Soyon Kim

TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells

BackgroundUnlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-β1 (TGF-β1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components.ResultsTGF-β1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-β1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels.ConclusionsThis study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects.

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

Photopolymerizable chitosan–collagen hydrogels for bone tissue engineering

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue‐engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi‐interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC–Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC–Col composite hydrogels may be useful in promoting bone regeneration. Copyright

Delivery of siRNA via cationic Sterosomes to enhance osteogenic differentiation of mesenchymal stem cells

Noggin is a specific antagonist of bone morphogenetic proteins (BMPs) that can prevent the interaction of BMPs with their receptors. RNA interfering molecules have been used to downregulate noggin expression and thereby stimulate BMP signaling and osteogenesis. Cationic liposomes are considered one of the most efficient non-viral systems for gene delivery. In the past decade, non-phospholipid liposomes (Sterosomes) formulated with single-chain amphiphiles and high content of sterols have been developed. In particular, Sterosomes composed of stearylamine (SA) and cholesterol (Chol) display distinct properties compared with traditional phospholipid liposomes, including increased positive surface charges and enhanced particle stability. Herein, we report SA/Chol Sterosome and small interfering RNA (siRNA) complexes that significantly enhanced cellular uptake and gene knockdown efficiencies in adipose derived mesenchymal stem cells with minimal cytotoxicity compared with commercially available lipofectamine 2000. Furthermore, we confirmed osteogenic efficacy of these Sterosomes loaded with noggin siRNA in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. The delivery of siRNA via novel SA/Chol Sterosomes presents a powerful method for efficient gene knockdown. These distinct nanoparticles may present a promising alternative approach for gene delivery.

Visible-light-initiated hydrogels preserving cartilage extracellular signaling for inducing chondrogenesis of mesenchymal stem cells.

Hydrogels have a unique opportunity to regenerate damaged cartilage tissues by introducing mesenchymal stem cells (MSCs) in a highly swollen environment similar to articular cartilage. During cartilage development, collagen-cell interactions play an important role in mediating early mesenchymal condensation and chondrogenesis with transforming growth factor-β1 (TGF-β1) stimulation. Here, a hydrogel environment that can enhance cell-matrix interactions and chondrogenesis by stabilizing type-II collagen (Col II) and TGF-β1 into photopolymerizable (methacrylated) chitosan (MeGC) with simple entrapment and affinity binding is demonstrated. The MeGC hydrogel was designed to gel upon initiation by exposure to visible blue light in the presence of riboflavin, an aqueous initiator from natural vitamin. The incorporation of Col II into MeGC hydrogels increased cellular condensation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. MeGC hydrogels containing Col II supported the release of TGF-β1 in a controlled manner over time in chondrogenic medium and the incorporated TGF-β1 further enhanced chondrogenesis of encapsulated chondrocytes and MSCs, especially synovial MSCs. Subcutaneous implantation of hydrogel cultures showed greatly improved neocartilage formation in constructs loaded with TGF-β1 compared with controls. These findings suggest that cartilage mimetic hydrogels have a high potential for cartilage repair.

Glutamine-chitosan modified calcium phosphate nanoparticles for efficient siRNA delivery and osteogenic differentiation

RNA interference (RNAi)-based therapy using small interfering RNA (siRNA) exhibits great potential to treat diseases. Although calcium phosphate (CaP)-based systems are attractive options to deliver nucleic acids due to their good biocompatibility and high affinity with nucleic acids, they are limited by uncontrollable particle formation and inconsistent transfection efficiencies. In this study, we developed a stable CaP nanocarrier system with enhanced intracellular uptake by adding highly cationic, glutamine-conjugated oligochitosan (Gln-OChi). CaP nanoparticles coated with Gln-OChi (CaP/Gln-OChi) significantly enhanced gene transfection and knockdown efficiency in both immortalized cell line (HeLa) and primary mesenchymal stem cells (MSCs) with minimal cytotoxicity. The osteogenic bioactivity of siRNA-loaded CaP/Gln-OChi particles was further confirmed in three-dimensional environments by using photocrosslinkable chitosan hydrogels encapsulating MSCs and particles loaded with siRNA targeting noggin, a bone morphogenetic protein antagonist. These findings suggest that our CaP/Gln-OChi nanocarrier provides an efficient and safe gene delivery system for therapeutic applications.

Photocrosslinkable chitosan hydrogels functionalized with the RGD peptide and phosphoserine to enhance osteogenesis

Hydrogels derived from naturally occurring polymers are attractive matrix for tissue engineering. Here, we report a biofunctional hydrogel for specific use in bone regeneration by introducing Arg-Gly-Asp (RGD)-containing cell adhesive motifs and phosphorylated serine residues, which are prevalent in native bone extracellular matrix and known to promote osteogenesis by enhancing cell-matrix interactions and hydroxyapatite nucleation, into photopolymerizable methacrylated glycol chitosan (MeGC). Incorporation of phosphoserine into MeGC hydrogels increased the ability of the hydrogels to nucleate mineral on their surfaces. RGD incorporation enhanced cell-matrix interactions by supporting attachment, spreading, and proliferation of bone marrow stromal cells (BMSCs) encapsulated in the hydrogels. Moreover, co-modification of MeGC hydrogels with RGD and phosphoserine synergistically increased osteogenic differentiation of encapsulated BMSCs in vitro. The bone healing capacity of the modified hydrogels was further confirmed in a mouse calvarial defect model. These findings suggest a promising hydrogel platform with a specific microenvironment tailored to promote osteogenesis for clinical bone repair.

Enhanced Osteogenesis of Adipose-Derived Stem Cells by Regulating Bone Morphogenetic Protein Signaling Antagonists and Agonists

Although adipose‐derived stem cells (ASCs) are an attractive cell source for bone tissue engineering, direct use of ASCs alone has had limited success in the treatment of large bone defects. Although bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors to promote osteogenic differentiation of ASCs, their clinical applications require supraphysiological dosage, leading to high medical burden and adverse side effects. In the present study, we demonstrated an alternative approach that can effectively complement the BMP activity to maximize the osteogenesis of ASCs without exogenous application of BMPs by regulating levels of antagonists and agonists to BMP signaling. Treatment of ASCs with the amiloride derivative phenamil, a positive regulator of BMP signaling, combined with gene manipulation to suppress the BMP antagonist noggin, significantly enhanced osteogenic differentiation of ASCs through increased BMP–Smad signaling in vitro. Furthermore, the combination approach of noggin suppression and phenamil stimulation enhanced the BMP signaling and bone repair in a mouse calvarial defect model by adding noggin knockdown ASCs to apatite‐coated poly(lactic‐coglycolic acid) scaffolds loaded with phenamil. These results suggest novel complementary osteoinductive strategies that could maximize activity of the BMP pathway in ASC bone repair while reducing potential adverse effects of current BMP‐based therapeutics.

Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct mesenchymal stem cell differentiation for bone repair

The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy. STATEMENT OF SIGNIFICANCE Application of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier with Osteoinductive Characteristics To Promote Bone Formation

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.