Neural cell integration into 3D bioprinted skeletal muscle constructs accelerates restoration of muscle function.

A bioengineered skeletal muscle construct that mimics structural and functional characteristics of native skeletal muscle is a promising therapeutic option to treat extensive muscle defect injuries. We previously showed that bioprinted human skeletal muscle constructs were able to form multi-layered bundles with aligned myofibers. In this study, we investigate the effects of neural cell integration into the bioprinted skeletal muscle construct to accelerate functional muscle regeneration in vivo. Neural input into this bioprinted skeletal muscle construct shows the improvement of myofiber formation, long-term survival, and neuromuscular junction formation in vitro. More importantly, the bioprinted constructs with neural cell integration facilitate rapid innervation and mature into organized muscle tissue that restores normal muscle weight and function in a rodent model of muscle defect injury. These results suggest that the 3D bioprinted human neural-skeletal muscle constructs can be rapidly integrated with the host neural network, resulting in accelerated muscle function restoration.

3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration.

A bioengineered skeletal muscle tissue as an alternative for autologous tissue flaps, which mimics the structural and functional characteristics of the native tissue, is needed for reconstructive surgery. Rapid progress in the cell-based tissue engineering principle has enabled in vitro creation of cellularized muscle-like constructs; however, the current fabrication methods are still limited to build a three-dimensional (3D) muscle construct with a highly viable, organized cellular structure with the potential for a future human trial. Here, we applied 3D bioprinting strategy to fabricate an implantable, bioengineered skeletal muscle tissue composed of human primary muscle progenitor cells (hMPCs). The bioprinted skeletal muscle tissue showed a highly organized multi-layered muscle bundle made by viable, densely packed, and aligned myofiber-like structures. Our in vivo study presented that the bioprinted muscle constructs reached 82% of functional recovery in a rodent model of tibialis anterior (TA) muscle defect at 8 weeks of post-implantation. In addition, histological and immunohistological examinations indicated that the bioprinted muscle constructs were well integrated with host vascular and neural networks. We demonstrated the potential of the use of the 3D bioprinted skeletal muscle with a spatially organized structure that can reconstruct the extensive muscle defects.

Optimization of gelatin-alginate composite bioink printability using rheological parameters: a systematic approach.

Three-dimensional bioprinting has emerged as a promising technique in tissue engineering applications through the precise deposition of cells and biomaterials in a layer-by-layer fashion. However, the limited availability of hydrogel bioinks is frequently cited as a major issue for the advancement of cell-based extrusion bioprinting technologies. It is well known that highly viscous materials maintain their structure better, but also have decreased cell viability due to the higher forces which are required for extrusion. However, little is known about the effect of the two distinct components of dynamic modulus of viscoelastic materials, storage modulus (G') and loss modulus (G″), on the printability of hydrogel-based bioinks. Additionally, 'printability' has been poorly defined in the literature, mostly consisting of gross qualitative measures which do not allow for direct comparison of bioinks. This study developed a framework for evaluating printability and investigated the effect of dynamic modulus, including storage modulus (G'), loss modulus (G″), and loss tangent (G″/G') on the printing outcome. Gelatin and alginate as model hydrogels were mixed at various concentrations to obtain hydrogel formulations with a wide range of storage and loss moduli. These formulations were then evaluated for the quantitatively defined values of extrudability, extrusion uniformity, and structural integrity. For extrudability, increasing either the loss or storage modulus increased the pressure required to extrude the bioink. A mathematical model relating the G' and G″ to the required extrusion pressure was derived based on the data. A lower loss tangent was correlated with increased structural integrity while a higher loss tangent correlated with increased extrusion uniformity. Gelatin-alginate composite hydrogels with a loss tangent in the range of 0.25-0.45 exhibited an excellent compromise between structural integrity and extrusion uniformity. In addition to the characterization of a common bioink, the methodology introduced in this paper could also be used to evaluate the printability of other bioinks in the future.

3D Bioprinted BioMask for Facial Skin Reconstruction.

