A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

Dual Growth Factor Delivery Using Photo-Cross-Linkable Gelatin Hydrogels for Effectively Reinforced Regeneration of the Rotator Cuff Tendon.

Rotator cuff tears are currently treated with drugs (steroids and nonsteroidal anti-inflammatory drugs) and surgery. However, the damaged rotator cuff requires a considerable amount of time to regenerate, and the regenerated tissue cannot restore the same level of function as that before the damage. Although growth factors can accelerate regeneration, they are difficult to be used alone because of the risk of degradation and the difficulties in ensuring their sustained release. Thus, hydrogels such as gelatin are used, together with growth factors. Gelatin is a biocompatible and biodegradable hydrogel derived from collagen; therefore, it closely resembles the components of native tissues and can retain water and release drugs continuously, while also showing easily tunable mechanical properties by simple modifications. Moreover, gelatin is a natural biopolymer that possesses the ability to form hydrogels of varying compositions, thereby facilitating effective cross-linking. Therefore, gelatin can be considered to be suitable for rotator-to-tendon healing. In this study, we designed photo-cross-linkable gelatin hydrogels to enhance spacing and adhesive effects for rotator cuff repair. We mixed a ruthenium complex (Ru(II)bpy32+) and sodium persulfate into gelatin-based hydrogels and exposed them to blue light to induce gelation. Basic fibroblast growth factor and bone morphogenetic protein-12 were encapsulated in the gelatin hydrogel for localized and sustained release into the wound, thereby enhancing the cell proliferation. The effects of these dual growth factor-loaded hydrogels on cell cytotoxicity and tendon regeneration in rotator cuff tear models were evaluated using mechanical and histological assessments. The findings confirmed that the gelatin hydrogel was biocompatible and that treatment with the dual growth factor-loaded hydrogels in in vivo rotator cuff tear models promoted regeneration and functional restoration in comparison with the findings in the nontreated group. Therefore, growth factor-loaded gelatin-based hydrogels may be suitable for the treatment of rotator cuff tears.

A Conductive and Adhesive Hydrogel Composed of MXene Nanoflakes as a Paintable Cardiac Patch for Infarcted Heart Repair.

Myocardial infarction (MI) is a major cause of death worldwide. After the occurrence of MI, the heart frequently undergoes serious pathological remodeling, leading to excessive dilation, electrical disconnection between cardiac cells, and fatal functional damage. Hence, extensive efforts have been made to suppress pathological remodeling and promote the repair of the infarcted heart. In this study, we developed a hydrogel cardiac patch that can provide mechanical support, electrical conduction, and tissue adhesiveness to aid in the recovery of an infarcted heart function. Specifically, we developed a conductive and adhesive hydrogel (CAH) by combining the two-dimensional titanium carbide (Ti3C2Tx) MXene with natural biocompatible polymers [i.e., gelatin and dextran aldehyde (dex-ald)]. The CAH was formed within 250 s of mixing the precursor solution and could be painted. The hydrogel containing 3.0 mg/mL MXene, 10% gelatin, and 5% dex-ald exhibited appropriate material characteristics for cardiac patch applications, including a uniform distribution of MXene, a high electrical conductivity (18.3 mS/cm), cardiac tissue-like elasticity (30.4 kPa), strong tissue adhesion (6.8 kPa), and resistance to various mechanical deformations. The CAH was cytocompatible and induced cardiomyocyte (CM) maturation in vitro, as indicated by the upregulation of connexin 43 expression and a faster beating rate. Furthermore, CAH could be painted onto the heart tissue and remained stably adhered to the beating epicardium. In vivo animal studies revealed that CAH cardiac patch treatment significantly improved cardiac function and alleviated the pathological remodeling of an infarcted heart. Thus, we believe that our MXene-based CAH can potentially serve as a promising platform for the effective repair of various electroactive tissues including the heart, muscle, and nerve tissues.

Self-assembled peptide-substance P hydrogels alleviate inflammation and ameliorate the cartilage regeneration in knee osteoarthritis.

