Simulated microgravity attenuates myogenesis and contractile function of 3D engineered skeletal muscle tissues.

While the effects of microgravity on inducing skeletal muscle atrophy have been extensively studied, the impacts of microgravity on myogenesis and its mechanisms remain unclear. In this study, we developed a microphysiological system of engineered muscle tissue (EMT) fabricated using a collagen / Matrigel composite hydrogel and murine skeletal myoblasts. This 3D EMT model allows non-invasive quantitative assessment of contractile function. After applying a 7-day differentiation protocol to induce myotube formation, the EMTs clearly exhibited sarcomerogenesis, myofilament formation, and synchronous twitch and tetanic contractions with electrical stimuli. Using this 3D EMT system, we investigated the effects of simulated microgravity at 10-3 G on myogenesis and contractile function utilizing a random positioning machine. EMTs cultured for 5 days in simulated microgravity exhibited significantly reduced contractile forces, myofiber size, and differential expression of muscle contractile, myogenesis regulatory, and mitochondrial biogenesis-related proteins. These results indicate simulated microgravity attenuates myogenesis, resulting in impaired muscle function.

Coagulant Protein-Free Blood Coagulation Using Catechol-Conjugated Adhesive Chitosan/Gelatin Double Layer.

Since the discovery of polyphenolic underwater adhesion in marine mussels, researchers strive to emulate this natural phenomenon in the development of adhesive hemostatic materials. In this study, bio-inspired hemostatic materials that lead to pseudo-active blood coagulation, utilizing traditionally passive polymer matrices of chitosan and gelatin are developed. The two-layer configuration, consisting of a thin, blood-clotting catechol-conjugated chitosan (CHI-C) layer and a thick, barrier-functioning gelatin (Geln) ad-layer, maximizes hemostatic capability and usability. The unique combination of coagulant protein-free condition with CHI-C showcases not only coagulopathy-independent blood clotting properties (efficacy) but also exceptional clinical potential, meeting all necessary biocompatibility evaluation (safety) without inclusion of conventional coagulation triggering proteins such as thrombin or fibrinogen. As a result, the CHI-C/Geln is approved by the Ministry of Food and Drug Safety (MFDS, Republic of Korea) as a class II medical device. Hemostatic efficacy observed in multiple animal models further demonstrates the superiority of CHI-C/Geln sponges in achieving quick hemostasis compared to standard treatments. This study not only enriches the growing body of research on mussel-inspired materials but also emphasizes the potential of biomimicry in developing advanced medical materials, contributing a promising avenue toward development of readily accessible and affordable hemostatic materials.

Efficient improvement of the proliferation, differentiation, and anti-arthritic capacity of mesenchymal stem cells by simply culturing on the immobilized FGF2 derived peptide, 44-ERGVVSIKGV-53.

INTRODUCTION: The stem cell microenvironment has been evidenced to robustly affect its biological functions and clinical grade. Natural or synthetic growth factors, especially, are essential for modulating stem cell proliferation, metabolism, and differentiation via the interaction with specific extracellular receptors. Fibroblast growth factor-2 (FGF-2) possesses pleiotropic functions in various tissues and organs. It interacts with the FGF receptor (FGFR) and activates FGFR signaling pathways, which involve numerous biological functions, such as angiogenesis, wound healing, cell proliferation, and differentiation. OBJECTIVES: Here, we aim to explore the molecular functions, mode of action, and therapeutic activity of yet undetermined function, FGF-2-derived peptide, FP2 (44-ERGVVSIKGV-53) in promoting the proliferation, differentiation, and therapeutic application of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) in comparison to other test peptides, canofin1 (FP1), hexafin2 (FP3), and canofin3 (FP4) with known functions. METHODS: The immobilization of test peptides that are fused with mussel adhesive proteins (MAP) on the culture plate was carried out via EDC/NHS chemistry. Cell Proliferation assay, colony-forming unit, western blotting analysis, gene expression analysis, RNA-Seq. analysis, osteogenic, and chondrogenic differentiation capacity were applied to test the activity of the test peptides. We additionally utilized three-dimensional (3D) structural analysis and artificial intelligence (AI)-based AlphaFold2 and CABS-dock programs for receptor interaction prediction of the peptide receptor. We also verified the in vivo therapeutic capacity of FP2-cultured hWJ-MSCs using an osteoarthritis mice model. RESULTS: Culture of hWJ-MSC onto an FP2-immobilized culture plate showed a significant increase in cell proliferation (n = 3; *p < 0.05, **p < 0.01) and the colony-forming unit (n = 3; *p < 0.05, **p < 0.01) compared with the test peptides. FP2 showed a significantly upregulated phosphorylation of FRS2α and FGFR1 and activated the AKT and ERK signaling pathways (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Interestingly, we detected efficient FP2 receptor binding that was predicted using AI-based tools. Treatment with an AKT inhibitor significantly abrogated the FP2-mediated enhancement of cell differentiation (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Intra-articular injection of FP2-cultured MSCs significantly mitigated arthritis symptoms in an osteoarthritis mouse model, as shown through the functional tests (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), modulation of the expression level of the pro-inflammatory and anti-inflammatory genes, and improved osteochondral regeneration as demonstrated by tissue sections. CONCLUSION: Our study identified the FGF-2-derived peptide FP2 as a promising candidate peptide to improve the therapeutic potential of hWJ-MSCs, especially in bone and cartilage regeneration.

