Effect of Morphological Characteristics and Biomineralization of 3D-Printed Gelatin/Hyaluronic Acid/Hydroxyapatite Composite Scaffolds on Bone Tissue Regeneration

The use of porous three-dimensional (3D) composite scaffolds has attracted great attention in bone tissue engineering applications because they closely simulate the major features of the natural extracellular matrix (ECM) of bone. This study aimed to prepare biomimetic composite scaffolds via a simple 3D printing of gelatin/hyaluronic acid (HA)/hydroxyapatite (HAp) and subsequent biomineralization for improved bone tissue regeneration. The resulting scaffolds exhibited uniform structure and homogeneous pore distribution. In addition, the microstructures of the composite scaffolds showed an ECM-mimetic structure with a wrinkled internal surface and a porous hierarchical architecture. The results of bioactivity assays proved that the morphological characteristics and biomineralization of the composite scaffolds influenced cell proliferation and osteogenic differentiation. In particular, the biomineralized gelatin/HA/HAp composite scaffolds with double-layer staggered orthogonal (GEHA20-ZZS) and double-layer alternative structure (GEHA20-45S) showed higher bioactivity than other scaffolds. According to these results, biomineralization has a great influence on the biological activity of cells. Hence, the biomineralized composite scaffolds can be used as new bone scaffolds in bone regeneration.     

Bioactive and Biocompatible Macroporous Scaffolds with Tunable Performances Prepared Based on 3D Printing of the Pre‐Crosslinked Sodium Alginate/Hydroxyapatite Hydrogel Ink

Bioactive and biocompatible porous scaffold materials with adjustable pore structures and drug delivery capability are one of the key elements in bone tissue engineering. In this work, bioactive and biocompatible sodium alginate (SA)/hydroxyapatite (HAP) macroporous scaffolds are facilely and effectively fabricated based on 3D printing of the pre‐crosslinked SA/HAP hydrogels followed by further crosslinking to improve the mechanical properties of scaffolds. The pore structures and porosity (>80%) of the porous scaffolds can be readily tailored by varying the formation conditions. Furthermore, the in vitro biomineralization tests show that the bioactivity of the porous scaffolds is effectively enhanced by the addition of HAP nanoparticles into the scaffold matrix. Furthermore, the anti‐inflammatory drug curcumin is loaded into the porous scaffolds and the in vitro release study shows the sustainable drug release function of the porous scaffolds. Moreover, mouse bone mesenchymal stem cells (mBMSCs) are cultured on the porous scaffolds, and the results of the in vitro biocompatibility experiment show that the mBMSCs can be adhered well on the porous scaffolds. All of the results suggest that the bioactive and biocompatible SA/HAP porous scaffolds have great application potential in bone tissue engineering.     

Active Materials for 3D Printing in Small Animals: Current Modalities and Future Directions for Orthopedic Applications

The successful clinical application of bone tissue engineering requires customized implants based on the receiver’s bone anatomy and defect characteristics. Three-dimensional (3D) printing in small animal orthopedics has recently emerged as a valuable approach in fabricating individualized implants for receiver-specific needs. In veterinary medicine, because of the wide range of dimensions and anatomical variances, receiver-specific diagnosis and therapy are even more critical. The ability to generate 3D anatomical models and customize orthopedic instruments, implants, and scaffolds are advantages of 3D printing in small animal orthopedics. Furthermore, this technology provides veterinary medicine with a powerful tool that improves performance, precision, and cost-effectiveness. Nonetheless, the individualized 3D-printed implants have benefited several complex orthopedic procedures in small animals, including joint replacement surgeries, critical size bone defects, tibial tuberosity advancement, patellar groove replacement, limb-sparing surgeries, and other complex orthopedic procedures. The main purpose of this review is to discuss the application of 3D printing in small animal orthopedics based on already published papers as well as the techniques and materials used to fabricate 3D-printed objects. Finally, the advantages, current limitations, and future directions of 3D printing in small animal orthopedics have been addressed.     

