Bioceramic-mediated chondrocyte hypertrophy promotes calcified cartilage formation for rabbit osteochondral defect repair.

Osteochondral tissue is a highly specialized and complex tissue composed of articular cartilage and subchondral bone that are separated by a calcified cartilage interface. Multilayered or gradient scaffolds, often in conjunction with stem cells and growth factors, have been developed to mimic the respective layers for osteochondral defect repair. In this study, we designed a hyaline cartilage-hypertrophic cartilage bilayer graft (RGD/RGDW) with chondrocytes. Previously, we demonstrated that RGD peptide-modified chondroitin sulfate cryogel (RGD group) is chondro-conductive and capable of hyaline cartilage formation. Here, we incorporated whitlockite (WH), a Mg2+-containing calcium phosphate, into RGD cryogel (RGDW group) to induce chondrocyte hypertrophy and form collagen X-rich hypertrophic cartilage. This is the first study to use WH to produce hypertrophic cartilage. Chondrocytes-laden RGDW cryogel exhibited significantly upregulated expression of hypertrophy markers in vitro and formed ectopic hypertrophic cartilage in vivo, which mineralized into calcified cartilage in bone microenvironment. Subsequently, RGD cryogel and RGDW cryogel were combined into bilayer (RGD/RGDW group) and implanted into rabbit osteochondral defect, where RGD layer supports hyaline cartilage regeneration and bioceramic-containing RGDW layer promotes calcified cartilage formation. While the RGD group (monolayer) formed hyaline-like neotissue that extends into the subchondral bone, the RGD/RGDW group (bilayer) regenerated hyaline cartilage tissue confined to its respective layer and promoted osseointegration for integrative defect repair.

Comparative analysis of supercritical fluid-based and chemical-based decellularization techniques for nerve tissue regeneration.

Axon regeneration and Schwann cell proliferation are critical processes in the repair and functional recovery of damaged neural tissues. Biomaterials can play a crucial role in facilitating cell proliferative processes that can significantly impact the target tissue repair. Chemical decellularization and supercritical fluid-based decellularization methods are similar approaches that eliminate DNA from native tissues for tissue-mimetic biomaterial production by using different solvents and procedures to achieve the final products. In this study, we conducted a comparative analysis of these two methods in the context of nerve regeneration and neuron cell differentiation efficiency. We evaluated the efficacy of each method in terms of biomaterial quality, preservation of extracellular matrix components, promotion of neuronal cell differentiation and nerve tissue repair ability in vivo. Our results indicate that while both methods produce high-quality biomaterials, supercritical fluid-based methods have several advantages over conventional chemical decellularization, including better preservation of extracellular matrix components and mechanical properties and superior promotion of cellular responses. We conclude that supercritical fluid-based methods show great promise for biomaterial production for nerve regeneration and neuron cell differentiation applications.

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.

Injectable anti-inflammatory hyaluronic acid hydrogel for osteoarthritic cartilage repair.

Osteoarthritis (OA) is one of the most common cartilage disorder that results from breakdown of joint cartilage and underlying bone tissues. Once OA is induced in the joint, subsequent immune reaction leads to chronic inflammation that results in the progression of OA and deconstruction of the cartilage. In this study, an injectable hydrogel with epigallocatechin-3-gallate (EGCG) is introduced to control inflammation and enhance cartilage regeneration. EGCG has intrinsic properties that can modulate inflammation and scavenge radical species. Hyaluronic acid (HA), as a major component of the cartilage ECM, is commonly used for cartilage tissue engineering. In this study, EGCG was combined with tyramine-conjugated HA and gelatin to create a composite hydrogel at an optimized concentration of 50 µM EGCG and 5% w/v HA. The composite hydrogel provided protection to chondrocytes against the pro-inflammatory factor, IL-1ß. Additionally, the composite hydrogel led to chondrogenic regeneration in vitro. Histological analysis in vivo showed that EGCG-HA/Gelatin hybrid hydrogel minimized cartilage loss in surgically induced OA model. This study demonstrates that inflammation-modulating HA-based hydrogel may provide a therapeutic option for OA treatment.

Inflammation-Modulating Hydrogels for Osteoarthritis Cartilage Tissue Engineering.

Osteoarthritis (OA) is the most common form of the joint disease associated with age, obesity, and traumatic injury. It is a disabling degenerative disease that affects synovial joints and leads to cartilage deterioration. Despite the prevalence of this disease, the understanding of OA pathophysiology is still incomplete. However, the onset and progression of OA are heavily associated with the inflammation of the joint. Therefore, studies on OA treatment have sought to intra-articularly deliver anti-inflammatory drugs, proteins, genes, or cells to locally control inflammation in OA joints. These therapeutics have been delivered alone or increasingly, in delivery vehicles for sustained release. The use of hydrogels in OA treatment can extend beyond the delivery of anti-inflammatory components to have inherent immunomodulatory function via regulating immune cell polarization and activity. Currently, such immunomodulatory biomaterials are being developed for other applications, which can be translated into OA therapy. Moreover, anabolic and proliferative levels of OA chondrocytes are low, except initially, when chondrocytes temporarily increase anabolism and proliferation in response to structural changes in their extracellular environment. Therefore, treatments need to restore matrix protein synthesis and proliferation to healthy levels to reverse OA-induced damage. In conjugation with injectable and/or adhesive hydrogels that promote cartilage tissue regeneration, immunomodulatory tissue engineering solutions will have robust potential in OA treatment. This review describes the disease, its current and future immunomodulatory therapies as well as cartilage-regenerative injectable and adhesive hydrogels.

Chondrogenically primed tonsil-derived mesenchymal stem cells encapsulated in riboflavin-induced photocrosslinking collagen-hyaluronic acid hydrogel for meniscus tissue repairs.

