3D Bioprinted Scaffolds Research Articles

Osteoimmunomodulatory bioinks for 3D bioprinting achieve complete regeneration of critical-sized bone defects

Regeneration the critical-sized bone defects remains a great challenge to clinical therapy due to the inflammatory microenvironment and lack of stem cells in the region of the bone defects. 3D bioprinted scaffolds based on bioactive ink and loaded active cells can promote the inflammatory microenvironment and cell viability, thereby enhancing bone regeneration. In this study, 10 % Gelatin-methacryloyl (GelMA)/5 %Sr substituted xonotlite (Sr-CSH) nanocomposite hydrogel was developed as a bioink to encapsulate bone marrow mesenchymal stem cells (BMSCs), and then constructed a biomimetic bone tissue by 3D bioprinting. The incorporation of Sr-CSH nanowires enhanced the printing accuracy and mechanical property of GelMA, and enhanced the osteogenic differentiation of BMSCs. In addition, Sr-CSH induced macrophage M2 polarization, which modulated the inflammatory microenvironment and further promoted osteogenic differentiation of BMSCs. In rat critical-sized calvarial defects model, 3D bioprinted scaffolds based on GelMA-Sr-CSH bioinks laden with BMSCs achieve complete bone repair. In summary, this study developed an osteoimmunomodulatory bioink, and 3D bioprinted scaffolds laden with stem cells may be an effective method for achieving complete regeneration of critical-sized bone defects.

Design and optimization of 3D-bioprinted cell-laden scaffolds in dynamic culture

Light-based 3D printing enables the fabrication of biological scaffolds with high precision, versatility and biocompatibility, particularly the cell-laden scaffolds with architecturally complex geometric features. However, many bioprinted tissue scaffolds suffer from low cell viability due to insufficient oxygen and nutrient supply, which is heavily influenced by scaffold structure and cultivation conditions. Current practice relies mainly on resource-intensive trial-and-error methods to optimize scaffolds’ structures and cultivation parameters. In this study, we developed a comprehensive multi-physics model integrating fluid dynamics, oxygen mass transfer, cell oxygen consumption, and cell growth processes to capture cell growth behaviors in scaffolds, establishing a robust theoretical foundation for scaffold structure optimization. The modeling results showed that a large number of parameters, such as system inlet flow rate, geometric feature size, cell parameters, and material properties, significantly impact oxygen concentration and cell growth within the scaffold. A two-step optimization strategy is proposed in this paper and was applied to obtain optimal geometric parameters of channeled scaffolds to demonstrate the model’s effectiveness for scaffold optimization. The model can be employed for scaffolds with arbitrary shapes and various materials, facilitating the optimal design of sophisticated scaffolds for more advanced tissue engineering.

3D-bioprinted bone scaffolds incorporating SR1 nanoparticles enhance blood vessel regeneration in rat calvarial defects

The inherent limitations of bone grafting in the treatment of critical-sized bone defects have led to a growing demand for bone repair implants. Three-dimensional (3D) bioprinting has emerged as a promising manufacturing technique for implants, offering flexibility in their structural design and the use of applicable materials. Although numerous 3D-bioprinted bone scaffolds have been developed to enhance osteogenesis, angiogenesis remains a challenge. Angiogenesis is crucial for successful bone healing because the process forms blood vessels to deliver essential nutrients and oxygen. Endothelial progenitor cells (EPCs) play a pivotal role in the early stages of vascularization. These cells, capable of differentiating into endothelial cells (ECs), are recruited from the bone marrow to the injured area during the healing process. CD34+ cells, a subset of EPCs, have gained attention because of their neovascularization potential and ability to contribute to bone regeneration. The incorporation of CD34+ cell-enhancing factors into 3D-printed bone scaffolds may facilitate successful bone healing in critical defects. StemRegenin-1 (SR1), a molecule that promotes CD34+ cell expansion, has shown promising results in increasing CD34+ hematopoietic stem and progenitor cell populations. This study aimed to investigate the sustained release of SR1 from a collagen-based scaffold integrated with mesoporous silica nanoparticles (MSNs) to promote angiogenesis and enhance bone healing. The sustained release of SR1 from the collagen scaffold is hypothesized to promote angiogenesis, thereby facilitating bone repair. In vitro studies have demonstrated the angiogenic potential of SR1; however, further in vivo investigations are required to establish its clinical efficacy. This study contributes to the development of novel therapies targeting CD34+ cells and demonstrates the potential of SR1 as a promising agent for promoting angiogenesis and enhancing bone healing in critical defects.

