Hydrogel-mediated delivery of microRNA-92a inhibitor polyplex nanoparticles induces localized angiogenesis.

Localized stimulation of angiogenesis is an attractive strategy to improve the repair of ischemic or injured tissues. Several microRNAs (miRNAs) such as miRNA-92a (miR-92a) have been reported to negatively regulate angiogenesis in ischemic disease. To exploit the clinical potential of miR-92a inhibitors, safe and efficient delivery needs to be established. Here, we used deoxycholic acid-modified polyethylenimine polymeric conjugates (PEI-DA) to deliver a locked nucleic acid (LNA)-based miR-92a inhibitor (LNA-92a) in vitro and in vivo. The positively charged PEI-DA conjugates condense the negatively charged inhibitors into nano-sized polyplexes (135 ± 7.2 nm) with a positive net charge (34.2 ± 10.6 mV). Similar to the 25 kDa-branched PEI (bPEI25) and Lipofectamine RNAiMAX, human umbilical vein endothelial cells (HUVECs) significantly internalized PEI-DA/LNA-92a polyplexes without any obvious cytotoxicity. Down-regulation of miR-92a following the polyplex-mediated delivery of LNA-92a led to a substantial increase in the integrin subunit alpha 5 (ITGA5), the sirtuin-1 (SIRT1) and Krüppel-like factors (KLF) KLF2/4 expression, formation of capillary-like structures by HUVECs, and migration rate of HUVECs in vitro. Furthermore, PEI-DA/LNA-92a resulted in significantly enhanced capillary density in a chicken chorioallantoic membrane (CAM) model. Localized angiogenesis was substantially induced in the subcutaneous tissues of mice by sustained release of PEI-DA/LNA-92a polyplexes from an in situ forming, biodegradable hydrogel based on clickable poly(ethylene glycol) (PEG) macromers. Our results indicate that PEI-DA conjugates efficiently deliver LNA-92a to improve angiogenesis. Localized delivery of RNA interference (RNAi)-based therapeutics via hydrogel-laden PEI-DA polyplex nanoparticles appears to be a safe and effective approach for different therapeutic targets.

Mesenchymal stem cell-derived extracellular vesicles alone or in conjunction with a SDKP-conjugated self-assembling peptide improve a rat model of myocardial infarction.

PURPOSE: The aim of this study was to investigate the cardiac repair effect of human bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) after intramyocardial injection in free form or encapsulated within a self-assembling peptide hydrogel modified with SDKP motif, in a rat model of myocardial infarction (MI). METHODS: MSC-EVs were isolated by ultracentrifuge and characterized for physical parameters and surface proteins. Furthermore, cellular uptake and cardioprotective effects of MSC-EVs were evaluated in vitro using neonatal mouse cardiomyocytes (NMCMs). In vivo effects of MSC-EVs on cardiac repair were studied in rat MI model by comparing the vehicle group (injected with PBS), EV group (injected with MSC-EVs) and Gel + EV group (injected with MSC-EVs encapsulated in (RADA)4-SDKP hydrogel) with respect to cardiac function and fibrotic area using echocardiography and Masson's trichrome staining, respectively. Histological sections were assessed by α-SMA and CD68 immunostaining to investigate the angiogenic and anti-inflammatory effects of the MSC-EVs. RESULTS: We observed the uptake of MSC-EVs into NMCMs which led to NMCMs protection against H2O2-induced oxidative stress by substantial reduction of apoptosis. In myocardial infarcted rats, cardiac function was improved after myocardial injection of MSC-EVs alone or in conjunction with (RADA)4-SDKP hydrogel. This functional restoration coincided with promotion of angiogenesis and decrement of fibrosis and inflammation. CONCLUSION: These data demonstrated that MSC-EVs can be used alone as a potent therapeutic agent for improvement of myocardial infarction.

In Situ Forming, Cytocompatible, and Self-Recoverable Tough Hydrogels Based on Dual Ionic and Click Cross-Linked Alginate.

