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Intraoperative bioprinting
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Limitations of monolayer culture conditions have motivated scientists to explore new models that can recapitulate the architecture and function of human organs more accurately. Recent advances in the improvement of protocols have resulted in establishing three-dimensional (3D) organ-like architectures called 'organoids' that can display the characteristics of their corresponding real organs, including morphological features, functional activities, and personalized responses to specific pathogens. We discuss different organoid-based 3D models herein, which are classified based on their original germinal layer. Studies of organoids simulating the complexity of real tissues could provide novel platforms and opportunities for generating practical knowledge along with preclinical studies, including drug screening, toxicology, and molecular pathophysiology of diseases. This paper also outlines the key challenges, advantages, and prospects of current organoid systems.
Graphic abstract:
Heart diseases remain the top threat to human health, and the treatment of heart diseases changes with each passing day. Convincing evidence shows that three-dimensional (3D) printing allows for a more precise understanding of the complex anatomy associated with various heart diseases. In addition, 3D-printed models of cardiac diseases may serve as effective educational tools and for hands-on simulation of surgical interventions. We introduce examples of the clinical applications of different types of 3D printing based on specific cases and clinical application scenarios of 3D printing in treating heart diseases. We also discuss the limitations and clinically unmet needs of 3D printing in this context.
Drop-on-demand (DOD) bioprinting has been widely used in tissue engineering due to its high-throughput efficiency and cost effectiveness. However, this type of bioprinting involves challenges such as satellite generation, too-large droplet generation, and too-low droplet speed. These challenges reduce the stability and precision of DOD printing, disorder cell arrays, and hence generate further structural errors. In this paper, a multi-objective optimization (MOO) design method for DOD printing parameters through fully connected neural networks (FCNNs) is proposed in order to solve these challenges. The MOO problem comprises two objective functions: to develop the satellite formation model with FCNNs; and to decrease droplet diameter and increase droplet speed. A hybrid multi-subgradient descent bundle method with an adaptive learning rate algorithm (HMSGDBA), which combines the multi-subgradient descent bundle (MSGDB) method with Adam algorithm, is introduced in order to search for the Pareto-optimal set for the MOO problem. The superiority of HMSGDBA is demonstrated through comparative studies with the MSGDB method. The experimental results show that a single droplet can be printed stably and the droplet speed can be increased from 0.88 to 2.08 m·s⁻¹ after optimization with the proposed method. The proposed method can improve both printing precision and stability, and is useful in realizing precise cell arrays and complex biological functions. Furthermore, it can be used to obtain guidelines for the setup of cell-printing experimental platforms.
Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-Assisted Bioprinting (LAB) was used to pattern Red Fluorescent Protein (RFP)-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor (VEGF). LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the "random seeding" condition and both "disc" pattern (vr=+203.6%; br=+294.1%) and "crossed circle" pattern (vr=+355%; br=+602.1%). These results indicate that in vivo Laser-Assisted Bioprinting is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.
The early treatment and rapid closure of acute or chronic wounds is essential for normal healing and prevention of hypertrophic scarring. The use of split thickness autografts is often limited by the availability of a suitable area of healthy donor skin to harvest. Cellular and non-cellular biological skin-equivalents are commonly used as an alternative treatment option for these patients, however these treatments usually involve multiple surgical procedures and associated with high costs of production and repeated wound treatment. Here we describe a novel design and a proof-of-concept validation of a mobile skin bioprinting system that provides rapid on-site management of extensive wounds. Integrated imaging technology facilitated the precise delivery of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into an injured area, replicating the layered skin structure. Excisional wounds bioprinted with layered autologous dermal fibroblasts and epidermal keratinocytes in a hydrogel carrier showed rapid wound closure, reduced contraction and accelerated re-epithelialization. These regenerated tissues had a dermal structure and composition similar to healthy skin, with extensive collagen deposition arranged in large, organized fibers, extensive mature vascular formation and proliferating keratinocytes.
Biomimicry, adapting and implementing nature’s designs provides an adequate first-order solution to achieving superior mechanical properties. However, the design space is too vast even using biomimetic designs as prototypes for optimization. Here, we propose a new approach to design hierarchical materials using machine learning, trained with a database of hundreds of thousands of structures from finite element analysis, together with a self-learning algorithm for discovering high-performing materials where inferior designs are phased out for superior candidates. Results show that our approach can create microstructural patterns that lead to tougher and stronger materials, which are validated through additive manufacturing and testing. We further show that machine learning can be used as an alternative method of coarse-graining – analyzing and designing materials without the use of full microstructural data. This novel paradigm of smart additive manufacturing can aid in the discovery and fabrication of new material designs boasting orders of magnitude increase in computational efficacy over conventional methods.
Conventional 3D printing technologies typically rely on open‐loop, calibrate‐then‐print operation procedures. An alternative approach is adaptive 3D printing, which is a closed‐loop method that combines real‐time feedback control and direct ink writing of functional materials in order to fabricate devices on moving freeform surfaces. Here, it is demonstrated that the changes of states in the 3D printing workspace in terms of the geometries and motions of target surfaces can be perceived by an integrated robotic system aided by computer vision. A hybrid fabrication procedure combining 3D printing of electrical connects with automatic pick‐and‐placing of surface‐mounted electronic components yields functional electronic devices on a free‐moving human hand. Using this same approach, cell‐laden hydrogels are also printed on live mice, creating a model for future studies of wound‐healing diseases. This adaptive 3D printing method may lead to new forms of smart manufacturing technologies for directly printed wearable devices on the body and for advanced medical treatments.
