Subcortical Backup Tibial Fixation in Anterior Cruciate Ligament Reconstruction Has Similar Maximal Strength to Current Techniques.

Purpose: To evaluate the biomechanical profile of subcortical backup fixation (subcortical button [SB]) in anterior cruciate ligament (ACL) reconstruction as compared with a bicortical post and washer (BP) and suture anchor (SA) when used with interference screw (IS) primary fixation and to evaluate the utility of backup fixation for tibial fixation with extramedullary cortical button primary fixation. Methods: Fifty composite tibias with polyester webbing-simulated graft were used to test constructs across 10 methods. Specimens were separated into the following groups (n = 5): 9-mm IS only, BP (with and without graft and IS), SB (with and without graft and IS), SA (with and without graft and IS), extramedullary suture button (with and without graft and IS), and extramedullary suture button with BP as backup fixation. Specimens were tested under cyclic loading and then loaded to failure. Maximal load at failure, displacement, and stiffness were compared. Results: Without a graft, the SB and BP had similar maximal loads (802.46 ± 185.18 N vs 785.67 ± 100.96 N, P = .560), and both were stronger than the SA (368.13 ± 77.26 N, P < .001). With graft and an IS, there was no significant difference in maximal load between the BP (1,461.27 ± 173.75 N), SB (1,362.46 ± 80.47 N), and SA (1,334.52 ± 195.80 N). All backup fixation groups were stronger than the control group with IS fixation only (932.91 ± 99.86 N, P < .001). There was no significant difference in outcome measures between the extramedullary suture button groups with and without the BP (failure loads of 721.39 ± 103.32 N and 718.15 ± 108.61 N, respectively). Conclusions: Subcortical backup fixation in ACL reconstruction has similar biomechanical properties to current methods and is a viable backup fixation alternative. Backup fixation methods work synergistically with IS primary fixation to strengthen the construct. There is no advantage to adding backup fixation to extramedullary button (all-inside) primary fixation when all suture strands are secured to the extramedullary button. Clinical Relevance: This study provides evidence that subcortical backup fixation is a viable alternative for surgeons during ACL reconstruction.

3D Bioprinting-Tunable Small-Diameter Blood Vessels with Biomimetic Biphasic Cell Layers.

Blood vessel damage resulting from trauma or diseases presents a serious risk of morbidity and mortality. Although synthetic vascular grafts have been successfully commercialized for clinical use, they are currently only readily available for large-diameter vessels (>6 mm). Small-diameter vessel (<6 mm) replacements, however, still present significant clinical challenges worldwide. The primary objective of this study is to create novel, tunable, small-diameter blood vessels with biomimetic two distinct cell layers [vascular endothelial cell (VEC) and vascular smooth muscle cell (VSMC)] using an advanced coaxial 3D-bioplotter platform. Specifically, the VSMCs were laden in the vessel wall and VECs grew in the lumen to mimic the natural composition of the blood vessel. First, a novel bioink consisting of VSMCs laden in gelatin methacryloyl (GelMA)/polyethylene(glycol)diacrylate/alginate and lyase was designed. This specific design is favorable for nutrient exchange in an ambient environment and simultaneously improves laden cell proliferation in the matrix pore without the space restriction inherent with substance encapsulation. In the vessel wall, the laden VSMCs steadily grew as the alginate was gradually degraded by lyase leaving more space for cell proliferation in matrices. Through computational fluid dynamics simulation, the vessel demonstrated significantly perfusable and mechanical properties under various flow velocities, flow viscosities, and temperature conditions. Moreover, both VSMCs in the scaffold matrix and VECs in the lumen steadily proliferated over time creating a significant two-cell-layered structure. Cell proliferation was confirmed visually through staining the markers of alpha-smooth muscle actin and cluster of differentiation 31, commonly tied to angiogenesis phenomena, in the vessel matrices and lumen, respectively. Furthermore, the results were confirmed quantitatively through gene analysis which suggested good angiogenesis expression in the blood vessels. This study demonstrated that the printed blood vessels with two distinct cell layers of VECs and VSMCs could be potential candidates for clinical small-diameter blood vessel replacement applications.

In vitro and in vivo evaluation of 3D bioprinted small-diameter vasculature with smooth muscle and endothelium.

