Tissue engineering and regenerative medicine in applied research: a year in review of 2014.

Page 1 of 36

Tissue Engineering Part B: Reviews Tissue Engineering and Regenerative Medicine in Applied Research: A Year in Review of 2014 (doi: 10.1089/ten.TEB.2015.0004) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1

Tissue Engineering and Regenerative Medicine in Applied Research: A Year in Review of 2014 Xunxun Lin, M.D. 1, Jia Huang, M.D.1, Yuan Shi, M.D. 1, and Wei Liu, M.D., Ph.D.#

Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering Research, National Tissue Engineering Center of China

1

Authros who contributed equally

#Corresponding author Dr. Wei Liu Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital Shanghai Jiao Tong University School of Medicine 639 Zhi Zao Ju Rd Shanghai, 200011, P.R. China Tel: +86-21-63138341, Ext 5061 Fax: +86-21-53078128 Email: [email protected]

Running Title: TERM 2014: A YEAR IN REVIEW-Applied Research

Page 2 of 36


2 Abstract Tissue engineering and regenerative medicine (TERM) remains to be one of the fastest growing fields, which covers a wide scope of topics of both basic and applied biological researches. This overview article summarized the advancements in applied researches of TERM area including stem cells mediated tissue regeneration, material science and TERM clinical trial. These achievements demonstrated the great potentials of clinical regenerative therapy of tissue/organ disease or defect via stem cells and tissue engineering approaches.

Keywords: Pluripotent stem cells; Tissue regeneration; Biomimetic materials; Clinical trials

Page 3 of 36


3

The Aim and Methods This part serves as Part Two of "A Year in Review", the applied research. The same method was adopted to perform this review as described in Part One. Advancements in Stem Cell Mediated Tissue Regeneration In 2014, significant advancements were made in pluripotent stem cells (PSC)-mediated tissue and organ regeneration. Organ development is a complicated process and is difficult to define the exact network that regulates the development process. Using single cell as a model, Durruthy- Durruthy reported the reconstruction of mouse otocyst and early neuroblast lineages based on highly parallel quantitative RT-PCR measurements of 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling pathway associated transcripts, and novel otic-specific genes.1 Wen and Tang further proposed reconstructing complex tissues from single-cell analyses, because the techniques for profiling individual cells become available, such an approach provided an unprecedented opportunity for studying the genetic regulation of development and disease. Naturally, it also provides essential information to guide organ regeneration.2 This strategy is well presented in Figure 1. Induced PSCs (iPSCs) derived from a patient of genetic disease are an invaluable cell model to study genetic disorders in culture dish. Wang et al. reported that this could be combined with tissue engineering technique to generate an organ model on chip with a particular genetic disorder for studying the mechanism.3 They chose

Page 4 of 36


4 mitochondrial cardiomyopathy of Barth Syndrome (BTHS) as a disease model and generated patient derived iPSCs, which were further differentiated into BTHS iPSC-derived cardiomyocytes (iPSC-CMs). Using gene transfer to knock-down TAZ (encoding tafazzin) gene in normal iPSCs or overexpress the gene in BTHS-iPSCs, they were able to confirm the causative role of TAZ gene in BHHS with defined characters of metabolic, structural and functional abnormalities. More interestingly, the iPSC-CMs were able to self-organize into a laminar anisotropic myocardium, so called "heart on a chip", which allowed them observing sarcomere assembly and myocardial contraction at tissue level. Indeed, this is a good example how stem cells can be combined with tissue engineering to set up a clean in vitro tissue model for disease study, and this is certainly applicable to the mechanism study of tissue or organ regeneration. Chong et, al. reported the use of human ES cells derived myocardiocytes hESC-CMs to regenerate non-human primate hearts following myocardial ischemia.4 They showed that hESC-CM production could be scaled up more than one billion cells per batch, a suitable clinical scale. Intra-myocardial delivery of one billion hESC-CMs generated extensive remuscularization of the infarcted heart. hESC-CMs exhibited progressive but incomplete maturation over three month time period. More importantly, grafts could be perfused by host vasculature, and electromechanical junctions could be established between graft and host myocytes within 2 weeks of engraftment. Furthermore, the grafts revealed regular calcium transients that were synchronized to the host electrocardiogram, an evidence of electromechanical

Page 5 of 36


5 coupling. To enhance the proliferation of human PSCs, Sanz-Garcia et al. reported an interesting finding that the use of extracellular matrix (ECM) extracted from human fibroblasts as coating substance plus fibroblast derived conditional medium could efficiently maintain the pluripotency of expanded human embryonic stem cells for more than 40 passages without the need of feeder cells.5 For whole heart regeneration via recellularizing the decellularized heart, the real challenge is to rebuild a pacemaker and heart conduction system. Hu et al. reported an interesting work of creating a biological pacemaker by in vivo reprogramming.6 It has been shown that reexpression of the human embryonic transcription factor T-box 18 (TBX18) could convert ordinary ventricular cardiomyocytes into pacemaker cells, also called induced sinoatrial node (iSAN) cells. In this study, they employed minimally invasive percutaneous gene delivery to achieve in vivo somatic reprogramming in a porcine model of complete heart block via adenoviral vector mediated overexpression of TBX18 gene. They showed that TBX18 converted ventricular myocytes to iSAN cells in situ and the biological pacemaker activity originated at the focal injection site. TBX18 biological pacemaker not only supported daily physical activity, but also did not increase arrhythmic risk. In the future, the possible strategies for cardiac regeneration and repair were proposed by Lin and Pu.7 Whereas Matsa et al. proposed the use of iPSC-derived cardiomyocytes for screen of cardiac drug and toxicity testing and in vitro modeling of cardiac disease such as cardiac channelopathies and cardiomyopathies.8