Background:
The incidence of surgical site infection (SSI) across surgical procedures, specialties, and conditions is reported to vary from 0.1% to 50%. Operative duration is often cited as an independent and potentially modifiable risk factor for SSI. The objective of this systematic review was to provide an in-depth understanding of the relation between operating time and SSI.
Patients and methods:
This review included 81 prospective and retrospective studies. Along with study design, likelihood of SSI, mean operative times, time thresholds, effect measures, confidence intervals, and p values were extracted. Three meta-analyses were conducted, whereby odds ratios were pooled by hourly operative time thresholds, increments of increasing operative time, and surgical specialty.
Results:
Pooled analyses demonstrated that the association between extended operative time and SSI typically remained statistically significant, with close to twice the likelihood of SSI observed across various time thresholds. The likelihood of SSI increased with increasing time increments; for example, a 13%, 17%, and 37% increased likelihood for every 15 min, 30 min, and 60 min of surgery, respectively. On average, across various procedures, the mean operative time was approximately 30 min longer in patients with SSIs compared with those patients without.
Conclusions:
Prolonged operative time can increase the risk of SSI. Given the importance of SSIs on patient outcomes and health care economics, hospitals should focus efforts to reduce operative time.
Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called “Biopen”. To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10 seconds of exposure to 700 mW/cm² of 365 nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering.
Biofabrication as an interdisciplinary area is fostering new knowledge and integration of areas like nanotechnology, chemistry, biology, physics, materials science, control systems, among many others, necessary to accomplish the challenge of bioengineering functional complex tissues. The emergence of integrated platforms and systems biology to understand complex biological systems in multiscale levels will enable the prediction and creation of biofabricated biological structures. This systematic analysis (meta-analysis) or integrated platforms for estimating biological process have been named as BioCAE, which will become the key for important steps of the biofabrication processes. Biological Computational Aided Engineering (BioCAE) is a new computational approach to understanding and bioengineer complex tissues (biofabrication) using a combination of different methods as multiscale modelling, computer simulations, data mining and systems biology. In addition, multi-agent systems (MAS), which are composed of different interacting computing entities called agents, also provide an interesting way to
design and implement simulations of biological systems, integrating them with all steps of the BioCAE. MAS as a part of computational science has become a growing area to manipulate and solve complex problems. This paper presents an approach that will allow predicting the development and behavior of different biological processes such as molecular networks, gene interactions, cells, tissues and organs due to its flexibility, beyond to provide a new outlook in the biofabrication of tissues and organs.
Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ 3D printing is an exciting and innovative bio-fabrication technology that enables the surgeon to deliver tissue- engineering techniques at the time and location of need. We have created a hand- held 3D printing device (Biopen) that allows the simultaneous co-axial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single session surgery. This pilot study assesses the ability of the Biopen to repair a full thickness chondral defect and the early outcomes in cartilage regeneration, and compares these results to other treatments in a large animal model. A standardised critical-sized full thickness chondral defect was created in the weight-bearing surface of the lateral and medial condyles of both femurs of 6 sheep. Each defect was treated with one of the following treatments: (i) hand- held in situ 3D printed bioscaffold using the Biopen (HH group), (ii) pre- constructed bench-based printed bioscaffolds (BB group), (iii) micro-fractures (MF group) or (iv) untreated (Control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bio-printing was performed in all animals without any intra- or post- operative complication. The HH Biopen allowed early cartilage regeneration. Results of this study show that real-time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage. This article is protected by copyright. All rights reserved.
Statement of significance:
Present advances in tissue biofabrication have crucial role to play in aiding the pharmaceutical development process achieve its objectives. Advent of three-dimensional (3D) models, in particular, is viewed with immense interest by the community due to their ability to mimic in vivo hierarchical tissue architecture and heterogeneous composition. Successful realization of 3D models will not only provide greater in vitro-in vivo correlation compared to the two-dimensional (2D) models, but also eventually replace pre-clinical animal testing, which has their own shortcomings. Amongst all fabrication techniques, bioprinting- comprising all the different modalities (extrusion-, droplet- and laser-based bioprinting), is emerging as the most viable fabrication technique to create the biomimetic tissue constructs. Notwithstanding the interest in bioprinting by the pharmaceutical development researchers, it can be seen that there is a limited availability of comparative literature which can guide the proper selection of bioprinting processes and associated considerations, such as the bioink selection for a particular pharmaceutical study. Thus, this work emphasizes these aspects of bioprinting and presents them in perspective of differential requirements of different pharmaceutical studies like in vitro predictive toxicology, high-throughput screening, drug delivery and tissue-specific efficacies. Moreover, since bioprinting techniques are mostly applied in regenerative medicine and tissue engineering, a comparative analysis of similarities and differences are also expounded to help researchers make informed decisions based on contemporary literature.
Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.
In-situ printing is a promising injury repair technique that can be directly applied during surgical operations. This paper features a potential in-situ printing platform based on a small-scale robotic arm with a micro-sized dispenser valve. A double-light-source curing method was applied to print poly(ethylene glycol) diacrylate (PEGDA) with a 20% (weight/volume) ratio and the entire process was controlled automatically by a computer interface where droplet diameter, curing time, mechanical properties were measured and essential printing parameters (e.g., nozzle velocity, nozzle frequency) were determined. Three different two-dimensional (2D) plane models (namely, square, circular, and heart-shaped) were printed during initial printing trials. The feasibility study of in-situ printing on curved surfaces was tested using a three-dimensional (3D) printed defect model. The defect was successfully filled using both parallel and ring printing paths. In conclusion, the robotic arm printing platform and its forming method can achieve a rapid curing of PEGDA hydrogel on a curved surface and has the potential to be applied to in-situ printing.
