Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

The onset of osteoporosis leads to a gradual decrease in bone density due to an imbalance between bone formation and resorption. To achieve optimal drug efficacy with minimal side effects, targeted drug delivery to the bone is necessary. Previous studies have utilized peptides that bind to hydroxyapatite, a mineral component of bone, for bone-targeted drug delivery. In this study, a hydroxyapatite binding (HAB) tag is fused to 30Kc19α-Runt-related transcription factor 2 (RUNX2) for bone-targeting. This recombinant protein can penetrate the nucleus of human mesenchymal stem cells (hMSCs) and act as a master transcription factor for osteogenesis. The HAB tag increases the binding affinity of 30Kc19α-RUNX2 to mineral deposition in mature osteoblasts and bone tissue, without affecting its osteogenic induction capability. In the osteoporosis mouse model, intravenous injection of HAB-30Kc19α-RUNX2 results in preferential accumulation in the femur and promotes bone formation while reducing toxicity in the spleen. These findings suggest that HAB-30Kc19α-RUNX2 may be a promising candidate for bone-targeted therapy in osteoporosis.

Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor.

BACKGROUND: The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. METHODS: OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. RESULTS: OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. CONCLUSIONS: Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.

RGD-incorporated biomimetic cryogels for hyaline cartilage regeneration.

Maintaining the integrity of articular cartilage is paramount to joint health and function. Under constant mechanical stress, articular cartilage is prone to injury that often extends to the underlying subchondral bone. In this study, we incorporated arginine-aspartate-glycine (RGD) peptide into chondroitin sulfate-based cryogel for hyaline cartilage regeneration. Known to promote cell adhesion and proliferation, RGD peptide is a double-edged sword for cartilage regeneration. Depending on the peptide availability in the microenvironment, RGD may aid in redifferentiation of dedifferentiated chondrocytes by mimicking physiological cell-matrix interaction or inhibit chondrogenic phenotype via excessive cell spreading. Here, we observed an increase in chondrogenic phenotype with RGD concentration. The group containing the highest RGD concentration (3 mM; RGD group) experienced a 24-fold increase inCOL2expression in the 1st week ofin vitroculture and formed native cartilage-resembling ectopic tissuein vivo. No sign of dedifferentiation (COL1) was observed in all groups. Within the concentration range tested (0-3 mM RGD), RGD promotes chondrocyte redifferentiation after monolayer expansion and thus, formation of hyaline cartilage tissue.

Sequential growth factor releasing double cryogel system for enhanced bone regeneration.

Bone regeneration is a complicated physiological process regulated by several growth factors. In particular, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-4 (BMP-4) are regarded as key factors that induce bone regeneration by angiogenesis and osteogenesis. In this study, we developed a double cryogel system (DC) composed of gelatin/chitosan cryogel (GC) surrounded by gelatin/heparin cryogel (GH) for dual drug delivery with different release kinetics. VEGF was loaded in GH (outer layer of DC) for the initial release of VEGF to induce angiogenesis and provide blood supply in the defect area, while BMP-4 was loaded in GC (inner layer of DC) that leads to sustained release for continuous osteogenic induction. After analyzing characteristics of the double cryogel system such as porosity, degradation rate, swelling ratio, and mechanical properties, we evaluated release kinetics of VEGF (initial release) and BMP-4 (sustained-release) by ELISA. Then, the timely release of VEGF and BMP from DC synergistically induced in vitro osteogenic differentiation as confirmed by alkaline phosphatase staining, Alizarin Red S staining, and real-time PCR analysis. Finally, a critical-sized cranial defect model confirmed the enhanced bone regeneration as a result of dual release growth factor mechanisms.

Ectopic transient overexpression of OCT-4 facilitates BMP4-induced osteogenic transdifferentiation of human umbilical vein endothelial cells.