Determination of cytocompatibility and osteogenesis properties of in situ forming collagen-based scaffolds loaded with bone synthesizing drug for bone tissue engineering

Bone tissue engineering using in situ forming 3D scaffolds can be an alternative to surgically treated scaffolds. This work aimed to develop in situ forming scaffolds using poly (lactic-co-glycolic acid) and a bone synthesizing drug (risedronate) with or without the porogenic agent (collagen). Hybrid scaffolds were formed through solvent-induced phase inversion technique and were morphologically evaluated using scanning electron microscopy (SEM). The effect of scaffolds on Saos-2 cell line viability using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test besides their effect on cell growth using fluorescence microscope was assessed. Furthermore, alkaline phosphatase (ALP) activity as well as Ca²⁺ deposition on the scaffolds was evaluated. SEM images revealed the porous structure for collagen-based scaffolds. Saos-2 cell proliferation was significantly enhanced with risedronate-loaded scaffolds compared to those lacking the drug. Porous collagen-based scaffolds were more favorable for both the cell growth and the promotion of ALP activity. Furthermore, collagen-based scaffolds promoted the Ca²⁺ deposition compared to their counterparts without collagen. Such results suggest that collagen-based scaffolds offer excellent biocompatibility for bone regeneration, where this biocompatible nature of scaffold leads to the proliferation of cells that lead to the deposition of mineral on the scaffold. Such in situ forming 3D scaffolds provide a promising noninvasive approach for bone tissue engineering.     

In vitro and in vivo properties of graphene-incorporated scaffolds for bone defect repair

The employment of graphene and its derivatives, graphene oxide and reduced graphene oxide, is extending from bioimaging and fabrications of biosensors to drug delivery and tissue engineering in the biomedical area. Graphene family-incorporated scaffolds, used in bone tissue engineering and bone regenerative medicine, profit superior properties of these materials, such as enhanced mechanical properties, large surface area, and the existence of functional groups. At the same time, problems related to cytotoxicity and adverse immune response of graphene family are solved when they are applied to produce 3-dimensional scaffolds. The objective of this review is to focus on in vitro properties of scaffolds consisting of graphene or its derivatives, especially osteogenic and antibacterial properties, as well as the influence of graphene and its derivatives on in vivo performances of implanted bone scaffolds. The positive effect of graphene and its two derivatives on attachment, and cell proliferation, as well as in vitro osteogenic differentiation of different cells was undeniable. Besides, the synergetic outcome of using graphene family on the antibacterial feature of scaffolds, especially incorporation with the silver element, was effective. Moreover, successful treatment of critical-sized bone defects was reported during in vivo preclinical tests when graphene or its derivatives-incorporated scaffolds were used. However, the limited number of in vivo studies should be considered as one of the main shortcomings to use graphene as a promising candidate for treating bone defects. It is anticipated that the increased number of well-designed preclinical studies could improve the applications of graphene incorporated scaffolds in bone tissue engineering/regeneration, and find out explanations and appropriate solutions to possible long-term toxicity and nonbiodegradability of these materials.     

Three-dimensional printing of triply periodic minimal surface structured scaffolds for load-bearing bone defects

The porous and cellular architecture of scaffolds plays a significant role in mechanical strength and bone regeneration during the healing of fractured bones. In this present study, triply periodic minimal surface (TPMS)-based gyroid and primitive lattice structures were used to design the cellular porous biomimetic scaffolds with different unit cell sizes (4, 5, and 6). The fused filament fabrication-based 3D printing technology was used for the fabrication of polylactic acid scaffolds. The surface morphology and mechanical compressive strength of differently structured scaffolds were observed using scanning electron microscopy and a universal testing machine. The unit cell size of 4 showed higher compressive strength in both gyroid and primitive structured scaffolds compared to unit cell sizes 5 and 6. Moreover, the gyroid structured scaffolds have higher compressive strengths as compared to primitive structured scaffolds due to the higher bonding surface area at the intercalated layers of the scaffold. Hence, the mechanical strength of scaffolds can be tailored by varying the unit cell size and cellular structures to avoid stress shielding and ensure implant safety. These TPMS-based scaffolds are promising and can be used as bone substitute materials in tissue engineering and orthopedic applications.     

Functionalized tricalcium phosphate and poly(3-hydroxyoctanoate) derived composite scaffolds as platforms for the controlled release of diclofenac

Novel bone substitutes such as highly porous ceramic scaffolds can serve as platforms for delivering active molecules. A common problem is to control the release of the drug, therefore, it is beneficial to use a drug-functionalized polymer coating. In this study, β-tricalcium phosphate-based porous scaffolds were obtained and coated with diclofenac-functionalized biopolymer – poly(3-hydroxyoctanoate) – P(3HO). To the best of our knowledge, studies using P(3HO) as a component in ceramic-polymer based drug delivery system for bone tissue regeneration have not yet been reported. Presented materials were comprehensively investigated by various techniques such as powder X-ray diffraction, scanning electron microscopy with energy dispersive spectroscopy, hydrostatic weighing and compression tests, pH and ionic conductivity measurements, high-performance liquid chromatography and in vitro cytotoxicity studies. The obtained diclofenac-loaded composite was not only characterised by controlled and sustained drug release, but also possessed improved mechanical properties. Moreover, the precipitation of apatite-like forms on its surface was observed after incubation in simulated body fluid, which indicates its bioactive potential. After 24 hours no cytotoxic effect on MC3T3-E1 mouse preosteoblastic cells was confirmed using indirect cytotoxicity studies. Thus, this promising multifunctional composite scaffold can be a promising candidate as an anti-inflammatory drug-delivery system in bone tissue engineering.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