Bioprinting is an emerging technology that allows the assembling of both living and non-living biological materials into an ideal complex layout for further tissue maturation. Bioprinting aims to produce engineered tissue or organ in a mechanized, organized, and optimized manner. Various biomaterials and techniques have been utilized to bioprint biological constructs in different shapes, sizes and resolutions. There is a need to systematically discuss and analyze the reported strategies employed to fabricate these constructs. We identified and discussed important design factors in bioprinting, namely shape and resolution, material heterogeneity, and cellular-material remodeling dynamism. Each design factors are represented by the corresponding process capabilities and printing parameters. The process-design map will inspire future biomaterials research in these aspects. Design considerations such as data processing, bio-ink formulation and process selection are discussed. Various printing and crosslinking strategies, with relevant applications, are also systematically reviewed. We categorized them into 5 general bioprinting strategies, including direct bioprinting, in-process crosslinking, post-process crosslinking, indirect bioprinting and hybrid bioprinting. The opportunities and outlook in 3D bioprinting are highlighted. This review article will serve as a framework to advance computer-aided design in bioprinting technologies.
The skin is the largest organ of the body, having a complex multi-layered structure and guards the underlying muscles, bones, ligaments, and internal organs. It serves as the first line of defence to any external stimuli, hence it is the most vulnerable to injury and warrants the need for rapid and reliable regeneration methods. Tissue engineered skin substitutes help overcome the limitations of traditional skin treatment methods, in terms of technology, time, and cost. While there is commendable progress in the treating of superficial wounds and injuries with skin substitutes, treatment of full-thickness injuries, especially with third or fourth degree burns, still looks murkier. Engineering multi-layer skin architecture, conforming to the native skin structure is a tougher goal to achieve with the current tissue engineering methods, if not impossible, restoring all the functions of the native skin. The testing of drugs and cosmetics is another area, where engineered skins are very much needed, with bans being imposed on product testing on animals. Given this greater need, 3D bioprinting is a promising technology that can achieve rapid and reliable production of biomimetic cellular skin substitutes, satisfying both clinical and industrial needs. This paper reviews all aspects related to the 3D bioprinting of skin, right from imaging the injury site, 3D model creation, biomaterials that are used and their suitability, types of cells and their functions, actual bioprinting technologies, along with the challenges and future prospects.
We present a new approach which aims to translate freeform biofabrication into the surgical field, while staying true to the practical constraints of the operating theatre. Herein we describe the development of a handheld biofabrication tool, dubbed the ‘biopen’, which enables the deposition of living cells and biomaterials in a manual, direct-write fashion. A gelatin–methacrylamide/hyaluronic acid–methacrylate (GelMa/HAMa) hydrogel was printed and UV crosslinked during the deposition process to generate surgically sculpted 3D structures. Custom titanium nozzles were fabricated to allow printing of multiple ink formulations in a collinear (side-by-side) geometry. Independently applied extrusion pressure for both chambers allows for geometric control of the printed structure and for the creation of compositional gradients. In vitro experiments demonstrated that human adipose stem cells maintain high viability (>97%) one week after biopen printing in GelMa/HAMa hydrogels. The biopen described in this study paves the way for the use of 3D bioprinting during the surgical process. The ability to directly control the deposition of regenerative scaffolds with or without the presence of live cells during the surgical process presents an exciting advance not only in the fields of cartilage and bone regeneration but also in other fields where tissue regeneration and replacement are critical.
Bioprinting is a rapidly developing technique for biofabrication. Because of its high resolution and the ability to print living cells, bioprinting has been widely used in artificial tissue and organ generation as well as microscale living cell deposition. In this paper, we present a low-cost stereolithography-based bioprinting system that uses visible light crosslinkable bioinks. This low-cost stereolithography system was built around a commercial projector with a simple water filter to prevent harmful infrared radiation from the projector. The visible light crosslinking was achieved by using a mixture of polyethylene glycol diacrylate (PEGDA) and gelatin methacrylate (GelMA) hydrogel with eosin Y based photoinitiator. Three different concentrations of hydrogel mixtures (10% PEG, 5% PEG + 5% GelMA, and 2.5% PEG + 7.5% GelMA, all w/v) were studied with the presented systems. The mechanical properties and microstructure of the developed bioink were measured and discussed in detail. Several cell-free hydrogel patterns were generated to demonstrate the resolution of the solution. Experimental results with NIH 3T3 fibroblast cells show that this system can produce a highly vertical 3D structure with 50 μm resolution and 85% cell viability for at least five days. The developed system provides a low-cost visible light stereolithography solution and has the potential to be widely used in tissue engineering and bioengineering for microscale cell patterning.
Arterio-venous anastomoses (AVAs) are direct connections between small arteries and small veins. In humans they are numerous in the glabrous skin of the hands and feet. The AVAs are short vessel segments with a large inner diameter and a very thick muscular wall. They are densely innervated by adrenergic axons. When they are open, they provide a low-resistance connection between arteries and veins, shunting blood directly into the venous plexuses of the limbs.The AVAs play an important role in temperature regulation in humans in their thermoneutral zone, which for a naked resting human is about 26°C to 36°C, but lower when active and clothed. From the temperature control center in the hypothalamus, bursts of nerve impulses are sent simultaneously to all AVAs. The AVAs are all closed near the lower end and all open near the upper end of the thermoneutral zone. The small veins in the skin of the arms and legs are also contracted near the lower end of the thermoneutral zone and relax to a wider cross section as the ambient temperature rises. At the cold end of the thermoneutral range, the blood returns to the heart through the deep veins and cools the arterial blood through a countercurrent mechanism. As the ambient temperature rises, more blood is returned through the superficial venous plexuses and veins and heats the skin surface of the full length of the four limbs. This skin surface is responsible for a large part of the loss of heat from the body towards the upper end of the thermoneutral zone.