3D-bioprinted alginate-based bioink scaffolds with β-tricalcium phosphate for bone regeneration applications

Background/purpose3D-printed bone tissue engineering is becoming recognized as a key approach in dentistry for creating customized bone regeneration treatments fitting patients bone defects requirements. 3D bioprinting offers an innovative method to fabricate detailed 3D structures, closely emulating the native bone micro-environment and better bone regeneration. This study aimed to develop an 3D-bioprintable scaffold using a combination of alginate and β-tricalcium phosphate (β-TCP) with the Cellink® BioX printer, aiming to advance the field of tissue engineering. Materials and methodsThe physical and biological properties of the resulting 3D-printed scaffolds were evaluated at 10 %, 12 %, and 15 % alginate combined with 10 % β-TCP. The scaffolds were characterized through printability, swelling behavior, degradability, and element analysis. The biological assessment included cell viability, alkaline phosphatase (ALP) activity. Results10 % alginate/β-TCP 3D printed at 25 °C scaffold demonstrated the optimal condition for printability, swelling capability, and degradability of cell growth and nutrient diffusion. Addition of β-TCP particles significantly improved the 3D printed material viscosity over only alginate (P < 0.05). 10 % alginate/β-TCP enhanced MG-63 cell's proliferation (P < 0.05) and alkaline phosphatase activity (P < 0.001). ConclusionThis study demonstrated in vitro that 10 % alginate/β-TCP bioink characteristic for fabricating 3D acellular bioprinted scaffolds was the best approach. 10 % alginate/β-TCP bioink 3D-printed scaffold exhibited superior physical properties and promoted enhanced cell viability and alkaline phosphatase activity, showing great potential for personalized bone regeneration treatments.

A microbial infection‐resistant 3D bioprinted material for corneal stroma regeneration

Aims/Purpose: To assess whether 3D bioprinted, scaffold‐immobilized antimicrobial peptides (AMPs) derivatives, BMAP27(1–18) and hLF(1–11) can inhibit microbial colonization to obtain infection‐resistant 3D bioprinted hemi‐corneas.Methods: The research followed the tenets of the Declaration of Helsinki. 3D bioprinting was used to produce transparent nanocellulose‐ and alginate‐based 3D constructs. Functionalized 3D bioprinted scaffolds with AMPs were characterized by contact angle and transparency measurements. The antimicrobial and antifungal activity of the peptides in solution were investigated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods against Staphylococcus epidermidis ATCC 35984, S. aureus ATCC 25923, S. epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Candida albicans ATCC 90028. The antimicrobial activity of the AMP‐modified scaffolds was studied against P. aeruginosa and C. albicans reference strains by colony forming unit (CFU) counts, and their biocompatibility was investigated by measuring the viability of primary human/cadaver‐isolated corneal stromal‐mesenchymal stromal cells (CS‐MSCs).Results: The original and modified peptides showed MIC (MBC) values of 1–4 (2–8) μM and 16 – &gt;128 (32 – &gt;128) μM, respectively, against S. epidermidis ATCC 35984, S. aureus, S. epidermidis ATCC 12228, P. aeruginosa, E. coli and C. albicans, while ncBP‐BMAP27(1–18) and ncBP‐hLF(1–11) values were 1–16 (2–32) μM 4–128 (4 – &gt;128) μM, respectively, against all the pathogens. The modified scaffolds containing the AMPs attached to the scaffolds, demonstrated the ability to kill 20–96% of P. aeruginosa, and between 60–70% of C. albicans. All the 3D bioprinted scaffolds showed no cytotoxicity towards CS‐MSCs.Conclusions: We could successfully combine 3D bioprinting with immobilization of AMPs that show promising antimicrobial activity against two of the most dangerous pathogens to the cornea, while a good cytocompatibility for the CS‐MSCs could be achieved. Optimal bioink composition and cellularization of tissue equivalents are essential in fine‐tuning a method to promote the current 3D bioprinting technique as a future treatment modality for corneal regeneration.