BACKGROUND: Self-assembled peptide (SAP)-substance P (SP) hydrogels can be retained in the joint cavity longer than SP alone, and they can alleviate inflammation and ameliorate cartilage regeneration in knee osteoarthritis (OA). We conducted a preclinical study using diverse animal models of OA and an in vitro study using human synoviocytes and patient-derived synovial fluids to demonstrate the effect of SAP-SP complex on the inflammation and cartilage regeneration. METHODS: Surgical induction OA model was prepared with New Zealand white female rabbits and chemical induction, and naturally occurring OA models were prepared using Dunkin Hartely female guinea pigs. The SAP-SP complex or control (SAP, SP, or saline) was injected into the joint cavities in each model. We performed micro-computed tomography (Micro-CT) analysis, histological evaluation, immunofluorescent analysis, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay and analyzed the recruitment of intrinsic mesenchymal stem cells (MSCs), macrophage activity, and inflammatory cytokine in each OA model. Human synoviocytes were cultured in synovial fluid extracted from human OA knee joints injected with SAP-SP complexes or other controls. Proliferative capacity and inflammatory cytokine levels were analyzed. RESULTS: Alleviation of inflammation, inhibition of apoptosis, and enhancement of intrinsic MSCs have been established in the SAP-SP group in diverse animal models. Furthermore, the inflammatory effects on human samples were examined in synoviocytes and synovial fluid from patients with OA. In this study, we observed that SAP-SP showed anti-inflammatory action in OA conditions and increased cartilage regeneration by recruiting intrinsic MSCs, inhibiting progression of OA. CONCLUSIONS: These therapeutic effects have been validated in diverse OA models, including rabbits, Dunkin Hartley guinea pigs, and human synoviocytes. Therefore, we propose that SAP-SP may be an effective injectable therapeutic agent for treating OA. In this manuscript, we report a preclinical study of novel self-assembled peptide (SAP)-substance P (SP) hydrogels with diverse animal models and human synoviocytes and it displays anti-inflammatory effects, apoptosis inhibition, intrinsic mesenchymal stem cells recruitments and cartilage regeneration.

Effect of Electrical Stimulation on Nerve-Guided Facial Nerve Regeneration.

This study aimed to investigate the effect of electrical stimulation on poly(d,l-lactide-co-ε-caprolactone) nerve guidance conduits (NGCs) in promoting the recovery of facial function and nerve regeneration after facial nerve (FN) injury in a rat model. In the experimental group, both the NGC and transcutaneous electrical nerve stimulation (ES) were used simultaneously; in the control group, only NGC was used. ES groups were divided into two groups, and direct current (DC) and charge-balanced pulse stimulation (Pulse) were applied. The ES groups showed significantly improved whisker movement than the NGC-only group. The number of myelinated neurons was higher in ES groups, and the myelin sheath was also thicker and more uniform. In addition, the expression of neurostructural proteins was also higher in ES groups than in the NGC-only group. This study revealed that FN regeneration and functional recovery occurred more efficiently when ES was applied in combination with NGCs.

Three-dimensional bioprinting of mesenchymal stem cells using an osteoinductive bioink containing alginate and BMP-2-loaded PLGA nanoparticles for bone tissue engineering.

Hydrogels mimicking the physicochemical properties of the native extracellular matrix have attracted great attention as bioinks for three-dimensional (3D) bioprinting in tissue engineering applications. Alginate is a widely used bioink with beneficial properties of fast gelation and biocompatibility; however, bioprinting using alginate-based bioinks has several limitations, such as poor printability, structural instability, and limited biological activities. To address these issues, we formulated various bioinks using bone morphogenetic protein-2 (BMP-2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and alginate for mesenchymal stem cell (MSC) printing and induction of osteogenic differentiation. Incorporation of PLGA nanoparticles into alginate could enhance the mechanical properties and printability of the bioink. In particular, Alg/NPN30 (30 mg/mL PLGA nanoparticles and 3% w/v alginate) was most suitable for 3D printing with respect to printability and stability. BMP-2-loaded PLGA nanoparticles (NPBMP-2) displayed sustained in vitro release of BMP-2 for up to two weeks. Further in vitro studies indicated that bioinks composed of alginate and NPBMP-2 significantly induced osteogenesis of the MSCs compared with other controls, evidenced by enhanced calcium deposition, alkaline phosphatase activity, and gene expression of osteogenic markers. Our novel bioink consisting of widely used biocompatible components displays good printability, stability, and osteogenic inductivity, and holds strong potential for cell printing and bone tissue engineering applications.