A dual cross-linking strategy was developed to answer the urgent need for fatigue-resistant, cytocompatible, and in situ forming tough hydrogels. Clickable, yet calcium-binding derivatives of alginate were synthesized by partial substitution of its carboxyl functionalities with furan, which could come into Diels-Alder click reaction with maleimide end groups of a four arm poly(ethylene glycol) cross-linker. Tuning the cooperative viscoelastic action of transient ionic and permanent click cross-links within the single network of alginate provided a soft tough hydrogel with a set of interesting features: (i) immediate self-recovery under cyclic loading, (ii) highly efficient and autonomous self-healing upon fracture, (iii) in situ forming ability for molding and minimally invasive injection, (iv) capability for viable cell encapsulation, and (v) reactivity for on-demand biomolecule conjugation. The facile strategy is applicable to a wide range of natural and synthetic polymers by introducing the calcium binding and click reacting functional groups and can broaden the use of tough hydrogels in load-bearing, cell-laden applications such as soft tissue engineering and bioactuators.

A self-gelling hydrogel based on thiolated hyaluronic acid for three-dimensional culture of ovine preantral follicles.

Three-dimensional (3D) ovarian follicle culture offers a promising option for fertility preservation in patients who cannot receive ovarian tissue transplantation. Our research evaluated the potential of a hydrogel composed of thiolated hyaluronic acid (HA-SH) for ovine preantral follicle development compared to routinely used alginate hydrogel (ALG). Synthesized via a carbodiimide reaction, HA-SH facilitated a self-crosslinking hydrogel through disulfide bond formation. Ovine preantral follicles (200-300 µm) retrieved through mechanical and enzymatic methods were encapsulated individually in either ALG or HA-SH hydrogels. Although both hydrogels adequately supported follicle survival, 3D integrity, and antrum formation over a 17-day in vitro culture, follicle growth was significantly higher within the HA-SH hydrogel. Gene expression analysis underscored that some folliculogenesis-related genes (ZP3, BMP7, and GJA1) and a steroidogenic gene (CYP19A1) demonstrated higher expression levels in HA-SH encapsulated follicles versus ALG. Collectively, our findings advocate for HA-SH hydrogel as a potent biomaterial for in vitro follicle cultures, attributing its efficacy to facile gelation, bio-responsiveness, and superior support for follicle growth.

Chemical modification of hyaluronic acid improves its supportive action on embryo implantation.

Hyaluronic acid (HA) is a supplement of the embryo transfer medium that improves embryo implantation. We have suggested that the supportive action of HA can be promoted by introducing additional artificial binding sites on the HA structure. HA was modified at carboxyl sites separately with thiol (SH) and N-hydroxysuccinimide (NHS), as mucoadhesive and amine-reactive groups, respectively. The mouse blastocysts were incubated with HA derivatives for 15 min. The HA coatings maintained their potential for enzymatic degradation and showed no detrimental effect on embryonic viability and developmental potential. After in vivo transfer, a significantly higher implantation rate was attained by HA-NHS treatment (80 %) compared with the HA-SH (53 %) and the commercial transfer medium, EmbryoGlue® (56 %). The HA-NHS was produced by a slight modification on the native structure of HA using a simple, fast, non-expensive and scalable chemistry which all promise applicability of this new HA derivative in assisted reproductive technologies.

Earlier Detection of Alzheimer's Disease Based on a Novel Biomarker cis P-tau by a Label-Free Electrochemical Immunosensor.

Early detection of cis phosphorylated tau (cis P-tau) may help as an effective treatment to control the progression of Alzheimer's disease (AD). Recently, we introduced for the first time a monoclonal antibody (mAb) with high affinity against cis P-tau. In this study, the cis P-tau mAb was utilized to develop a label-free immunosensor. The antibody was immobilized onto a gold electrode and the electrochemical responses to the analyte were acquired by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The immunosensor was capable of selective detection of cis P-tau among non-specific targets like trans P-tau and major plasma proteins. A wide concentration range (10 × 10-14 M-3.0 × 10-9 M) of cis P-tau was measured in PBS and human serum matrices with a limit of detection of 0.02 and 0.05 pM, respectively. Clinical applicability of the immunosensor was suggested by its long-term storage stability and successful detection of cis P-tau in real samples of cerebrospinal fluid (CSF) and blood serum collected from human patients at different stages of AD. These results suggest that this simple immunosensor may find great application in clinical settings for early detection of AD which is an unmet urgent need in today's healthcare services.

Tissue-Specific Microparticles Improve Organoid Microenvironment for Efficient Maturation of Pluripotent Stem-Cell-Derived Hepatocytes.

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.

Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies.