Functionalized silver doped hydroxyapatite scaffolds for controlled simultaneous silver ion and drug delivery

Bacterial infections are a major problem in bone tissue regeneration, thus it is essential to incorporate antibacterial properties within the bone scaffolds. Silver compounds are frequently used as antibacterial agents to prevent bacterial infections and numerous studies have shown that silver ions can be incorporated within the biocompatible and osteoconductive biomaterial hydroxyapatite (HAp) structure, but, so far, no study has thoroughly evaluated silver ion release rates in long term. Therefore, we have established a novel carrier system for local drug delivery based on functionalized silver doped hydroxyapatite with determined long term silver ion release rates. Silver ions from prepared scaffolds were released with a rate of 0.001±0.0005 wt%/h taking into account the incorporated silver amount. Moreover, lidocaine hydrochloride was incorporated in the prepared scaffolds, to provide local anesthetic effect. These scaffolds were functionalized with sodium alginate and chitosan and in vitro drug release rate in simulated body fluid was evaluated. The results suggested that the developed novel composite scaffolds possess the antibacterial activity up to one year as well as controlled anesthetic drug delivery up to two weeks.     

Facile fabrication of near-infrared light-responsive shape memory nanocomposite scaffolds with hierarchical porous structures

In this work, the near-infrared (NIR) light-responsive shape memory scaffolds with hierarchical porous structures are designed and facilely formed by freeze drying of 3D printed viscous gel-like pickering emulsions, which are stabilized by hydrophobically modified graphene oxide (g-GO) and silica nanoparticles, and contain thermo-responsive poly(d , l -lactic acid-co-trimethylene carbonate) (PLMC) in the oil phase. The prepared scaffolds display an interconnected filament structure with hierarchical pores and high porosity. The incorporation of g-GO nanoparticles into PLMC matrix prompts that the scaffold shape memory can be triggered by NIR light with fast shape recovery. Moreover, the in vitro mineralization experiment shows that the scaffolds have biological activity, and the drug release study demonstrates that the scaffolds can be used as drug carriers with efficient drug release capacity. Furthermore, cell culture assays based on mouse bone mesenchymal stem cells exhibit that the scaffolds own good cytocompatibility. Therefore, the facile preparation and remote activation of the shape memory nanocomposite scaffolds with hierarchical porous structure and multifunctionality represents a highly attractive candidate as minimally invasive implantation scaffolds for bone tissue engineering applications.     

3D printed polylactide-based zirconia-toughened alumina composites: fabrication,mechanical, and in vitro evaluation

Fused deposition modeling (FDM) has been a commonly used technique in the fabrication of geometrically complex biodegradable scaffolds for bone tissue engineering. Generally, either individual polylactide (PLA) or its combination with calcium phosphates or bioglass has been employed to design scaffolds through the principles of FDM. In this study, FDM protocol has been employed to design 3D printed PLA/zirconia-toughened alumina (ZTA). A series of PLA/ZTA combinations have been attempted to determine the feasibility of the resultant in filament extrusion and their subsequent capacity to obtain a stable 3D printed component. A maximum of 80 wt.% PLA and 20 wt.% ZTA has been determined as an optimum combination to yield a stable 3D structure beyond which an enhanced ZTA content in the PLA matrix yielded a fragile filament that lacked effectiveness in 3D printing. 5 and 10 wt.% of ZTA addition in the PLA matrix produced a better 3D design that reasonably displayed good mechanical properties. Depending on the ceramic content, a homogeneous dispersion of the constituent elements representative of ZTA has been determined throughout the PLA matrix. Simulation studies through finite element analysis (FEA) exhibited good corroboration with the test results obtained from the mechanical studies.     

