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In order to recreate the complexity of human organs, the field of tissue engineering and regenerative medicine has been focusing on methods to build organs from the bottom up by assembling distinct small functional units consisting of a biomaterial and cells. This bottom-up engineering requires bioinks that can be assembled by 3D bioprinting and that permit fast vascularization of the construct to ensure survival of embedded cells. To this end, a small molecular weight alginate (SMWA) gel porogen is presented herein. Alginate is a biocompatible biomaterial, which can be easily converted into small porogen gels with the procedure reported in this article. The SMWA porogen is mixed with photo-crosslinkable hydrogels and leached from the hydrogel post-crosslinking to increase porosity and facilitate vascularization. As a proof of concept, this system is tested with the commonly used biomaterial Gelatin Methacryloyl (GelMA). The SMWA porogen-GelMA blend is proven to be bioprintable. Incubating the blend for 20 min in a low concentration phosphate buffered saline and sodium citrate solution significantly reduces the remaining porogen in the hydrogel . The intent to completely leach the porogen from the hydrogel was abandoned, as longer incubation times and higher concentrations of phosphate and citrate were detrimental to endothelial proliferation. Nonetheless, even with remnants of the porogen left in the hydrogel, the created porosity significantly improves viability, growth factor signaling, vasculogenesis, and angiogenesis in 3D bioprinted structures. This article concludes that the usage of the SMWA porogen can improve the assembly of microvasculature in 3D bioprinted structures. This technology can benefit the bottom-up assembly of large scaffolds with high cell density through 3D bioprinting by improving cell viability and allowing faster vascularization.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold’s architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds’ architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01 kPa, 100.59 ± 31.15 kPa and 245.78 ± 33.54 kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69 kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5–15] kPa for COL, [80–350] kPa for SES, [20–70] kPa for 3DP and [100–190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes’ architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds’ architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

Skeletal muscle tissue engineering (SMTE) aims at the in vitro generation of 3D skeletal muscle engineered constructs which mimic the native muscle structure and function. Although native skeletal muscle is a highly dynamic tissue, most research approaches still focus on static cell culture methods, while research on stimulation protocols indicates a positive effect, especially on myogenesis. A more mature muscle construct may be needed especially for the potential applications for regenerative medicine purposes, disease or drug disposition models. Most efforts towards dynamic cell or tissue culture methods have been geared towards mechanical or electrical stimulation or a combination of those. In the context of dynamic methods, pulsed electromagnetic field (PEMF) stimulation has been extensively used in bone tissue engineering, but the impact of PEMF on skeletal muscle development is poorly explored. Here, we evaluated the effects of PEMF stimulation on human skeletal muscle cells both in 2D and 3D experiments. First, PEMF was applied on 2D cultures of human myoblasts during differentiation. In 2D, enhanced myogenesis was observed, as evidenced by an increased myotube diameter and fusion index. Second, 2D results were translated towards 3D bioartificial muscles (BAMs). BAMs were subjected to PEMF for varying exposure times, where a 2-h daily stimulation was found to be effective in enhancing 3D myotube formation. Third, applying this protocol for the entire 16-days culture period was compared to a stimulation starting at day 8, once the myotubes were formed. The latter was found to result in significantly higher myotube diameter, fusion index, and increased myosin heavy chain 1 expression. This work shows the potential of electromagnetic stimulation for enhancing myotube formation both in 2D and 3D, warranting its further consideration in dynamic culturing techniques.

Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human and equine athletes, due to the cumulative degenerative damage. These injuries present significant challenges as the healing process often results in the formation of inferior scar tissue. The poor success with conventional therapy supports the need to search for novel treatments to restore functionality and regenerate tissue as close to native tendon as possible. Mesenchymal stem cell (MSC)-based strategies represent promising therapeutic tools for tendon repair in both human and veterinary medicine. The translation of tissue engineering strategies from basic research findings, however, into clinical use has been hampered by the limited understanding of the multifaceted MSC mechanisms of action. In vitro models serve as important biological tools to study cell behavior, bypassing the confounding factors associated with in vivo experiments. Controllable and reproducible in vitro conditions should be provided to study the MSC healing mechanisms in tendon injuries. Unfortunately, no physiologically representative tendinopathy models exist to date. A major shortcoming of most currently available in vitro tendon models is the lack of extracellular tendon matrix and vascular supply. These models often make use of synthetic biomaterials, which do not reflect the natural tendon composition. Alternatively, decellularized tendon has been applied, but it is challenging to obtain reproducible results due to its variable composition, less efficient cell seeding approaches and lack of cell encapsulation and vascularization. The current review will overview pros and cons associated with the use of different biomaterials and technologies enabling scaffold production. In addition, the characteristics of the ideal, state-of-the-art tendinopathy model will be discussed. Briefly, a representative in vitro tendinopathy model should be vascularized and mimic the hierarchical structure of the tendon matrix with elongated cells being organized in a parallel fashion and subjected to uniaxial stretching. Incorporation of mechanical stimulation, preferably uniaxial stretching may be a key element in order to obtain appropriate matrix alignment and create a pathophysiological model. Together, a thorough discussion on the current status and future directions for tendon models will enhance fundamental MSC research, accelerating translation of MSC therapies for tendon injuries from bench to bedside.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor–ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant in vitro vascular models, and the future trend in this field of research will be presented and discussed.