Living bacterial sacrificial porogens to engineer decellularized porous scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.     

Proliferation and Differentiation Potential of Human Adipose-Derived Stem Cells Grown on Chitosan Hydrogel

Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.     

Bioconjugation of hydrogels for tissue engineering

Success of tissue engineered constructs in regenerative medicine is limited by the lack of cellmatrix interactions to guide devleopment of the seeded cells into the desired tissue. This review highlights the most exciting developments in bioconjugation of synthetic hydrogels targeted to tissue engineering. Application of conjugation techniques has resulted in the synthesis of novel biomimetic cell-responsive hydrogels to control the cascade of cell migration, adhesion, survival, differentiation, and maturation to the desired lineage concurrent with matrix remodeling. The future outlook includes developing conjugated patterned hydrogel matrices, developing novel hydrogel matrices to support self-renewal and pluripotency of embryonic and adult stem cells, and merging 3D printing with bioconjugation to fabricate hydrogels with anatomical arrangement of cells and biomolecules.     

Three-dimensional bioprinting in tissue engineering and regenerative medicine

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.     

The effects of mechanical stretch on the biological characteristics of human adipose‐derived stem cells

Adipose‐derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs), which have promised a vast therapeutic potential in tissue regeneration. Recent studies have demonstrated that combining stem cells with mechanical stretch may strengthen the efficacy of regenerative therapies. However, the exact influences of mechanical stretch on MSCs still remain inconclusive. In this study, human ADSCs (hADSCs) were applied cyclic stretch stimulation under an in vitro stretching model for designated duration. We found that mechanical stretch significantly promoted the proliferation, adhesion and migration of hADSCs, suppressing cellular apoptosis and increasing the production of pro‐healing cytokines. For differentiation of hADSCs, mechanical stretch inhibited adipogenesis, but enhanced osteogenesis. Long‐term stretch could promote ageing of hADSCs, but did not alter the cell size and typical immunophenotypic characteristics. Furthermore, we revealed that PI3K/AKT and MAPK pathways might participate in the effects of mechanical stretch on the biological characteristics of hADSCs. Taken together, mechanical stretch is an effective strategy for enhancing stem cell behaviour and regulating stem cell fate. The synergy between hADSCs and mechanical stretch would most likely facilitate tissue regeneration and promote the development of stem cell therapy.     

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

Engineering inkjet bioprinting processes toward translational therapies

Bioprinting is the assembly of three-dimensional (3D) tissue constructs by layering cell-laden biomaterials using additive manufacturing techniques, offering great potential for tissue engineering and regenerative medicine. Such a process can be performed with high resolution and control by personalized or commercially available inkjet printers. However, bioprinting's clinical translation is significantly limited due to process engineering challenges. Upstream challenges include synthesis, cellular incorporation, and functionalization of “bioinks,” and extrusion of print geometries. Downstream challenges address sterilization, culture, implantation, and degradation. In the long run, bioinks must provide a microenvironment to support cell growth, development, and maturation and must interact and integrate with the surrounding tissues after implantation. Additionally, a robust, scaleable manufacturing process must pass regulatory scrutiny from regulatory bodies such as U.S. Food and Drug Administration, European Medicines Agency, or Australian Therapeutic Goods Administration for bioprinting to have a real clinical impact. In this review, recent advances in inkjet-based 3D bioprinting will be presented, emphasizing on biomaterials available, their properties, and the process to generate bioprinted constructs with application in medicine. Current challenges and the future path of bioprinting and bioinks will be addressed, with emphasis in mass production aspects and the regulatory framework bioink-based products must comply to translate this technology from the bench to the clinic.     

Bioactive nanoparticles stimulate bone tissue formation in bioprinted three‐dimensional scaffold and human mesenchymal stem cells

Bioprinting based on thermal inkjet printing is a promising but unexplored approach in bone tissue engineering. Appropriate cell types and suitable biomaterial scaffolds are two critical factors to generate successful bioprinted tissue. This study was undertaken in order to evaluate bioactive ceramic nanoparticles in stimulating osteogenesis of printed bone marrow‐derived human mesenchymal stem cells (hMSCs) in poly(ethylene glycol)dimethacrylate (PEGDMA) scaffold. hMSCs suspended in PEGDMA were co‐printed with nanoparticles of bioactive glass (BG) and hydroxyapatite (HA) under simultaneous polymerization so the printed substrates were delivered with highly accurate placement in three‐dimensional (3D) locations. hMSCs interacted with HA showed the highest cell viability (86.62 ± 6.02%) and increased compressive modulus (358.91 ± 48.05 kPa) after 21 days in culture among all groups. Biochemical analysis showed the most collagen production and highest alkaline phosphatase activity in PEG‐HA group, which is consistent with gene expression determined by quantitative PCR. Masson's trichrome staining also showed the most collagen deposition in PEG‐HA scaffold. Therefore, HA is more effective comparing to BG for hMSCs osteogenesis in bioprinted bone constructs. Combining with our previous experience in vasculature, cartilage, and muscle bioprinting, this technology demonstrates the capacity for both soft and hard tissue engineering with biomimetic structures.     