Biomolecules-Loading of 3D-Printed Alginate-Based Scaffolds for Cartilage Tissue Engineering Applications: A Review on Current Status and Future Prospective.

Osteoarthritis (OA) can arise from various factor including trauma, overuse, as well as degeneration resulting from age or disease. The specific treatment options will vary based on the severity of the condition, and the affected joints. Some common treatments for OA include lifestyle modifications, medications, physical therapy, surgery and tissue engineering (TE). For cartilage tissue engineering (CTE), three-dimension (3D) scaffolds are made of biocompatible natural polymers, which allow for the regeneration of new cartilage tissue. An ideal scaffold should possess biological and mechanical properties that closely resemble those of the cartilage tissue, and lead to improved functional of knee. These scaffolds are specifically engineered to serve as replacements for damaged and provide support to the knee joint. 3D-bioprinted scaffolds are made of biocompatible materials natural polymers, which allow for the regeneration of new cartilage. The utilization of 3D bioprinting method has emerged as a novel approach for fabricating scaffolds with optimal properties for CTE applications. This method enables the creation of scaffolds that closely mimic the native cartilage in terms of mechanical characteristics and biological functionality. Alginate, that has the capability to fabricate a cartilage replacement customized for each individual patient. This polymer exhibits hydrophilicity, biocompatibility, and biodegradability, along with shear-thinning properties. These unique properties enable Alginate to be utilized as a bio-ink for 3D bioprinting method. Furthermore, chondrogenesis is the complex process through which cartilage is formed via a series of cellular and molecular signaling. Signaling pathway is as a fundamental mechanism in cartilage formation, enhanced by the incorporation of biomolecules and growth factors that induce the differentiation of stem cells. Accordingly, ongoing review is focusing to promote of 3D bioprinting scaffolds through the utilization of advanced biomolecules-loading of Alginate-based that has the capability to fabricate a cartilage replacement tailored specifically to each patient's unique needs and anatomical requirements.

3D-Bioprinted GelMA Scaffold with ASCs and HUVECs for Engineering Vascularized Adipose Tissue.

The purpose of tissue engineering is to reconstruct parts of injured tissues and to resolve the shortage of organ donations. However, the main concern is the limited size of engineered tissue due to insufficient oxygen and nutrition distribution in large three-dimensional (3D) tissue constructs. To provide better support for cells inside the scaffolds, the vascularization of blood vessels within the scaffold could be a solution. This study compared the effects of different culturing systems using human adipose tissue-derived stem/stromal cells (ASCs), human umbilical vein endothelial cells (HUVECs), and coculture of ASCs and HUVECs in 3D-bioprinted gelatin methacrylate (GelMA) hydrogel constructs. The in vitro results showed that the number of live cells was highest in the coculture of ASCs and HUVECs in the GelMA hydrogel after culturing for 21 days. Additionally, the tubular structure was the most abundant in the GelMA hydrogel, containing both ASCs and HUVECs. In the in vivo test, blood vessels were present in both the HUVECs and the coculture of ASCs and HUVECs hydrogels implanted in mice. However, the blood vessel density was the highest in the HUVEC and ASC coculture groups. These findings indicate that the 3D-bioprinted GelMA hydrogel coculture system could be a promising biomaterial for large tissue engineering applications.

Vascularization ability of glioma stem cells in different three-dimensional microenvironments.

Glioblastoma (GBM) is among the most common and aggressive adult central nervous system tumors. One prominent characteristic of GBM is the presence of abnormal microvessels. A significant correlation between angiogenesis and prognosis has been observed. Accurately reconstructing this neovascularization and tumor microenvironment through personalized in vitro disease models presents a significant challenge. However, it is crucial to develop new anti-angiogenic therapies for GBM. In this study, 3D bioprinted glioma stem cell (GSC)-laden hydrogel scaffolds, hybrid GSC hydrogels and cell-free hydrogel scaffolds were manufactured to investigate the vascularization ability of GSCs in varying 3D microenvironments. Our results demonstrated that the bioactivity of GSCs in the 3D bioprinted GSC-laden hydrogel scaffold was preferable and stable, and the amounts of vascular endothelial growth factor A and basic fibroblast growth factor were the highest in the microenvironment. When the three different models were co-cultured with human umbilical vein endothelial cells, the expression of angiogenesis-related markers was the most abundant in the bioprinted GSC-laden hydrogel scaffold. Additionally, xenograft tumors formed by bioprinted GSC-laden hydrogel scaffolds more closely resembled human gliomas regarding color, texture and vascularization. Notably, in xenograft tumors derived from 3D bioprinted GSC-laden hydrogel scaffolds, the number of human CD105+ cells was significantly higher, and human endothelial vascular lumen-like structures were observed. This indicates that the 3D bioprinted GSC-laden hydrogel scaffold is a suitable model for mimicking the glioma microenvironment and studying tumor angiogenesis.