The ability to fabricate perfusable, small-diameter vasculature is a foundational step toward generating human tissues/organs for clinical applications. Currently, it is highly challenging to generate vasculature integrated with smooth muscle and endothelium that replicates the complexity and functionality of natural vessels. Here, a novel method for directly printing self-standing, small-diameter vasculature with smooth muscle and endothelium is presented through combining tailored mussel-inspired bioink and unique 'fugitive-migration' tactics, and its effectiveness and advantages over other methods (i.e. traditional alginate/calcium hydrogel, post-perfusion of endothelial cells) are demonstrated. The biologically inspired, catechol-functionalized, gelatin methacrylate (GelMA/C) undergoes rapid oxidative crosslinking in situ to form an elastic hydrogel, which can be engineered with controllable mechanical strength, high cell/tissue adhesion, and excellent bio-functionalization. The results demonstrate the bioprinted vascular construct possessed numerous favorable, biomimetic characteristics such as proper biomechanics, higher tissue affinity, vascularized tissue manufacturing ability, beneficial perfusability and permeability, excellent vasculoactivity, and in vivo autonomous connection (∼2 weeks) as well as vascular remodeling (∼6 weeks). The advanced achievements in creating biomimetic, functional vasculature illustrate significant potential toward generating a complicated vascularized tissue/organ for clinical transplantation.

4D anisotropic skeletal muscle tissue constructs fabricated by staircase effect strategy.

Like the morphology of native tissue fiber arrangement (such as skeletal muscle), unidirectional anisotropic scaffolds are highly desired as a means to guide cell behavior in anisotropic tissue engineering. In contrast, contour-like staircases exhibit directional topographical cues and are judged as an inevitable defect of fused deposition modeling (FDM). In this study, we will translate this staircase defect into an effective bioengineering strategy by integrating FDM with surface coating technique (FCT) to investigate the effect of topographical cues on regulating behaviors of human mesenchymal stem cells (hMSCs) toward skeletal muscle tissues. This integrated approach serves to fabricate shape-specific, multiple dimensional, anisotropic scaffolds using different biomaterials. 2D anisotropic scaffolds, first demonstrated with different polycaprolactone concentrations herein, efficiently direct hMSC alignment, especially when the scaffold is immobilized on a support ring. By surface coating the polymer solution inside FDM-printed sacrificial structures, 3D anisotropic scaffolds with thin wall features are developed and used to regulate seeded hMSCs through a self-established rotating bioreactor. Using layer-by-layer coating, along with a shape memory polymer, smart constructs exhibiting shape fix and recovery processes are prepared, bringing this study into the realm of 4D printing. Immunofluorescence staining and real-time quantitative polymerase chain reaction analysis confirm that the topographical cues created via FCT significantly enhance the expression of myogenic genes, including myoblast differentiation protein-1, desmin, and myosin heavy chain-2. We conclude that there are broad application potentials for this FCT strategy in tissue engineering as many tissues and organs, including skeletal muscle, possess highly organized and anisotropic extracellular matrix components.

Photolithographic-stereolithographic-tandem fabrication of 4D smart scaffolds for improved stem cell cardiomyogenic differentiation.

4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

Three-Dimensional-Bioprinted Dopamine-Based Matrix for Promoting Neural Regeneration.

Central nerve repair and regeneration remain challenging problems worldwide, largely because of the extremely weak inherent regenerative capacity and accompanying fibrosis of native nerves. Inadequate solutions to the unmet needs for clinical therapeutics encourage the development of novel strategies to promote nerve regeneration. Recently, 3D bioprinting techniques, as one of a set of valuable tissue engineering technologies, have shown great promise toward fabricating complex and customizable artificial tissue scaffolds. Gelatin methacrylate (GelMA) possesses excellent biocompatible and biodegradable properties because it contains many arginine-glycine-aspartic acids (RGD) and matrix metalloproteinase sequences. Dopamine (DA), as an essential neurotransmitter, has proven effective in regulating neuronal development and enhancing neurite outgrowth. In this study, GelMA-DA neural scaffolds with hierarchical structures were 3D-fabricated using our custom-designed stereolithography-based printer. DA was functionalized on GelMA to synthesize a biocompatible printable ink (GelMA-DA) for improving neural differentiation. Additionally, neural stem cells (NSCs) were employed as the primary cell source for these scaffolds because of their ability to terminally differentiate into a variety of cell types including neurons, astrocytes, and oligodendrocytes. The resultant GelMA-DA scaffolds exhibited a highly porous and interconnected 3D environment, which is favorable for supporting NSC growth. Confocal microscopy analysis of neural differentiation demonstrated that a distinct neural network was formed on the GelMA-DA scaffolds. In particular, the most significant improvements were the enhanced neuron gene expression of TUJ1 and MAP2. Overall, our results demonstrated that 3D-printed customizable GelMA-DA scaffolds have a positive role in promoting neural differentiation, which is promising for advancing nerve repair and regeneration in the future.