Bioprinting is one of several newly emerged tissue engineering strategies that hold great promise in alleviating of organ shortage crisis. To date, a range of living biological constructs have already been fabricated in vitro using this technology. However, an in vitro approach may have several intrinsic limitations regarding its clinical applicability in some cases. A possible solution is in vivo bioprinting, in which the de novo tissues/organs are to be directly fabricated and positioned at the damaged site in the living body. This strategy would be particularly effective in the treatment of tissues/organs that can be safely arrested and immobilized during bioprinting. Proof-of-concept studies on in vivo bioprinting have been reported recently, on the basis of which this paper reviews the current state-of-the-art bioprinting technologies with a particular focus on their advantages and challenges for the in vivo application.
Provision of supplemental oxygen to maintain soft tissue viability acutely following trauma in which vascularization has been compromised would be beneficial for limb and tissue salvage. For this application, an oxygen generating biomaterial that may be injected directly into the soft tissue could provide an unprecedented treatment in the acute trauma setting. The purpose of the current investigation was to determine if sodium percarbonate (SPO), an oxygen generating biomaterial, is capable of maintaining resting skeletal muscle homeostasis under otherwise hypoxic conditions. In the current studies, a biologically and physiologically compatible range of SPO (1-2 mg/mL) was shown to: 1) improve the maintenance of contractility and attenuate the accumulation of HIF1α, depletion of intramuscular glycogen, and oxidative stress (lipid peroxidation) that occurred following ∼30 minutes of hypoxia in primarily resting (duty cycle = 0.2 s train/120 s contraction interval <0.002) rat extensor digitorum longus (EDL) muscles in vitro (95% N2-5% CO2, 37°C); 2) attenuate elevations of rat EDL muscle resting tension that occurred during contractile fatigue testing (3 bouts of 25 100 Hz tetanic contractions; duty cycle = 0.2 s/2 s = 0.1) under oxygenated conditions in vitro (95% O2-5% CO2, 37°C); and 3) improve the maintenance of contractility (in vivo) and prevent glycogen depletion in rat tibialis anterior (TA) muscle in a hindlimb ischemia model (i.e., ligation of the iliac artery). Additionally, injection of a commercially available lipid oxygen-carrying compound or the components (sodium bicarbonate and hydrogen peroxide) of 1 mg/mL SPO did not improve EDL muscle contractility under hypoxic conditions in vitro. Collectively, these findings demonstrate that a biological and physiological concentration of SPO (1-2 mg/mL) injected directly into rat skeletal muscle (EDL or TA muscles) can partially preserve resting skeletal muscle homeostasis under hypoxic conditions.
Stem cells obtained from amniotic fluid show high proliferative capacity in culture and multilineage differentiation potential. Because of the lack of significant immunogenicity and the ability of the amniotic fluid-derived stem (AFS) cells to modulate the inflammatory response, we investigated whether they could augment wound healing in a mouse model of skin regeneration. We used bioprinting technology to treat full-thickness skin wounds in nu/nu mice. AFS cells and bone marrow-derived mesenchymal stem cells (MSCs) were resuspended in fibrin-collagen gel and "printed" over the wound site. At days 0, 7, and 14, AFS cell- and MSC-driven wound closure and re-epithelialization were significantly greater than closure and re-epithelialization in wounds treated by fibrin-collagen gel only. Histological examination showed increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels. However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue. These observations suggest that the increased wound closure rates and angiogenesis may be due to delivery of secreted trophic factors, rather than direct cell-cell interactions. Accordingly, we performed proteomic analysis, which showed that AFS cells secreted a number of growth factors at concentrations higher than those of MSCs. In parallel, we showed that AFS cell-conditioned media induced endothelial cell migration in vitro. Taken together, our results indicate that bioprinting AFS cells could be an effective treatment for large-scale wounds and burns.
We have developed an injectable foam suspension containing self-assembling, lipid-based microparticles encapsulating a core of pure oxygen gas for intravenous injection. Prototype suspensions were manufactured to contain between 50 and 90 ml of oxygen gas per deciliter of suspension. Particle size was polydisperse, with a mean particle diameter between 2 and 4 μm. When mixed with human blood ex vivo, oxygen transfer from 70 volume % microparticles was complete within 4 s. When the microparticles were infused by intravenous injection into hypoxemic rabbits, arterial saturations increased within seconds to near-normal levels; this was followed by a decrease in oxygen tensions after stopping the infusions. The particles were also infused into rabbits undergoing 15 min of complete tracheal occlusion. Oxygen microparticles significantly decreased the degree of hypoxemia in these rabbits, and the incidence of cardiac arrest and organ injury was reduced compared to controls. The ability to administer oxygen and other gases directly to the bloodstream may represent a technique for short-term rescue of profoundly hypoxemic patients, to selectively augment oxygen delivery to at-risk organs, or for novel diagnostic techniques. Furthermore, the ability to titrate gas infusions rapidly may minimize oxygen-related toxicity.