Adipose-Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold.

Intra-portal islet transplantation is currently the only clinically approved beta cell replacement therapy, but its outcome is hindered by limited cell survival due to a multifactorial reaction against the allogeneic tissue in liver. Adipose-derived stromal cells (ASCs) can potentially improve the islet micro-environment by their immunomodulatory action. The challenge is to combine both islets and ASCs in a relatively easy and consistent long-term manner in a deliverable scaffold. Manufacturing the 3D bioprinted double-layered scaffolds with primary islets and ASCs using a mix of alginate/nanofibrillated cellulose (NFC) bioink is reported. The diffusion properties of the bioink and the supportive effect of human ASCs on islet viability, glucose sensing, insulin secretion, and reducing the secretion of pro-inflammatory cytokines are demonstrated. Diabetic mice transplanted with islet-ASC scaffolds reach normoglycemia seven days post-transplantation with no significant difference between this group and the group received islets under the kidney capsules. In addition, animals transplanted with islet-ASC scaffolds stay normoglycemic and show elevated levels of C-peptide compared to mice transplanted with islet-only scaffolds. The data present a functional 3D bioprinted scaffold for islets and ASCs transplanted to the extrahepatic site and suggest a possible role of ASCs on improving the islet micro-environment.

Alginate-waterborne polyurethane 3D bioprinted scaffolds for articular cartilage tissue engineering

Articular cartilage defects comprise a spectrum of diseases associated with degeneration or damage of the connective tissue present in particular joints, presenting progressive osteoarthritis if left untreated. In vitro tissue regeneration is an innovative treatment for articular cartilage injuries that is attracting not only clinical attention, but also great interest in the development of novel biomaterials, since this procedure involves the formation of a neotissue with the help of material support. In this work, functional alginate and waterborne polyurethane (WBPU) scaffolds have been developed for articular cartilage regeneration using 3D bioprinting technology. The particular properties of alginate-WBPU blends, like mechanical strength, elasticity and moistening, mimic the original cartilage tissue characteristics, being ideal for this application. To fabricate the scaffolds, mature chondrocytes were loaded into different alginate-WBPU inks with rheological properties suitable for 3D bioprinting. Bioinks with high alginate content showed better 3D printing performance, as well as structural integrity and cell viability, being most suitable for scaffolds fabrication. After 28 days of in vitro cartilage formation experiments, scaffolds containing 3.2 and 6.4 % alginate resulted in the maintenance of cell number in the range of 104 chondrocytes/scaffold in differentiated phenotypes, capable of synthesizing specialized extracellular matrix (ECM) up to 6 μg of glycosaminoglycans (GAG) and thus, showing a potential application of these scaffolds for in vitro regeneration of articular cartilage tissue.

In Vitro and In Vivo Biological Assessments of 3D-Bioprinted Scaffolds for Dental Applications.

Three-dimensional (3D) bioprinting is a unique combination of technological advances in 3D printing and tissue engineering. It has emerged as a promising approach to address the dilemma in current dental treatments faced by clinicians in order to repair or replace injured and diseased tissues. The exploration of 3D bioprinting technology provides high reproducibility and precise control of the bioink containing the desired cells and biomaterial over the architectural and dimensional features of the scaffolds in fabricating functional tissue constructs that are specific to the patient treatment need. In recent years, the dental applications of different 3D bioprinting techniques, types of novel bioinks, and the types of cells used have been extensively explored. Most of the findings noted significant challenges compared to the non-biological 3D printing approach in constructing the bioscaffolds that mimic native tissues. Hence, this review focuses solely on the implementation of 3D bioprinting techniques and strategies based on cell-laden bioinks. It discusses the in vitro applications of 3D-bioprinted scaffolds on cell viabilities, cell functionalities, differentiation ability, and expression of the markers as well as the in vivo evaluations of the implanted bioscaffolds on the animal models for bone, periodontal, dentin, and pulp tissue regeneration. Finally, it outlines some perspectives for future developments in dental applications.