Stereolithographic 4D Bioprinting of Multiresponsive Architectures for Neural Engineering.

4D printing represents one of the most advanced fabrication techniques for prospective applications in tissue engineering, biomedical devices, and soft robotics, among others. In this study, a novel multiresponsive architecture is developed through stereolithography-based 4D printing, where a universal concept of stress-induced shape transformation is applied to achieve the 4D reprogramming. The light-induced graded internal stress followed by a subsequent solvent-induced relaxation, driving an autonomous and reversible change of the programmed configuration after printing, is employed and investigated in depth and details. Moreover, the fabricated construct possesses shape memory property, offering a characteristic of multiple shape change. Using this novel multiple responsive 4D technique, a proof-of-concept smart nerve guidance conduit is demonstrated on a graphene hybrid 4D construct providing outstanding multifunctional characteristics for nerve regeneration including physical guidance, chemical cues, dynamic self-entubulation, and seamless integration. By employing this fabrication technique, creating multiresponsive smart architectures, as well as demonstrating application potential, this work paves the way for truly initiation of 4D printing in various high-value research fields.

3D printing nano conductive multi-walled carbon nanotube scaffolds for nerve regeneration.

OBJECTIVE: Nanomaterials, such as carbon nanotubes (CNTs), have been introduced to modify the surface properties of scaffolds, thus enhancing the interaction between the neural cells and biomaterials. In addition to superior electrical conductivity, CNTs can provide nanoscale structures similar to those present in the natural neural environment. The primary objective of this study is to investigate the proliferative capability and differential potential of neural stem cells (NSCs) seeded on a CNT incorporated scaffold. APPROACH: Amine functionalized multi-walled carbon nanotubes (MWCNTs) were incorporated with a PEGDA polymer to provide enhanced electrical properties as well as nanofeatures on the surface of the scaffold. A stereolithography 3D printer was employed to fabricate a well-dispersed MWCNT-hydrogel composite neural scaffold with a tunable porous structure. 3D printing allows easy fabrication of complex 3D scaffolds with extremely intricate microarchitectures and controlled porosity. MAIN RESULTS: Our results showed that MWCNT-incorporated scaffolds promoted neural stem cell proliferation and early neuronal differentiation when compared to those scaffolds without the MWCNTs. Furthermore, biphasic pulse stimulation with 500 µA current promoted neuronal maturity quantified through protein expression analysis by quantitative polymerase chain reaction. SIGNIFICANCE: Results of this study demonstrated that an electroconductive MWCNT scaffold, coupled with electrical stimulation, may have a synergistic effect on promoting neurite outgrowth for therapeutic application in nerve regeneration.

Integrating three-dimensional printing and nanotechnology for musculoskeletal regeneration.

The field of tissue engineering is advancing steadily, partly due to advancements in rapid prototyping technology. Even with increasing focus, successful complex tissue regeneration of vascularized bone, cartilage and the osteochondral interface remains largely illusive. This review examines current three-dimensional printing techniques and their application towards bone, cartilage and osteochondral regeneration. The importance of, and benefit to, nanomaterial integration is also highlighted with recent published examples. Early-stage successes and challenges of recent studies are discussed, with an outlook to future research in the related areas.

Development of Novel 3-D Printed Scaffolds With Core-Shell Nanoparticles for Nerve Regeneration.

A traumatic injury of peripheral nerves is serious clinical problem that may lead to major loss of nerve function, affecting quality of patient's life. Currently, nerve autograft is widely used to reconstruct the nerve gap. However, such surgical procedure suffers from many disadvantages including donor site morbidity and limited availability. In order to address these issues, neural tissue engineering has focused on the development of synthetic nerve scaffolds to support bridging a larger gap and improving nerve generation. For this purpose, we fabricated a novel 3-D biomimetic scaffold, which has tunable porous structure and embedded core-shell nanoparticles with sustained neurogenic factor delivery system, using stereolithography based 3-D printing and coaxial electrospraying techniques. Our results showed that scaffolds with larger porosity significantly improve PC-12 neural cell adhesion compared to ones with smaller porosity. Furthermore, scaffolds embedded with bovine serum albumin containing nanoparticles showed an enhancement in cell proliferation relative to bared control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with nerve growth factor nanoparticles greatly increased the length of neurites and directed neurite extension of PC-12 cells along the fiber. In addition, the 3-D printed nanocomposite scaffolds also improved the average neurite length of primary cortical neurons. The results in this study demonstrate the potential of this 3-D printed scaffold in improving neural cell function and nerve growth.