Skin injury to the face remains one of the greatest challenges in wound care due to the varied contours and complex movement of the face. Current treatment strategies for extensive facial burns are limited to the use of autografts, allografts, and skin substitutes, and these often result in scarring, infection, and graft failure. Development of an effective treatment modality will greatly improve the quality of life and social integration of the affected individuals. In this proof of concept study, we developed a novel strategy, called "BioMask", which is a customized bioengineered skin substitute combined with a wound dressing layer that snugly fits onto the facial wounds. To achieve this goal, three-dimensional (3D) bioprinting principle was used to fabricate the BioMask that could be customized by patients' clinical images such as computed tomography (CT) data. Based on a face CT image, a wound dressing material and cell-laden hydrogels were precisely dispensed and placed in a layer-by-layer fashion by the control of air pressure and 3-axis stage. The resulted miniature BioMask consisted of three layers; a porous polyurethane (PU) layer, a keratinocyte-laden hydrogel layer, and a fibroblast-laden hydrogel layer. To validate this novel concept, the bioprinted BioMask was applied to a skin wound on a pre-fabricated face-shaped structure in mice. Through this in vivo study using the 3D BioMask, skin contraction and histological examination showed the regeneration of skin tissue, consisting of epidermis and dermis layers, on the complex facial wounds. Consequently, effective and rapid restoration of aesthetic and functional facial skin would be a significant improvement to the current issues a facial wound patient experience.

Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases - before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

A 3D bioprinted complex structure for engineering the muscle-tendon unit.

Three-dimensional integrated organ printing (IOP) technology seeks to fabricate tissue constructs that can mimic the structural and functional properties of native tissues. This technology is particularly useful for complex tissues such as those in the musculoskeletal system, which possess regional differences in cell types and mechanical properties. Here, we present the use of our IOP system for the processing and deposition of four different components for the fabrication of a single integrated muscle-tendon unit (MTU) construct. Thermoplastic polyurethane (PU) was co-printed with C2C12 cell-laden hydrogel-based bioink for elasticity and muscle development on one side, while poly(ϵ-caprolactone) (PCL) was co-printed with NIH/3T3 cell-laden hydrogel-based bioink for stiffness and tendon development on the other. The final construct was elastic on the PU-C2C12 muscle side (E = 0.39 ± 0.05 MPa), stiff on the PCL-NIH/3T3 tendon side (E = 46.67 ± 2.67 MPa) and intermediate in the interface region (E = 1.03 ± 0.14 MPa). These constructs exhibited >80% cell viability at 1 and 7 d after printing, as well as initial tissue development and differentiation. This study demonstrates the versatility of the IOP system to create integrated tissue constructs with region-specific biological and mechanical characteristics for MTU engineering.

Development of hybrid scaffolds using ceramic and hydrogel for articular cartilage tissue regeneration.

The regeneration of articular cartilage consisting of hyaline cartilage and hydrogel scaffolds has been generally used in tissue engineering. However, success in in vivo studies has been rarely reported. The hydrogel scaffolds implanted into articular cartilage defects are mechanically unstable and it is difficult for them to integrate with the surrounding native cartilage tissue. Therefore, it is needed to regenerate cartilage and bone tissue simultaneously. We developed hybrid scaffolds with hydrogel scaffolds for cartilage tissue and with ceramic scaffolds for bone tissue. For in vivo study, hybrid scaffolds were press-fitted into osteochondral tissue defects in a rabbit knee joints and the cartilage tissue regeneration in blank, hydrogel scaffolds, and hybrid scaffolds was compared. In 12th week after implantation, the histological and immunohistochemical analyses were conducted to evaluate the cartilage tissue regeneration. In the blank and hydrogel scaffold groups, the defects were filled with fibrous tissues and the implanted hydrogel scaffolds could not maintain their initial position; in the hybrid scaffold group, newly generated cartilage tissues were morphologically similar to native cartilage tissues and were smoothly connected to the surrounding native tissues. This study demonstrates hybrid scaffolds containing hydrogel and ceramic scaffolds can provide mechanical stability to hydrogel scaffolds and enhance cartilage tissue regeneration at the defect site.

Short-term evaluation of electromagnetic field pretreatment of adipose-derived stem cells to improve bone healing.