Halloysite clay nanotube in regenerative medicine for tissue and wound healing

The vital necessity of effective treatment at damaged tissue or wound site has resulted in emerging tissue engineering and regenerative medicine. Tissue engineering has been introduced as an alternative approach for common available therapeutic strategies in the terms of restoring deformed tissue structure and its functionality via the developing of new bio-scaffold. Designed three-dimensional (3D) scaffolds, alone or in combination with bioactive agents, should be able to stimulate and accelerate the development of engineered tissues and provide proper mechanical support during in-vivo implantation and later regeneration process. To cover it up, a series of new bio-structures with higher mechanical strength were designed through the combination of halloysite nanotubes (HNTs) into 3D bio-polymeric networks. HNTs clay mineral with its unique rod-like structure and distinctive chemical surface features, exhibits excellent biocompatibility and biosafety for doping into regenerative scaffolds to enhance their mechanical stiffness and biological performance. In this paper, the ongoing procedures of bone/cartilage tissue engineering and wound healing strategies focusing on the designing of 3D-HNTs bio-composites and their multi-cellular interactions in-vitro and in-vivo preclinical studies are reviewed. Furthermore, the challenges and prospects of 3D-HNTs and HNTs-based functional bio-devices for regenerative medicine are also discussed.     

Fabrication of forsterite scaffolds with photothermal-induced antibacterial activity by 3D printing and polymer-derived ceramics strategy

Bacterial infection of the implanting materials is one of the greatest challenges in bone tissue engineering. In this study, porous forsterite scaffolds with antibacterial activity have been fabricated by combining 3D printing and polymer-derived ceramics (PDCs) strategy, which effectively avoided the generation of MgSiO₃ and MgO impurities. Forsterite scaffolds sintered in an argon atmosphere can generate free carbon in the scaffolds, which exhibited excellent photothermal effect and could inhibit the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in vitro. In addition, forsterite scaffolds have uniform macroporous structure, high compressive strength (>30 MPa) and low degradation rate. Hence, forsterite scaffolds fabricated by combining 3D printing and PDCs strategy would be a promising candidate for bone tissue engineering.     

Electrospun polycaprolactone scaffolds for tissue engineering: a review

Polycaprolactone is a biodegradable and biocompatible polyester which has a wide range of applications in tissue engineering. Electrospinning, the versatile technique, used for the fabrication of fibrous scaffolds, which is widely used in tissue engineering, due to the ability of fabrication of nano/micro-scale fiber scaffolds. Polycaprolactone nanofiber scaffolds are widely used in tissue engineering and drug delivery. Polycaprolactone can be used in a wide variety of scaffolds construction. In this review, we will discuss the recent advances in the electrospinning of polycaprolactone nanofiber scaffolds in bone, cardiovascular, nerve, and skin tissue engineering.     

Application of 3D-Printed,PLGA-Based Scaffolds in Bone Tissue Engineering

Polylactic acid–glycolic acid (PLGA) has been widely used in bone tissue engineering due to its favorable biocompatibility and adjustable biodegradation. 3D printing technology can prepare scaffolds with rich structure and function, and is one of the best methods to obtain scaffolds for bone tissue repair. This review systematically summarizes the research progress of 3D-printed, PLGA-based scaffolds. The properties of the modified components of scaffolds are introduced in detail. The influence of structure and printing method change in printing process is analyzed. The advantages and disadvantages of their applications are illustrated by several examples. Finally, we briefly discuss the limitations and future development direction of current 3D-printed, PLGA-based materials for bone tissue repair.     

Biomimetic Polycaprolactone-Graphene Oxide Composites for 3D Printing Bone Scaffolds

Bone shows a radial gradient architecture with the exterior densified cortical bone and the interior porous cancellous bone. However, previous studies presented uniform designs for bone scaffolds that do not mimic natural bone's gradient structure. Hence, mimicking native bone structures is still challenging in bone tissue engineering. In this study, a novel biomimetic bone scaffold with Haversian channels is designed, which approximates mimicking the native bone structure. Also, the influence of adding graphene oxide (GO) to polycaprolactone (PCL)-based scaffolds are investigated by preparing PCL/GO composite ink containing 0.25% and 0.75% GO and then 3D printing scaffolds by an extrusion-based machine. Scanning electron microscopy (SEM) is used for morphological analysis. SEM reveals good printability and interconnected pore structure. The contact angle test shows that wettability reinforces with the increase of GO content. The mechanical behavior of the scaffolds under compression is examined numerically and experimentally. The results indicate that incorporation of GO can affect bone scaffolds' Young's modulus and von Mises stress distribution. Moreover, the biodegradation rates accelerate in the PCL/GO scaffolds. Biological characterizations, such as cell growth, viability, and attachment, are performed utilizing osteoblast cells. Compared to pure PCL, an enhancement is observed in cell viability in the PCL/GO scaffolds.     