Fabrication of a Highly Aligned Neural Scaffold via a Table Top Stereolithography 3D Printing and Electrospinning.

Three-dimensional (3D) bioprinting is a rapidly emerging technique in the field of tissue engineering to fabricate extremely intricate and complex biomimetic scaffolds in the range of micrometers. Such customized 3D printed constructs can be used for the regeneration of complex tissues such as cartilage, vessels, and nerves. However, the 3D printing techniques often offer limited control over the resolution and compromised mechanical properties due to short selection of printable inks. To address these limitations, we combined stereolithography and electrospinning techniques to fabricate a novel 3D biomimetic neural scaffold with a tunable porous structure and embedded aligned fibers. By employing two different types of biofabrication methods, we successfully utilized both synthetic and natural materials with varying chemical composition as bioink to enhance biocompatibilities and mechanical properties of the scaffold. The resulting microfibers composed of polycaprolactone (PCL) polymer and PCL mixed with gelatin were embedded in 3D printed hydrogel scaffold. Our results showed that 3D printed scaffolds with electrospun fibers significantly improve neural stem cell adhesion when compared to those without the fibers. Furthermore, 3D scaffolds embedded with aligned fibers showed an enhancement in cell proliferation relative to bare control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with PCL/gelatin fibers greatly increased the average neurite length and directed neurite extension of primary cortical neurons along the fiber. The results of this study demonstrate the potential to create unique 3D neural tissue constructs by combining 3D bioprinting and electrospinning techniques.

3D Bioprinting for Organ Regeneration.

Regenerative medicine holds the promise of engineering functional tissues or organs to heal or replace abnormal and necrotic tissues/organs, offering hope for filling the gap between organ shortage and transplantation needs. Three-dimensional (3D) bioprinting is evolving into an unparalleled biomanufacturing technology due to its high-integration potential for patient-specific designs, precise and rapid manufacturing capabilities with high resolution, and unprecedented versatility. It enables precise control over multiple compositions, spatial distributions, and architectural accuracy/complexity, therefore achieving effective recapitulation of microstructure, architecture, mechanical properties, and biological functions of target tissues and organs. Here we provide an overview of recent advances in 3D bioprinting technology, as well as design concepts of bioinks suitable for the bioprinting process. We focus on the applications of this technology for engineering living organs, focusing more specifically on vasculature, neural networks, the heart and liver. We conclude with current challenges and the technical perspective for further development of 3D organ bioprinting.

4D printing of polymeric materials for tissue and organ regeneration.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex, stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applications. Although the 4D concept was first highlighted in 2013, extensive research has rapidly developed, along with more-in-depth understanding and assertions regarding the definition of 4D. In this review, we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in the field. Both transformation-preprogrammed 4D printing and 4D printing of shape memory polymers are intensively surveyed. Afterwards we will explore and discuss the applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissues and implantable scaffolds, as well as future perspectives and conclusions.

3D Bioprinting a Cell-Laden Bone Matrix for Breast Cancer Metastasis Study.

Metastasis is one of the deadliest consequences of breast cancer, with bone being one of the primary sites of occurrence. Insufficient 3D biomimetic models currently exist to replicate this process in vitro. In this study, we developed a biomimetic bone matrix using 3D bioprinting technology to investigate the interaction between breast cancer (BrCa) cells and bone stromal cells (fetal osteoblasts and human bone marrow mesenchymal stem cells (MSCs)). A tabletop stereolithography 3D bioprinter was employed to fabricate a series of bone matrices consisting of osteoblasts or MSCs encapsulated in gelatin methacrylate (GelMA) hydrogel with nanocrystalline hydroxyapatite (nHA). When BrCa cells were introduced into the stromal cell-laden bioprinted matrices, we found that the growth of BrCa cells was enhanced by the presence of osteoblasts or MSCs, whereas the proliferation of the osteoblasts or MSCs was inhibited by the BrCa cells. The BrCa cells co-cultured with MSCs or osteoblasts presented increased vascular endothelial growth factor (VEGF) secretion in comparison to that of monocultured BrCa cells. Additionally, the alkaline phosphatase activity of MSCs or osteoblasts was reduced after BrCa cell co-culture. These results demonstrate that the 3D bioprinted matrix, with BrCa cells and bone stromal cells, provides a suitable model with which to study the interactive effects of cells in the context of an artificial bone microenvironment and thus may serve as a valuable tool for the investigation of postmetastatic breast cancer progression in bone.

3D printing of novel osteochondral scaffolds with graded microstructure.