An osteogenic bioink composed of alginate, cellulose nanofibrils, and polydopamine nanoparticles for 3D bioprinting and bone tissue engineering.

Bioprinting is an emerging technology for manufacturing cell-laden three-dimensional (3D) scaffolds, which are used to fabricate complex 3D constructs and provide specific microenvironments for supporting cell growth and differentiation. The development of bioinks with appropriate printability and specific bioactivities is crucial for bioprinting and tissue engineering applications, including bone tissue regeneration. Therefore, to produce functional bioinks for osteoblast printing and bone tissue formation, we formulated various nanocomposite hydrogel-based bioinks using natural and biocompatible biomaterials (i.e., alginate, tempo-oxidized cellulose nanofibrils (TOCNF), and polydopamine nanoparticles (PDANPs)). Rheological studies and printability tests revealed that bioinks containing 1.5% alginate and 1.5% TOCNF in the presence or absence of PDANP (0.5%) are suitable for 3D printing. Furthermore, in vitro studies of 3D-printed osteoblast-laden scaffolds indicated that the 0.5% PDANP-incorporated bioink induced significant osteogenesis. Overall, the bioink consisting of alginate, TOCNF, and PDANPs exhibited excellent printability and bioactivity (i.e., osteogenesis).

In Situ Formation of Proangiogenic Mesenchymal Stem Cell Spheroids in Hyaluronic Acid/Alginate Core-Shell Microcapsules.

Mesenchymal stem-cell (MSC)-based therapies have been recognized as promising strategies for the treatment of various injuries or diseases because of their unique characteristics, such as self-renewal, differentiation potential, and secretion of various bioactive molecules. However, MSC transplantation often results in low efficacy, including a cell viability loss and a low therapeutic activity. Alternatively, MSC spheroids have been studied to improve the viability and therapeutic activity of MSCs. Also, microencapsulation of cells can protect and retain the cells from harsh environments after transplantation. Here, MSC spheroids were formed in hyaluronic acid/alginate (HA@Alg) core-shell microcapsules and employed for neovascularization. A well-defined core-shell structure of HA@Alg microcapsules was produced by optimizing various electrospraying conditions. MSC spheroids could be spontaneously formed in the HA core of the microcapsules after 1 day of incubation. Enhanced secretion of various growth factors was found from MSC spheroids in HA@Alg. In vivo plug assay revealed the significant promotion of angiogenesis by MSC spheroids in HA@Alg compared to that by the controls (i.e., MSCs and MSC spheroids), which is likely because of the better retention of MSC spheroid forms in the microcapsules. Thus, the HA@Alg microcapsules embedding MSC spheroids will be greatly beneficial for various stem cell-based therapies.

Graphene oxide/alginate composites as novel bioinks for three-dimensional mesenchymal stem cell printing and bone regeneration applications.

Three-dimensional (3D) cell printing is a versatile technique enabling the creation of 3D constructs containing hydrogel and cells in the desired shape or pattern. Bioinks exhibiting appropriate mechanical properties and biological activities to support cell growth and/or differentiation toward a specific lineage play critical roles in 3D cell printing and tissue engineering applications. Herein, we explored alginate/graphene oxide (GO) composites as bioinks for their potential to improve printability, structural stability, and osteogenic activities for osteogenic tissue engineering applications. The addition of GO (0.05-1.0 mg mL-1) to 3% alginate significantly enhanced the printing performances of the alginate bioink. In addition, mesenchymal stem cells (MSCs) printed with alginate/GO showed good proliferation and higher survival in an oxidative stress environment. The 3D scaffolds printed with MSCs and alginate/GO demonstrated significantly enhanced osteogenic differentiation compared with those printed with MSCs and alginate. Overall, a bioink of 3% alginate and 0.5 mg mL-1 GO showed the most balanced characteristics in terms of printability, structural stability, and osteogenic induction of the printed MSCs. Alginate/GO composite bioinks will be useful for bioprinting research for various tissue engineering applications.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