Limitation in cell sources for autologous cell therapy has been a recent focus in stem cell therapy and tissue engineering. Among various research advances, direct conversion, or transdifferentiation, is a notable and feasible strategy for the generation and acquirement of wanted cell source. So far, utilizing cell transdifferentiation technology in tissue engineering was mainly restricted at achieving single wanted cell type from diverse cell types with high efficiency. However, regeneration of a complete tissue always requires multiple cell types which poses an intrinsic complexity. In this study, enhanced osteogenic differentiation was achieved by transient ectopic expression of octamer-binding transcription factor 4 (OCT-4) gene followed by bone morphogenetic protein 4 treatment on human umbilical vein endothelial cells. OCT-4 transfection and bone morphogenetic protein 4 treatment resulted in enhanced expression of osteogenic markers such as core-binding factor alpha 1, alkaline phosphatase, and collagen 1 compared with bone morphogenetic protein 4 treatment alone. Furthermore, we employed gelatin-heparin cryogel in cranial defect model for in vivo bone formation. Micro-computed tomography and histological analysis of in vivo samples showed that OCT-4 transfection followed by bone morphogenetic protein 4 treatment resulted in efficient transdifferentiation of endothelial cells to osteogenic cells. These results suggest that the combination of OCT-4 and bone morphogenetic protein 4 on endothelial cells would be a reliable multicellular transdifferentiation model which could be applied for bone tissue engineering.

Intracellular Delivery of Recombinant RUNX2 Facilitated by Cell-Penetrating Protein for the Osteogenic Differentiation of hMSCs.

Human mesenchymal stem cells (hMSCs) are a commonly used cell source for cell therapy and tissue engineering because of their easy accessibility and multipotency. Runt-related transcription factor 2 (RUNX2) is a master regulator of the osteogenic commitment of hMSCs. Either recombinant plasmid delivery or viral transduction has been utilized to activate RUNX2 gene expression for effective hMSC differentiation. In this study, recombinant RUNX2 fused with cell-penetrating 30Kc19α protein (30Kc19α-RUNX2) was delivered into hMSCs for osteogenic commitment. Fusion of recombinant RUNX2 with 30Kc19α resulted in successful delivery of the protein into cells and enhanced soluble expression of the protein. Intracellular delivery of the 30Kc19α-RUNX2 fusion protein enhanced the osteogenic differentiation of hMSCs in vitro. 30Kc19α-RUNX2 treatment resulted in increased ALP accumulation and elevated calcium deposition. Finally, implantation of hMSCs treated with 30Kc19α-RUNX2 showed osteogenesis via cell delivery into the subcutaneous tissue and bone regeneration in a cranial defect mouse model. Therefore, we suggest that 30Kc19α-RUNX2, an osteoinductive recombinant protein, is an efficient tool for bone tissue engineering.

Cardiac-mimetic cell-culture system for direct cardiac reprogramming.

Rationale: Cardiovascular diseases often cause substantial heart damage and even heart failure due to the limited regenerative capacity of adult cardiomyocytes. The direct cardiac reprogramming of fibroblasts could be a promising therapeutic option for these patients. Although exogenous transcriptional factors can induce direct cardiac reprogramming, the reprogramming efficiency is too low to be used clinically. Herein, we introduce a cardiac-mimetic cell-culture system that resembles the microenvironment in the heart and provides interactions with cardiomyocytes and electrical cues to the cultured fibroblasts for direct cardiac reprogramming. Methods: Nano-thin and nano-porous membranes and heart like electric stimulus were used in the cardiac-mimetic cell-culture system. The human neonatal dermal fibroblasts containing cardiac transcription factors were plated on the membrane and cultured with the murine cardiomyocyte in the presence of the electric stimulus. The reprogramming efficiency was evaluated by qRT-PCR and immunocytochemistry. Results: Nano-thin and nano-porous membranes in the culture system facilitated interactions between fibroblasts and cardiomyocytes in coculture. The cellular interactions and electric stimulation supplied by the culture system dramatically enhanced the cardiac reprogramming efficiency of cardiac-specific transcriptional factor-transfected fibroblasts. Conclusion: The cardiac-mimetic culture system may serve as an effective tool for producing a feasible number of reprogrammed cardiomyocytes from fibroblasts.

