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Thomas Lewis
Thomas Lewis

Building Coronary Heart Disease Layer By Layer Using Superior 3D Bioprinting Strategies



In the past few decades, 3D bioprinting has garnered extensive attention[15-17]. 3D bioprinting possesses superior flexibility and controllability on the spatial arrangement of biomaterials and cells, which has been expansively applied to tumor-related studies including TME mimicking, tumor angiogenesis, tumor metastasis, and antitumor drug screening using individual cells and miscellaneous biomaterials[18-21]. Nevertheless, individually dispersed cells within the hydrogel matrix are insufficient in faithfully recapitulating specific disease states either indicating fibrosis or tumor propagation[22]. In contrast, spheroids could be a perfect alternative and implementable approach. Despite the high potential in building tissue constructs by combining 3D bioprinting and spheroidal assembly, 3D printing or positioning spheroids with high precision remains challenging.




Building coronary heart disease layer by layer using superior 3D bioprinting strategies


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Schematic illustration of using spheroids as building blocks in 3D bioprinting for healthy/disease tissue construction. (A) Overview of spheroid formation techniques. (B) 3D Printing and its adaptions in assisting spheroid assembly.


Using spheroids as building blocks in facile macro-sized tissue construction has been proposed[163], the most straightforward method in assembling these spheroids is spontaneous fusion[164-167]. Fleming et al. had reported a spheroidal fusion between unilluminated vascular spheroids through liquid-like coalescence. Specifically, 2 juxtaposed spheroids were fused when positioned close enough, forming a larger spheroid with preserved composite structural characteristics[168]. These features have legitimized a notion of suitability for using spheroids as building modules in engineering large-scale tissues for tissue regeneration and/or disease modeling. Early studies have also demonstrated the feasibility of assembling spheroids through directed fusion, achieving microtissues with prescribed patterns including honeycomb, rods, and tori[169,170]. Briefly, spheroids are manually placed in a pre-designed mold/template containing the prescribed topography. These spheroids will then gradually fuse together and achieve the specified pattern over time. This manual processing has shed light on a great possibility of generating tissue constructs by spheroidal modular assembly, despite its limited control and low throughput. For overcoming such challenge, several bioengineering strategies have been exploited for spheroid assembly, potentiating its large-scale functional tissue manufacturing with improvements in accuracy through a high-throughput manner.


Abstract: Heart failure (HF) is the terminal state of cardiovascular disease (CVD), leading numerous patients to death every year. Cardiac tissue engineering is a multidisciplinary field of creating functional cardiac patches in vitro to promote cardiac function after transplantation onto damaged zone, giving the hope for patients with end-stage HF. However, the limited thickness of cardiac patches results in the graft failure of survival and function due to insufficient blood supply. To date, prevascularized cardiac tissue, with the use of circulatory scaffolds, holds the promise to be inosculated and perfused with host vasculature to eventually promote cardiac pumping function. Circulatory scaffolds play its role to provide oxygen and nutrients and take metabolic wastes away, and achieve anastomosis with host vasculature in vivo. Of worth note, heart-on-a-chip based on circulatory scaffolds now has been considered as a valuable unit to broaden the research for building cardiac tissue. In this review, we will present recent different strategies to engineer circulatory scaffolds for building cardiac tissue with microvasculature, followed by its current state and future direction.


The source of ECs could be categorized into two groups, primary ECs and EPCs induced by pluripotent stem cells (PSCs). Primary ECs mainly consist of human umbilical vein ECs (HUVECs) and human microvascular ECs (HMVECs), enabling self-assembly to form vascular networks encompassed with CMs. HUVECs are the most usually utilized in the building of circulatory scaffolds (42,43). Lesman et al. constructed tissue-engineered human vascularized cardiac muscle ex vivo using the triculture of human embryonic stem cell (hESC)-derived CMs, HUVECs and embryonic fibroblasts (EmFs) onto biodegradable porous scaffolds (42), which then were transplanted to the in vivo rat heart and confirmed the functional integration between in vitro blood vessels and host coronary vasculature, forming stable grafts. Moreover, Valarmathi et al. engineered a 3D prevascularized collagen cell carrier (CCC) scaffold by the combination of human cardiac microvascular endothelial cells (hCMVECs) and human mesenchymal stem cells (hMSCs) for co-culture of the human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and the human adipose-derived multipotent mesenchymal stem cells (MSCs) (44). Regardless of the advanced results in primary ECs, the fact considered to be tough is the reproducible capacity of HUVECs and hCMVECs in a great amount. Alternatively, hESC-derived ECs (45,46) has been being attracted by the investigators. Caspi et al. (45) have successfully engineered vascularized cardiac tissue with tri-culture of hESC-CMs, hESC-ECs and EmFs, demonstrating large number of organized vessels similar to the addition of HUVECs. Furthermore, as the successful inducement and isolation of human induced pluripotent stem cells (hiPSCs) (47,48), hiPSC-derived ECs (49,50) have been brought into focus for the sake of patient-specific medicine. Samuel et al. (50) dramatically generation of functional blood vessels in vivo by hiPSC-derived ECs from the patients, which remarked the possibility of treatment for ischemic diseases.