Mechanical Properties Affect Primary T Cell Activation in 3D Bioprinted Hydrogels.

T cells play a critical role in the adaptive immune response of the body, especially against intracellular pathogens and cancer. In vitro, T cell activation studies typically employ planar (two-dimensional, 2D) culture systems that do not mimic native cell-to-extracellular matrix (ECM) interactions, which influence activation. The goal of this work was to study T cell responses in a cell line (EL4) and primary mouse T cells in three-dimensional (3D) bioprinted matrices of varied stiffness. Cell-laden hydrogels were 3D bioprinted from gelatin methacryloyl (GelMA) using a digital light processing (DLP)-based 3D bioprinter operated with visible light (405 nm). Mechanical characterization revealed that the hydrogels had pathophysiologically relevant stiffnesses for a lymph node-mimetic tissue construct. EL4, a mouse T cell lymphoma line, or primary mouse T cells were 3D bioprinted and activated using a combination of 10 ng/mL of phorbol myristate acetate (PMA) and 0.1 μM of ionomycin. Cellular responses revealed differences between 2D and 3D cultures and that the biomechanical properties of the 3D bioprinted hydrogel influence T cell activation. Cellular responses of the 2D and 3D cultures in a soft matrix (19.83 ± 2.36 kPa) were comparable; however, they differed in a stiff matrix (52.95 ± 1.36 kPa). The fraction of viable EL4 cells was 1.3-fold higher in the soft matrix than in the stiff matrix. Furthermore, primary mouse T cells activated with PMA and ionomycin showed 1.35-fold higher viable cells in the soft matrix than in the stiff matrix. T cells bioprinted in a soft matrix and a stiff matrix released 7.4-fold and 5.9-fold higher amounts of interleukin-2 (IL-2) than 2D cultured cells, respectively. Overall, the study demonstrates the changes in the response of T cells in 3D bioprinted scaffolds toward engineering an ex vivo lymphoid tissue-mimetic system that can faithfully recapitulate T cell activation and unravel pathophysiological characteristics of T cells in infectious biology, autoimmunity, and cancers.

A Semi-Three-Dimensional Bioprinted Neurocardiac System for Tissue Engineering of a Cardiac Autonomic Nervous System Model.

In this study, we designed a tissue-engineered neurocardiac model to help us examine the role of neuronal regulation and confirm the importance of neural innervation techniques for the regeneration of cardiac tissue. A three-dimensional (3D) bioprinted neurocardiac scaffold composed of a mixture of gelatin-alginate and alginate-genipin-fibrin hydrogels was developed with a 2:1 ratio of AC16 cardiomyocytes (CMs) and retinoic acid-differentiated SH-SY5Y neuronal cells (NCs) respectively. A unique semi-3D bioprinting approach was adopted, where the CMs were mixed in the cardiac bioink and printed using an anisotropic accordion design to mimic the physiological tissue architecture in vivo. The voids in this 3D structure were methodically filled in using a NC-gel mixture and crosslinked. Confocal fluorescent imaging using microtubule-associated protein 2 (MAP-2) and anticholine acetyltransferase (CHAT) antibodies for labeling the NCs and the MyoD1 antibody for the CMs revealed functional coupling between the two cell types in the final crosslinked structure. These data confirmed the development of a relevant neurocardiac model that could be used to study neurocardiac modulation under physiological and pathological conditions.