Osteogenic Effects of VEGF-Overexpressed Human Adipose-Derived Stem Cells with Whitlockite Reinforced Cryogel for Bone Regeneration.

Bone is a vascularized tissue that is comprised of collagen fibers and calcium phosphate crystals such as hydroxyapatite (HAp) and whitlockite (WH). HAp and WH are known to elicit bone regeneration by stimulating osteoblast activities and osteogenic commitment of stem cells. In addition, vascular endothelial growth factor (VEGF) is shown to promote osteogenesis and angiogenesis which is considered as an essential process in bone repair by providing nutrients. In this study, VEGF-secreting human adipose-derived stem cells (VEGF-ADSCs) are developed by transducing ADSCs with VEGF-encoded lentivirus. Additionally, WH-reinforced gelatin/heparin cryogels (WH-C) are fabricated by loading WH into gelatin/heparin cryogels. VEGF-ADSC secrete tenfold more VEGF than ADSC and show increased VEGF secretion with cell growth. Also, incorporation of WH into cryogels provides a mineralized environment with ions secreted from WH. When the VEGF-ADSCs are seeded on WH-C, sustained release of VEGF is observed due to the specific affinity of VEGF to heparin. Finally, the synergistic effect of VEGF-ADSC and WH on osteogenesis is successfully confirmed by alkaline phosphatase and real-time polymerase chain reaction analysis. In vivo bone formation is demonstrated via implantation of VEGF-ADSC seeded WH-C into mouse calvarial bone defect model, resulted in enhanced bone development with the highest bone volume/total volume.

Biomaterials for Stem Cell Therapy for Cardiac Disease.

Myocardial Infarction (MI) in cardiac disease is the result of heart muscle losses due to a wide range of factors. Cardiac muscle failure is a crucial condition that provokes life-threatening outcomes. Heretofore, regeneration therapies in MI have used stem-cell-based therapy instantly after a myocardial injury to prevent the disease process and tissue malfunction. Despite the therapeutic utility of stem-cell-based regenerative therapy, barriers to successful treatment have been addressed. In this chapter, we illustrate a variety of emerging biomaterial strategies for enhancing the function of therapeutic stem cells, such as cell surface modification to synthetically endowing stem cells with new characteristics and hydrogels with its biological and mechanical properties. These investments offer a potential accompaniment to traditional stem-cell-based therapies for enhancing the efficacy of stem cell therapy to design properly activating cardiac tissues.

Self-Healing and Adhesive Artificial Tissue Implant for Voice Recovery.

Loss of voice after vocal fold resection due to laryngeal cancer is a significant problem resulting in a low quality of life. Although there were many attempts to achieve a functional restoration of voice, challenges to regenerate vocal fold still remain due to its unique tissue mechanical characteristics such as pliability that produces phonation via vibration. In this study, we developed a mechanically compliant interpenetrating polymer network (IPN) hydrogel based on polyacrylamide (PAAM) and gelatin that matches physical and functional properties with native vocal fold tissue. The mechanical properties of this PAAM/gelatin (PG) hydrogel were optimized to have an elastic modulus of 5.4 kPa by adjusting the PAAM/gelatin ratio. In addition, the PG hydrogel demonstrated a minimal foreign body reaction upon implantation, and the hydrogel displayed a strong resistance to dehydration conditions that can last 40 days in the chamber with 60% humidity. Furthermore, the PG hydrogel demonstrated a self-healing ability that may allow ad-hoc implant augmentation. In addition, tough adhesion of the PG hydrogel resulted in stable attachment to vocal fold tissues. Finally, we demonstrated the functional restoration of voice on an ex vivo canine model by implanting the PG hydrogel as an artificial vocal fold tissue.

Bioglass-Incorporated Methacrylated Gelatin Cryogel for Regeneration of Bone Defects.