Scaffold‐free inkjet printing of three‐dimensional zigzag cellular tubes

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160. © 2012 Wiley Periodicals, Inc.     

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105⁺/CD45,⁻ 10.3% HLA-DR,⁺ 100.0% CD90,⁺ and 99.2% CD73⁺/CD31⁻ expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 ± 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

Vascular engineering using human embryonic stem cells

Engineering vascularized tissue constructs remains a major problem in regenerative medicine. The formation of such a microvasculature—like the vasculogenesis in early embryogenesis that it closely resembles—is guided by biochemical and biophysical cues, such as growth factors, extracellular matrix proteins, hypoxia, and hydrodynamic shear. As they undergo spontaneous and directed vascular differentiation, human embryonic stem cells can be used as a model system to explore central issues in engineering vascularized tissue constructs and, potentially, to elucidate vasculogenic and angiogenic mechanisms involved in such vascular diseases as limb and cardiac ischemia. Because the conventional spontaneous differentiation approach can only isolate small quantities of vascular cells, recent efforts have sought to develop controlled approaches, including the development of three‐dimensional scaffolds to reengineer the microenvironments of early embryogenesis. This review focuses on emerging approaches to deriving and directing vasculatures from human embryonic stem cells and efforts to engineer 3D vasculatures from such derivatives. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

ECM concentration and cell-mediated traction forces play a role in vascular network assembly in 3D bioprinted tissue

Tissue vascularization is critical to enable oxygen and nutrient supply. Therefore, establishing expedient vasculature is necessary for the survival of tissue after transplantation. The use of biomechanical forces, such as cell-induced traction forces, may be a promising method to encourage growth of the vascular network. Three-dimensional (3D) bioprinting, which offers unprecedented versatility through precise control over spatial distribution and structure of tissue constructs, can be used to generate capillary-like structures in vitro that would mimic microvessels. This study aimed to develop an in vitro, 3D bioprinted tissue model to study the effect of cellular forces on the spatial organization of vascular structures and tissue maturation. The developed in vitro model consists of a 3D bioprinted polycaprolactone (PCL) frame with a gelatin spacer hydrogel layer and a gelatin–fibrin–hyaluronic acid hydrogel layer containing normal human dermal fibroblasts and human umbilical vein endothelial cells printed as vessel lines on top. The formation of vessel-like networks and vessel lumens in the 3D bioprinted in vitro model was assessed at different fibrinogen concentrations with and without inhibitors of cell-mediated traction forces. Constructs containing 5 mg/ml fibrinogen had longer vessels compared to the other concentrations of fibrinogen used. Also, for all concentrations of fibrinogen used, most of the vessel-like structures grew parallel to the direction the PCL frame-mediated tensile forces, with very few branching structures observed. Treatment of the 3D bioprinted constructs with traction inhibitors resulted in a significant reduction in length of vessel-like networks. The 3D bioprinted constructs also had better lumen formation, increased collagen deposition, more elaborate actin networks, and well-aligned matrix fibers due to the increased cell-mediated traction forces present compared to the non-anchored, floating control constructs. This study showed that cell traction forces from the actomyosin complex are critical for vascular network assembly in 3D bioprinted tissue. Strategies involving the use of cell-mediated traction forces may be promising for the development of bioprinting approaches for fabrication of vascularized tissue constructs.     

Three-dimensional printing biotechnology for the regeneration of the tooth and tooth-supporting tissues

The tooth and its supporting tissues are organized with complex three-dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth-supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration.     