Another issue that should be concerned about is the efficiency of recellularization. The fact presented by previous studies was that the dispersion of seeded cells could not uniformly achieve throughout the whole heart, resulting in varying cell density (28,87), thereby the mechanical force and electrical synchronization have been affected (84). Therefore, optimal reseeding strategies also should be employed to promote the dispersion of cells uniformly. Moreover, given the complicity of the whole decellularized scaffold, some investigators pursued simple strategy, using part of decellularized scaffold to reconstruct cardiac patches in vitro for transplantation (88,89). The biggest hurdle in the field is the dearth of human donor for decellularization, restraining the clinical translation, but the xenografts, rat or porcine, were also shown to be enough to replace human heart for decellularization. Collectively, the decellularized scaffolds hold a promise for building perfusable cardiac tissue in theory, but the breakthrough among the decellularization methodology, reseeded strategy and the source of donor should be made.


Furthermore, Fleischer et al. developed a comprehensive bioreactor based on the electrospinning technology, which were involved in three layers: (I) microgroove scaffolds to promote alignment of heart cells; (II) microtunnels to reside ECs with VEGF-embedded microparticles in a controlled release; (III) microcage-like scaffolds embedded with poly (lactic-co-glycolic acid) (PLGA) microparticles to control the release of anti-inflammatory drugs into the ambient microenvironment (80). Each layer was expected to play an individual pivotal role in the process of building vascularized cardiac tissue. After transplantation in vivo, the heart chip manifested excellent aligned and elongated cardiac bundles, endothelialization of microtunnels and high density of blood vessels under the controlled release of VEGF (80).


It is acknowledged that the heart is an electro-mechanical coupled living organ. With the electrical stimulus from cardiac conduction system, cardiac myocytes exhibited synchronous contraction, causing the cyclic mechanical loading to cardiac tissue to sustain homeostasis and efficient functioning. In vitro studies have shown that proper mechanical (132,133) and electrical stimulus (134,135) could contribute to differentiation and maturation of heart cells, eventually performing efficient synchronous contraction (136). To date, numerous studies introduced mechanical and electrical stimulus into microfluidic bioreactor to promote the development of heart-on-a-chip system (137). Cyclic mechanical stimulation was usually employed (124,138). Marsano et al. developed an innovative microfluidic platform stimulated by uniaxial cyclic strain, presenting superior cardiac differentiation, early spontaneous synchronous beating and better contractile capability (124). As to such bioreactor in details, the system contained two layers separated by the PDMS membrane, which the top layer was compartmentalized by two rows of hanging posts into three channels, central part filled with fibrin gel embedded cardiac cells and two lateral channels perfused with culture medium, and the bottom layer was connected to an electro-pneumatic controller as an actuation compartment. Once pressure signal was released, the middle membrane deformed and abutted onto the posts ends, and then returned previous state (124). Under the cyclic mechanical pressure, mimicking the physiological systole and diastole, the features of seeded cells show great similarities to the native counterpart. Moreover, electrical stimulus also has served as great potential to build mature cardiac tissue combined with microfluidic channels (139,140). Xiao et al. microfabricated a perfusable biowire to test pharmacological agents (139). The bioreactor comprised of a drug reservoir, a perfusable biowire (141) and a channel connecting to external peristaltic pump. The study found that CMs in the parallel- and perpendicular-stimulated biowires showed stronger expression of Cx43 compared to the control biowires, indicating better coupling between the CMs in the stimulated groups. In addition, the simutaneous performance of medium perfusion and electrical stimulation dramatically resulted in better performance. Furthermore, simultaneous mechanical and electrical stimulation have also been presented (142,143). Obviously, the comprehensive stimulation could promote maturation and contractivity. 041b061a72


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