A major hindrance in engineering tissues containing highly metabolically active cells is the insufficient oxygenation of these implants, which results in dying or dysfunctional cells in portions of the graft. The development of methods to increase oxygen availability within tissue-engineered implants, particularly during the early engraftment period, would serve to allay hypoxia-induced cell death. Herein, we designed and developed a hydrolytically activated oxygen-generating biomaterial in the form of polydimethylsiloxane (PDMS)-encapsulated solid calcium peroxide, PDMS-CaO(2). Encapsulation of solid peroxide within hydrophobic PDMS resulted in sustained oxygen generation, whereby a single disk generated oxygen for more than 6 wk at an average rate of 0.026 mM per day. The ability of this oxygen-generating material to support cell survival was evaluated using a β cell line and pancreatic rat islets. The presence of a single PDMS-CaO(2) disk eliminated hypoxia-induced cell dysfunction and death for both cell types, resulting in metabolic function and glucose-dependent insulin secretion comparable to that in normoxic controls. A single PDMS-CaO(2) disk also sustained enhanced β cell proliferation for more than 3 wk under hypoxic culture conditions. Incorporation of these materials within 3D constructs illustrated the benefits of these materials to prevent the development of detrimental oxygen gradients within large implants. Mathematical simulations permitted accurate prediction of oxygen gradients within 3D constructs and highlighted conditions under which supplementation of oxygen tension would serve to benefit cellular viability. Given the generality of this platform, the translation of these materials to other cell-based implants, as well as ischemic tissues in general, is envisioned.
Bioprinting technologies have been advancing at the convergence of automation, digitalization, and new tissue engineering (TE) approaches. In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when de novo tissues are to be printed directly on the intended anatomical location in the living body. To date, few attempts have been made to fabricate in situ tissues, which can be safely arrested and immobilized while printing in preclinical living models. In this review, we have explained the need and utility for in situ bioprinting with regard to the conventional bioprinting approach. The two main in situ bioprinting approaches, namely, robotic arm and handheld approaches, have been defined and differentiated. The various studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. Finally, we have also discussed the advantages, challenges, and the prospects in the field of in situ bioprinting modalities in line with parallel technological advancements. STATEMENT OF SIGNIFICANCE: In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when tissues are to be fabricated or repaired directly on the intended anatomical location in the living body, using the body as a bioreactor. However, the technology requires a lot more improvement to fabricate complex tissues in situ, which could eventually be possible through the multi-disciplinary innovations in tissue engineering. This review explains the need and utility and current approaches by handheld and robotic modes for in situ bioprinting. The latest studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. The review also covers the background studies, advantages, technical and ethical challenges, and possible suggestions for future improvements.
Additive manufacturing of soft materials requires optimization of printable inks, formulations of these feedstocks, and complex printing processes that must balance a large number of disparate but highly correlated variables. Here, hierarchical machine learning (HML) is applied to 3D printing of silicone elastomer via freeform reversible embedding (FRE), which is challenging because it involves depositing a Newtonian prepolymer liquid phase within a Bingham plastic support bath. The advantage of the HML algorithm is that it can predict the behavior of complex physical systems using sparse data sets through integration of physical modeling in a framework of statistical learning. Here, it is shown that this algorithm can be used to simultaneously optimize material, formulation, and processing variables. The FRE method for 3D printing silicone parts was optimized based on a training set with 38 trial runs. Compared with the previous results from iterative optimization approaches using design-of-experiment and steepest-ascent methods, HML increased printing speed by up to 2.5 × while retaining print fidelity and also identified a unique silicone formulation and printing parameters that had not been found previously through trial-and-error approaches. These results indicate that HML is an effective tool with the potential for broad application for planning and optimizing in additive manufacturing of soft materials via the FRE method.
Three-dimensional (3D) bioprinting is an emerging biofabrication technology, driving many innovations and opening new avenues in regenerative therapeutics. The aim of 3D bioprinting is to fabricate grafts in vitro, which can then be implanted in vivo. However, the tissue culture ex vivo carries safety risks and thereby complicated manufacturing equipment and practice are required for tissues to be implanted in the humans. The implantation of printed tissues also adds complexities due to the difficulty in maintaining the structural integrity of fabricated constructs. To tackle this challenge, the concept of in situ 3D bioprinting has been suggested in which tissues are directly printed at the site of injury or defect. Such approach could be combined with cells freshly isolated from patients to produce custom-made grafts that resemble target tissue and fit precisely to target defects. Moreover, the natural cellular microenvironment in the body can be harnessed for tissue maturation resulting in the tissue regeneration and repair. Here, we discuss literature reports on in situ 3D printing and we describe future directions and challenges for in situ 3D bioprinting. We expect that this novel technology would find great attention in different biomedical fields in near future.
Three‐dimensional (3D) bioprinting of cell‐laden biomaterials is used to fabricate constructs that can mimic the structure of native tissues. The main techniques used for 3D bioprinting include microextrusion, inkjet, and laser‐assisted bioprinting. Bioinks used for bone bioprinting include hydrogels loaded with bioactive ceramics, cells, and growth factors. In this review, a critical overview of the recent literature on various types of bioinks used for bone bioprinting is presented. Major challenges, such as the vascularity, clinically relevant size, and mechanical properties of 3D printed structures, that need to be addressed to successfully use the technology in clinical settings, are discussed. Emerging approaches to solve these problems are reviewed, and future strategies to design customized 3D printed structures are proposed.