BACKGROUND: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). METHODS: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. RESULTS: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. CONCLUSION: Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Dual functional construct containing kartogenin releasing microtissues and curcumin for cartilage regeneration.

BACKGROUND: Regeneration of articular cartilage poses a tremendous challenge due to its limited self-repair capability and inflammation at the damaged site. To generate the desired structures that mimic the structure of native tissue, microtissues with repeated functional units such as cell aggregates have been developed. Multicellular aggregates of mesenchymal stem cells (MSCs) can be used as microscale building blocks of cartilage due to their potential for cell-cell contact, cell proliferation, and differentiation. METHODS: Chondrogenic microtissues were developed through incorporation of kartogenin-releasing poly (lactic-co-glycolic acid) (PLGA) microparticles (KGN-MP) within the MSC aggregates. The chondrogenic potential of KGN-MP treated MSC aggregates was proven in vitro by studying the chondrogenic markers at the RNA level and histological analysis. In order to address the inflammatory responses at the defect site, the microtissues were delivered in vivo via an injectable, anti-inflammatory hydrogel that contained gelatin methacryloyl (GelMA) loaded with curcumin (Cur). RESULTS: The KGN-MPs were fabricated to support MSCs during cartilage differentiation. According to real-time RT-PCR analysis, the presence of KGN in the aggregates led to the expression of cartilage markers by the MSCs. Both toluidine blue (TB) and safranin O (SO) staining demonstrated homogeneous glycosaminoglycan production throughout the KGN-MP incorporated MSC aggregates. The curcumin treatment efficiently reduced the expressions of hypertrophy markers by MSCs in vitro. The in vivo results showed that implantation of chondrogenic microtissues (KGN-MP incorporated MSC aggregates) using the curcumin loaded GelMA hydrogel resulted in cartilage tissue regeneration that had characteristic features close to the natural hyaline cartilage according to observational and histological results. CONCLUSIONS: The use of this novel construct that contained chondrogenic cell blocks and curcumin is highly desired for cartilage regeneration.

A Cell-Free SDKP-Conjugated Self-Assembling Peptide Hydrogel Sufficient for Improvement of Myocardial Infarction.

Biomaterials in conjunction with stem cell therapy have recently attracted attention as a new therapeutic approach for myocardial infarction (MI), with the aim to solve the delivery challenges that exist with transplanted cells. Self-assembling peptide (SAP) hydrogels comprise a promising class of synthetic biomaterials with cardiac-compatible properties such as mild gelation, injectability, rehealing ability, and potential for sequence modification. Herein, we developed an SAP hydrogel composed of a self-assembling gel-forming core sequence (RADA) modified with SDKP motif with pro-angiogenic and anti-fibrotic activity to be used as a cardioprotective scaffold. The RADA-SDKP hydrogel was intramyocardially injected into the infarct border zone of a rat model of MI induced by left anterior descending artery (LAD) ligation as a cell-free or a cell-delivering scaffold for bone marrow mesenchymal stem cells (BM-MSCs). The left ventricular ejection fraction (LVEF) was markedly improved after transplantation of either free hydrogel or cell-laden hydrogel. This cardiac functional repair coincided very well with substantially lower fibrotic tissue formation, expanded microvasculature, and lower inflammatory response in the infarct area. Interestingly, BM-MSCs alone or in combination with hydrogel could not surpass the cardiac repair effects of the SDKP-modified SAP hydrogel. Taken together, we suggest that the RADA-SDKP hydrogel can be a promising cell-free construct that has the capability for functional restoration in the instances of acute myocardial infarction (AMI) that might minimize the safety concerns of cardiac cell therapy and facilitate clinical extrapolation.

Cannabidiol-loaded microspheres incorporated into osteoconductive scaffold enhance mesenchymal stem cell recruitment and regeneration of critical-sized bone defects.

