Showing papers in "Tissue Engineering Part A in 2017"

MicroRNA-183-5p Increases with Age in Bone-Derived Extracellular Vesicles, Suppresses Bone Marrow Stromal (Stem) Cell Proliferation, and Induces Stem Cell Senescence.

Abstract: Microvesicle- and exosome-mediated transport of microRNAs (miRNAs) represents a novel cellular and molecular pathway for cell-cell communication. In this study, we tested the hypothesis that these extracellular vesicles (EVs) and their miRNAs might change with age, contributing to age-related stem cell dysfunction. EVs were isolated from the bone marrow interstitial fluid (supernatant) of young (3-4 months) and aged (24-28 months) mice to determine whether the size, concentration, and miRNA profile of EVs were altered with age in vivo. Results show that EVs isolated from bone marrow are CD63 and CD9 positive, and the concentration and size distribution of bone marrow EVs are similar between the young and aged mice. Bioanalyzer data indicate that EVs from both young and aged mice are highly enriched in miRNAs, and the miRNA profile of bone marrow EVs differs significantly between the young and aged mice. Specifically, the miR-183 cluster (miR-96/-182/-183) is highly expressed in aged EVs. In vitro assays demonstrate that aged EVs are endocytosed by primary bone marrow stromal cells (BMSCs), and these aged EVs inhibit the osteogenic differentiation of young BMSCs. Transfection of BMSCs with miR-183-5p mimic reduces cell proliferation and osteogenic differentiation, increases senescence, and decreases protein levels of the miR-183-5p target heme oxygenase-1 (Hmox1). In vitro assays utilizing H2O2-induced oxidative stress show that H2O2 treatment of BMSCs increases the abundance of miR-183-5p in BMSC-derived EVs, and Amplex Red assays demonstrate that H2O2 is elevated in the bone marrow microenvironment with age. Together, these data indicate that aging and oxidative stress can significantly alter the miRNA cargo of EVs in the bone marrow microenvironment, which may in turn play a role in stem cell senescence and osteogenic differentiation by reducing Hmox1 activity.

Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells.

Abstract: Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stromal cells (MSCs) promote the regeneration of kidneys in different animal models of acute kidney injury (AKI) in a manner comparable with the cells of origin. However, due to the heterogeneity observed in the EVs isolated from MSCs, it is unclear which population is responsible for the proregenerative effects. We therefore evaluated the effect of various EV populations separated by differential ultracentrifugation (10K population enriched with microvesicles and 100K population enriched with exosomes) on AKI recovery. Only the exosomal-enriched population induced an improvement of renal function and morphology comparable with that of the total EV population. Interestingly, the 100K EVs exerted a proproliferative effect on murine tubular epithelial cells, both in vitro and in vivo. Analysis of the molecular content from the different EV populations revealed a distinct profile. The 100K population, for instance, was enriched in specific mRNAs (CCNB1, CDK8, CDC6) reported to influence cell cycle entry and progression; miRNAs involved in regulating proliferative/antiapoptotic pathways and growth factors (hepatocyte growth factor and insulin-like growth factor-1) that could explain the effect of renal tubular cell proliferation. On the other hand, the EV population enriched in microvesicles (10K) was unable to induce renal regeneration and had a molecular profile with lower expression of proproliferative molecules. In conclusion, the different molecular composition of exosome- and microvesicle-enriched populations may explain the regenerative effect of EVs observed in AKI.

Priming Adipose Stem Cells with Tumor Necrosis Factor-Alpha Preconditioning Potentiates Their Exosome Efficacy for Bone Regeneration.

Abstract: Mesenchymal stem cells (MSCs) have been widely used for tissue repair and regeneration. However, the inherent drawbacks, including limited cell survival after cell transplantation, have hindered direct MSC transplantation for tissue repair and regeneration. The aim of this study was to investigate if exosomes isolated from MSCs can promote the proliferation and differentiation of human primary osteoblastic cells (HOBs) and be potentially used for bone tissue regeneration. We showed that adipose tissue-derived MSC (ASC)-derived exosomes (ASC-EXO) were able to promote the proliferation and osteogenic differentiation in HOBs; and the trophic effects of ASC-EXO on HOBs were further harnessed when ASCs were preconditioned with tumor necrosis factor-alpha (TNF-α) for 3 days, which mimics the acute inflammatory phase upon bone injury. In addition, we showed that Wnt-3a content was elevated in ASC-EXO when ASCs were preconditioned by TNF-α, and inhibiting Wnt signaling decreased the osteogenic gene expression levels in HOBs which were cultured in TNF-α preconditioned ASCs conditioned medium. In conclusion, it was demonstrated that ASC-EXO, especially primed by TNF-α preconditioning on ASCs, offer a promising approach to replace direct stem cell transplantation for bone repair and regeneration.

* Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering.

Abstract: Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Roughness and Hydrophilicity as Osteogenic Biomimetic Surface Properties.

Abstract: Successful dental and orthopedic implant outcomes are determined by the degree of osseointegration. Over the last 60 years, endosseous implants have evolved to stimulate osteogenesis without the need for exogenous biologics such as bone morphogenetic proteins. An understanding of the interaction between cells and the physical characteristics of their environments has led to development of bioactive implants. Implant surfaces that mimic the inherent chemistry, topography, and wettability of native bone have shown to provide cells in the osteoblast lineage with the structural cues to promote tissue regeneration and net new bone formation. Studies show that attachment, proliferation, differentiation, and local factor production are sensitive to these implant surface characteristics. This review focuses on how surface properties, including chemistry, topography, and hydrophilicity, modulate protein adsorption, cell behavior, biological reactions, and signaling pathways in peri-implant bone tissue, allowing the development of true biomimetics that promote osseointegration by providing an environment suitable for osteogenesis.

Extracellular Matrix Bioscaffolds as Immunomodulatory Biomaterials

Abstract: Suppression of the recipient immune response is a common component of tissue and organ transplantation strategies and has also been used as a method of mitigating the inflammatory and scar tissue response to many biomaterials. It is now recognized, however, that long-term functional tissue replacement not only benefits from an intact host immune response but also depends upon such a response. The present article reviews the limitations associated with the traditionally held view of avoiding the immune response, the ability of acellular biologic scaffold materials to modulate the host immune response and promote a functional tissue replacement outcome, and current strategies within the fields of tissue engineering and biomaterials to develop immune-responsive and immunoregulatory biomaterials.

The Immunomodulatory Role of BMP-2 on Macrophages to Accelerate Osteogenesis.

Abstract: Bone morphogenetic protein-2 (BMP-2) is one of the most frequently used osteogenic factors for osteogenesis. Macrophages, acting as both immune cells and osteoclast precursors, have an indispensable functional input during multiple stages of bone healing. This study aims to investigate the immunoregulatory role of BMP-2 on macrophages and the subsequent effects on osteogenesis. In the subcutaneous implantation study, gelatin sponge incorporated with 20 μg/mL BMP-2 rendered significantly enhanced macrophage infiltration compared with the gelatin sponge control. Further in vitro study using murine macrophage cell line has shown that BMP-2 could function as a potent attractant for macrophage recruitment. The supplementation of BMP-2 dramatically diminished the expression of M1 phenotypic markers, including interleukin (IL)-1β, IL-6, and iNOS in M1 polarized macrophages, indicating a positive immunoregulatory role of BMP-2 under inflammatory status. In addition, BMP-2 alone could robustly activate macrophages through pSmad1/5/8 signaling pathway and generate a positive feedback loop by increasing the expression of angiogenic factors. Conditioned medium collected from BMP2-stimulated macrophages accelerated the osteogenic differentiation of bone marrow stromal cells. Our finding suggests that BMP-2-induced osteogenesis may also involve the regulation of the local osteoimmune environment. The positive effect of BMP-2 on regulating immune response makes it possible to utilize the immunomodulatory properties of BMP-2 to manipulate the osteoimmune environment for favorable bone regeneration.

Biomimetic Mineralization of Biomaterials Using Simulated Body Fluids for Bone Tissue Engineering and Regenerative Medicine .

Abstract: Development of synthetic biomaterials imbued with inorganic and organic characteristics of natural bone that are capable of promoting effective bone tissue regeneration is an ongoing goal of regenerative medicine. Calcium phosphate (CaP) has been predominantly utilized to mimic the inorganic components of bone, such as calcium hydroxyapatite, due to its intrinsic bioactivity and osteoconductivity. CaP-based materials can be further engineered to promote osteoinductivity through the incorporation of osteogenic biomolecules. In this study, we briefly describe the microstructure and the process of natural bone mineralization and introduce various methods for coating CaP onto biomaterial surfaces. In particular, we summarize the advantages and current progress of biomimetic surface-mineralizing processes using simulated body fluids for coating bone-like carbonated apatite onto various material surfaces such as metals, ceramics, and polymers. The osteoinductive effects of integrating biomolecules such as prote...