Engineering approaches for effective therapeutic applications based on extracellular vesicles.

The biological significance of extracellular vesicles (EVs) as intercellular communication mediators has been increasingly revealed in a wide range of normal physiological processes and disease pathogenesis. In particular, regenerative and immunomodulatory EVs hold potential as innate biotherapeutics, whereas pathological EVs are considered therapeutic targets for inhibiting their bioactivity. Given their ability to transport functional cargos originating from the source cells to target cells, EVs can also be used as a therapeutic means to deliver drug molecules. This review aims to provide an updated overview of the key engineering approaches for better exploiting EVs in disease intervention. The emphasis is lying on the preconditioning methods for therapeutic EVs, drug loading and targeting technologies for carrier EVs, and activity control strategies for pathological EVs.

Developmental role of hyaluronic acid and its application in salivary gland tissue engineering.

Dry mouth, or xerostomia, caused by salivary gland dysfunction significantly impacts oral/systemic health and quality of life. Although in vitro-generated artificial salivary glands have been considered as the fundamental solution, its structural complexity is difficult to reproduce using current biomaterials. Therefore, understanding and recapitulating the roles of biomacromolecules in salivary gland organogenesis is needed to solve these problems. Hyaluronic acid (HA) is a macromolecule abundant during salivary gland organogenesis, but its role remains unknown. Here, we verify the effects of HA on salivary gland organogenesis and artificial organ germ formation in solubilized and substrate-immobilized forms. In embryonic submandibular glands (eSMG), we found dense HA layers encapsulating proliferative c-Kit+ progenitor cells that were expressing CD44, an HA receptor. The blockage of HA synthesis, or degradation of HA, impaired eSMG growth by ablating the c-Kit+ progenitor cell population. We also found that high-molecular-weight (HMW) HA has a significant role in eSMG growth. Based on these findings, we discovered that HA is also crucial for in vitro formation of salivary gland organ germs, one of the most promising candidates for salivary gland tissue regeneration. We significantly enhanced salivary gland organ germ formation by supplementing HMW HA in solution; this effect was further increased when the HMW HA was immobilized on the substrate by polydopamine/HA co-immobilization. Our study suggests that the current use of HA in salivary gland tissue engineering can be further optimized.

Catechin solubilization by spontaneous hydrogen bonding with poly(ethylene glycol) for dry eye therapeutics.

Catechin exhibits various pharmacological effects, yet its poor aqueous solubility limits its clinical use. Here, we investigate a facile catechin solubilization method via spontaneous hydrogen bonding between catechin and poly(ethylene glycol) (PEG). The method is extremely simple in that mixing PEG with catechin followed by lyophilization completely converts insoluble catechin to soluble PEG/catechin nanoscale complexes. This solubilized catechin formulation is useful for preparing eyedrop medicine, and we demonstrate that the solubilized catechin exhibits therapeutic effect upon dry eye diseases.

Targeting protein and peptide therapeutics to the heart via tannic acid modification.