Page 6 of 36


6 There are several reports of reprogrammed organ regeneration with significant progress. Kidney is a complex organ gradually developed from pluripotent stem cells of an early embryo through a series of developmental stages. Therefore, defining the exact roles of PSCs in kidney development will provide an insight into the strategy of designing PSC-mediated kidney regeneration. Taguchi et al.’s report represents such an example of combining developmental biology and stem cell biology for organ regeneration.9 In the process of kidney development, intermediate mesoderm that expresses transcription factor Osr1 appears around embryonic day 8.5 (E8.5), which gives rise to metanephric mesenchyme (MM) and the ureteric bud. The reciprocally inductive interactions between these two precursor tissues lead to the formation of embryonic metanephros, which further develop into the complex structure of kidney. In their study, they defined the origins of the metanephric mesenchyme, and found that posteriorly located T+ MM precursors were developmentally distinct from Osr1+ ureteric bud progenitors during the postgastrulation stage, and phasic Wnt stimulation along with stage-specific growth factor addition could promote their development into MM. With the derived information, they were further able to induce mouse and human PSCs into metanephric nephron progenitors, which formed the structure of nephric tubules and glomeruli. Although this work represents an initial trial, it can be considered as a big step toward kidney reconstruction in vitro.9 In another study, Takasato et al. reported that directing differentiation of human ESCs into a renal lineage could generate a self-organizing kidney.10 This approach was nicely depicted in Figure 2 by Humphreys in his commentary article.11

Page 7 of 36


7 Liver is also a vital organ that is possible to be regenerated by PSCs. Takebe et al. reported a protocol for in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after its transplantation at various ectopic sites.12 Briefly, hepatic cells derived form iPSCs were mixed with endothelial cells and mesenchymal stem cells, and these mixed cells formed a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1-2 days. Interestingly, after in vivo implantation into nude mice, 4 day old iPSC-LB became further mature with host blood perfusion into human vascular networks and exhibited multiple hepatic functions in a chronological manner in vivo. Zhu et al. reported the repopulation of mouse liver with hepatocytes generated from human fibroblasts.13 They first generated an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-HEPs) were generated with the assistance of identified small molecules. Following in vivo implantation into an immune-deficient mouse model of human liver failure, iMPC-HEPs repopulated mouse livers and proliferated extensively with acquired levels of hepatocyte function. Thymus is an organ essential for the development of immune system, particularly for T cell maturation via it’s interaction with thymic epithelial cells (TECs). In a report, Bredenkamp et al. discovered that overexpression of FOXN1, a transcription factor critically required for TEC development, could directly reprogram moue embryonic fibroblasts (MEFs) into TECs (iTECs).14 They further demonstrated that iTECs could support T-cell development in vitro. When aggregated with fetal

Page 8 of 36


8 thymocytes and fetal thymic mesenchymal cells and upon in vivo grafting under kidney capsule, iTECs formed a functional thymus in vivo, which supported intrathymic T-cell development and generated a diverse and functional peripheral T-cell repertoire. This achievement represents the first demonstration of the generation of a complete, organized, and functional complex organ from reprogrammed cells.14 Likewise, lung is another major organ that might be regenerated via pluripotent stem cells. What has been achieved and how to translate were well summarized in the papers.15,16 Platelet is a crucial component of blood, which is frequently insufficient to meet transfusion needs. Nakamura et al. reported the establishment of stably immortalized megakaryocyte progenitor cell lines (imMKCLs). Both human ESCs and human iPSCs were used to derive human megakaryocyte progenitor cells. Further, they immortalized them into a stable cell line via overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. Interestingly, imMKCLs could be expanded for 4-5 months to produce a large quantity of megakaryocytes in vitro. More importantly, the expanded and even cryopreserved imMKCLs could generate human platelet functionality comparable to that of native platelets as confirmed by a range of assays in vitro and in vivo.17 Cartilage and muscle are the indispensable components of musculoskeletal system, thus the mimicking of developmental processes of these tissues may provide appropriate strategies for regenerating these tissues. Takebe et al. made the progress

Page 9 of 36


9 in cartilage engineering with such an approach.18 This was done by mimicking early stage of chondrogenesis in which transient vascularization could trigger expansion of cartilage progenitor cells (CPCs) and promote self-aggregation of a 3D condensation of progenitors called cellular condensation, eventually leading to cartilage formation. They isolated ear–derived CPCs and mixed them with HUVECs (human vascular endothelial cells) and cultured them in growth factor free and scaffold free condition to allow for self-formation of 3D immature condensed tissue. After in vivo implantation, this condensed tissue further developed into cartilage tissue. The results of this study indicate the importance of intercellular communication as a principal approach for regenerating simple and avascular tissues which is usually composed of single cell type. In another paper related to cartilage regeneration, Pelttari et al. discovered that adult human neuroectoderm-derived nasal chondrocytes (NCs) could be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes. Additionally, NCs exhibited more potent self-renewal and environmental plasticity following serial cloning. Upon transplantation to articular cartilage environment, NCs could acquire HOX expression pattern similar to that of chondrocytes of recipient site.19 The scientific finding revealed in this study sketches the feasibility of repairing articular cartilage with nasal cells. Human satellite cells are scarce and in vitro expansion leads to phenotype drifting and functional loss. In a study reported by Marg et al., they discovered that PAX7+

Page 10 of 36


10 satellite cells became activated and proliferated efficiently in culture, only when they were attached to the human muscle fibers. They isolated human muscle fiber filaments (HMFF) along with attached satellite cells and cultured them in vitro. Surprisingly, through this in vitro culture, as few as 2 to 4 PAX7+ satellite cells could give rise to several thousand myoblasts. More importantly, implantation of in vitro cultured HMFFs in vivo resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to stem cell niche. Thus, gene manipulation of attached satellite cells represents a novel approach of gene therapy of muscular disease in the future.20 In addition to the potential of regenerating muscle tissue, muscle derived stem/progenitor cells can also be applied to peripheral nerve regeneration. Lavasani et al. reported that human muscle-derived stem/progenitor cells could adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model and reduced the atrophy of degenerated muscle.21 Koehler and Hashino reported the generation of inner ear organoids via 3D mouse embryonic stem cell culture.22 They have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids, and the developed method provided a reproducible and scalable means to generate inner ear sensory tissue in vitro. In 2014’s literature, researchers reported an influential finding of stem cell mediated tissue repair and regeneration called adaptive transition of quiescent stem