Connective tissues within the synovial joints are characterized by their dense extracellular matrix and sparse cellularity. With injury or disease, however, tissues commonly experience an influx of cells owing to proliferation and migration of endogenous mesenchymal cell populations, as well as invasion of the tissue by other cell types, including immune cells. Although this process is critical for successful wound healing, aberrant immune-mediated cell infiltration can lead to pathological inflammation of the joint. Importantly, cells of mesenchymal or haematopoietic origin use distinct modes of migration and thus might respond differently to similar biological cues and microenvironments. Furthermore, cell migration in the physiological microenvironment of musculoskeletal tissues differs considerably from migration in vitro. This Review addresses the complexities of cell migration in fibrous connective tissues from three separate but interdependent perspectives: physiology (including the cellular and extracellular factors affecting 3D cell migration), pathophysiology (cell migration in the context of synovial joint autoimmune disease and injury) and tissue engineering (cell migration in engineered biomaterials). Improved understanding of the fundamental mechanisms governing interstitial cell migration might lead to interventions that stop invasion processes that culminate in deleterious outcomes and/or that expedite migration to direct endogenous cell-mediated repair and regeneration of joint tissues.
Bioprinted skin tissue has the potential for aiding drug screening, formulation development, clinical transplantation, chemical and cosmetic testing, as well as basic research. Limitations of conventional skin tissue engineering approaches have driven the development of biomimetic skin equivalent via 3D bioprinting. A key hope for bioprinting skin is the improved tissue authenticity over conventional skin equivalent construction, enabling the precise localization of multiple cell types and appendages within a construct. The printing of skin faces challenges broadly associated with general 3D bioprinting, including the selection of cell types and biomaterials, and additionally requires in vitro culture formats that allow for growth at an air-liquid interface. This paper provides a thorough review of current 3D bioprinting technologies used to engineer human skin constructs and presents the overall pipelines of designing a biomimetic artificial skin via 3D bioprinting from the design phase (i.e. pre-processing phase) through the tissue maturation phase (i.e. post-processing) and into final product evaluation for drug screening, development, and drug delivery applications.
We present the first cell attachable and visible light crosslinkable hydrogels based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography 3D bioprinting. In order to develop visible a light crosslinkable hydrogel, we systematically studied five combinations of the GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentration on the characteristics of the hydrogel. Experimental results show that the compressive Young's Modulus and pore size are positively affected by the concentration of EY, while the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps to improve the compressive Young's Modulus and cell attachment. We further employed the developed visible light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.
3D bioprinting is a pioneering technology that enables fabrication of biomimetic, multiscale, multi-cellular tissues with highly complex tissue microenvironment, intricate cytoarchitecture, structure-function hierarchy, and tissue-specific compositional and mechanical heterogeneity. Given the huge demand for organ transplantation, coupled with limited organ donors, bioprinting is a potential technology that could solve this crisis of organ shortage by fabrication of fully-functional whole organs. Though organ bioprinting is a far-fetched goal, there has been a considerable and commendable progress in the field of bioprinting that could be used as transplantable tissues in regenerative medicine. This paper presents a first-time review of 3D bioprinting in regenerative medicine, where the current status and contemporary issues of 3D bioprinting pertaining to the eleven organ systems of the human body including skeletal, muscular, nervous, lymphatic, endocrine, reproductive, integumentary, respiratory, digestive, urinary, and circulatory systems were critically reviewed. The implications of 3D bioprinting in drug discovery, development, and delivery systems are also briefly discussed, in terms of in vitro drug testing models, and personalized medicine. While there is a substantial progress in the field of bioprinting in the recent past, there is still a long way to go to fully realize the translational potential of this technology. Computational studies for study of tissue growth or tissue fusion post-printing, improving the scalability of this technology to fabricate human-scale tissues, development of hybrid systems with integration of different bioprinting modalities, formulation of new bioinks with tuneable mechanical and rheological properties, mechanobiological studies on cell-bioink interaction, 4D bioprinting with smart (stimuli-responsive) hydrogels, and addressing the ethical, social, and regulatory issues concerning bioprinting are potential futuristic focus areas that would aid in successful clinical translation of this technology.
We present a handheld skin printer that enables the in-situ formation of biomaterial and skin tissue sheets of different homogeneous and architected compositions. When manually positioned above a target surface, the compact instrument (weight <0.8kg) conformally deposits a biomaterial or tissue sheet from a microfluidic cartridge. Consistent sheet formation is achieved by coordinating the flow rates at which bioink and cross-linker solution are delivered, with the speed at which a pair of rollers actively translate the cartridge along the surface. We demonstrate compatibility with dermal and epidermal cells embedded in ionically cross-linkable biomaterials (e.g., alginate), enzymatically cross-linkable proteins (e.g., fibrin), as well as their mixtures with collagen type I and hyaluronic acid. Upon rapid crosslinking, biomaterial and skin cell-laden sheets of consistent thickness, width and composition were obtained. Sheets deposited onto horizontal, agarose-coated surfaces were used for physical and in-vitro characterization. Proof-of-principle demonstrations for the in-situ formation of biomaterial sheets in murine and porcine excisional wound models illustrate the capacity of depositing onto inclined and compliant wound surfaces that are subject to respiratory motion. We expect the presented work will enable the in-situ delivery of a wide range of different cells, biomaterials, and tissue adhesives, as well as the in-situ fabrication of spatially organized biomaterials, tissues, and biohybrid structures.
The purpose of 3D bioprinting technology is to design and create functional 3D tissues or organs in situ for in vivo applications. 3D cell-printing, or additive biomanufacturing, allows the selection of biomaterials and cells (bioink), and the fabrication of cell-laden structures in high resolution. 3D cell-printed structures have also been used for applications such as research models, drug delivery and discovery, and toxicology. Recently, numerous attempts have been made to fabricate tissues and organs by using various 3D printing techniques. However, challenges such as vascularization are yet to be solved. This article reviews the most commonly used 3D cell-printing techniques with their advantages and drawbacks. Furthermore, up-to-date achievements of 3D bioprinting in in vivo applications are introduced, and prospects for the future of 3D cell-printing technology are discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.
