Effect of Valproic Acid on Promoting the Differentiation of Human Embryonic Stem Cells Into Cholangiocyte-Like Cells.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production.

Oral Delivery of Bioactive Glass-Loaded Core-Shell Hydrogel Microspheres for Effective Treatment of Inflammatory Bowel Disease.

Resolving inflammation and promoting intestinal tissue regeneration are critical for inflammatory bowel disease (IBD) treatment. Bioactive glass (BG) is a clinically approved bone graft material and has been shown to modulate inflammatory response, but it is unknown whether BG can be applied to treat IBD. Here, it is reported that BG attenuates pro-inflammatory response of lipopolysaccharide (LPS)-stimulated macrophages and hence reduces inflammatory damage to intestinal organoids in vitro. In addition, zein/sodium alginate-based core-shell microspheres (Zein/SA/BG) are developed for oral delivery of BG, which helps prevent premature dissolution of BG in the stomach. The results show that Zein/SA/BG protects BG from a gastric-simulated environment while dissolved in an intestinal-simulated environment. When administered to acute and chronic colitis mice model, Zein/SA/BG significantly reduces intestinal inflammation, promotes epithelial tissue regeneration, and partially restores microbiota homeostasis. These findings are the first to reveal the therapeutic efficacy of BG against IBD, which may provide a new therapeutic approach at low cost for effective IBD treatment.

Injectable bioactive glass/sodium alginate hydrogel with immunomodulatory and angiogenic properties for enhanced tendon healing.

Tendon healing is a complex process involving inflammation, proliferation, and remodeling, eventually achieving a state of hypocellularity and hypovascularity. Currently, few treatments can satisfactorily restore the structure and function of native tendon. Bioactive glass (BG) has been shown to possess immunomodulatory and angiogenic properties. In this study, we investigated whether an injectable hydrogel fabricated of BG and sodium alginate (SA) could be applied to enhance tenogenesis following suture repair of injured tendon. We demonstrated that BG/SA hydrogel significantly accelerated tenogenesis without inducing heterotopic ossification based on histological analysis. The therapeutic effect could attribute to increased angiogenesis and M1 to M2 phenotypic switch of macrophages within 7 days post-surgery. Morphological characterization demonstrated that BG/SA hydrogel partially reverted the pathological changes of Achilles tendon, including increased length and cross-sectional area (CSA). Finally, biomechanical test showed that BG/SA hydrogel significantly improved ultimate load, failure stress, and tensile modulus of the repaired tendon. In conclusion, administration of an injectable BG/SA hydrogel can be a novel and promising therapeutic approach to augment Achilles tendon healing in conjunction with surgical intervention.

Bioactive glass-elicited stem cell-derived extracellular vesicles regulate M2 macrophage polarization and angiogenesis to improve tendon regeneration and functional recovery.

Effective countermeasures for tendon injury remains unsatisfactory. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs)-based therapy via regulation of Mφ-mediated angiogenesis has emerged as a promising strategy for tissue regeneration. Still, approaches to tailor the functions of EVs to treat tendon injuries have been limited. We reported a novel strategy by applying MSC-EVs boosted with bioactive glasses (BG). BG-elicited EVs (EVB) showed up-regulation of medicinal miRNAs, including miR-199b-3p and miR-125a-5p, which play a pivotal role in M2 Mφ-mediated angiogenesis. EVB accelerated angiogenesis via the reprogrammed anti-inflammatory M2 Mφs compared with naïve MSC-EVs (EVN). In rodent Achilles tendon rupture model, EVB local administration activated anti-inflammatory responses via M2 polarization and led to a spatial correlation between M2 Mφs and newly formed blood vessels. Our results showed that EVB outperformed EVN in promoting tenogenesis and in reducing detrimental morphological changes without causing heterotopic ossification. Biomechanical test revealed that EVB significantly improved ultimate load, stiffness, and tensile modulus of the repaired tendon, along with a positive correlation between M2/M1 ratio and biomechanical properties. On the basis of the boosted nature to reprogram regenerative microenvironment, EVB holds considerable potential to be developed as a next-generation therapeutic modality for enhancing functional regeneration to achieve satisfying tendon regeneration.

Efficient hepatic differentiation of hydrogel microsphere-encapsulated human pluripotent stem cells for engineering prevascularized liver tissue.