Current meniscus tissue repairing strategies involve partial or total meniscectomy, followed by allograft transplantation or synthetic material implantation. However, allografts and synthetic implants have major drawbacks such as the limited supply of grafts and lack of integration into host tissue, respectively. In this study, we investigated the effects of conditioned medium (CM) from meniscal fibrochondrocytes and TGF-ß3 on tonsil-derived mesenchymal stem cells (T-MSCs) for meniscus tissue engineering. CM-expanded T-MSCs were encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogels and cultured in chondrogenic medium containing TGF-ß3. In vitro results indicate that CM-expanded cells followed by TGF-ß3 exposure stimulated the expression of fibrocartilage-related genes (COL2, SOX9, ACAN, COL1) and production of extracellular matrix components. Histological assessment of in vitro and subcutaneously implanted in vivo constructs demonstrated that CM-expanded cells followed by TGF-ß3 exposure resulted in highest cell proliferation, GAG accumulation, and collagen deposition. Furthermore, when implanted into meniscus defect model, CM treatment amplified the potential of TGF-ß3 and induced complete regeneration. STATEMENT OF SIGNIFICANCE: Conditioned medium derived from chondrocytes have been reported to effectively prime mesenchymal stem cells toward chondrogenic lineage. Type I collagen is the main component of meniscus extracellular matrix and hyaluronic acid is known to promote meniscus regeneration. In this manuscript, we investigated the effects of conditioned medium (CM) and transforming growth factor-ß3 (TGF-ß3) on tonsil-derived mesenchymal stem cells (T-MSCs) encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogel. We employed a novel source of conditioned medium, derived from meniscal fibrochondrocytes. Our in vitro and in vivo results collectively illustrate that CM-expanded cells followed by TGF-ß3 exposure have the best potential for meniscus regeneration. This manuscript highlights a novel stem cell commitment strategy combined with biomaterials designs for meniscus regeneration.

Enhanced Osteogenic Commitment of Human Mesenchymal Stem Cells on Polyethylene Glycol-Based Cryogel with Graphene Oxide Substrate.

Graphene oxide (GO) is considered a comparatively recent biomaterial with enormous potential because of its nontoxicity, high dispersity, and enhanced interaction with biomolecules. These characteristics of GO can promote the interactions between the substrates and cell surfaces. In this study, we incorporated GO in a cryogel-based scaffold system to observe their influence on the osteogenic responses of human tonsil-derived mesenchymal stem cells (hTMSCs). Compared to polyethylene glycol (PEG)-based cryogel scaffold, GO-embedded PEG-based (PEGDA-GO) cryogels not only showed improved cell attachment and focal adhesion kinase (FAK) signaling activation but also enhanced cell viability. Taken together, we demonstrated that PEGDA-GO cryogels can stimulate osteogenic differentiation under an osteoinductive condition and enhance osteogenic phenotypes compared to the control group. In summary, we demonstrate that GO embedded in cryogels system is an effective biofunctionalizing scaffold to control osteogenic commitment of stem cells.

High throughput approaches for controlled stem cell differentiation.

Stem cells have unique ability to undergo self-renewal indefinitely in culture and potential to differentiate into almost all cell types in the human body. However, the developing a method for efficiently differentiating or manipulating these stem cells for therapeutic purposes remains a challenging problem. Pluripotent stem cells, as well as adult stem cells, require biological cues for their proliferation and differentiation. These cues are largely controlled by cell-cell, cell-insoluble factors (such as extracellular matrix), and cell-soluble factors (such as cytokine or growth factors) interactions. In this review, we describe a state of research on various stem cell-based tissue engineering applications and high throughput strategies for developing synthetic or biosynthetic microenvironments to allow efficient commitments in stem cells. STATEMENT OF SIGNIFICANCE: Nowadays, pluripotency of stem cells have received much attention to use therapeutic purpose. However, a major difficulty with stem cell therapy is to control its differentiation through desired cells or tissues. In other words, various microenvironment factors are involved during stem cell differentiation, including dimensionality, growth factors, cell junctions, nutritional status, matrix stiffness, matrix composition, mechanical stress, and cell-matrix adhesion. Therefore, researchers have engineered a variety of platforms to enable controlling and monitoring bioactive factors to induce stem cell commitment. In this review, we report on recent advancements in a novel technology based on high-throughput strategies for stem cell-based tissue engineering applications.

Riboflavin-induced photo-crosslinking of collagen hydrogel and its application in meniscus tissue engineering.

A meniscus tear is a common knee injury, but its regeneration remains a clinical challenge. Recently, collagen-based scaffolds have been applied in meniscus tissue engineering. Despite its prevalence, application of natural collagen scaffold in clinical setting is limited due to its extremely low stiffness and rapid degradation. The purpose of the present study was to increase the mechanical properties and delay degradation rate of a collagen-based scaffold by photo-crosslinking using riboflavin (RF) and UV exposure. RF is a biocompatible vitamin B2 that showed minimal cytotoxicity compared to conventionally utilized photo-initiator. Furthermore, collagen photo-crosslinking with RF improved mechanical properties and delayed enzyme-triggered degradation of collagen scaffolds. RF-induced photo-crosslinked collagen scaffolds encapsulated with fibrochondrocytes resulted in reduced scaffold contraction and enhanced gene expression levels for the collagen II and aggrecan. Additionally, hyaluronic acid (HA) incorporation into photo-crosslinked collagen scaffold showed an increase in its retention. Based on these results, we demonstrate that photo-crosslinked collagen-HA hydrogels can be potentially applied in the scaffold-based meniscus tissue engineering.