Page 11 of 36


11 cells from G0 to GAlert via systemic signals triggered by a remote injury. Generally, most stem cells are in a quiescent state and become activated upon tissue injury. Rodgers et al. used satellite cells as a model to demonstrate that quiescent satellite cells were composed of two distinct functional phases, G0 and GAlert phases. When a tissue was injured in a distant site, mTORC1 activity could cause the transition of satellite cells from G0 into GAlert via the signaling mediated by HGF (hepatic growth factor) receptor cMet. The ability of this dynamic transition between these two phases is critical to the positioning of stem cell populations to be able to respond rapidly in tissue homeostasis and repair while maintaining a pool of deeply quiescent and reserved stem cells.23 In fact, such a transition may also exist in other tissues and represent a universal mechanism of stem cell mediated tissue regeneration.24 Advancements in Engineered Tissue/Organ Regeneration Generation of tissue/organ via decellularization and recellularization is likely to be advanced with improved technical protocol. Jungebluth et al. described a detailed protocol of generating natural decellularized trachea or artificial electrospun nanofiber scaffolds and in vivo transplantation in a rat model, which allowed for reproducible analysis of the critical aspects for in situ tracheal tissue restoration and regeneration.25 Jacques et al. presented a protocol of obtaining decellularized complex organs (i.e., hearts, lungs and kidneys) by applying antegrade perfusion of detergents and subsequent washes to arterial vasculature at low physiological pressures,26 which also served as important models for pre-clinical studies.

Page 12 of 36


12 One impressive achievement in this field is the generation of bioengineered skin containing lymphatic plexuses as reported by Marino et al.27 They isolated human dermal microvascular endothelial cells (HDMECs, containing both lymphatic and blood vascular endothelial cells) from foreskin, and embedded them in three-dimensional fibrin or collagen hydrogels along with keratinocytes and dermal fibroblasts, thus creating a bioartificial skin with lymphatic capillary. These capillaries could branch, form lumen, and take up fluid in vitro and in vivo after transplantation into immunocompromised rodents. This artificial skin is likely to serve as a model for in vitro testing as the formation of lymphatic capillaries could be modulated by both lymphangiogenic and anti-lymphangiogenic stimuli. Interestingly, upon transplantation on a wound, engineered lymphatic capillary could be connected with recipient’s lymphatic plexus and fulfill the function of lymphatic drainage. In another study, Sánchez-Muñoz et al. reported the engineering of endothelialized skin structure using adipose mesenchymal stem cells and human umbilical vascular endothelial cells along with a fibrin matrix, and thus provided a novel therapeutic approach to different skin defects and a useful tool for regenerative medicine.28 Advancements in Materials Science Hydrogels are one of excellent scaffold materials for tissue engineering and regenerative medicine, and significant achievements were accomplished in the past years in both technical innovation and wide ranged applications.29 Photo-crosslinking of hydrogel is a mature technique, but creates significant impact on cell biology study

Page 13 of 36


13 once it is applied to cell culture system. Liu et al. demonstrated a new concept of photo-crosslinking mechanism, in which light controls where and when thiol-Michael interaction occurs in a 3D gel and light dose controls gel stiffness, thus providing a unique approach particularly suitable for fabricating 3D cell-culture hydrogel matrix.30 The freely tunable mechanical properties, fast gelation time, broad gelation conditions, highly precise photo-controllability, and excellent cytocompatibility make such synthetic hydrogels a powerful tool for important applications ranging from basic cell biology to 3D bioprinting and regenerative medicine. Lin et al. reported the development of an injectable, biodegradable methacrylated gelatin (mGL)–based hydrogel that was capable of rapid gelation via visible light (VL)–activated crosslinking in air or aqueous solution. Further, this hydrogel with mild photocrosslinking conditions permitted the encapsulation of human bone marrow derived mesenchymal stem cells and supported their chondrogenesis. Thus, it represented a promising scaffold for cell-based repair and resurfacing of articular cartilage defects.31 Smith et al. reported the development of an injectable, triple-interpenetrating-network hydrogel comprised of dextran, chitosan, and teleostean followed by a series of in vitro characterization after seeding with mesenchymal stem cells. The results of the study revealed the therapeutic potential of this novel hydrogel for functional regeneration of the NP.32 One exciting achievement of last year material science research is the use of high quantum bulk heterojunctions (BHJ) polymer structure as an active photosensitive,

Page 14 of 36


14 wiring free epiretinal interface to stimulate an embryonic-stage blind chick retina as reported by Gautam et al.33 They showed that the BHJ layer could initiate photoelectric signals and evoke neuronal activity similarly as the natural response of the retina to light stimulation. Then these responses were also investigated as the functions of light intensity, color, light pulse-width and repetition rate, indicating the possibility of using moderate light levels to generate visual signal with encoded information. The bulk heterojunction layer can be further patterned and become a component of retinal prosthesis without an external power source. Bioinspired materials reflect the trend in the combination of material science and tissue regeneration, which are gradually evolved from one to three dimensions with significantly enhanced biomimetic functions.34 One example of applied bioinspired material research is the superhydrophobic surface with special wetting and adhesion properties, which has gained attention due to its potential for biological applications. Seo et al. presented a novel and facile strategy to potentiate the therapeutic efficacy of 3D stem cell spheroids using bio-inspired superhydrophobic surfaces.35 The authors used a switchable water-adhesive, super-hydrophobic nanowire surface for the formation of functional human adipose-derived stem cell spheroids, of which the sizes were readily controllable on the surface. It increased cell-cell and cell-matrix interaction, and thus improved viability and paracrine secretion of the spheroids. They also observed that human adipose-derived stem cell spheroids produced on the surface exhibited significantly enhanced angiogenic efficacy. To generate bioinspired materials, technical development is another major pass.