Liver tissue engineering is promising as an alternative strategy to treat liver failure. However, generating functional hepatocytes from stem cells is conventionally restricted by the immature status of differentiated cells. Besides, embedding hepatocytes in bulk scaffold is limited by a lack of vascularity and low cell-packing density. Here, we fabricate collagen type I (COL1) microspheres for efficient hepatic differentiation of pluripotent stem cells and subsequent assembly of prevascularized liver tissue (PLT). Using a microfluidic platform, we demonstrate that hydrogel COL1 microspheres (mCOL1) encapsulating human embryonic stem cells (hESCs) can be reproducibly generated and efficiently differentiated into hepatocyte-like cells (HLCs) microspheres for the first time. Compared with other culture configurations such as encapsulation of hESC in a bulk COL1 hydrogel and 2D monolayer culture, mCOL1 with high uniformity produce HLC microspheres of improved maturity based on comprehensive analyses of cell morphology, transcriptome profile, hepatic marker expression and hepatic functions. In addition, these HLC microspheres can be applied as building blocks to self-assemble with endothelial cells to construct a dense PLT. The PLT resembles native liver tissue with high cell-packing density, shows successful engraftment in mice liver following implantation, and exhibits improved hepatic functionin vivo. Overall, it is believed that this multiscale technology will advance the fabrication of stem cell-based liver tissue for regenerative medicine, drug screening, andin vitroliver modeling.

Efficient fabrication of monodisperse hepatocyte spheroids and encapsulation in hybrid hydrogel with controllable extracellular matrix effect.

Three-dimensional (3D) culture techniques, such as spheroid and organoid cultures, have gained increasing interest in biomedical research. However, the understanding and control of extracellular matrix (ECM) effect in spheroid and organoid culture has been limited. Here, we report a biofabrication approach to efficiently form uniform-sized 3D hepatocyte spheroids and encapsulate them in a hybrid hydrogel composed of alginate and various ECM molecules. Cells were seeded in a microwell platform to form spheroid before being encapsulated directly in a hybrid hydrogel containing various ECM molecules, including collagen type I (COL1), collagen type IV (COL4), fibronectin (FN), and laminin (LM). A systematic analysis of the effect of ECM molecules on the primary mouse hepatocyte phenotype was then performed. Our results showed that hydrogel encapsulation of hepatocyte spheroid promoted hepatic marker expression and secretory functions. In addition, different ECM molecules elicited distinct effects on hepatic functions in 3D encapsulated hepatocyte spheroids, but not in 2D hepatocyte and 3D non-encapsulated spheroids. When encapsulated in hybrid hydrogel containing LM alone or COL1 alone, hepatocyte spheroids exhibited improved hepatic functions overall. Analysis of gene and protein expression showed an upregulation of integrinα1 and integrinα6 when LM was introduced in the hybrid hydrogel, suggesting a possible role of integrin signaling involved in the ECM effect. Finally, a combinatorial screening was performed to demonstrate the potential to screen a multitude of 3D microenvironments of varying ECM combinations that exhibited synergistic influence, indicating a strong positive effect of COL1 and a negative interaction effect of COL1·LM on both albumin and urea secretion. These findings illustrate the broad application potential of this biofabrication approach in identifying optimal ECM composition(s) for engineering 3D tissue, and elucidating defined ECM cues for tissue engineering and regenerative medicine.

Deciphering and engineering tissue folding: A mechanical perspective.

The folding of tissues/organs into complex shapes is a common phenomenon that occurs in organisms such as animals and plants, and is both structurally and functionally important. Deciphering the process of tissue folding and applying this knowledge to engineer folded systems would significantly advance the field of tissue engineering. Although early studies focused on investigating the biochemical signaling events that occur during the folding process, the physical or mechanical aspects of the process have received increasing attention in recent years. In this review, we will summarize recent findings on the mechanical aspects of folding and introduce strategies by which folding can be controlled in vitro. Emphasis will be placed on the folding events triggered by mechanical effects at the cellular and tissue levels and on the different cell- and biomaterial-based approaches used to recapitulate folding. Finally, we will provide a perspective on the development of engineering tissue folding toward preclinical and clinical translation. STATEMENT OF SIGNIFICANCE: Tissue folding is a common phenomenon in a variety of organisms including human, and has been shown to serve important structural and functional roles. Understanding how folding forms and applying the concept in tissue engineering would represent an advance of the research field. Recently, the physical or mechanical aspect of tissue folding has gained increasing attention. In this review, we will cover recent findings of the mechanical aspect of folding mechanisms, and introduce strategies to control the folding process in vitro. We will also provide a perspective on the future development of the field towards preclinical and clinical translation of various bio fabrication technologies.