Statement of significance:
Biofabrication of living tissues and organs at the clinically-relevant volumes vitally depends on the integration of vascular network. Despite the great progress in traditional biofabrication approaches, building perfusable hierarchal vascular network is a major challenge. Bioprinting is an emerging technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision, which holds a great promise in fabrication of vascular or vascularized tissues for transplantation use. Although a great progress has recently been made on building perfusable tissues and branched vascular network, a comprehensive review on the state-of-the-art in vascular and vascularized tissue bioprinting has not reported so far. This contribution is thus significant because it discusses the use of three major bioprinting modalities in vascular tissue biofabrication for the first time in the literature and compares their strengths and limitations in details. Moreover, the use of scaffold-based and scaffold-free bioprinting is expounded within the domain of vascular tissue fabrication.
An in situ crosslinking strategy is used for 3D bioprinting of nonviscous photo-crosslinkable hydrogels. This method can be generalized to various photo-crosslinkable formulations, maintaining high embedded cell viability and tunable cell behavior. Heterogeneous and hollow filaments can be printed using this strategy, allowing for fabrication of complex engineered cell-laden constructs.
In order to bioprint living tissue and organ constructs, patient-specific anatomical models need to be acquired; however, these models mainly provide external surface information only. The internal architecture of tissue constructs plays a crucial role as it provides a porous environment for media exchange, vascularization, tissue growth and engraftment. This review presents design requirements for bioprinting and discusses currently available medical imaging techniques used in acquisition of anatomical models including magnetic resonance imaging (MRI) and computed tomography (CT), and compares their strengths and limitations. Then, consideration for design architecture is discussed and various approaches in blueprint modeling of tissue constructs are presented for creation of porous architectures. Next, existing toolpath planning approaches for bioprinting of tissues and organs are presented. Design limitations for bioprinting are discussed and future perspectives are provided to the reader.
Oxygen inhibition is a phenomenon that directly impacts the print fidelity of 3D biofabricated and photo-polymerised hydrogel constructs. It typically results in the undesirable physical collapse of fabricated constructs due to impaired crosslinking, and is an issue that generally remains unreported in the literature. In this study, we describe a systematic approach to minimising oxygen inhibition in photo-polymerised gelatine-methacryloyl (Gel-MA) based hydrogel constructs, by comparing a new visible light initiating system, Vis + ruthenium (Ru)/sodium persulfate (SPS) to more conventionally adopted ultraviolet (UV) + Irgacure® 2959 system. For both systems, increasing photo-initiator concentration and light irradiation intensity successfully reduced oxygen inhibition. However, the UV + I2959 system was detrimental to cells at both high I2959 concentrations and UV light irradiation intensities. The Vis + Ru/SPS system yielded better cell cyto-compatibility, where encapsulated cells remained >85% viable even at high Ru/SPS concentrations and visible light irradiation intensities for up to 21 days, further highlighting the potential of this system to biofabricate cell-laden constructs with high shape fidelity, cell viability and metabolic activity.
For many cellular therapies being evaluated in preclinical and clinical trials, the mechanisms behind their therapeutic effects appear to be the secretion of growth factors and cytokines, also known as paracrine activity. Often, delivered cells are transient, and half-lives of the growth factors that they secrete are short, limiting their long-term effectiveness. The goal of this study was to optimize a hydrogel system capable of in situ cell delivery that could sequester and release growth factors secreted from those cells after the cells were no longer present. Here, we demonstrate the use of a fast photocross-linkable heparin-conjugated hyaluronic acid (HA-HP) hydrogel as a cell delivery vehicle for sustained growth factor release, which extends paracrine activity. The hydrogel could be modulated through cross-linking geometries and heparinization to support sustained release proteins and heparin-binding growth factors. To test the hydrogel in vivo, we used it to deliver amniotic fluid-derived stem (AFS) cells, which are known to secrete cytokines and growth factors, in full thickness skin wounds in a nu/nu murine model. Despite transience of the AFS cells in vivo, the HA-HP hydrogel with AFS cells improved wound closure and reepithelialization and increased vascularization and production of extracellular matrix in vivo. These results suggest that HA-HP hydrogel has the potential to prolong the paracrine activity of cells, thereby increasing their therapeutic effectiveness in wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.
Droplet-based bioprinting (DBB) offers greater advantages due to its simplicity and agility with precise control on deposition of biologics including cells, growth factors, genes, drugs and biomaterials, and has been a prominent technology in the bioprinting community. Due to its immense versatility, DBB technology has been adopted by various application areas, including but not limited to, tissue engineering and regenerative medicine, transplantation and clinics, pharmaceutics and high-throughput screening, and cancer research. Despite the great benefits, the technology currently faces several challenges such as a narrow range of available bioink materials, bioprinting-induced cell damage at substantial levels, limited mechanical and structural integrity of bioprinted constructs, and restrictions on the size of constructs due to lack of vascularization and porosity. This paper presents a first-time review of DBB and comprehensively covers the existing DBB modalities including inkjet, electrohydrodynamic, acoustic, and micro-valve bioprinting. The recent notable studies are highlighted, the relevant bioink biomaterials and bioprinters are expounded, the application areas are presented, and the future prospects are provided to the reader.