Osteochondral tissue has a complex graded structure where biological, physiological, and mechanical properties vary significantly over the full thickness spanning from the subchondral bone region beneath the joint surface to the hyaline cartilage region at the joint surface. This presents a significant challenge for tissue-engineered structures addressing osteochondral defects. Fused deposition modeling (FDM) 3D bioprinters present a unique solution to this problem. The objective of this study is to use FDM-based 3D bioprinting and nanocrystalline hydroxyapatite for improved bone marrow human mesenchymal stem cell (hMSC) adhesion, growth, and osteochondral differentiation. FDM printing parameters can be tuned through computer aided design and computer numerical control software to manipulate scaffold geometries in ways that are beneficial to mechanical performance without hindering cellular behavior. Additionally, the ability to fine-tune 3D printed scaffolds increases further through our investment casting procedure which facilitates the inclusion of nanoparticles with biochemical factors to further elicit desired hMSC differentiation. For this study, FDM was used to print investment-casting molds innovatively designed with varied pore distribution over the full thickness of the scaffold. The mechanical and biological impacts of the varied pore distributions were compared and evaluated to determine the benefits of this physical manipulation. The results indicate that both mechanical properties and cell performance improve in the graded pore structures when compared to homogeneously distributed porous and non-porous structures. Differentiation results indicated successful osteogenic and chondrogenic manipulation in engineered scaffolds.

Hierarchical Fabrication of Engineered Vascularized Bone Biphasic Constructs via Dual 3D Bioprinting: Integrating Regional Bioactive Factors into Architectural Design.

A biphasic artificial vascularized bone construct with regional bioactive factors is presented using dual 3D bioprinting platform technique, thereby forming a large functional bone grafts with organized vascular networks. Biocompatible mussel-inspired chemistry and "thiol-ene" click reaction are used to regionally immobilize bioactive factors during construct fabrication for modulating or improving cellular events.

4D printing smart biomedical scaffolds with novel soybean oil epoxidized acrylate.

Photocurable, biocompatible liquid resins are highly desired for 3D stereolithography based bioprinting. Here we solidified a novel renewable soybean oil epoxidized acrylate, using a 3D laser printing technique, into smart and highly biocompatible scaffolds capable of supporting growth of multipotent human bone marrow mesenchymal stem cells (hMSCs). Porous scaffolds were readily fabricated by simply adjusting the printer infill density; superficial structures of the polymerized soybean oil epoxidized acrylate were significantly affected by laser frequency and printing speed. Shape memory tests confirmed that the scaffold fixed a temporary shape at -18 °C and fully recovered its original shape at human body temperature (37 °C), which indicated the great potential for 4D printing applications. Cytotoxicity analysis proved that the printed scaffolds had significant higher hMSC adhesion and proliferation than traditional polyethylene glycol diacrylate (PEGDA), and had no statistical difference from poly lactic acid (PLA) and polycaprolactone (PCL). This research is believed to significantly advance the development of biomedical scaffolds with renewable plant oils and advanced 3D fabrication techniques.

Human papillomavirus testing with conventional Pap smear screening in three inner London community clinics.

BACKGROUND AND METHODOLOGY: This observational study aimed to establish prevalence of high-risk human papillomaviruses (hrHPV) in women attending three inner London community clinics for routine screening and to pilot hrHPV testing in the triage of either borderline or negative cytology after previous abnormalities. Hybrid Capture 2 was carried out on brush samples taken alongside conventional smears from 1434 women aged 20-49 years. hrHPV positivity prompted earlier referral of women with previous abnormalities and either low-grade or negative cytology. Outcome at colposcopy was compared with the records of 1871 women aged 20-49 years attending colposcopy during the same period of time (routine colposcopies). RESULTS: hrHPV was detected in 111/161 (68.9%) women with abnormal cytology, 76/460 (16.5%) with negative cytology after previous abnormalities and 105/813 (12.9%) with negative cytology and no previous abnormalities. Overall, hrHPV was detected in 292/1434 (20.4%) women in the study (95% CI 18.3-22.5). hrHPV prevalence increased with severity of cytological abnormality (p<0.001) and decreased with age both with negative and low-grade cytology (p<0.001). High-grade cervical intraepithelial neoplasia (CIN) biopsies were found more frequently in women in the study groups with low-grade (p<0.001) or negative cytology than in routine colposcopies, but more women in the study groups attended colposcopy (8.2% compared with 4.1% routine colposcopies, p<0.001). CONCLUSIONS: hrHPV positivity increased detection of high-grade CIN in the study groups at the expense of more colposcopies. hrHPV negativity could reduce the need for investigation of low-grade cytology in women aged over 35 years and for surveillance after previous abnormalities.