Macrophages activated by akermanite/alginate composite hydrogel stimulate migration of bone marrow-derived mesenchymal stem cells.

Akermanite (Aker) has been widely used for bone regeneration through regulating osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Previously, we developed an injectable Aker/sodium alginate (Aker/SA) hydrogel to facilitate bone regeneration. However, the effect of this injectable hydrogel on thein vivoresponse, particularly the inflammatory response, has not been fully understood. Here, to elucidate the response following the implantable of Aker/SA hydrogel, we investigated the interaction among Aker/SA hydrogel, inflammatory cells and cells involved in bone regeneration (BMSCs). Specifically, we cultured macrophages (RAW 264.7 cell line) with the extract liquid of Aker/SA and assessed their phenotypic changes. Subsequently, BMSCs (2 × 105cells per 24 well) were cultured with different conditioned media including that of Aker/SA hydrogel-activated macrophages to investigate their effect on cell migration. Finally, Aker/SA hydrogel was injected subcutaneously (1 × 106cells ml-1) in rat to verify its effectin vivo. Thein vitroresults indicated that Aker/SA hydrogel activated macrophages towards M2 phenotype and stimulated macrophages to express anti-inflammatory factors. In addition, the conditioned medium collected from Aker-activated macrophages could accelerate the migration of BMSCs in 24 h. Consistent with thein vitroresults, when the Aker/SA hydrogel was injected subcutaneously, more M2 macrophages could be observed than when the SA solution was injected after 7 d. Besides, when BMSCs were delivered via subcutaneous injection, more BMSCs were recruited by the Aker/SA hydrogel than the SA solution. All these results suggest that the Aker/SA hydrogel can modulate the immune environment at the implantation site and subsequently recruit BMSCs, which can be one of the mechanisms through which the Aker/SA hydrogel accelerates new bone formation.

Modulation of macrophages by bioactive glass/sodium alginate hydrogel is crucial in skin regeneration enhancement.

Inflammatory response is a critical stage in typical wound healing. Although studies have reported that some bioactive materials can modulate the polarization of macrophages to benefit tissue regeneration, the roles of the inflammatory responses, especially the crucial roles of macrophages, in tissue regeneration stimulated by biomaterials remains unclear. Bioactive glass (BG) and hydrogel containing BG have been reported to be able to promote both hard and soft tissue regeneration. However, the critical roles of macrophages in tissue regeneration enhanced by BG have not been fully elucidated. In this study, the effects of BG/sodium alginate (SA) hydrogel (BG/SA hydrogel) on the behaviors of macrophages as well as on the interactions between macrophages and repairing cells were investigated. In addition, macrophage-depleted mice were used to investigate the necessity of macrophages in the regeneration of full-thickness skin wounds treated with BG/SA hydrogel. Our results indicated that BG/SA hydrogel could polarize macrophages towards M2 phenotype in vitro and in vivo and upregulate the expression of anti-inflammatory genes. In addition, the M2 polarized macrophages could further recruit fibroblasts and endothelial cells as well as enhance the extracellular matrix (ECM) synthesis of fibroblasts and vascularization of endothelial cells in vitro and in vivo. Depletion of macrophages in the wound sites impeded the recruitment of repairing cells and reduced the formation of blood vessels and ECM, slowing down skin regeneration. These results provide an insight into the biomaterial-immune system interactions and demonstrate that modulation of macrophages by BG/SA hydrogel in the inflammatory response is crucial in skin regeneration enhanced by the hydrogel.

Local intramyocardial delivery of bioglass with alginate hydrogels for post-infarct myocardial regeneration.