Page 15 of 36


15 Nanofibers or hydrogels with non-covalently assembled functional peptides, such as ligands or epitopes fused to fibrillizing peptides, are promising materials with great potentials for biomedical and biotechnological applications including tissue engineering and regenerative medicine as well as biosensor and drug delivery, etc. The key point is that, when desired sets of expressed functional proteins are induced to assemble directly onto nanofibres or gels, gradated control over the amount of each protein ultimately being incorporated is needed. Since most peptides will become self-assembly or aggregated during their expression, it is necessary to maintain them in a soluble state during purification and storage, and then subsequently induce their co-assembly at a desired time and place. To achieve this, Hudalla et al. reported the use of -tail tag fused to engineered proteins, which allowed proteins to be expressed and purified in a soluble form and then induced to assemble at a desired time point via mixing them with additional fibrillizing peptides. Consequently, multiple different β-Tail fusion proteins could be inserted at predictable, smoothly gradated concentrations, providing a simple yet versatile route for installing precise combination of proteins into nanomaterials.36 It is believed that this advanced technique will be able to generate bioactive functional scaffold materials that can regulate stem cells' function and tissue regeneration. In a review by Murphy et al., they discussed recent evidence showing that inherent material properties might be engineered to dictate stem cell fate decisions, and also overviewed a subset of operative signal transduction mechanisms that had begun to emerge. Besides, they pointed out that rapid progress in stem cell engineering and

Page 16 of 36


16 mechanotransduction tended to have substantial implications for stem cell biology and regenerative medicine.37 Indeed, using designed and tailored materials to direct the fate of stem cells is currently an important subject in material science as well as in stem cell biology. There are numerous examples that can illustrate how to combine insoluble and soluble factors to steer stem cell fate, in which properly designed metatarsal can serve the insoluble part.38 One typical example will be the influence of material mechanical properties on stem cells through their mechanical memory as reported by Yang et al. By culturing human mesenchymal stem cells on substrates with different stiffness, they found that mechanical property could direct specific lineage differentiation of human mesenchymal stem cells, and stem cells possessed mechanical memory and could store information from past physical environments.39 Interestingly, the report by Wen et al. implied that the stiffness of planar matrices could regulate stem cell differentiation independently of protein tethering and porosity.40 The topography of nanoscale surface also significantly influences stem cell fate. Particularly, the interaction of nanotopographical features with integrin receptors in the cells focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. Exploitation of these interactions enables researchers to direct stem cell fate and understand the ways of stem cell behavior in their niche, which can be properly controlled.41 Several research papers in last year literature provided such examples. Gautrot et al. showed that the nanoscale geometry of the adhesive landscape dictated the fate

Page 17 of 36


17 decision of epidermal stem cells via the control of cell shape and associated change in activator protein 1 transcription activity.42 The authors found that cells responded to such hard nanopatterns similarly to their behavior on soft hydrogels and cellular responses were observed to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction. This could contribute to the design of novel materials and engineered platforms for regenerative medicine applications. Shah et al. also reported an interesting finding that neuronal stem cells could be guided for differentiation into oligodendrocytes simply using graphene-nanofiber hybrid scaffolds. Polymeric nanofibers (composed of polycaprolactone) generated using electrospinning were subsequently coated with graphene oxide to form a hybrid scaffold. Then neuronal stem cells were cultured on the scaffold without adding any inducers. Interestingly, when examined with gene expression of markers indicative of neurons (TuJ1), astrocytes (GFAP) and oligodendrocytes (MBP), this particular material induced the highest expression of MBP, indicating a specific lineage differentiation likely to be induced via the particular topography of nanofibers. This finding may also be translated into the regeneration of central nervous system in the future.43 The other application of materials in stem cell biology is to serve as the substrate for large scale stem cell production, so as to translate it from laboratory-based research to industrial-scale biomedicine.44 Similarly, materials can also be applied to generate in vivo biomimetic environment for cell and tissue growth. Different from in

Page 18 of 36


18 vitro culture environment, in vivo cells reside in a highly crowded extracellular space and therefore the conversion of the de novo synthesized procollagen to collagen I is rapid. To imitate native tissue localized density, Satyam et al. inserted polydispersed macromolecules in the culture media and termed this macromolecular crowding (MMC).45 The thermodynamic activities and biological processes of the cultured cells were increased by several orders of magnitude in MMC environment, and MMC ultimately produced ECM-rich cell substitutes at low serum concentration within hours without compromising fundamental cellular functions. Such system can be applied to mature engineered tissue in vitro by mimicking in vivo MMC microenvironments. Since that traditional tissue engineering approach needs to seed cells into a bulk scaffold to engineer a live tissue with a desired size, it usually causes non-even distribution of seeded cells and central tissue necrosis. Reversely, a material-based bottom-up tissue engineering approach was proposed towards an assembly of cells and engineered micro-objects at the macroscale as illustrated in Figure 3. Leferink et al. showed that shape, size and wettability of engineered micro-objects play important roles in the behavior of cells on these objects.46 Ultimately, this approach can not only be used as a tool to engineer complex 3D tissues of clinically relevant size, but also find its application in clinical injectable system, or in the pharmaceutical industry as a 3D model to study tissue pathology and discover potentially new drugs. 3D printing technique is primarily used to create acellular 3D scaffolds and molds, which require post-fabrication cell seeding. Direct deposition of cells or cell

Page 19 of 36


19 aggregates called bioprinting can create a live tissue, but still faces the challenge of central area necrosis due to short of blood supply. To overcome this, Kolesky et al. reported a 3D bioprinting technique that was able to integrate vascular structure and multiple tissue cell types into vascularized and heterogeneous cell-laden tissue constructs.47 They used Pluronic as the cell carrier and neonatal dermal fibroblasts and human vein endothelial cells as the cell source to reveal the feasibility of creating such a construct. This highly scalable platform permits the production of engineered tissue constructs containing both vasculature and multiple cell types, which are programmably placed within extracellular matrices. Obviously, this technique paves the ways for various applications including drug screening, fundamental studies of wound healing, angiogenesis, stem cell niches and also rapid manufacturing of functional 3D tissues and even functional organs. Holmes et al. reported the development of a novel 3D printed bi-phasic scaffold that mimicked the osteochondral region of articulate joints. They further linked acetylated collagen to the 3D printed bi-phasic key scaffold. This scaffold exhibited enhanced mechanical property and could mediate MSC proliferation and chondrogenic differentiation in vitro.48 Shtrichman et al. reported the generation hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, which could serve as a "off-the-shelf" implantable product for tissue regeneration.49 Gene delivery is also an important application of biomaterials by using material-based non-viral vectors. Different from viral vector which has a core DNA

