentiation by supporting M2 polarization of macrophages. EM/nHA/SA/Gel hydrogel scaffolds are potential bone tissue engineering materials for bone regeneration.

Abstract
The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca2+ ions from hydroxyapatite structure with cerium ions (Ca10-xCex(PO4)6(OH)2, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.

Abstract
As bone diseases and defects are constantly increasing, the improvement of bone regeneration techniques is constantly evolving. The main purpose of this scientific study was to obtain and investigate biomaterials that can be used in tissue engineering. In this respect, nanocomposite inks of GelMA modified with hydroxyapatite (HA) substituted with Mg and Zn were developed. Using a 3D bioprinting technique, scaffolds with varying shapes and dimensions were obtained. The following analyses were used in order to study the nanocomposite materials and scaffolds obtained by the 3D printing technique: Fourier transform infrared spectrometry and X-ray diffraction (XRD), scanning electron microscopy (SEM), and micro-computed tomography (Micro-CT). The swelling and dissolvability of each scaffold were also studied. Biological studies, osteopontin (OPN), and osterix (OSX) gene expression evaluations were confirmed at the protein levels, using immunofluorescence coupled with confocal microscopy. These findings suggest the positive effect of magnesium and zinc on the osteogenic differentiation process. OSX fluorescent staining also confirmed the capacity of GelMA-HM5 and GelMA-HZ5 to support osteogenesis, especially of the magnesium enriched scaffold.

Abstract
Triply periodic minimal surfaces (TPMS) are gaining popularity as scaffolds for bioapplications due to their unique structure, offering strong mechanical properties and biomorphic surfaces which enhance cell attachment and proliferation. In this work, polymer TPMS sheet lattices were printed using a well-known yet unprecedented technique of manufacturing such structures; which is material extrusion (specifically, pneumatic melt extrusion). This method offers a one step, straightforward yet reliable way to print complex porous structures while retaining design accuracy and significantly simplifying the process. Multiple primitive, gyroid and cubic structures were designed using MSLattice and Solidworks with 70% porosity and 2×2×3 unit cells. The scaffolds were printed by melt extrusion of polycaprolactone (PCL) at different parameters to establish the optimal settings. Morphological features (pore size and strut thickness) were determined using scanning electron microscopy (SEM) and the accuracy of print was determined by comparing to the design, showing high print accuracy and minimal percentage errors of less than 15% in all prints. Uniaxial compression testing was used to demonstrate the different deformation processes of the scaffolds and evaluate their mechanical properties, with primitive having the highest modulus and gyroid the highest yield strength. Finally, cell viability was quantified by alamar blue cell viability assay and visualized by SEM, displaying significant increase in cell proliferation and attachment, specifically in the primitive structure. Herein we will explain the challenges faced with design and print optimization and how we overcame them, making this work the first of its kind in material extrusion (pneumatic melt extrusion) printing of TPMS scaffolds.

Abstract
Methacryloyl gelatin (GelMA) is a versatile material for bioprinting because of its tunable physical properties and inherent bioactivity. Bioprinting of GelMA is often met with challenges such as lower viscosity of GelMA inks due to higher methacryloyl substitution and longer physical gelation time at room temperature. In this study, a tunable interpenetrating polymer network (IPN) hydrogel was prepared from gelatin-hyaluronan dialdehyde (Gel-HDA) Schiff’s polymer, and 100% methacrylamide substituted GelMA for biofabrication through extrusion based bioprinting. Temperature sweep rheology measurements show a higher sol-gel transition temperature for IPN (30 °C) compared to gold standard GelMA (27 °C). Furthermore, to determine the tunability of the IPN hydrogel, several IPN samples were prepared by combining different ratios of Gel-HDA and GelMA achieving a compressive modulus ranging from 20.6 ± 2.48 KPa to 116.7 ± 14.80 KPa. Our results showed that the mechanical properties and printability at room temperature could be tuned by adjusting the ratios of GelMA and Gel-HDA. To evaluate cell response to the material, MC3T3-E1 mouse pre-osteoblast cells were embedded in hydrogels and 3D-printed, demonstrating excellent cell viability and proliferation after 10 d of 3D in vitro culture, making the IPN an interesting bioink for the fabrication of 3D constructs for tissue engineering applications.