Computational modeling of probiotic recovery from 3D-bioprinted scaffolds for localized vaginal application

Lactobacilli, play a beneficial role in the female reproductive tract (FRT), regulating pH via lactic acid metabolism to help maintain a healthy environment. Bacterial vaginosis (BV) is characterized by a dysregulated flora in which anaerobes such as Gardnerella vaginalis (Gardnerella) create a less acidic environment. Current treatment focuses on antibiotic administration, including metronidazole, clindamycin, or tinidazole; however, lack of patient compliance as wel as antibiotic resistance may contribute to 50% recurrence within a year. Recently, locally administered probiotic such as Lactobacillus crispatus (L. crispatus) have been evaluated as a prophylactic against recurrence. To mitigate the lack of patient compliance, sustained probiotic delivery has been proposed via 3D-bioprinted delivery vehicles. Successful delivery depends on a variety of vehicle fabrication parameters influencing timing and rate of probiotic recovery; detailed evaluation of these parameters would benefit from computational modeling complementary to experimental evaluation. This study implements a novel simulation platform to evaluate sustained delivery of probiotics from 3D-bioprinted scaffolds, taking into consideration bacterial lactic acid production and associated pH changes. The results show that the timing and rate of probiotic recovery can be realistically simulated based on fabrication parameters that affect scaffold degradation and probiotic survival. Longer term, the proposed approach could help personalize localized probiotic delivery to the FRT to advance women's health.

Flow cytometry as an analytical method of drug-induced apoptosis in 3D bioprinted melanoma cells

Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to its inherent advantages in providing more physiologically relevant information and more predictive data for in vivo tests. Along with the development of more physiologically relevant 3D cell culture models, researchers bear the responsibility to validate new cell assay techniques capable of measuring and evaluating constructs that are physically larger and more complex compared to two-dimensional cell cultures. It is important to note that assays based on monolayer cultures may be insufficient for the use in 3D cell cultures models. In this study we firstly fabricated a 3D bioprinted hydrogel melanoma scaffold. This was used to validate a flow cytometry-based analytical method as a tool for 3D bioprinted structures to assess drug-induced apoptosis. The results indicated high robustness, reproducibility and sensitivity of the flow cytometric method established on the 3D cell-laden A375 melanoma hydrogel scaffolds. Over and above this, it was possible to determine the effect of etoposide on A375 melanoma cells using Annexin V and propidium iodide apoptosis assay.

Visible light-induced 3D bioprinted injectable scaffold for minimally invasive tissue regeneration

Pre-formed hydrogel scaffolds have emerged as favorable vehicles for tissue regeneration, promoting minimally invasive treatment of native tissue. However, due to the high degree of swelling and inherently poor mechanical properties, development of complex structural hydrogel scaffolds at different dimensional scales has been a continuous challenge. Herein, we take a novel approach at the intersections of engineering design and bio-ink chemistry to develop injectable pre-formed structural hydrogel scaffolds fabricated via visible light (VL) induced digital light processing (DLP). In this study, we first determined the minimum concentration of poly(ethylene glycol) diacrylate (PEGDA) to be added to the gelatin methacrylate (GelMA) bio-ink in order to achieve scalable and high printing-fidelity with desired cell adhesion, viability, spreading, and osteogenic differentiation characteristics. Despite the advantages of hybrid GelMA-PEGDA bio-ink in improving scalability and printing-fidelity, compressibility, shape-recovery, and injectability of the 3D bioprinted scaffolds were compromised. To restore these needed characteristics for minimally invasive tissue regeneration applications, we performed topological optimization to design highly compressible and injectable pre-formed (i.e., 3D bioprinted) microarchitectural scaffolds. The designed injectable pre-formed microarchitectural scaffolds showed a great capacity to retain the viability of the encapsulated cells (>72 % after 10 cycles of injection). Lastly, ex ovo chicken chorioallantoic membrane (CAM) studies revealed that the optimized injectable pre-formed hybrid hydrogel scaffold is biocompatible and supports angiogenic growth.

3D bioprinted scaffolds based on functionalized gelatin for soft tissue engineering