Studies on the effects of microencapsulated human mesenchymal stem cells in RGD-modified alginate on cardiomyocytes under oxidative stress conditions using in vitro biomimetic co-culture system.

Stem cell therapy has been recognized as a promising approach for myocardium regeneration post myocardial infarction (MI); however, it unfortunately often remains a challenge because of poor survival of transplanted cells and a lack of clear understanding of their interactions with host cells. High oxidative stress at heart tissues post MI is considered one of the important factors damaging transplanted cells and native cells/tissues. Here, we employed an in vitro co-culture system, capable of mimicking cases of stem cell transplantation into the myocardium presenting high oxidative stress, using human mesenchymal stem cells (hMSCs) encapsulated in alginate or cell interactive Arg-Gly-Asp (RGD) peptide-modified alginate micro-hydrogels. Under H2O2-induced oxidative stress conditions, viabilities of hMSCs and CMs were significantly higher in their co-culture than in their individual monolayer cultures. Expression of cardiac muscle markers remained high even with H2O2 treatment when cardiomyocytes (CMs) were co-cultured with hMSCs in RGD-alginate. Higher levels of various growth factors (associated with angiogenesis, cardiac regeneration, and contractility) were found in co-culture (noticeably with RGD-alginate) compared to monolayer cultures of CMs or hMSCs. These results can benefit the study of in vivo MI progression with transplanted stem cells and the development of effective stem cell-based therapeutic strategies for various oxidative stress-related diseases.

Hydrogel Biomaterials for Stem Cell Microencapsulation.

Stem cell transplantation has been recognized as a promising strategy to induce the regeneration of injured and diseased tissues and sustain therapeutic molecules for prolonged periods in vivo. However, stem cell-based therapy is often ineffective due to low survival, poor engraftment, and a lack of site-specificity. Hydrogels can offer several advantages as cell delivery vehicles, including cell stabilization and the provision of tissue-like environments with specific cellular signals; however, the administration of bulk hydrogels is still not appropriate to obtain safe and effective outcomes. Hence, stem cell encapsulation in uniform micro-sized hydrogels and their transplantation in vivo have recently garnered great attention for minimally invasive administration and the enhancement of therapeutic activities of the transplanted stem cells. Several important methods for stem cell microencapsulation are described in this review. In addition, various natural and synthetic polymers, which have been employed for the microencapsulation of stem cells, are reviewed in this article.

Polypyrrole-incorporated conductive hyaluronic acid hydrogels.

BACKGROUND: Hydrogels that possess hydrophilic and soft characteristics have been widely used in various biomedical applications, such as tissue engineering scaffolds and drug delivery. Conventional hydrogels are not electrically conductive and thus their electrical communication with biological systems is limited. METHOD: To create electrically conductive hydrogels, we fabricated composite hydrogels of hyaluronic acid and polypyrrole. In particular, we synthesized and used pyrrole-hyaluronic acid-conjugates and further chemically polymerized polypyrrole with the conjugates for the production of conductive hydrogels that can display suitable mechanical and structural properties. RESULTS: Various characterization methods, using a rheometer, a scanning electron microscope, and an electrochemical analyzer, revealed that the PPy/HA hydrogels were soft and conductive with ~ 3 kPa Young's modulus and ~ 7.3 mS/cm conductivity. Our preliminary in vitro culture studies showed that fibroblasts were well attached and grew on the conductive hydrogels. CONCLUSION: These new conductive hydrogels will be greatly beneficial in fields of biomaterials in which electrical properties are important such as tissue engineering scaffolds and prosthetic devices.