Abstract
Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs’ potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

Abstract
Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of β-tricalcium phosphate (β-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified β-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

Abstract
Fabrication of three-dimensional (3D) scaffolds using natural biomaterials introduces valuable opportunities in bone tissue reconstruction and regeneration. The current study aimed at the development of paste-like 3D printing inks with an extracellular matrix-inspired formulation based on marine materials: sodium alginate (SA), cuttlebone (CB), and fish gelatin (FG). Macroporous scaffolds with microporous biocomposite filaments were obtained by 3D printing combined with post-printing crosslinking. CB fragments were used for their potential to stimulate biomineralization. Alginate enhanced CB embedding within the polymer matrix as confirmed by scanning electron microscopy (ESEM) and micro-computer tomography (micro-CT) and improved the deformation under controlled compression as revealed by micro-CT. SA addition resulted in a modulation of the bulk and surface mechanical behavior, and lead to more elongated cell morphology as imaged by confocal microscopy and ESEM after the adhesion of MC3T3-E1 preosteoblasts at 48 h. Formation of a new mineral phase was detected on the scaffold’s surface after cell cultures. All the results were correlated with the scaffolds’ compositions. Overall, the study reveals the potential of the marine materials-containing inks to deliver 3D scaffolds with potential for bone regeneration applications.

Abstract
Bioactive composites made of ∽85 wt% poly(ε-caprolactone) (PCL) and ∽15 wt% nanometric hydroxyapatite (HA) produced from biogenic sources were 3D printed by an extrusion-based process to obtain porous scaffolds suitable for bone regeneration. Three different composite formulations were considered by using HA synthesized from three distinct natural sources, which were collected as food wastes: cuttlefish bones, mussel shells and chicken eggshells. Composition and thermal properties of the materials were analysed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and x-ray spectroscopy (XRD), while the morphological and mechanical properties of the 3D scaffolds were studied by means of electron microscopy (SEM) and compression tests. Bioactivity was tested by seeding human osteoblast cell line (MG63) onto the scaffolds which were analysed by confocal microscopy and Alamar Blue and PicoGreen® tests after 1 to 7 culture days. The elastic modulus (177–316 MPa) is found to be within the range reported for typical trabecular bones being increased by the presence of the bio-HA particles. Moreover, cells adhesion, viability and proliferation are largely promoted in the scaffolds containing nanometric HA with respect to pure PCL, the best results being revealed when mussel shell-derived HA is used. Indeed, different biological sources result in different cell proliferation rates, pointing that the biological origin has an impact on the cells-scaffold interaction. In general, the results show that PCL/bio-HA scaffolds possess improved mechanical properties and enhanced bioactivity when compared with pure PCL ones.

Abstract
Critical bone defects are considered one of the major clinical challenges in reconstructive bone surgery. The combination of 3D printed conductive scaffolds and exogenous electrical stimulation (ES) is a potential favorable approach for bone tissue repair. In this study, 3D conductive scaffolds made with biocompatible and biodegradable polycaprolactone (PCL) and multi-walled carbon nanotubes (MWCNTs) were produced using the extrusion-based additive manufacturing to treat large calvary bone defects in rats. Histology results show that the use of PCL/MWCNTs scaffolds and ES contributes to thicker and increased bone tissue formation within the bone defect. Angiogenesis and mineralization are also significantly promoted using high concentration of MWCNTs (3 wt%) and ES. Moreover, scaffolds favor the tartrate-resistant acid phosphatase (TRAP) positive cell formation, while the addition of MWCNTs seems to inhibit the osteoclastogenesis but present limited effects on the osteoclast functionalities (receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) expressions). The use of ES promotes the osteoclastogenesis and RANKL expressions, showing a dominant effect in the bone remodeling process. These results indicate that the combination of 3D printed conductive PCL/MWCNTs scaffold and ES is a promising strategy to treat critical bone defects and provide a cue to establish an optimal protocol to use conductive scaffolds and ES for bone tissue engineering.

Abstract
Background: One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using three-dimensional printing (3DP). Herein, we aimed to determine whether the much tighter control of microstructure of 3DP poly(lactic-co-glycolic) acid/β-tricalcium phosphate (PLGA/β-TCP) scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods: Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results: The 3DP scaffolds had a smoother surface (Ra: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (Ra: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. Alkaline phosphatase (ALP) activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion: The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation.

Abstract
In tissue engineering, cellularization of scaffolds has typically been performed by seeding the cells after scaffold fabrication. 3D-printing technology now allows bioprinting of cells encapsulated in a hydrogel simultaneously with the scaffold material. Here, we aimed to investigate whether bioprinting or cell seeding post-printing is more effective in enhancing responses of pre-osteoblastic MC3T3-E1 cell line derived from mouse calvaria.