Recruitment of mesenchymal stem cells (MSCs) to an injury site and their differentiation into the desired cell lineage are implicated in deficient bone regeneration. To date, there is no ideal structure that provides these conditions for bone regeneration. In the current study, we aim to develop a novel scaffold that induces MSC migration towards the defect site, followed by their differentiation into an osteogenic lineage. We have fabricated a gelatin/nano-hydroxyapatite (G/nHAp) scaffold that delivered cannabidiol (CBD)-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres to critical size radial bone defects in a rat model. The fabricated scaffolds were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), and then analyzed for porosity and degradation rate. The release profile of CBD from the PLGA microsphere and CBD-PLGA-G/nHAp scaffold was analyzed by fluorescence spectroscopy. We performed an in vitro assessment of the effects of CBD on cellular behaviors of viability and osteogenic differentiation. Radiological evaluation, histomorphometry, and immunohistochemistry (IHC) analysis of all defects in the scaffold and control groups were conducted following transplantation into the radial bone defects. An in vitro migration assay showed that CBD considerably increased MSCs migration. qRT-PCR results showed upregulated expression of osteogenic markers in the presence of CBD. Histological and immunohistochemical findings confirmed new bone formation and reconstruction of the defect at 4 and 12 week post-surgery (WPS) in the CBD-PLGA-G/nHAp group. Immunofluorescent analysis revealed enhanced migration of MSCs into the defect areas in the CBD-PLGA-G/nHAp group in vivo. Based on the results of the current study, we concluded that CBD improved bone healing and showed a critical role for MSC migration in the bone regeneration process.

Hydrogel-Mediated Sustained Systemic Delivery of Mesenchymal Stem Cell-Derived Extracellular Vesicles Improves Hepatic Regeneration in Chronic Liver Failure.

Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have been widely reported as promising cell-free products that show therapeutic effects of the parental cells but not their limitations. Due to the intrinsic liver tropism of MSC-EVs, they have been widely used as therapeutics or drug carriers for treatment of liver diseases. However, rapid clearance from the target site may attenuate the efficiency of systemically administered MSC-EVs. Herein, sustained release into the peritoneum has been proposed as a new strategy to prolong the bioavailability of the MSC-EVs in the target liver. During intraperitoneal injection, clickable polyethylene glycol (PEG) macromeres were mixed with MSC-EVs to form EV-encapsulated PEG hydrogels via a fast, biocompatible click reaction. Upon biodegradation, the EV-laden hydrogels were swollen gradually to release EVs in a sustained manner over 1 month. In vivo tracking of the labeled EVs revealed that the accumulation of EVs in the liver was extended by hydrogel-mediated delivery for 1 month. Four weeks after injection in a rat model of chronic liver fibrosis, the physical and histopathological investigations of the harvested liver showed superior antifibrosis, anti-apoptosis, and regenerative effects of the EVs when delivered by the sustained systemic release (Gel-EV) to the conventional bolus injection (Free-EV). Specifically, the Gel-EV system improved the antifibrosis, anti-inflammation, anti-apoptosis, and regenerative effects of the EVs to nearly 40, 50, 40, and 50% compared to Free-EV, respectively, as was specified by quantification of the fibrotic area, α-SMA density, and caspase-3 density in the harvested tissues and ALT enzyme in serum. This study may potentiate the use of MSC-EVs as cell-free therapeutics for chronic liver failure. The sustained systemic delivery strategy may open a new paradigm to extend the effects of disease-targeting EVs over time.

Fabrication of microporous inorganic microneedles by centrifugal casting method for transdermal extraction and delivery.

Microneedle patches have been widely used as transdermal transport systems because of their painless and easy application. Marked rigidity, strength, biocompatibility, and physiological stability are unique features of microneedles fabricated from ceramic materials to be used as microneedle patches. However, the conventional ceramic microneedles are typically dense structures with limited free space for biomolecule loading. A facile method is required for fabrication of biocompatible ceramic microneedles with interconnected porosity. Herein, the simple method of centrifugal casting was developed for fabrication of microporous microneedles from alumina suspensions. The slurry or resin-based alumina suspensions were casted into micromolds under centrifugal force, followed by sintering at high temperatures. The effects of particle size, solvent type, binder amount, resin content and sintering temperature on the microstructure and mechanical properties of microneedles were investigated. By optimizing the process parameters, highly porous (up to 60%) microneedles with interconnected micropores (of diameter ∼1-1.5 µm) were produced. The microporous microneedles were biocompatible and mechanically strong for skin penetration. The potential use of the microneedles for transdermal transportation of biomolecules was shown by fast and accurate extraction of glucose from a skin model and efficient loading and fast release of insulin under physiological conditions. The results suggested that the microporous alumina microneedles may serve as molecular transport systems in transdermal biosensing and drug delivery.

Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids.