Page 20 of 36


20 inside an envelope protein that protects the DNA, non-viral vector is usually degraded and removed by the cells, generally triggered by one specific enzyme, acid or reductive. To develop virus-mimicking gene carriers via reduction-controlled hierarchical unpacking, He et al. mimicked the virus’ “Trojan Horse” strategy to build a dual reduction-sensitive system for programmed gene transfection. They produced a polyplexes called DOS (DNA/OEI-SeSex/HA -SS-COOH) by depositing disulfide-bond-modified hyaluronic acid onto the surface of diselenide-conjugated oligoethylenimine/DNA polyplexes. With this strategy, the core DNA could be protected and would not be exposed until the cleavage of the disulfide and diselenide bonds triggered by the gradient GSH level at the tumor site and inside the cells. As a result, the transfection efficiency of DOS showed significant enhancement over DNA/polyethyleneimine in vitro and in vivo.50 Among other non-viral methods, there was a notable development reported by Kurz et al. They used a synthetic nanopore to construct a single cell gene delivery system based on electroporation, which was not only highly specific and efficient but also transfected with single molecule resolution at low voltage (1V) with minimal perturbation to the cell. The main advantage of this system is that it can be used to control gene expression with unprecedented precision.51 Rajala et al. reported a biomaterial based non-viral vector gene deliver for tissue regeneration. They created a nanoparticle, liposome-protamine-DNA complex (LPD), modified with a cell permeable peptide and a nuclear localization signaling (NLS) peptide.52 The authors used this artificial virus to deliver a functional gene of retinal

Page 21 of 36


21 pigment epithelium protein 65 (Rpe65), which is the key enzyme for regulating the availability of photochemical for eye disease treatment, to the Rpe65 gene lacking mice. LPD promoted efficient delivery in a cell specific-manner, and a long-term expression of Rpe65 was observed, leading to in vivo correction of blindness. Thus, LPD nanoparticles could provide a promising and efficient nonviral method for gene delivery with clinical applications. Clinical Trials of Tissue Engineering and Regenerative Medicine Raya-Rivera et al. reported a pilot cohort study of clinical trial of tissue engineered vaginal organ. 53 In this report, 4 patients with congenital vaginal aplasia were involved with up to 8 years follow-up. Briefly, autologous epithelial and muscle cells were isolated, expanded and seeded onto biodegradable scaffold for in vitro culture and maturation followed by in vivo implantation. Yearly biopsy demonstrated a tri-layered structure composed of an epithelial cell-lined lumen surrounded by matrix and muscle, with expected components of vaginal tissue present. MRI and vaginoscopy revealed no abnormality of virginal structure. In addition, female Sexual Function Index questionnaire indicated normal virginal functions. This is the first report of reconstructing human sexual organs. Fulco et al. reported engineered autologous cartilage tissue for nasal reconstruction after tumor resection.54 Five cases were enrolled for engineered reconstruction of alar lobule cartilage defects resulted from tumor excision. In the report, the authors harvested nasal septum during tumor biopsy and in vitro expanded isolated chondrocytes. The cells were then seeded onto porcine collagen membrane

Page 22 of 36


22 and in vitro cultured to form cartilage membrane tissue. After tumor excision, engineered cartilage was implanted and covered with a skin flap. At 6 months post-surgery, biopsy demonstrated the presence of all three layers of the nasal tissues. One year follow-up demonstrated the safety and feasibility of engineered repair of nasal tissue, a significant achievement in plastic and reconstructive surgery as shown in Figure 4. Conclusion Remark This article serves as an overview of applied research advancements in TERM field with particular focus on stem cell mediated tissue regeneration, engineered tissue/organ regeneration and material-based tissue regeneration. The impressive achievements can be summarized in these aspects: (1) Significant advancements were

achieved in puluripotent potent stem cells mediated

tissue/organ regeneration, such as human ES cell mediated heart regeneration in non-human primate model, creation of a biological pacemaker by in vivo reprogramming; iPS mediated liver regeneration; generation of complete, organized, and functional thymus organ from reprogrammed cells; iPSCs generated and immortalized megakaryocyte progenitor cell lines, which could produce large quantities of functional human platelets in vitro; and mouse ES generated inner ear organoids. (2) Achievements were made in engineered tissue/organ regeneration, such as the generation of bioengineered skin containing lymphatic plexuses and endothelialized skin structure. (3) Material mediated tissue regeneration including hydrogel mediated chondrogenesis and

Page 23 of 36


23 bioinspired materials mediated stem cell differentiation and expansion; (4) New concepts and approaches such as bottom-up tissue engineering approach; developmental biology oriented approaches for organ regeneration and chondrogenesis; reconstructing complex tissues from single-cell analyses; “heart on a chip” and systemic signals triggered transition of quiescent stem cells from G0 to GAlert. All these advancements represent the significant achievements in TERM applied researches and will have fundamental impact on TERM translation.

Acknowledgment This was supported by National Natural Science Foundation (31170937, 81071590).

Page 24 of 36


24

Reference 1.

2. 3.

4.

5.

6.

7. 8. 9.

10.

11. 12.