*Matrix-Bound Nanovesicles Recapitulate Extracellular Matrix Effects on Macrophage Phenotype

Abstract: The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a regulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype

Braided and Stacked Electrospun Nanofibrous Scaffolds for Tendon and Ligament Tissue Engineering

Abstract: Tendon and ligament injuries are a persistent orthopedic challenge given their poor innate healing capacity. Nonwoven electrospun nanofibrous scaffolds composed of polyesters have been used to mimic the mechanics and topographical cues of native tendons and ligaments. However, nonwoven nanofibers have several limitations that prevent broader clinical application, including poor cell infiltration, as well as tensile and suture-retention strengths that are inferior to native tissues. In this study, multilayered scaffolds of aligned electrospun nanofibers of two designs-stacked or braided-were fabricated. Mechanical properties, including structural and mechanical properties and suture-retention strength, were determined using acellular scaffolds. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on scaffolds for up to 28 days, and assays for tenogenic differentiation, histology, and biochemical composition were performed. Braided scaffolds exhibited improved tensile and suture-retention strengths, but reduced moduli. Both scaffold designs supported expression of tenogenic markers, although the effect was greater on braided scaffolds. Conversely, cell infiltration was superior in stacked constructs, resulting in enhanced cell number, total collagen content, and total sulfated glycosaminoglycan content. However, when normalized against cell number, both designs modulated extracellular matrix protein deposition to a similar degree. Taken together, this study demonstrates that multilayered scaffolds of aligned electrospun nanofibers supported tenogenic differentiation of seeded MSCs, but the macroarchitecture is an important consideration for applications of tendon and ligament tissue engineering.

Mechanically Oriented 3D Collagen Hydrogel for Directing Neurite Growth.

Abstract: Recent studies in the field of neuro-tissue engineering have demonstrated the promising effects of aligned contact guidance cue to scaffolds of enhancement and direction of neuronal growth. In vivo, neurons grow and develop neurites in a complex three-dimensional (3D) extracellular matrix (ECM) surrounding. Studies have utilized hydrogel scaffolds derived from ECM molecules to better simulate natural growth. While many efforts have been made to control neuronal growth on 2D surfaces, the development of 3D scaffolds with an elaborate oriented topography to direct neuronal growth still remains a challenge. In this study, we designed a method for growing neurons in an aligned and oriented 3D collagen hydrogel. We aligned collagen fibers by inducing controlled uniaxial strain on gels. To examine the collagen hydrogel as a suitable scaffold for neuronal growth, we evaluated the physical properties of the hydrogel and measured collagen fiber properties. By combining the neuronal culture in 3D collagen hydrogels with strain-induced alignment, we were able to direct neuronal growth in the direction of the aligned collagen matrix. Quantitative evaluation of neurite extension and directionality within aligned gels was performed. The analysis showed neurite growth aligned with collagen matrix orientation, while maintaining the advantageous 3D growth.

Extracellular Matrices for Bone Regeneration: A Literature Review.

Abstract: The gold standard material for bone regeneration is still autologous bone, a mesenchymal tissue that consists mainly of extracellular matrix (ECM) (90% v/v) and little cellular content (10% v/v). However, the fact that decellularized allogenic bone grafts often present a clinical performance comparable to autologous bone grafts demonstrates the crucial role of ECM in bone regeneration. For long, the mechanism by which bone allografts function was not clear, but recent research has unveiled many unique characteristics of ECM that seem to play a key role in tissue regeneration. This is further confirmed by the fact that synthetic biomaterials with composition and properties resembling bone ECM present excellent bone regeneration properties. In this context, ECM molecules such as glycosaminoglycans (GAGs) and self-assembly peptides (SAPs) can improve the performance of bone regeneration biomaterials. Moreover, decellularized ECM derived either from native tissues such as bone, cartilage, skin, and tooth germs or from cells such as osteoblasts, chondrocytes, and stem cells has shown promising results in bone regeneration applications. Understanding the role of ECM in bone regeneration is crucial for the development of the next generation of biomaterials for bone tissue engineering. In this sense, this review addresses the state-of-the-art on this subject matter.