Heart failure (HF) is a common and serious manifestation after myocardial infarction (MI). Despite their clinical importance, current treatments for MI still have several limitations. Revascularization has been proven to have positive effects on MI-induced damage. Currently biomaterial-based angiogenesis strategies represent potential candidates for MI treatment. Bioglass (BG) is a commercially available family of bioactive glasses. BG has angiogenic properties and thus might be an attractive alternative for MI treatments. Here, we loaded BG in sodium alginate (BGSA), locally injected it into peri-infarct myocardial tissue and examined its suitability for inducing cardiac angiogenesis and eventually improving cardiac function following MI. Cardiac function was evaluated via echocardiography. Infarct morphometry, angiogenesis, apoptosis and angiogenic protein expression were all analysed 4 weeks after BGSA injection. Compared with the control treatment, BGSA was sufficient to prompt angiogenesis, suppress apoptosis, up-regulate the expression of angiogenic proteins, attenuate infarct size, preserve wall thickness and eventually improve cardiac function. Our results demonstrate the feasibility and effectiveness of BGSA in myocardial regeneration via angiogenesis, suggesting that BGSA is a potential therapeutic strategy for post-infarct myocardial regeneration.

Hepatic Differentiation of Stem Cells in 2D and 3D Biomaterial Systems.

A critical shortage of donor livers for treating end-stage liver failure signifies the urgent need for alternative treatment options. Hepatocyte-like cells (HLC) derived from various stem cells represent a promising cell source for hepatocyte transplantation, liver tissue engineering, and development of a bioartificial liver assist device. At present, the protocols of hepatic differentiation of stem cells are optimized based on soluble chemical signals introduced in the culture medium and the HLC produced typically retain an immature phenotype. To promote further hepatic differentiation and maturation, biomaterials can be designed to recapitulate cell-extracellular matrix (ECM) interactions in both 2D and 3D configurations. In this review, we will summarize and compare various 2D and 3D biomaterial systems that have been applied to hepatic differentiation, and highlight their roles in presenting biochemical and physical cues to different stem cell sources.

Injectable Quercetin-Loaded Hydrogel with Cartilage-Protection and Immunomodulatory Properties for Articular Cartilage Repair.

Articular cartilage plays an important role in human body. How to repair articular cartilage defects when they appear due to various factors has always been a major clinical challenge. Recently, studies have shown that slowing the degradation of cartilage extracellular matrix (ECM) and modulating the inflammatory response of the host thereby promoting cartilage tissue regeneration are important in the cartilage repair process. In this study, a drug-loaded injectable hydrogel was constructed for repairing articular cartilage. This hydrogel could not only maintain the phenotype of chondrocytes but also regulate the inflammatory response of the host. The injectable sodium alginate (SA)/bioglass (BG) hydrogel was mixed with the injectable thermal-responsive SA/agarose (AG)/quercetin (Que) hydrogel to obtain an injectable hydrogel containing both Que and BG (Que-BG hydrogel) for articular cartilage regeneration. The Que-BG hydrogel has a proper swelling ratio that can promote integration between the formed tissue and host tissue, and it allows Que to release slowly in situ to improve its bioavailability. The Que-BG hydrogel could upregulate SRY-box 9 (SOX9), aggrecan (ACAN), and collagen type II alpha 1 chain (COL2A1) of normal chondrocytes to maintain the normal chondrocyte phenotype. In addition, it could promote macrophage M2 polarization, reduce inflammation, and inhibit ECM degradation by downregulating the expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-13 (MMP13), and matrix metalloproteinase-1 (MMP1) in degenerative chondrocytes. After injecting the Que-BG hydrogel into a rat cartilage defect model, the formed tissue was observed to be similar to the normal tissue and was highly integrated with the surrounding tissue. Therefore, the injectable Que-BG hydrogel improves Que bioavailability, maintains chondrocyte phenotype, inhibits ECM degradation, and reduces inflammatory response.

Stimulation of osteogenesis and angiogenesis by micro/nano hierarchical hydroxyapatite via macrophage immunomodulation.

Biomaterial topography-based strategies are regarded as an effective way to regulate the osteoimmune environment which plays an indispensable role in the bone regeneration process. The rapid development of manufacture techniques makes it possible to investigate the cell-topography interactions by preparing various micro and nano-topographical surfaces on biomaterials. Still, it is a challenge to prepare well-defined micro/nano hierarchical structures of bioceramics due to the inherent brittleness of ceramic materials. Also, the correlation between osteoimmunomodulation initiated by micro/nano hierarchical topographies and the tissue regeneration outcomes is unclear. In this study, we prepared well-defined micro/nano hierarchical structures on hydroxyapatite (HA) bioceramics through the combination of the photolithography and hydrothermal techniques. Three different microscale circular patterns (4 µm, 12 µm and 36 µm) and nanotopographies (nanoneedle, nanosheet and nanorod) were fabricated by changing the size of the mask and the condition of the hydrothermal reaction. The macrophage responses on the nanoneedle structures with different micropatterns were investigated and the micro/nano hierarchical structures with appropriate pattern sizes could either promote or alleviate the macrophage polarization, which further affected the outcomes of the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and angiogenic activity of human umbilical vein endothelial cells (HUVECs). Our study demonstrated that osteoimmunomodulation could be manipulated via tuning the micro/nano hierarchical structures, which could lead to a new strategy for the development of bone biomaterials with favorable osteoimmunomodulatory properties.