Abstract
Local tissue microenvironment is able to regulate cell-to-cell interaction that leads to effective tissue repair. This study aims to demonstrate a tunable magnesium ionic (Mg2+) microenvironment in bony tissue that can significantly induce bone defect repair. The concept can be realized by using a newly fabricated nanocomposite comprising of custom-made copolymer polycaprolactone-co-poly(ethylene glycol)-co-polycaprolactone (PCL-PEG-PCL) and surface-modified magnesium oxide (MgO) nanoparticles. In this study, additive manufacturing (AM) technology had been adopted to help design the porous three-dimensional (3D) scaffolds with tunable Mg2+ microenvironment. We found that the wettability and printability of new copolymer had been improved as compared with that of PCL polymer. Additionally, when MgO nanoparticles incorporated into the newly synthesized hydrophilic copolymer matrix, it could lead to increased compressive moduli significantly. In the in vitro studies, the fabricated nanocomposite scaffold with low concentration of Mg2+ microenvironment not only demonstrated better cytocompatibility, but also remarkably enhanced osteogenic differentiation in vitro as compared with the pure PCL and PCL-PEG-PCL co-polymer controls. In the animal studies, we also found that superior and early bone formation and tissue mineralization could be observed in the same 3D printed scaffold. However, the nanocomposite scaffold with high concentration of Mg2+ jeopardized the in situ bony tissue regeneration capability due to excessive magnesium ions in bone tissue microenvironment. Lastly, this study demonstrates that the nanocomposite 3D scaffold with controlled magnesium concentration in bone tissue microenvironment can effectively promote bone defect repair.

Abstract
Scaffolds are important physical substrates for cell attachment, proliferation and differentiation. Multiple factors could influence the optimal design of scaffolds for a specific tissue, such as the geometry, the materials used to modulate cell proliferation and differentiation, its biodegradability and biocompatibility. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Previous studies of human adipose-derived stem cells (hADSCs) seeded on poly(ε-caprolactone) (PCL)/graphene scaffolds have proved that the addition of small concentrations of graphene to PCL scaffolds improves cell proliferation. Based on such results, this paper further investigates, for the first time, both in vitro and in vivo characteristics of 3D printed PCL/graphene scaffolds. Scaffolds were evaluated from morphological, biological and short term immune response points of view. Results show that the produced scaffolds induce an acceptable level of immune response, suggesting high potential for in vivo applications. Finally, the scaffolds were used to treat a rat calvaria critical size defect with and without applying micro electrical stimulation (10 μA). Quantification of connective and new bone tissue formation and the levels of ALP, RANK, RANKL, OPG were considered. Results show that the use of scaffolds containing graphene and electrical stimulation seems to increase cell migration and cell influx, leading to new tissue formation, well-organized tissue deposition and bone remodelling.

Abstract
In bone tissue engineering, the intrinsic hydrophobicity and surface smoothness of three-dimensional (3D)-printed poly(ε-caprolactone) scaffolds hamper cell attachment, proliferation and differentiation. This intrinsic hydrophobicity of poly(ε-caprolactone) can be overcome by surface modifications, such as surface chemical modification or immobilization of biologically active molecules on the surface. Moreover, surface chemical modification may alter surface smoothness. Whether surface chemical modification or immobilization of a biologically active molecule on the surface is more effective to enhance pre-osteoblast proliferation and differentiation is currently unknown. Therefore, we aimed to investigate the osteogenic response of MC3T3-E1 pre-osteoblasts to chemically surface-modified and RGD-immobilized 3D-printed poly(ε-caprolactone) scaffolds. Poly(ε-caprolactone) scaffolds were 3D-printed consisting of strands deposited layer by layer with alternating 0°/90° lay-down pattern. 3D-printed poly(ε-caprolactone) scaffolds were surface-modified by either chemical modification using 3 M sodium hydroxide (NaOH) for 24 or 72 h, or by RGD-immobilization. Strands were visualized by scanning electron microscopy. MC3T3-E1 pre-osteoblasts were seeded onto the scaffolds and cultured up to 14 d. The strands of the unmodified poly(ε-caprolactone) scaffold had a smooth surface. NaOH treatment changed the scaffold surface topography from smooth to a honeycomb-like surface pattern, while RGD immobilization did not alter the surface topography. MC3T3-E1 pre-osteoblast seeding efficiency was similar (44%–54%) on all scaffolds after 12 h. Cell proliferation increased from day 1 to day 14 in unmodified controls (1.9-fold), 24 h NaOH-treated scaffolds (3-fold), 72 h NaOH-treated scaffolds (2.2-fold), and RGD-immobilized scaffolds (4.5-fold). At day 14, increased collagenous matrix deposition was achieved only on 24 h NaOH-treated (1.8-fold) and RGD-immobilized (2.2-fold) scaffolds compared to unmodified controls. Moreover, 24 h, but not 72 h, NaOH-treated scaffolds, increased alkaline phosphatase activity by 5-fold, while the increase by RGD immobilization was only 2.5-fold. Only 24 h NaOH-treated scaffolds enhanced mineralization (2.0-fold) compared to unmodified controls. In conclusion, RGD immobilization (0.011 μg mg−1 scaffold) on the surface and 24 h NaOH treatment of the surface of 3D-printed PCL scaffold both enhance pre-osteoblast proliferation and matrix deposition while only 24 h NaOH treatment results in increased osteogenic activity, making it the treatment of choice to promote bone formation by osteogenic cells.