Extrusion-based bioprinting (EBB) is a rapidly growing technology that has made substantial progress during the last decade. It has great versatility in printing various biologics, including cells, tissues, tissue constructs, organ modules and microfluidic devices, in applications from basic research and pharmaceutics to clinics. Despite the great benefits and flexibility in printing a wide range of bioinks, including tissue spheroids, tissue strands, cell pellets, decellularized matrix components, micro-carriers and cell-laden hydrogels, the technology currently faces several limitations and challenges. These include impediments to organ fabrication, the limited resolution of printed features, the need for advanced bioprinting solutions to transition the technology bench to bedside, the necessity of new bioink development for rapid, safe and sustainable delivery of cells in a biomimetically organized microenvironment, and regulatory concerns to transform the technology into a product. This paper, presenting a first-time comprehensive review of EBB, discusses the current advancements in EBB technology and highlights future directions to transform the technology to generate viable end products for tissue engineering and regenerative medicine.
A 3D printable and highly stretchable tough hydrogel is developed by combining poly(ethylene glycol) and sodium alginate, which synergize to form a hydrogel tougher than natural cartilage. Encapsulated cells maintain high viability over a 7 d culture period and are highly deformed together with the hydrogel. By adding biocompatible nanoclay, the tough hydrogel is 3D printed in various shapes without requiring support material.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Bioprinting is an emerging field that is having a revolutionary impact on the medical sciences. It offers great precision for the spatial placement of cells, proteins, genes, drugs, and biologically active particles to better guide tissue generation and formation. This emerging biotechnology appears to be promising for advancing tissue engineering toward functional tissue and organ fabrication for transplantation, drug testing, research investigations, and cancer or disease modeling, and has recently attracted growing interest worldwide among researchers and the general public. In this Opinion, I highlight possibilities for the bioprinting scale-up of functional tissue and organ constructs for transplantation and provide the reader with alternative approaches, their limitations, and promising directions for new research prospects.
Copyright © 2015 Elsevier Ltd. All rights reserved.
ImportanceThere is an ever-increasing drive to improve surgical patient outcomes. Given the benefits which robotics has bestowed upon a wide range of industries, from vehicle manufacturing to space exploration, robots have been highlighted by many as essential for continued improvements in surgery.Objective
The goal of this review is to outline the history of robotic surgery, and detail the key studies which have investigated its effects on surgical outcomes. Issues of cost-effectiveness and patient acceptability will also be discussed.Results and conclusionRobotic surgery has been shown to shorten hospital stays, decrease complication rates and allow surgeons to perform finer tasks, when compared to the traditional laparoscopic and open approaches. These benefits, however, must be balanced against increased intraoperative times, vast financial costs and the increased training burden associated with robotic techniques. The outcome of such a cost-benefit analysis appears to vary depending on the procedure being conducted; indeed the strongest evidence in favour of its use comes from the fields of urology and gynaecology. It is hoped that with the large-scale, randomised, prospective clinical trials underway, and an ever-expanding research base, many of the outstanding questions surrounding robotic surgery will be answered in the near future.
Significance
Effective restoration of large soft tissue defects requires the use of tissue flaps, with viability that is largely determined by degree of vascularization. In view of the tedious transfer procedures and donor site morbidity associated with autologous flaps, this work set out to design and evaluate an engineered muscle flap featuring a robust vascular port formed from preseeded endothelial cells and host vasculature. The implanted flap was highly vascularized, well-perfused, and anastomosed with host vessels. Engineered flaps of this nature promise to circumvent the need to harvest and transfer massive tissue volumes, while avoiding the consequential complications.
Laser cooling of solids, sometimes also known as optical refrigeration, is a fast developing area of optical science, investigating the interaction of light with condensed matter. Apart from being of fundamental scientific interest, this topic addresses a very important practical issue: design and construction of laser pumped solid-state cryocoolers, which are compact, free from mechanical vibrations, moving parts, fluids and can cause only low electromagnetic interference in the cooled area. The optical cryocooler has a broad area of applications such as in the development of magnetometers for geophysical sensors, in biomedical sensing and can be beneficial for satellite instrumentations and small sensors, where compactness and the lack of vibrations are very important. Simply, a laser cooler works on the conversion of low energy pump photons into high-energy anti-Stokes fluorescence photons by extracting some of the phonons (heat energy) in a material. That is, the process of laser cooling of solids is based on anti-Stokes fluorescence also known as luminescence upconversion, when light quanta in the red tail of the absorption spectrum are absorbed from a pump laser, and blue-shifted photons are spontaneously emitted. The extra energy extracted from the solid-state lattice in the form of the phonons is the quanta of vibrational energy which generates heat. The idea to cool solids with anti-Stokes fluorescence was proposed in 1929 by Peter Pringsheim and first demonstrated experimentally by Epstein's research team in 1995. In 1999, Steven Bowman proposed to use the optical refrigeration by anti-Stokes fluorescence within the laser medium to balance the heat generated by the Stokes shifted stimulated emission in a high-power solid-state bulk laser. Such a laser without internal heating named radiation-balanced or athermal laser was experimentally demonstrated for the first time in 2002. At the present time laser cooling of solids can be largely divided into three main areas: laser cooling of rare-earth doped solids, laser cooling in semiconductors and radiation-balanced lasers. All three areas are very interesting and important and will be considered in this paper.
Articular cartilage was predicted to be one of the first tissues to successfully be regenerated, but this proved incorrect. In contrast, bone (but also vasculature and cardiac tissues) has seen numerous successful reparative approaches, despite consisting of multiple cell and tissue types and, thus, possessing more complex design requirements. Here, we use bone-regeneration successes to highlight cartilage-regeneration challenges: such as selecting appropriate cell sources and scaffolds, creating biomechanically suitable tissues, and integrating to native tissue. We also discuss technologies that can address the hurdles of engineering a tissue possessing mechanical properties that are unmatched in human-made materials and functioning in environments unfavorable to neotissue growth.