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and ß-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Engineering mesenchymal stem cell spheroids by incorporation of mechanoregulator microparticles.

Mechanical forces throughout human mesenchymal stem cell (hMSC) spheroids (mesenspheres) play a predominant role in determining cellular functions of cell growth, proliferation, and differentiation through mechanotransductional mechanisms. Here, we introduce microparticle (MP) incorporation as a mechanical intervention method to alter tensional homeostasis of the mesensphere and explore MSC differentiation in response to MP stiffness. The microparticulate mechanoregulators with different elastic modulus (34 kPa, 0.6 MPa, and 2.2 MPa) were prepared by controlled crosslinking cell-sized microdroplets of polydimethylsiloxane (PDMS). Preparation of MP-MSC composite spheroids enabled us to study the possible effects of MPs through experimental and computational assays. Our results showed that MP incorporation selectively primed MSCs toward osteogenesis, yet hindered adipogenesis. Interestingly, this behavior depended on MP mechanics, as the spheroids that contained MPs with intermediate stiffness behaved similar to control MP-free mesenspheres with more tendencies toward chondrogenesis. However, by using the soft or stiff MPs, the MP-mesenspheres significantly showed signs of osteogenesis. This could be explained by the complex of forces which acted in the cell spheroid and, totally, provided a homeostasis situation. Incorporation of cell-sized polymer MPs as mechanoregulators of cell spheroids could be utilized as a new engineering toolkit for multicellular organoids in disease modeling and tissue engineering applications.

In situ formation of interpenetrating polymer network using sequential thermal and click crosslinking for enhanced retention of transplanted cells.

Injectable hydrogels, which are used as scaffolds in cell therapy, provide a minimally invasive strategy to enhance cell retention and survival at injection site. However, till now, slow in situ gelation, undesired mechanical properties, and weak cell adhesion characteristics of reported hydrogels, have led to improper results. Here, we developed an injectable fully-interpenetrated polymer network (f-IPN) by integration of Diels-Alder (DA) crosslinked network and thermosensitive injectable hydrogel. The proposed DA hydrogels were formed in a slow manner showing robust mechanical properties. Interpenetration of thermosensitive network into DA hydrogel accelerated in situ gel-formation and masked the slow reaction rate of DA crosslinking while keeping its unique features. Two networks were formed by simple syringe injection without the need of any initiator, catalyst, or double barrel syringe. The DA and f-IPN hydrogels showed comparable viscoelastic properties along with outstanding load-bearing and shape-recovery even under high levels of compression. The subcutaneous administration of cardiomyocytes-laden f-IPN hydrogel into nude mice revealed high cell retention and survival after two weeks. Additionally, the cardiomyocyte's identity of retained cells was confirmed by detection of human and cardiac-related markers. Our results indicate that the thermosensitive-covalent networks can open a new horizon within the injection-based cell therapy applications.

Efficient and cost-effective generation of hepatocyte-like cells through microparticle-mediated delivery of growth factors in a 3D culture of human pluripotent stem cells.

Biomedical application of human pluripotent stem cell-derived hepatocyte-like cells (hPSC-HLCs) relies on efficient large-scale differentiation, which is commonly performed by a suspension culture of three-dimensional (3D) multicellular spheroids in bioreactors. However, this approach requires large amounts of growth factors (GFs) and the need to overcome limited diffusional transport posed by the inherent 3D structure of hPSC spheroids. Here, we have hypothesized that localized delivery of GFs by incorporation of GF-laden degradable polymeric microparticles (MPs) within the hPSC spheroids would circumvent such limitations. In this study, GFs for hepatocytic differentiation were encapsulated in gelatin-coated poly (l-lactic acid)/poly (DL-lactic-co-glycolic acid) (PLLA/PLGA) MPs which were subsequently incorporated into the hPSC spheroids. Gene expression analyses demonstrated that MP delivery of the GFs resulted in similar expression levels of hepatocytic markers despite the use of 10-fold less total GFs. The differentiated HLCs in the MP group exhibited ultrastructure and functional characteristics comparable with the conventional soluble GF group. The generated HLCs in the MP group were successfully engrafted in an acute liver injury mouse model and maintained hepatocytic function after implantation. These results suggested that sustained and localized delivery of GFs using MPs might offer a novel approach towards scalable technologies for hepatocytic differentiation and engineer a better 3D microenvironment for cells.