Durruthy-Durruthy, R., Gottlieb, A., Hartman, B.H., Waldhaus, J., Laske, R.D., Altman, R., and Heller, S. Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution. Cell 157, 964, 2014. Wen, L., and Tang, F. Reconstructing complex tissues from single-cell analyses. Cell 157, 771, 2014. Wang, G., McCain, M.L., Yang, L., He, A., Pasqualini, F.S., Agarwal, A., Yuan, H., Jiang, D., Zhang, D., Zangi, L., Geva, J., Roberts, A.E., Ma, Q., Ding, J., Chen, J., Wang, D.Z., Li, K., Wang, J., Wanders, R.J., Kulik, W., Vaz, F.M., Laflamme, M.A., Murry, C.E., Chien, K.R., Kelley, R.I., Church, G.M., Parker, K.K., and Pu, W.T. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat Med 20, 616, 2014. Chong, J.J.H., Yang, X., Don, C.W., Minami, E., Liu, Y.-W., Weyers, J.J., Mahoney, W.M., Van Biber, B., Cook, S.M., Palpant, N.J., Gantz, J.A., Fugate, J.A., Muskheli, V., Gough, G.M., Vogel, K.W., Astley, C.A., Hotchkiss, C.E., Baldessari, A., Pabon, L., Reinecke, H., Gill, E.A., Nelson, V., Kiem, H.-P., Laflamme, M.A., and Murry, C.E. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510, 273, 2014. Sanz-Garcia, A., Stojkovic, M., and Escobedo-Lucea, C. Growth of human pluripotent stem cells using functional human extracellular matrix. Methods Mol Biol 2014. Hu, Y.F., Dawkins, J.F., Cho, H.C., Marban, E., and Cingolani, E. Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block. Sci Transl Med 6, 245, 2014. Lin, Z., and Pu, W.T. Strategies for cardiac regeneration and repair. Sci Transl Med 6, 239, 2014. Matsa, E., Burridge, P.W., and Wu, J.C. Human stem cells for modeling heart disease and for drug discovery. Sci Transl Med 6, 239ps6, 2014. Taguchi, A., Kaku, Y., Ohmori, T., Sharmin, S., Ogawa, M., Sasaki, H., and Nishinakamura, R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 14, 53, 2014. Takasato, M., Er, P.X., Becroft, M., Vanslambrouck, J.M., Stanley, E.G., Elefanty, A.G., and Little, M.H. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 16, 118, 2014. Humphreys, B.D. Kidney structures differentiated from stem cells. Nat Cell Biol 16, 19, 2014. Takebe, T., Zhang, R.R., Koike, H., Kimura, M., Yoshizawa, E., Enomura, M., Koike, N., Sekine, K., and Taniguchi, H. Generation of a vascularized and

Page 25 of 36


25

13.

14.

15.

16. 17.

18.

19.

20.

21.

22. 23.

24.

functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 9, 396, 2014. Zhu, S., Rezvani, M., Harbell, J., Mattis, A.N., Wolfe, A.R., Benet, L.Z., Willenbring, H., and Ding, S. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93, 2014. Bredenkamp, N., Ulyanchenko, S., O'Neill, K.E., Manley, N.R., Vaidya, H.J., and Blackburn, C.C. An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts. Nat Cell Biol 16, 902, 2014. Hogan, B.L., Barkauskas, C.E., Chapman, H.A., Epstein, J.A., Jain, R., Hsia, C.C., Niklason, L., Calle, E., Le, A., Randell, S.H., Rock, J., Snitow, M., Krummel, M., Stripp, B.R., Vu, T., White, E.S., Whitsett, J.A., and Morrisey, E.E. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123, 2014. Kotton, D.N., and Morrisey, E.E. Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat Med 20, 822, 2014. Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K., Koike, T., Harimoto, K., Dohda, T., Watanabe, A., Okita, K., Takahashi, N., Sawaguchi, A., Yamanaka, S., Nakauchi, H., Nishimura, S., and Eto, K. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell 14, 535, 2014. Takebe, T., Kobayashi, S., Suzuki, H., Mizuno, M., Chang, Y.M., Yoshizawa, E., Kimura, M., Hori, A., Asano, J., Maegawa, J., and Taniguchi, H. Transient vascularization of transplanted human adult-derived progenitors promotes self-organizing cartilage. J Clin Invest 124, 4325, 2014. Pelttari, K., Pippenger, B., Mumme, M., Feliciano, S., Scotti, C., Mainil-Varlet, P., Procino, A., von Rechenberg, B., Schwamborn, T., Jakob, M., Cillo, C., Barbero, A., and Martin, I. Adult human neural crest-derived cells for articular cartilage repair. Sci Transl Med 6, 251ra119, 2014. Marg, A., Escobar, H., Gloy, S., Kufeld, M., Zacher, J., Spuler, A., Birchmeier, C., Izsvak, Z., and Spuler, S. Human satellite cells have regenerative capacity and are genetically manipulable. J Clin Invest 124, 4257, 2014. Lavasani, M., Thompson, S.D., Pollett, J.B., Usas, A., Lu, A., Stolz, D.B., Clark, K.A., Sun, B., Peault, B., and Huard, J. Human muscle-derived stem/progenitor cells promote functional murine peripheral nerve regeneration. J Clin Invest 124, 1745, 2014. Koehler, K.R., and Hashino, E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9, 1229, 2014. Rodgers, J.T., King, K.Y., Brett, J.O., Cromie, M.J., Charville, G.W., Maguire, K.K., Brunson, C., Mastey, N., Liu, L., Tsai, C.R., Goodell, M.A., and Rando, T.A. mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510, 393, 2014. Malam, Z., and Cohn, R.D. Stem cells on alert: priming quiescent stem cells after remote injury. Cell Stem Cell 15, 7, 2014.

Page 26 of 36


26 25.

26. 27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37. 38.