An injectable continuous stratified structurally and functionally biomimetic construct for enhancing osteochondral regeneration.

Osteochondral regeneration with the formation of hyaline cartilage and subchondral bone as well as the integration between the newly formed tissues with the host tissue still remains a great challenge. In this study, a construct containing an injectable continuous stratified scaffold and multiple cell systems was designed for enhancing osteochondral regeneration. Briefly, an injectable sodium alginate(SA)/bioglass (BG) composite hydrogel containing bone marrow stem cells (BMSCs) (SA/BG + BMSCs) was used for subchondral bone regeneration and an injectable thermosensitive SA/agarose (AG) composite hydrogel with co-culture of BMSCs and articular chondrocytes (ACs) (SA/AG + ACs/BMSCs) was applied for articular cartilage regeneration. The continuous SA phase and the stratified structure enable the scaffold to mimic the natural osteochondral structure. In addition, the SA/BG + BMSCs hydrogel could enhance the osteoblast differentiation of BMSCs by upregulating their alkaline phosphatase and collagen I gene expressions, and the SA/AG + ACs/BMSCs hydrogel could promote the chondrocyte differentiation of BMSCs by upregulating their Acan and collagen II gene expressions, which indicated that this stratified scaffold could mimic the natural osteochondral function. Furthermore, after the stratified construct was injected into a rat osteochondral defect model, obvious neonatal articular cartilage tissues and subchondral bone tissues with regular surface and highly integration with normal tissues could be observed. This structural and functional biomimetic construct, together with its proper swelling ratio, could not only stimulate the hyaline cartilage and subchondral bone regeneration in an entire osteochondral unit but also promote the integration between the newly formed tissues and the host tissue.

Alginate-aker injectable composite hydrogels promoted irregular bone regeneration through stem cell recruitment and osteogenic differentiation.

Recent studies have unveiled the unique osteogenesis and angiogenesis abilities of akermanite. However, such bioceramics rarely fit well into irregularly shaped bony cavities (such as bone defects and the maxillary sinus). In this study, an injectable hydrogel SAG system containing sodium alginate, akermanite and glutamate was prepared and evaluated in such defects. Cell proliferation experiments showed that except for stoste, the other extracts showed no cytotoxicity at various concentrations. In addition, the gene expression of Runx2 (runt-related transcription factor-2), BMP-2 (bone morphogenetic protein-2), ALP (alkaline phosphatase), and BSP (bone sialoprotein) and the activity of ALP were significantly higher in human bone marrow stromal cells (hBMSCs) cultured with 1/2 hydrogel extracts containing 66.9 ppm calcium ions, 23.8 ppm magnesium ions and 33.5 ppm silicon ions compared to those in hBMSCs cultured with the control medium, which indicated that the hydrogel extracts could stimulate the osteogenic differentiation of hBMSCs. Further experiments revealed that the ERK signaling pathway was engaged in osteogenic differentiation as early as 15 minutes after incubation with the hydrogel extracts. In addition, hBMSCs incubated in half-diluted extracts exhibited an almost doubled migration ability compared with the hBMSCs in the control group. More specifically, compared to the control group, hBMSCs cultured with 1/2 hydrogel extracts showed a 7% increase and a 10-fold increase in the protein and gene levels of CXCR4 (C-X-C chemokine receptor type 4), respectively. In vivo tests demonstrated that the bone formation rate of SAG hydrogels injected alone was similar to that of its counterpart seeded with BMSCs, reaching 24% at three months after operation. Therefore, it could be concluded that SAG hydrogels contribute to bone regeneration by not only promoting osteogenic differentiation but also by enhancing the recruitment of BMSCs to defect sites, making the injectable SAG hydrogels competent for the regeneration of irregular bony cavities.