Cryogels have recently gained interest in the field of tissue engineering as they inherently possess an interconnected macroporous structure. Considered to be suitable for scaffold cryogel fabrication, methacrylated gelatin (GelMA) is a modified form of gelatin valued for its ability to retain cell adhesion site. Bioglass nanoparticles have also attracted attention in the field due to their osteoinductive and osteoconductive behavior. Here, we prepare methacrylated gelatin cryogel with varying concentration of bioglass nanoparticles to study its potential for bone regeneration. We demonstrate that an increase in bioglass concentration in cryogel leads to improved mechanical property and augmented osteogenic differentiation of mesenchymal cells during in vitro testing. Furthermore, in vivo testing in mice cranial defect model shows that highest concentration of bioglass nanoparticles (2.5 w/w %) incorporated in GelMA cryogel induces the most bone formation compared to the other tested groups, as studied by micro-CT and histology. The in vitro and in vivo results highlight the potential of bioglass nanoparticles incorporated in GelMA cryogel for bone regeneration.

Non-viral approaches for direct conversion into mesenchymal cell types: Potential application in tissue engineering.

Acquiring adequate number of cells is one of the crucial factors to apply tissue engineering strategies in order to recover critical-sized defects. While the reprogramming technology used for inducing pluripotent stem cells (iPSCs) opened up a direct path for generating pluripotent stem cells, a direct conversion strategy may provide another possibility to obtain desired cells for tissue engineering. In order to convert a somatic cell into any other cell type, diverse approaches have been investigated. Conspicuously, in contrast to traditional viral transduction method, non-viral delivery of conversion factors has the merit of lowering immune responses and provides safer genetic manipulation, thus revolutionizing the generation of directly converted cells and its application in therapeutics. In addition, applying various microenvironmental modulations have potential to ameliorate the conversion of somatic cells into different lineages. In this review, we discuss the recent progress in direct conversion technologies, specifically focusing on generating mesenchymal cell types.

Injectable PLGA microspheres encapsulating WKYMVM peptide for neovascularization.

Formyl peptide receptor-2 (FPR-2) is expressed in various cell types, such as phagocytes, fibroblasts, and endothelial cells. FPR-2 has been reported to play a significant role in inflammation and angiogenic response, and synthetic WKYMVm peptide has been identified as a novel peptide agonist for the FPR-2. In this study, we demonstrate that WKYMVm peptides stimulate the angiogenic potential of outgrowth endothelial cells (OECs). Upon WKYMVm peptide exposure, migration and proliferation of OECs were stimulated. WKYMVm effectively stimulated angiogenesis in tube formation assay and aortic ring assay. Furthermore, we fabricated injectable poly (lactide-co-glycolide) (PLGA) microspheres encapsulating WKYMVm peptides, which showed sustained release of cargo molecule. When WKYMVm peptide encapsulated microspheres were injected into the hind limb ischemia model, a single injection of microspheres was as effective as multiple injections of WKYMVm peptide in restoring blood flow from ischemic injury and promoting capillary growth. These results demonstrate that sustained release of WKYMVm peptide from microspheres in the application to ischemic hind limb extended angiogenic stimulation. STATEMENT OF SIGNIFICANCE: Formyl peptide receptor (FPR) has been reported to play an important role in inflammation and angiogenic response. A synthetic WKYMVm peptide has been identified as a novel peptide activating the FPR-2 that is expressed in a various cell types, such as phagocytes, fibroblasts, and endothelial cells. In this manuscript we explored a unique property of high-affinity ligand for formyl peptide receptors-2 (FPR-2) (i.e., WKYMVm). WKYMVm-induced activation of FPR2 has been reported to be crucial in host defense and inflammation by activation of phagocytes, monocytes, and lymphocytes. In this study, highlight the efficacy of WKYMVm peptide's role in inducing neovascularization in vivo hind limb ischemia model when the peptide was released from injected PLGA microspheres in sustained manner. Our results demonstrate that sustained release of WKYMVm peptide from microspheres have extended angiogenic stimulation capacity.