Jungebluth, P., Haag, J.C., Sjoqvist, S., Gustafsson, Y., Beltran Rodriguez, A., Del Gaudio, C., Bianco, A., Dehnisch, I., Uhlen, P., Baiguera, S., Lemon, G., Lim, M.L., and Macchiarini, P. Tracheal tissue engineering in rats. Nat Protoc 9, 2164, 2014. Guyette, J.P., Gilpin, S.E., Charest, J.M., Tapias, L.F., Ren, X., and Ott, H.C. Perfusion decellularization of whole organs. Nat Protoc 9, 1451, 2014. Marino, D., Luginbuhl, J., Scola, S., Meuli, M., and Reichmann, E. Bioengineering dermo-epidermal skin grafts with blood and lymphatic capillaries. Sci Transl Med 6, 221ra14, 2014. Sanchez-Munoz, I., Granados, R., Holguin Holgado, P., Garcia-Vela, J.A., Casares, C., and Casares, M. The use of adipose mesenchymal stem cells and human umbilical vascular endothelial cells on a fibrin matrix for endothelialized skin substitute. Tissue Eng Part A 2014. Annabi, N., Tamayol, A., Uquillas, J.A., Akbari, M., Bertassoni, L.E., Cha, C., Camci-Unal, G., Dokmeci, M.R., Peppas, N.A., and Khademhosseini, A. 25th Anniversary Article: Rational design and applications of hydrogels in regenerative medicine. Adv Mater 26, 85, 2014. Liu, Z., Lin, Q., Sun, Y., Liu, T., Bao, C., Li, F., and Zhu, L. Spatiotemporally controllable and cytocompatible approach builds 3D cell culture matrix by photo-uncaged-thiol Michael addition reaction. Adv Mater 26, 3912, 2014. Lin, H., Cheng, A.W., Alexander, P.G., Beck, A.M., and Tuan, R.S. Cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution. Tissue Eng Part A 20, 2402, 2014. Smith, L.J., Gorth, D.J., Showalter, B.L., Chiaro, J.A., Beattie, E.E., Elliott, D.M., Mauck, R.L., Chen, W., and Malhotra, N.R. In vitro characterization of a stem-cell-seeded triple-interpenetrating-network hydrogel for functional regeneration of the nucleus pulposus. Tissue Eng Part A 20, 1841, 2014. Gautam, V., Rand, D., Hanein, Y., and Narayan, K.S. A Polymer optoelectronic interface provides visual cues to a blind retina. Adv Mater 26, 1751, 2014. Zhao, N., Wang, Z., Cai, C., Shen, H., Liang, F., Wang, D., Wang, C., Zhu, T., Guo, J., Wang, Y., Liu, X., Duan, C., Wang, H., Mao, Y., Jia, X., Dong, H., Zhang, X., and Xu, J. Bioinspired materials: from low to high dimensional structure. Adv Mater 26, 6994, 2014. Seo, J., Lee, J.S., Lee, K., Kim, D., Yang, K., Shin, S., Mahata, C., Jung, H.B., Lee, W., Cho, S.W., and Lee, T. Switchable water-adhesive, superhydrophobic palladium-layered silicon nanowires potentiate the angiogenic efficacy of human stem cell spheroids. Adv Mater 26, 7043, 2014. Hudalla, G.A., Sun, T., Gasiorowski, J.Z., Han, H., Tian, Y.F., Chong, A.S., and Collier, J.H. Gradated assembly of multiple proteins into supramolecular nanomaterials. Adv Mater 13, 829, 2014. Murphy, W.L., McDevitt, T.C., and Engler, A.J. Materials as stem cell regulators. Adv Mater 13, 547, 2014. Dingal, P.C., and Discher, D.E. Combining insoluble and soluble factors to steer

Page 27 of 36


27

39. 40.

41. 42.

43.

44.

45.

46.

47.

48.

49.

50.

51. 52.

stem cell fate. Adv Mater 13, 532, 2014. Yang, C., Tibbitt, M.W., Basta, L., and Anseth, K.S. Mechanical memory and dosing influence stem cell fate. Adv Mater 13, 645, 2014. Wen, J.H., Vincent, L.G., Fuhrmann, A., Choi, Y.S., Hribar, K.C., Taylor-Weiner, H., Chen, S., and Engler, A.J. Interplay of matrix stiffness and protein tethering in stem cell differentiation. Adv Mater 13, 979, 2014. Dalby, M.J., Gadegaard, N., and Oreffo, R.O. Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Adv Mater 13, 558, 2014. Gautrot, J.E., Malmstrom, J., Sundh, M., Margadant, C., Sonnenberg, A., and Sutherland, D.S. The nanoscale geometrical maturation of focal adhesions controls stem cell differentiation and mechanotransduction. Nano Lett 14, 3945, 2014. Shah, S., Yin, P.T., Uehara, T.M., Chueng, S.T., Yang, L., and Lee, K.B. Guiding stem cell differentiation into oligodendrocytes using graphene-nanofiber hybrid scaffolds. Adv Mater 26, 3673, 2014. Celiz, A.D., Smith, J.G., Langer, R., Anderson, D.G., Winkler, D.A., Barrett, D.A., Davies, M.C., Young, L.E., Denning, C., and Alexander, M.R. Materials for stem cell factories of the future. Adv Mater 13, 570, 2014. Satyam, A., Kumar, P., Fan, X., Gorelov, A., Rochev, Y., Joshi, L., Peinado, H., Lyden, D., Thomas, B., Rodriguez, B., Raghunath, M., Pandit, A., and Zeugolis, D. Macromolecular crowding meets tissue engineering by self-assembly: a paradigm shift in regenerative medicine.Adv Mater 26, 3024, 2014. Leferink, A., Schipper, D., Arts, E., Vrij, E., Rivron, N., Karperien, M., Mittmann, K., van Blitterswijk, C., Moroni, L., and Truckenmuller, R. Engineered micro-objects as scaffolding elements in cellular building blocks for bottom-up tissue engineering approaches. Adv Mater 26, 2592, 2014. Kolesky, D.B., Truby, R.L., Gladman, A.S., Busbee, T.A., Homan, K.A., and Lewis, J.A. 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater 26, 3124, 2014. Holmes, B., Zhu, W., Li, J., Lee, J.D., and Zhang, L.G. Development of novel three-dimensional printed scaffolds for osteochondral regeneration. Tissue Eng Part A 2014. Shtrichman, R., Zeevi-Levin, N., Zaid, R., Barak, E., Fishman, B., Ziskind, A., Shulman, R., Novak, A., Avrahami, R., Livne, E., Lowenstein, L., Zussman, E., and Itskovitz-Eldor, J. The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications. Tissue Eng Part A 20, 2756, 2014. He, Y., Nie, Y., Cheng, G., Xie, L., Shen, Y., and Gu, Z. Viral mimicking ternary polyplexes: a reduction-controlled hierarchical unpacking vector for gene delivery. Adv Mater 12,1534, 2014 Kurz, V., Tanaka, T., and Timp, G. Single cell transfection with single molecule resolution using a synthetic nanopore. Nano Lett 14, 604, 2014. Rajala, A., Wang, Y., Zhu, Y., Ranjo-Bishop, M., Ma, J.X., Mao, C., and Rajala,

Page 28 of 36


28

53.

54.