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell-based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose-derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor ß, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three-dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short-term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment.

Bioprinting technology and its applications.

Bioprinting technology has emerged as a powerful tool for building tissue and organ structures in the field of tissue engineering. This technology allows precise placement of cells, biomaterials and biomolecules in spatially predefined locations within confined three-dimensional (3D) structures. Various bioprinting technologies have been developed and utilized for applications in life sciences, ranging from studying cellular mechanisms to constructing tissues and organs for implantation, including heart valve, myocardial tissue, trachea and blood vessels. In this article, we introduce the general principles and limitations of the most widely used bioprinting technologies, including jetting- and extrusion-based systems. Application-based research focused on tissue regeneration is presented, as well as the current challenges that hamper clinical utility of bioprinting technology.

Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration.

Improvement of bone regeneration capability of ceramic scaffolds by accelerated release of their calcium ions.

To regenerate the bone tissue, the fabrication of scaffolds for better tissue regeneration has attracted a great deal of attention. In fact, growth factors are already used in clinical practice and are being investigated for enhancing the capacity for bone tissue regeneration. However, despite their strong osteoinductive activity, these growth factors have several limitations: safety issues, high treatment costs, and the potential for ectopic bone formation. The aim of this study was therefore to develop ceramic scaffolds that could promote the capacity for bone regeneration without growth factors. Three-dimensional ceramic scaffolds were successfully fabricated from hydroxyapatite (HA) and tricalcium phosphate (TCP) using projection-based microstereolithography, which is an additive manufacturing technology. The effects of calcium ions released from ceramic scaffolds on osteogenic differentiation and bone regeneration were evaluated in vitro and in vivo. The osteogenesis-related gene expression and area of new bone formation in the HA/TCP scaffolds was higher than those in the HA scaffolds. Moreover, regenerated bone tissue in HA/TCP scaffolds were more matured than that in HA scaffolds. Through this study, we were able to enhance the bone regeneration capacity of scaffolds not by growth factors but by calcium ions released from the scaffolds. Ceramic scaffolds developed in this study might be useful for enhancing the capacity for regeneration in complex bone defects.

Combined effect of three types of biophysical stimuli for bone regeneration.

Pretreatment using various types of biophysical stimuli could provide appropriate potential to cells during construction of the engineered tissue in vitro. We hypothesized that multiple combinations of these biophysical stimuli could enhance osteogenic differentiation in vitro and bone formation in vivo. Cyclic strain, an electromagnetic field, and ultrasound were selected and combined as effective stimuli for osteogenic differentiation using a developed bioreactor. Here we report the experimental evaluation of the osteogenic effects of various combinations of three different biophysical stimuli in vitro and in vivo using human adipose-derived stem cells (ASCs). Osteogenic differentiation of ASCs was accelerated by multiple-combination biophysical stimulation in vitro. However, both single stimulation and double-combination stimulation were sufficient to accelerate bone regeneration in vivo, while the osteogenic marker expression of those groups was not as high as that of triple-combination stimulation in vitro. We inferred from these data that ASCs appropriately differentiated into the osteogenic lineage by biophysical stimulation could be a better option for accelerating bone formation in vivo than relatively undifferentiated or completely differentiated ASCs. Although many questions remain about the mechanisms of combined effects of various biophysical stimuli, this approach could be a more powerful tool for bone tissue regeneration.

A new method of fabricating robust freeform 3D ceramic scaffolds for bone tissue regeneration.

Fabrication of three-dimensional (3D) scaffolds with appropriate mechanical properties and desired architecture for promoting cell growth and new tissue formation is one of the most important efforts in tissue engineering field. Scaffolds fabricated from bioactive ceramic materials such as hydroxyapatite and tricalcium phosphate show promise because of their biological ability to support bone tissue regeneration. However, the use of ceramics as scaffold materials is limited because of their inherent brittleness and difficult processability. The aim of this study was to create robust ceramic scaffolds, which have a desired architecture. Such scaffolds were successfully fabricated by projection-based microstereolithography, and dilatometric analysis was conducted to study the sintering behavior of the ceramic materials. The mechanical properties of the scaffolds were improved by infiltrating them with a polycaprolactone solution. The toughness and compressive strength of these ceramic/polymer scaffolds were about twice those of ceramic scaffolds. Furthermore, the osteogenic gene expression on ceramic/polymer scaffolds was better than that on ceramic scaffolds. Through this study, we overcame the limitations of previous research on fabricating ceramic scaffolds and these new robust ceramic scaffolds may provide a much improved 3D substrate for bone tissue regeneration.