*Fabrication and Characterization of Biphasic Silk Fibroin Scaffolds for Tendon/Ligament-to-Bone Tissue Engineering

Abstract: Tissue engineering is an attractive strategy for tendon/ligament-to-bone interface repair. The structure and extracellular matrix composition of the interface are complex and allow for a gradual mechanical stress transfer between tendons/ligaments and bone. Thus, scaffolds mimicking the structural features of the native interface may be able to better support functional tissue regeneration. In this study, we fabricated biphasic silk fibroin scaffolds designed to mimic the gradient in collagen molecule alignment present at the interface. The scaffolds had two different pore alignments: anisotropic at the tendon/ligament side and isotropic at the bone side. Total porosity ranged from 50% to 80% and the majority of pores (80-90%) were <100-300 μm. Young's modulus varied from 689 to 1322 kPa depending on the type of construct. In addition, human adipose-derived mesenchymal stem cells were cultured on the scaffolds to evaluate the effect of pore morphology on cell proliferation and gene expression. Biphasic scaffolds supported cell attachment and influenced cytoskeleton organization depending on pore alignment. In addition, the gene expression of tendon/ligament, enthesis, and cartilage markers significantly changed depending on pore alignment in each region of the scaffolds. In conclusion, the biphasic scaffolds fabricated in this study show promising features for tendon/ligament-to-bone tissue engineering.

Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss.

Abstract: Volumetric muscle loss (VML) injuries exceed the considerable intrinsic regenerative capacity of skeletal muscle, resulting in permanent functional and cosmetic deficits. VML and VML-like injuries occur in military and civilian populations, due to trauma and surgery as well as due to a host of congenital and acquired diseases/syndromes. Current therapeutic options are limited, and new approaches are needed for a more complete functional regeneration of muscle. A potential solution is human hair-derived keratin (KN) biomaterials that may have significant potential for regenerative therapy. The goal of these studies was to evaluate the utility of keratin hydrogel formulations as a cell and/or growth factor delivery vehicle for functional muscle regeneration in a surgically created VML injury in the rat tibialis anterior (TA) muscle. VML injuries were treated with KN hydrogels in the absence and presence of skeletal muscle progenitor cells (MPCs), and/or insulin-like growth factor 1 (IGF-1), and/or basic fib...

Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

Abstract: Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

CRISPR-based epigenome editing of cytokine receptors for the promotion of cell survival and tissue deposition in inflammatory environments

Abstract: Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investig...

Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats.

Abstract: A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CP...

* Collagen Cross-Linking: Biophysical, Biochemical, and Biological Response Analysis.

Abstract: Extracted forms of collagen are subjected to chemical cross-linking to enhance their stability. However, traditional cross-linking approaches are associated with toxicity and inflammation. This work investigates the stabilization capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 4-arm polyethylene glycol (PEG) succinimidyl glutarate (4SP), genipin (GEN), and oleuropein. Although all cross-linking methods reduced free amine groups, variable data were obtained with respect to denaturation temperature, resistance to collagenase digestion, and mechanical properties. With respect to biological analysis, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human-derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of GTA, THP-1 cultures supplemented with conditioned medium from the various groups showed no significant diff...

Mimicking Cartilage Tissue Zonal Organization by Engineering Tissue-Scale Gradient Hydrogels as 3D Cell Niche.

Abstract: Zonal organization plays an important role in cartilage structure and function, whereas most tissue-engineering strategies developed to date have only allowed the regeneration of cartilage with homogeneous biochemical and mechanical cues. To better restore tissue structure and function, there is a strong need to engineer materials with biomimetic gradient niche cues that recapitulate native tissue organization. To address this critical unmet need, in this study, we report a method for rapid formation of tissue-scale gradient hydrogels as a three-dimensional (3D) cell niche with tunable biochemical and physical properties. When encapsulated in stiffness gradient hydrogels, both chondrocytes and mesenchymal stem cells demonstrated zone-specific response and extracellular deposition that mimics zonal organization of articular cartilage. Blocking cell mechanosensing using blebbistatin abolished the zonal response of chondrocytes in 3D hydrogels with a stiffness gradient. Such tissue-scale gradient hydrogels can provide a 3D artificial cell niche to enable tissue engineering of various tissue types with zonal organizations or tissue interfaces.

Pivotal Role of Non-cardiomyocytes in Electromechanical and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissue.