Systemic injection into blood vessels is the most common method of drug administration. However, targeting drugs to the heart is challenging, owing to its dynamic mechanical motions and large cardiac output. Here, we show that the modification of protein and peptide therapeutics with tannic acid-a flavonoid found in plants that adheres to extracellular matrices, elastins and collagens-improves their ability to specifically target heart tissue. Tannic-acid-modified (TANNylated) proteins do not adsorb on endothelial glycocalyx layers in blood vessels, yet they penetrate the endothelium to thermodynamically bind to myocardium extracellular matrix before being internalized by myoblasts. In a rat model of myocardial ischaemia-reperfusion injury, TANNylated basic fibroblast growth factor significantly reduced infarct size and increased cardiac function. TANNylation of systemically injected therapeutic proteins, peptides or viruses may enhance the treatment of heart diseases.

BiFACIAL ( Biomimetic Freestanding Anisotropic Catechol- Interfaces with Asymmetrically Layered) Films as Versatile Extracellular Matrix Substitutes.

Biological naïve extracellular matrices (ECMs) exhibit anisotropic functions in their physical, chemical, and morphological properties. Representative examples include anisotropic skin layers or blood vessels simultaneously facing multiphasic environments. Here, anisotropically multifunctional structures called BiFACIAL ( biomimetic freestanding anisotropic catechol- interfaces with asymmetrically layered) films were developed simply by contacting two polysaccharide solutions of heparin-catechol (Hep-C) and chitosan-catechol (Chi-C). Such anisotropic characters were due to controlling catechol cross-linking by alkaline pH, resulting in a trimodular structure: a rigid yet porous Hep-C exterior, nonporous interfacial zone, and soft/highly porous Chi-C interior. The anisotropic features of each layer, including the porosity, rigidity, rheology, composition, and ionic strength, caused the BiFACIAL films to show spontaneously biased stimuli responses and differential behaviors against biological substances (e.g., blood plasma). The films could be created in situ in live animals and imitated the structural/functional aspects of the representative anisotropic tissues (e.g., skin and blood vessels), providing valuable ECM-like platforms for the creation of favorable environments or for tissue regeneration or disease treatment by effectively manipulating cellular behaviors.

M13 bacteriophage displaying DOPA on surfaces: fabrication of various nanostructured inorganic materials without time-consuming screening processes.

M13 bacteriophage (phage) was engineered for the use as a versatile template for preparing various nanostructured materials via genetic engineering coupled to enzymatic chemical conversions. First, we engineered the M13 phage to display TyrGluGluGlu (YEEE) on the pVIII coat protein and then enzymatically converted the Tyr residue to 3,4-dihydroxyl-l-phenylalanine (DOPA). The DOPA-displayed M13 phage could perform two functions: assembly and nucleation. The engineered phage assembles various noble metals, metal oxides, and semiconducting nanoparticles into one-dimensional arrays. Furthermore, the DOPA-displayed phage triggered the nucleation and growth of gold, silver, platinum, bimetallic cobalt-platinum, and bimetallic iron-platinum nanowires. This versatile phage template enables rapid preparation of phage-based prototype devices by eliminating the screening process, thus reducing effort and time.

Development of animal experimental periodontitis models.

PURPOSE: An animal periodontitis model is essential for research on the pathogenesis and treatment of periodontal disease. In this study, we have introduced a lipopolysaccharide (LPS) of a periodontal pathogen to the alveolar bone defect of experimental animals and investigated its suitability as a periodontitis model. METHODS: Alveolar bone defects were made in both sides of the mandibular third premolar region of nine beagle dogs. Then, the animals were divided into the following groups: silk ligature tied on the cervical region of tooth group, Porphyromonas gingivalis LPS (P.g. LPS)-saturated collagen with silk ligature group, and no ligature or P.g. LPS application group as the control. The plaque index and gingival index were measured at 0 and 4 weeks postoperatively. The animals were then euthanized and prepared for histologic evaluation. RESULTS: The silk ligature group and P.g. LPS with silk ligature group showed a significantly higher plaque index at 4 weeks compared to the control (P<0.05). No significant difference was found in the plaque index between the silk ligature group and P.g. LPS with silk ligature group. The P.g. LPS with silk ligature group showed a significantly higher gingival index compared to the silk ligature group or the control at 4 weeks (P<0.05). Histologic examination presented increased inflammatory cell infiltration in the gingival tissue and alveolar bone of the P.g. LPS with silk ligature group. CONCLUSIONS: An additional P.g. LPS-saturated collagen with silk ligature ensured periodontal inflammation at 4 weeks. Therefore, P.g. LPS with silk ligature application to surgically created alveolar bone defects may be a candidate model for experimental periodontitis.