Human inferior turbinate: an alternative tissue source of multipotent mesenchymal stromal cells.

OBJECTIVE: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells in adult tissues. Current challenges for the clinical application of MSCs include donor site morbidity, which underscores the need to identify alternative sources of MSCs. This study aimed to explore potential new sources of multipotent MSCs for use in tissue regeneration and the functional restoration of organs. STUDY DESIGN: Mixed methods research. SETTING: Tertiary care center. SUBJECTS AND METHODS: The authors isolated MSCs from human inferior turbinate tissues discarded during turbinate surgery of 10 patients for nasal obstruction. The expression of surface markers for MSCs was assessed by fluorescence-activated cell sorting. The differentiation potential of human turbinate mesenchymal stromal cells (hTMSCs) was analyzed by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and Western blot analysis. RESULTS: Surface epitope analysis revealed that hTMSCs were negative for CD14, CD19, CD34, and HLA-DR and positive for CD29, CD73, and CD90, representing a characteristic phenotype of MSCs. Extracellular matrices with characteristics of cartilage, bone, and adipose tissue were produced by inducing the chondrogenic, osteogenic, and adipogenic differentiation of hTMSCs, respectively. The expression of neuron-specific markers in hTMSCs was confirmed immunocytochemically. CONCLUSION: The hTMSCs represent a new source of multipotent MSCs that are potentially applicable to tissue engineering and regenerative medicine. The availability of differentiated adult cells will allow the development of an effective tissue regeneration method.

Unit cell-based computer-aided manufacturing system for tissue engineering.

Scaffolds play an important role in the regeneration of artificial tissues or organs. A scaffold is a porous structure with a micro-scale inner architecture in the range of several to several hundreds of micrometers. Therefore, computer-aided construction of scaffolds should provide sophisticated functionality for porous structure design and a tool path generation strategy that can achieve micro-scale architecture. In this study, a new unit cell-based computer-aided manufacturing (CAM) system was developed for the automated design and fabrication of a porous structure with micro-scale inner architecture that can be applied to composite tissue regeneration. The CAM system was developed by first defining a data structure for the computing process of a unit cell representing a single pore structure. Next, an algorithm and software were developed and applied to construct porous structures with a single or multiple pore design using solid freeform fabrication technology and a 3D tooth/spine computer-aided design model. We showed that this system is quite feasible for the design and fabrication of a scaffold for tissue engineering.

Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification.

Poly(propylene fumarate) (PPF) is an ultraviolet-curable and biodegradable polymer with potential applications for bone regeneration. In this study, we designed and fabricated three-dimensional (3D) porous scaffolds based on a PPF polymer network using micro-stereolithography (MSTL). The 3D scaffold was well fabricated with a highly interconnected porous structure and porosity of 65%. These results provide a new scaffold fabrication method for tissue engineering. Surface modification is a commonly used and effective method for improving the surface characteristics of biomaterials without altering their bulk properties that avoids the expense and long time associated with the development of new biomaterials. Therefore, we examined surface modification of 3D scaffolds by applying accelerated biomimetic apatite and arginine-glycine-aspartic acid (RGD) peptide coating to promote cell behavior. The apatite coating uniformly covered the scaffold surface after immersion for 24 h in 5-fold simulated body fluid (5SBF) and then the RGD peptide was applied. Finally, the coated 3D scaffolds were seeded with MC3T3-E1 pre-osteoblasts and their biologic properties were evaluated using an MTS assay and histologic staining. We found that 3D PPF/diethyl fumarate (DEF) scaffolds fabricated with MSTL and biomimetic apatite coating can be potentially used in bone tissue engineering.