Abstract: Although engineered cardiac tissues (ECTs) derived from induced pluripotent stem cells (iPSCs) are promising for myocardial regenerative therapy, the appropriate ratio of cardiomyocytes to non-cardiomyocytes is not fully understood. Here, we determined whether ECT-cell content is a key determinant of its structure/function, thereby affecting ECT therapeutic potential for advanced heart failure. Scaffold-free ECTs containing different ratios (25%, 50%, 70%, or 90%) of iPSC-derived cardiomyocytes were generated by magnetic-activated cell sorting by using cardiac-specific markers. Notably, ECTs showed synchronized spontaneous beating when cardiomyocytes constituted ≥50% of total cells, with the electrical-conduction velocity increasing depending on cardiomyocyte ratio; however, ECTs containing 90% cardiomyocytes failed to form stable structures. ECTs containing 25% or 50% cardiomyocytes predominantly expressed collagen and fibronectin, whereas ECTs containing 70% cardiomyocytes predominantly expressed lamini...

Efficacy and Persistence of Allogeneic Adipose-Derived Mesenchymal Stem Cells Combined with Hyaluronic Acid in Osteoarthritis After Intra-articular Injection in a Sheep Model

Abstract: Although a number of studies have reported efficacy of autologous adipose-derived mesenchymal stem cells (AD-MSCs) in treating osteoarthritis (OA) no reliable evidences demonstrate whether allogene...

Three-Dimensional Bioprinting of Hepatic Structures with Directly Converted Hepatocyte-Like Cells

Abstract: Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8–10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 m...

*Thermosensitive Poly(N-vinylcaprolactam) Injectable Hydrogels for Cartilage Tissue Engineering

Abstract: Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and af...

Controlled Dual Delivery of Angiogenin and Curcumin by Electrospun Nanofibers for Skin Regeneration.

Abstract: In this study, we successfully fabricated a novel drug and plasmid DNA (pDNA) dual delivery system by electrospinning the dispersion composed of polyethyleneimine-carboxymethyl chitosan/pDNA-angiogenin (ANG) nanoparticles, curcumin (Cur), poly (D, L-lactic-co-glycolic acid) (PLGA), and cellulose nanocrystals (CNCs). In vitro release studies showed that the bioactivity of Cur and ANG was preserved in the nanofibers, and a sequential release pattern was achieved in which nearly 90% of the Cur was released in ca. 6 days and the ANG release lasted up to about 20 days. In vitro cell culture results suggested that the composite nanofibers exhibit excellent biocompatibility. To evaluate the in vivo angiogenesis and anti-infection properties, the PLGA/CNC/Cur/pDNA-ANG composite nanofibers were transplanted into the infected full-thickness burn wounds. Biopsy specimens were harvested for histology, immunohistochemistry, immunofluorescence, real-time quantitative PCR, and Western blotting analyses. The results indicated that the PLGA/CNC/Cur/pDNA-ANG composite nanofibers not only prevented local infection but also promoted skin regeneration.

*An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells

Abstract: At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly deliv...

*Biomimetic Versus Sintered Calcium Phosphates: The In Vitro Behavior of Osteoblasts and Mesenchymal Stem Cells

Abstract: The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area (SSA) and micro- or nanometric crystal size. The aim of this article is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium-deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate were subsequently obtained by sintering CDHA either at 1400°C or 1100°C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of SSA, skeletal density, porosity, and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips s...

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model

Abstract: Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injurie...

Hyaluronic Acid/Collagen Hydrogel as an Alternative to Alginate for Long-Term Immunoprotected Islet Transplantation*

Abstract: Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were n...

Exosome-Like Vesicles Derived from Adipose Tissue Provide Biochemical Cues for Adipose Tissue Regeneration.

Abstract: There is an emerging need for soft tissue replacements in the field of reconstructive surgery for the treatment of congenital deformities, posttraumatic repair, and cancer rehabilitation. Previous studies have shown that the bioactive adipose tissue extract can induce adipogenesis without additional stem cells or growth factors. In this study, we innovatively investigated whether exosome-like vesicles derived from adipose tissue (ELV-AT) could direct stem cell differentiation and trigger adipose tissue regeneration. In vitro, ELV-AT can induce adipogenesis of adipose-derived stem cells and promote proliferation, migration, and angiogenic potential of the aorta endothelial cells. In vivo, ELV-AT were transplanted to a chamber on the back of nude mice and neoadipose tissues were formed. Our findings indicated that ELV-AT could be used as a cell-free therapeutic approach for adipose tissue regeneration.