R.V. Nanoparticle-assisted targeted delivery of eye-specific genes to eyes significantly improves the vision of blind mice in vivo. Nano Lett 14, 5257, 2014. Raya-Rivera, A.M., Esquiliano, D., Fierro-Pastrana, R., López-Bayghen, E., Valencia, P., Ordorica-Flores, R., Soker, S., Yoo, J.J., and Atala, A. Tissue-engineered autologous vaginal organs in patients: a pilot cohort study. Lancet 384, 329, 2014. Fulco, I., Miot, S., Haug, M.D., Barbero, A., Wixmerten, A., Feliciano, S., Wolf, F., Jundt, G., Marsano, A., Farhadi, J., Heberer, M., Jakob, M., Schaefer, D.J., and Martin, I. Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial. Lancet 384, 337, 2014

Tissue Engineering Part B: Reviews Tissue Engineering and Regenerative Medicine in Applied Research: A Year in Review of 2014 (doi: 10.1089/ten.TEB.2015.0004) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 29 of 36

29

Figure legends

Page 30 of 36


30

Figure 1. Dissecting developmental gene regulatory networks in single cells. An approach for comprehensive analysis of developmental gene regulatory networks in single cells is depicted. First, all genes involved in a regulatory network for a developmental process are disrupted one by one by a gene editing approach. Then, single-cell omics analyses are performed to profile the genome, epigenome, and transcriptome simultaneously within an individual cell. Finally, the quantitative characteristics of the whole gene regulation network at single-cell resolution are established, and the temporal order as well as spatial structures of the developmental process are reconstructed by informatics analyses and mathematical modeling.


31

(Permitted reprinting from reference 2)

Page 32 of 36


32

Figure 2. Generation of kidney progenitor populations and nephron structures by directed differentiation. Schematic showing the developmental stages and markers expressed during differentiation of pluripotent stem cells (PSCs) to kidney cell fates. PSCs are induced to mesendoderm, then intermediate mesoderm, and then into either ureteric bud or metanephric mesenchyme. Dissociation and re-aggregation of these two cell populations leads to selforganization of a three-dimensional structure that undergoes mesenchymal-to-epithelial transition during formation of nephron-like structures. During reaggregation, nephron formation from metanephric mesenchyme can be accomplished either by induction from ureteric bud itself, or by an exogenous Wnt4 source such as embryonic spinal cord12. Different epithelial fates in each mature nephron segment are reflected by different colors. Oct4, octamer-binding transcription factor 4; T, brachyury; MIXL1, mix paired-like homeobox; OSR1, odd-skipped related transcription factor 1; WT1, Wilms tumour 1; HOXB7, homeobox B7; SIX2, sine oculis homeobox homologue 2. (Permitted reprinting from reference 11)

Page 33 of 36


33

Figure 3. Schematic representation of bottom-up TE approach with microfabricated solid objects. (A) Micro-objects and (B) cells are cultured in (C) arrays of micro-wells to pre-aggregate. (D) Micro-objects directly after seeding. (E, F) Aggregate formation and cell fate (represented by a color change) can be infl uenced by the geometrical, mechanical, physical and chemical surface and bulk properties of the objects. (G) After pre-aggregation, multiple micro-aggregates can be transferred and pooled into geometrically defined wells to fuse into larger sized tissue constructs. (H) Subsequently, constructs of different shapes and from various cell types can be combined to form controlled complex tissues. (I) A suspension of micro-objects and


Page 34 of 36

34

cells can be applied directly into a defect site where the objects act as a filler and as

mechanical support, and ideally comprises instructive or inductive properties for the

cells to grow and differentiate. (Permitted reprinting from reference 46)


35

Figure 4. Surgical procedure in one patient. (A) Two-layer defect after wide local

excision of the skin cancer on the alar lobule. (B) Tissue engineered cartilage cut to

the right shape and ready for implantation; this patient needed cartilage support to

Page 36 of 36


36 achieve stability in the alar lobule (labelled AC) and at the upper lateral site (labelled ULC). (C,D) Tissue engineered cartilage was inserted to replace the structural support and secured by absorbable sutures. (E) Reconstruction of the outer layer with a paramedian forehead fl ap. (F) Division of the flap pedicle 2 weeks after reconstruction. (G) Intraoperative appearance of the implanted engineered tissue during refinements 6 months after reconstruction. (H) Follow-up 1 year after reconstruction. (Permitted reprinting from reference 54)

Tissue engineering and regenerative medicine in basic research: a year in review of 2014.

Tissue Engineering and Regenerative Medicine 2015: A Year in Review.

Tissue engineering and regenerative medicine: a year in review.

Stem cell research and regenerative medicine in 2014: first year of regenerative medicine in Japan.

Osteoarthritis year in review 2014: highlighting innovations in basic research and clinical applications in regenerative medicine.

tissue microenvironment engineering and monitoring in tissue engineering, regenerative medicine, and in vitro tissue models.

Regulation challenge of tissue engineering and regenerative medicine in China.

A new age of regenerative medicine: fusion of tissue engineering and stem cell research.

A new age of regenerative medicine: fusion of tissue engineering and stem cell research.

MiRNA inhibition in tissue engineering and regenerative medicine.

Implementing tissue engineering and regenerative medicine solutions in medical implants.

Mechanical cues in orofacial tissue engineering and regenerative medicine.

Emerging nanotechnology approaches in tissue engineering and regenerative medicine.

MicroRNAs in tissue engineering & regenerative medicine. Preface.

Concise review: cell-based strategies in bone tissue engineering and regenerative medicine.

On the genealogy of tissue engineering and regenerative medicine.

Designing Biopolymer Microthreads for Tissue Engineering and Regenerative Medicine.

Advanced methods for tissue engineering and regenerative medicine.

Multiscale assembly for tissue engineering and regenerative medicine.

3D biofabrication strategies for tissue engineering and regenerative medicine.

Chitosan and Its Potential Use as a Scaffold for Tissue Engineering in Regenerative Medicine.

Cell microenvironment engineering and monitoring for tissue engineering and regenerative medicine: the recent advances.

Applications and implications of heparin and protamine in tissue engineering and regenerative medicine.

Recent developments in vascular imaging techniques in tissue engineering and regenerative medicine.