Cellules souches et biomatériaux injectables pour la médecine régénératrice du cartilage : le consortium « chondrograft »

Articular cartilage is a non innerved, nonvascularized and poorly cellularized connective tissue that is frequently damaged as a result of trauma or age-linked degenerative diseases. It hardly heals spontaneously and its alterations often lead to further extracellular matrix degradation and ultimately, to the loss of joint function. Past decades, many therapeutic approaches have been developed to improve the poor intrinsic self-repair properties of cartilage. Unfortunately, these techniques have not proved really satisfying. In this context, the regeneration of a functional cartilage through tissue engineering and regenerative medicine has recently been contemplated. In particular, the transplantation of autologous reparative cells using a synthetic biomaterial appears promising. We have thus developed and patented a biocompatible self-setting cellulose hydrogel that can be used as an injectable scaffold for cell-based regenerative medicine. Our studies associate this hydrogel with adult mesenchymal stem cells derived from adipose tissue, as a source of reparative cells for cartilage tissue engineering. In a first set of experiments, we have determined the optimal culture conditions required to induce the controlled chondrogenic commitment of stem cells (morphogens, hypoxia, three-dimensional environments…). The preclinical potential of hybrid constructs associating cells and hydrogel has then been assessed with success in animals (mouse, rabbit). Today, trauma and degenerative pathologies of joint tissues remain a major challenge for clinicians and cartilage engineers. Establishing the proof of concept of hydrogel-associated stem cells-based regenerative medicine could help us open new therapeutic windows in the treatment of joint disorders.     

3D bioprinting and the current applications in tissue engineering

Bioprinting as an enabling technology for tissue engineering possesses the promises to fabricate highly mimicked tissue or organs with digital control. As one of the biofabrication approaches, bioprinting has the advantages of high throughput and precise control of both scaffold and cells. Therefore, this technology is not only ideal for translational medicine but also for basic research applications. Bioprinting has already been widely applied to construct functional tissues such as vasculature, muscle, cartilage, and bone. In this review, the authors introduce the most popular techniques currently applied in bioprinting, as well as the various bioprinting processes. In addition, the composition of bioink including scaffolds and cells are described. Furthermore, the most current applications in organ and tissue bioprinting are introduced. The authors also discuss the challenges we are currently facing and the great potential of bioprinting. This technology has the capacity not only in complex tissue structure fabrication based on the converted medical images, but also as an efficient tool for drug discovery and preclinical testing. One of the most promising future advances of bioprinting is to develop a standard medical device with the capacity of treating patients directly on the repairing site, which requires the development of automation and robotic technology, as well as our further understanding of biomaterials and stem cell biology to integrate various printing mechanisms for multi‐phasic tissue engineering.     

Effect of neuron‐derived neurotrophic factor on rejuvenation of human adipose‐derived stem cells for cardiac repair after myocardial infarction

The decline of cell function caused by ageing directly impacts the therapeutic effects of autologous stem cell transplantation for heart repair. The aim of this study was to investigate whether overexpression of neuron‐derived neurotrophic factor (NDNF) can rejuvenate the adipose‐derived stem cells in the elderly and such rejuvenated stem cells can be used for cardiac repair. Human adipose‐derived stem cells (hADSCs) were obtained from donors age ranged from 17 to 92 years old. The effects of age on the biological characteristics of hADSCs and the expression of ageing‐related genes were investigated. The effects of transplantation of NDNF over‐expression stem cells on heart repair after myocardial infarction (MI) in adult mice were investigated. The proliferation, migration, adipogenic and osteogenic differentiation of hADSCs inversely correlated with age. The mRNA and protein levels of NDNF were significantly decreased in old (>60 years old) compared to young hADSCs (<40 years old). Overexpression of NDNF in old hADSCs significantly improved their proliferation and migration capacity in vitro. Transplantation of NDNF‐overexpressing old hADSCs preserved cardiac function through promoting angiogenesis on MI mice. NDNF rejuvenated the cellular function of aged hADSCs. Implantation of NDNF‐rejuvenated hADSCs improved angiogenesis and cardiac function in infarcted mouse hearts.     

Helical spring template fabrication of cell‐laden microfluidic hydrogels for tissue engineering

Cell‐laden microfluidic hydrogels find great potential applications in microfluidics, tissue engineering, and drug delivery, due to their ability to control mass transport and cell microenvironment. A variety of methods have been developed to fabricate hydrogels with microfluidic channels, such as molding, bioprinting, and photopatterning. However, the relatively simple structure available and the specific equipment required limit their broad applications in tissue engineering. Here, we developed a simple method to fabricate microfluidic hydrogels with helical microchannels based on a helical spring template. Results from both experimental investigation and numerical modeling revealed a significant enhancement on the perfusion ability and cell viability of helical microfluidic hydrogels compared to those with straight microchannels. The feasibility of such a helical spring template method was also demonstrated for microfluidic hydrogels with complex three‐dimensional channel networks such as branched helical microchannels. The method presented here could potentially facilitate the development of vascular tissue engineering and cell microenvironment engineering. Biotechnol. Bioeng. 2013; 110: 980–989. © 2012 Wiley Periodicals, Inc.