Novel 3D scaffolds of chitosan-PLLA blends for tissue engineering applications: Preparation and characterization

This work addresses the preparation of 3D porous scaffolds of blends of chitosan and poly(l-lactic acid), CHT and PLLA, using supercritical fluid technology. Supercritical assisted phase-inversion was used to prepare scaffolds for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage regeneration. On the other hand, PLLA is a synthetic biodegradable polymer widely used for biomedical applications. Supercritical assisted phase-inversion experiments were carried out in samples with different polymer ratios and different polymer solution concentrations. The effect of CHT:PLLA ratio and polymer concentration and on the morphology and topography of the scaffolds was assessed by SEM and Micro-CT. Infra-red spectroscopic imaging analysis of the scaffolds allowed a better understanding on the distribution of the two polymers within the matrix. This work demonstrates that supercritical fluid technology constitutes a new processing technology, clean and environmentally friendly for the preparation of scaffolds for tissue engineering using these materials.     

Novel 3D scaffolds of chitosan–PLLA blends for tissue engineering applications: Preparation and characterization

This work addresses the preparation of 3D porous scaffolds of blends of chitosan and poly(l-lactic acid), CHT and PLLA, using supercritical fluid technology. Supercritical assisted phase-inversion was used to prepare scaffolds for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage regeneration. On the other hand, PLLA is a synthetic biodegradable polymer widely used for biomedical applications. Supercritical assisted phase-inversion experiments were carried out in samples with different polymer ratios and different polymer solution concentrations. The effect of CHT:PLLA ratio and polymer concentration and on the morphology and topography of the scaffolds was assessed by SEM and Micro-CT. Infra-red spectroscopic imaging analysis of the scaffolds allowed a better understanding on the distribution of the two polymers within the matrix. This work demonstrates that supercritical fluid technology constitutes a new processing technology, clean and environmentally friendly for the preparation of scaffolds for tissue engineering using these materials.     

Reinforcement of electrospun poly(ε‐caprolactone) scaffold using diopside nanopowder to promote biological and physical properties

Considerable efforts have been devoted toward the development of electrospun scaffolds based on poly(ε‐caprolactone) (PCL) for bone tissue engineering. However, most of previous scaffolds have lacked the structural and mechanical strength to engineer bone tissue constructs with suitable biological functions. Here, we developed bioactive and relatively robust hybrid scaffolds composed of diopside nanopowder embedded PCL electrospun nanofibers. Incorporation of various concentrations of diopside nanopowder from 0 to 3 wt % within the PCL scaffolds notably improved tensile strength (eight‐fold) and elastic modulus (two‐fold). Moreover, the addition of diopside nanopowder significantly improved bioactivity and degradation rate compared to pure PCL scaffold which might be due to their superior hydrophilicity. We investigated the proliferation and spreading of SAOS‐II cells on electrospun scaffolds. Notably, electrospun PCL‐diopside scaffolds induced significantly enhanced cell proliferation and spreading. Overall, we concluded that PCL‐diopside scaffold could potentially be used to develop clinically relevant constructs for bone tissue engineering. However, the extended in vivo studies are essential to evaluate the role of PCL‐diopside fibrous scaffolds on the new bone growth and regeneration. Therefore, in vivo studies will be the subject of our future work. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 44433.     

3D Printed Poly(𝜀-caprolactone)/Hydroxyapatite Scaffolds for Bone Tissue Engineering: A Comparative Study on a Composite Preparation by Melt Blending or Solvent Casting Techniques and the Influence of Bioceramic Content on Scaffold Properties

Bone tissue engineering has been developed in the past decades, with the engineering of bone substitutes on the vanguard of this regenerative approach. Polycaprolactone-based scaffolds are fairly applied for bone regeneration, and several composites have been incorporated so as to improve the scaffolds’ mechanical properties and tissue in-growth. In this study, hydroxyapatite is incorporated on polycaprolactone-based scaffolds at two different proportions, 80:20 and 60:40. Scaffolds are produced with two different blending methods, solvent casting and melt blending. The prepared composites are 3D printed through an extrusion-based technique and further investigated with regard to their chemical, thermal, morphological, and mechanical characteristics. In vitro cytocompatibility and osteogenic differentiation was also assessed with human dental pulp stem/stromal cells. The results show the melt-blending-derived scaffolds to present more promising mechanical properties, along with the incorporation of hydroxyapatite. The latter is also related to an increase in osteogenic activity and promotion. Overall, this study suggests polycaprolactone/hydroxyapatite scaffolds to be promising candidates for bone tissue engineering, particularly when produced by the MB method.