Tolerance induction by surface immobilization of Jagged-1 for immunoprotection of pancreatic islets.

Although transplantation of pancreatic islets is a promising approach for treatment of type 1 diabetes mellitus, the engraftment efficiency of these islets is limited by host immune responses. Extensive efforts have been made to immunoisolate these islets by introducing barriers on the islet surface. To date, these barriers have not successfully protected islets from attack by the immune system. In addition, the inevitable permeability of an islet capsule cannot prevent filtration by proinflammatory cytokines and islet self-antigens. Thus, we have developed a surface engineering approach for localized immonumodulation of the islet microenvironment. Jagged-1 (JAG-1), as a potent immunomodulatory factor, was immobilized on the islet surface by mediation of a double-layer of heterobifunctional poly (ethylene glycol) (PEG). Immobilization and functionality of JAG-1 on PEGylated islet surfaces were established. When co-cultured with splenocytes, the JAG-1 conjugated islets induced a significant increase in regulatory T cells and regulated the cytokine levels produced by immune cells. The results demonstrated that JAG-1 immobilization could improve immunoprotection of pancreatic islets by localized modulation of the immune milieu from an inflammatory to an anti-inflammatory state. We also evaluated the effects of surface modification of these islets by JAG-1 in a xenotransplantation model. The transplanted JAG-1/PEG/islets group showed a significantly reduced blood glucose levels compared with the control group of diabetic mice during the acute phase of the immune response to the transplanted islets. Our results demonstrated that surface modification has the potential to shift the immune system from an inflammatory to anti-inflammatory milieu and may offer a new prospective for immunoprotection of pancreatic islets.

Facile synthesis of biphasic calcium phosphate microspheres with engineered surface topography for controlled delivery of drugs and proteins.

Biphasic calcium phosphate (BCP) microspheres are of great interest due to their high stability and osteoinductive properties at specific compositions. However, the need for optimal performance at a unique composition limits their flexibility for tuning drug release by modulation of bulk properties and presents the question of engineering surface topography as an alternative. It is necessary to have a facile method to control surface topography at a defined bulk composition. Here, we have produced BCP microspheres with different surface topographies that have the capability to be used as tunable drug release systems. We synthesized calcium deficient hydroxyapatite (CDHA) microparticles by precipitating calcium and phosphate ions onto ethylenediaminetetraacetic acid (EDTA) templates. The morphology and surface topography of CDHA microparticles were controlled using process parameters, which governed nucleation and growth. These parameters included template concentration, heat rate, and stirring speed. Under low heat rate and static conditions, we could obtain spherical microparticles with long and short nanosheets on their surfaces at low and high EDTA concentrations, respectively. These nanostructured microspheres were subsequently crystallized by thermal treatment to produce EDTA-free BCP microspheres with intact morphology. These biocompatible BCP microspheres were highly effective in loading and prolonged release of both small molecule [dexamethasone (Dex)] and protein [bovine serum albumin (BSA)] models. This strategy has enabled us to control the surface topography of BCP microspheres at defined compositions and holds tremendous promise for drug delivery and tissue engineering applications.

Microparticle-Mediated Delivery of BMP4 for Generation of Meiosis-Competent Germ Cells from Embryonic Stem Cells.

Producing meiosis-competent germ cells (GCs) from embryonic stem cells (ESCs) is essential for developing advanced therapies for infertility. Here, a novel approach is presented for generation of GCs from ESCs. In this regard, microparticles (MPs) have been developed from alginate sulfate loaded with bone morphogenetic protein 4 (BMP4). The results here show that BMP4 release from alginate sulfate MPs is significantly retarded by the sulfated groups compared to neat alginate. Then, BMP4-laden MPs are incorporated within the aggregates during differentiation of GCs from ESCs. It is observed that BMP4-laden MPs increase GC differentiation from ESCs at least twofold compared to the conventional soluble delivery method. Interestingly, following meiosis induction, Dazl, an intrinsic factor that enables GCs to enter meiosis, and two essential meiosis genes (Stra8 and Smc1b) are upregulated significantly in MP-induced aggregates compared to aggregates, which are formed by the conventional method. Together, these data show that controlled delivery of BMP4 during ESC differentiation into GC establish meiosis-competent GCs which can serve as an attractive GC source for reproductive medicine.