3D bioprinting application in the medical field requires optimized and adapted bioinks/biomaterials, along with processing as scaffolds with morphology and structure that mimic human tissues. Various polymeric hydrogels have been tested in bioinks elaboration in order to support the cells biology. In this context, the paper presents the preparation of versatile bioadhesive hydrogels based on methacrylated gelatin, moderate crosslinked with poly (ethylene glycol) diacrylate, and 3D bioprinted as bioadhesive scaffolds for skin tissue engineering. Gelatin with various methacrylation degrees, confirmed by FTIR and H1NMR and analytical methods (TNBS, nynhydrin assay), mixed with poly (ethylene glycol) diacrylate and biocompatible photoinitiator led to stable gels, with rheological characteristics (yield stress and shear-thinning behavior) beneficial for bioprinting. Scaffolds with controllable shape and morphology have been obtained by bioprinting and polymers UV crosslinking. The 3D scaffolds absorb fluids and their biostability increases in presence of poly (ethylene glycol) diacrylate. Cell cultures of fibroblasts/ keratinocytes demonstrated the scaffolds cytocompatibility. The cells preserved the normal morphology, were adherent to substrate and formed uniform monolayers. The bioadhesion characteristics increased by adding poly (ethylene glycol) diacrylate as macromolecular crosslinker. The scaffolds absorb the blood plasma, promote the local accumulation of fibrinogen, and accelerate the adhesion of the blood components, with impact on angiogenesis, suitability characteristic for skin tissue engineering applications.

Bioprinted Scaffold Remodels the Neuromodulatory Microenvironment for Enhancing Bone Regeneration

AbstractHerein, a 3D bioprinted scaffold is proposed, containing a calcitonin gene‐related peptide (CGRP) and the β‐adrenergic receptor blocker propranolol (PRN) as a new method to achieve effective repair of bone defects. By leveraging the neuromodulation mechanism of bone regeneration, CGRP and PRN loaded mesoporous silica nanoparticles are added into a hybrid bio‐ink, which initially contains gelatin methacrylate, Poly (ethylene glycol) diacrylate and bone marrow mesenchymal stem cells (BMSCs). Subsequently, the optimized bio‐ink is used for 3D bioprinting to create a composite scaffold with a pre‐designed micro‐nano hierarchical structure. The migration and tube formation of human umbilical vein endothelial cells (HUVECs) can be promoted by the scaffold, which is beneficial to the formation of a new capillary network during the bone repair process. With the release of CGRP from the scaffold, the secretion of neuropeptides by sensory nerves is simulated. Meanwhile, the release of PRN can inhibit the binding process of catecholamine to β‐adrenergic receptor, co‐promoting the osteogenic differentiation of BMSCs with CGRP and silicon ions, which will effectively enhance bone repair of a critical‐sized cranial defect in a rat model. In conclusion, this study provides a promising strategy for bone defect repair by understanding the neuromodulatory mechanisms during bone regeneration.

Inhibited astrocytic differentiation in neural stem cell-laden 3D bioprinted conductive composite hydrogel scaffolds for repair of spinal cord injury

The emergence of three-dimensional (3D) bioprinting technology has attracted ever-increasing attention in engineered tissue fabrication for stem cell-based tissue repair. However, the in vivo performance of transplanted stem cells in the tissue engineering scaffolds is still a major concern for regenerative medicine researches. Especially for neural stem cell (NSC) transplantation, the uncontrollable differentiation of the NSCs in host often leads to a poor therapeutic effect in nerve tissue repair, such as spinal cord injury (SCI) repair. To address this issue, we have fabricated a conductive composite hydrogel (CCH) scaffold loading with NSCs by 3D bioprinting, for delivering the NSCs to injured spinal cord and repairing the propriospinal nerve circuit. In our strategy, a novel conductive polymer (PEDOT:CSMA,TA) was synthesized and introduced into a photocrosslinkable gelatin/polyethylene glycol physical-gel matrix, thereby forming a composite bioink with well shear-thinning and self-healing properties. The composite bioink we prepared was then printed into the NSC-laden CCH scaffold with high shape fidelity and similar physicochemical properties to native spinal cord tissues. The NSCs encapsulated in the bioprinted CCH scaffold extended their neurites to form superior physical contact with the neighboring cells as well as the electroconductive matrix, and maintained a predominant in vivo neuronal differentiation, accompanying with few astrocytic production in the lesion area after transplantation into the SCI sites. As a result, the removal of glial scar tissues and the regeneration of well-developed nerve fibres sequentially happened, which not only facilitated nerve tissue development, but also accelerated locomotor function recovery in the SCI rats. By exploring the application of conductive biomaterials in stem cell-based SCI therapy, this work represents a feasible, new approach to precisely construct tissue engineering scaffolds for stem cell-based therapy in traumatic SCI and other nervous system diseases.

Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application

Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. Post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.